抗發炎中草藥之開發應用與機轉

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(1)國立臺灣師範大學生命科學系(所) 生理組博士畢業論文. 論文題目抗發炎中草藥之開發應用與機轉 The Development, Application, and Mechanism of Anti-inflammatory Traditional Herbal Medicine. 博士生：田安然 An-Jan Tien 指導教授：鄭劍廷院長 Chiang-Ting Chien. 中華民國 107 年 8 月.

(2) 1.Abstract ............................................................................................ 3 2. Introduction ..................................................................................... 5 3. Materials and Methods .................................................................... 9 3.1 Chemicals............................................................................................................... 9 3.2 Experimental model of liver injury ........................................................................ 10 3.3 In vivo and in vitro chemiluminescence recording for ROS activity ......................... 11 3.4 Inflammation, ED-1, 3-NT, 4-HNE, apoptosis and autophagy in the liver ................. 13 3.5 Western Blotting for p85, MAPK and CYP2E1 in the livers...................................... 15 3.6 Preparation of Monascus adlay (MA) .................................................................... 16 3.7 Animals and MA Treatments................................................................................. 17 3.8 FeCl3-induced carotid arterial time to occlusion (TTO) ........................................... 17 3.9 Grouping.............................................................................................................. 18 3.10 Lucigenin-Enhanced Chemiluminescence (CL) Counts........................................... 18 3.11 Soluble form of Vascular ICAM-1 and ROS assay .................................................. 19 3.12 In situ demonstration of ROS production and amount in the carotid artery .......... 19 3.13 Immunoblot analysis for NFκB, 3-NT, ICAM-1, CHOP, and Nrf2 ............................. 20 3.14 Preparation of Platelet Suspensions for Platelet activation and aggregation ......... 21 3.15 Expression of ICAM-1 and VCAM-1 on ROS-treated endothelial cells .................... 22 3.16 Statistical analyses.............................................................................................. 23. 4. Final Results ................................................................................... 23 4.1 Ingredients of A. cinnamomea fruiting body.......................................................... 23 4.2 Scavenging O2-., H2O2 and HOCl ability by ACW and AC........................................... 24 4.3 Exploring Anti-inflammatory effect of AC and ACW by 3-NT and 4-HNE stain and ED-1 infiltration............................................................................................................ 24 4.4 Anti-fibrosis effect of AC and ACW ........................................................................ 25 4.5 ACW or AC treatment inhibiting NF-κB mediated inflammation ............................. 25 4.6 inhibition of phosphorylated p85, phosphorylated MAPK and CYP2E1 expression by AC or ACW............................................................................................................ 26 4.7 Reducing DEN-evoked apoptosis and autophagy by AC and ACW in the livers......... 26 4.8 MA or MK pretreatment delays FeCl3-induced TTO ................................................ 42 4.9 MA or MK reduces FeCl3-induced arterial thrombosis and endothelial 3-NT and ICAM-1................................................................................................................. 43 4.10 MK or MK inhibited platelet activation and aggregation and endothelial ICAM-1 and VCAM-1 expression ...................................................................................... 45 4.11 Scavenging O2-., H2O2 and HOCl ability by MA and MK.......................................... 47 4.12 MA or MK treatment on FeCl3-enhanced arterial ROS and sICAM-1 levels............. 49 4.13 MA or MK on 3-NT, ICAM-1, CHOP and n-Nrf2 expression in FeCl3-treated arteries 51 1.

(3) 5. Discussion ...................................................................................... 52 6. Conclusion...................................................................................... 62 7.未來展望: ....................................................................................... 63 8. References ..................................................................................... 64 9、個人介紹...................................................................................... 70. 2.

(4) 1.Abstract Traditional Chinese herbal medicine has been widely used for thousands of years for health promotion, disease prevention and treatment. the clinical effect of Traditional Chinese herbal medicine is remarkable, but the mechanism of those herbal medicine are incompletely clear. In our study, we chose two famous and widely used herbal medicine to explore the mechanism by determine the anti-inflammatory and anti-oxidant effect. We chose Antrodia cinnamomea and Monascus Adlay as the study subject. Antrodia cinnamomea (A. cinnamomea), a popular medicinal mushroom in Taiwan, is widely used for prevention or treatment of liver diseases. Systematic studies on the anti-inflammatory effect of A. cinnamomea and its molecular mechanisms have not yet been fully investigated. HPLC fingerprint analysis identified seven ergostane-type triterpenoids from A. cinnamomea water extract (ACW) including highest content of Antcin K (AC), Antcin C, Antcin H, Dehydrosulphurenic acid, Antcin B, Antcin A and Dehydroeburicoic acid. Here, we explored the effects and mechanisms of ACW and the highest content AC on N-nitrosodiethylamine (DEN) induced liver inflammation, fibrosis and carcinogenesis in rats. In in vitro study, we found ACW and AC dose-dependently scavenged O2-., H2O2 and HOCl amount by a chemiluminescence analyzer. In in vivo experiment, oral intake ACW and AC significantly inhibited DEN-enhanced hepatocellular inflammation, fibrosis and carcinoma by pathologic observation, the elevated bile and liver reactive oxygen species (ROS) amounts, plasma γ-glutamyl transpeptidase, and oxidative stress including 3-nitrotyrosine, 4-hydroxynonenal and Kuppfer cell infiltration (ED-1 stains) in the inflammatory livers. DEN enhanced nuclear factor-κB (NF-κB) translocation, whereas ACW and AC suppressed DEN-enhanced NF-κB translocation through the inhibition of its upstream signaling of p85/phosphoinositide-3-kinase, mitogen activated protein 3.

(5) kinase and CYP2E1 expression. In conclusion, DEN can induce hepatocellular inflammation, fibrosis and carcinoma through the increase in NF-κB translocation to nucleus and oxidative injury. ACW and its active component, Antcin K, counteract DEN-induced hepatic injury and inflammation by the protective and therapeutic mechanisms of a direct scavenging ROS activity and an upregulation of antioxidant defense mechanisms. Monascus Adlay (MA) prepared from fungal fermentation of Monascus purpureus inoculating with cooked adlay contains high content of monakolin K (MK) and phenolic compounds. We explored whether MA and MK improve FeCl3-induced arterial thrombosis in rats. The rats were divided into control, FeCl3-treated rat carotid artery occlusion (TTO), TTO determined with one-week MA, and TTO determined with one-week MK. We compared MA or MK effects on oxidative stress by chemiluminescence amplification and immunohistochemistry, TTO by a transonic system, NFκB, ICAM-1, endoplasmic reticulum stress CHOP and Nrf2 signaling by western blotting. MA or MK efficiently depressed O2-, H2O2 and HOCl levels, platelet activation and aggregation and H2O2-enhanced ICAM-1 and VCAM-1 expression in the endothelial cells. FeCl3 significantly increased NFκB p65, 3-nitrotyrosine, CHOP and ICAM-1 expression, and decreased nuclear Nrf2 translocation and induces arterial thrombus formation. MA or MK pretreatment significantly elongated the level of FeCl3-induced TTO compared to TTO group, significantly decreased proinflammatory NF-κB/ICAM-1 signaling, endoplasmic reticulum stress CHOP expression and decreased thrombotic area. MA or MK significantly preserved nuclear Nrf2 translocation. MA and MK exerted a similar protective effect in attenuating thrombus formation. We suggest MA is better than MK to improve FeCl3-induced arterial thrombosis. 4.

(6) Through the study about Monascus Adlay (MA) and Antrodia cinnamomea, we found that anti-inflammatory and anti-oxidative effect are one of the main mechanism of Traditional herbal medicine. We can explore more about the mechanisms of the other traditional herbal medicine to discover the secret of Chinese Medicine.. Key word: Antrodia cinnamomea; Hepatic inflammation; Fibrosis; Hepatocarcinogenesis; Reactive oxygen species; Antcin K; N-nitrosodiethylamine； arterial thrombosis, ICAM-1, monacolin K, Monascus Adlay, platelet.. 2. Introduction. Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and. the most common type of liver cancer. The 5-year relative survival rate is about 7% and causes more than six leading deaths annually worldwide. It represents the third cause of cancer-related deaths and the first cause of death amongst cirrhotic patients. Hepatitis viral infection, food additives, alcohol, fungal toxins (afla toxins), toxic industrial chemicals, air and water pollutants are the major risk factors of HCC [1,2]. N-nitrosodiethylamine (DEN) is a potent hepatocarcinogenic nitrosamine present in tobacco smoke, water, cheddar cheese, cured and fried meals, occupational settings, cosmetics, agricultural chemicals and pharmaceutical agents [3,4]. DEN produces the pro-mutagenic products, O6-ethyl deoxy guanosine and O4 and O6-ethyl deoxy thymidine in livers leading to oxidative stress and inflammation in early stage and HCC in later status [3-5]. However, the detailed progress and mechanism for DEN-induced a simulated inflammation, fibrosis, and HCC in rats is not clearly defined. Inflammation and oxidative stress contribute to liver injury. Overproduction of reactive oxygen species (ROS) including O2-, H2O2 and NO, can cause lipid 5.

(7) peroxidation, protein oxidation, DNA damage and mutagenesis associated with various stages of liver injury [4,6]. The main sources of ROS may derive from the mitochondria of hepatocytes, the activated macrophages (ED-1; Kupffer cells), and the infiltrating neutrophils [7]. Induction of cytochrome P4502E1 (CYP2E1) and inducible nitric oxide synthase (iNOS) enhances further oxidative stress in the damaged liver [8]. ROS can also trigger translocation of nuclear factor-kappa B (NF-κB) to nucleus [9] and activation of several inflammatory signaling to release cytokines and adhesion molecules that contribute to further production of ROS, inflammatory response, and consecutive programmed apoptotic and autophagic cell death [2,10]. The increased ROS can enhance Bax/Bcl-2 ratio, caspase 3 expression, and poly-(ADP-ribose)-polymerase fragments subsequently resulting in apoptotic cell death and enhance the expression of the autophagy-promoting protein Beclin-1 expression leading to autophagy [5]. ROS accumulation leading to subsequent autophagy and apoptosis has been reported in the HCC [11]. The use of anti-inflammatory herbal medicine blocked the activation and translocation of NF-κB by suppressing the upstream kinases including Akt (protein kinase B), p85/phosphoinositide-3-kinase,. and. mitogen. activated. protein. kinase. (MAPK)/extracellular signal-regulated kinase (ERK) [12]. Therefore, the therapeutic strategy for inhibiting CYP2E1-mediated ROS production and p85/MAPK (ERK1/2)/NF-κB-mediated. inflammatory. signaling. protects. livers. against. DEN-induced injury. Antrodia cinnamomea (formerly named Antrodia camphorata), a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity by the fruiting bodies and liquid cultured mycelium. It has been widely used 6.

(8) to treated cancer and inflammation, and reported to possess antioxidative, vasorelaxative,. anti-inflammatory,. anti-angiogenic,. hepatoprotective,. and. anti-hepatitis B virus effects [13-20]. These reports suggest potential pharmaceutical applications for A. cinnamomea. However, A. cinnamomea can only grow on C. kanehirai Hayata, an endangered species native to Taiwan [13] and it grows at an extremely slow rate. Although laboratory culture systems can easily produce massive amounts. of. A.. cinnamomea. mycelium,. certain. specific. and. medical. compounds/metabolites are deficient in the artificial cultured mycelium. Furthermore, it is difficult to induce fruiting body formation [21,22]. Recently, a technique using basswood-cultivated A. cinnamomea successfully obtained massive production of fruiting bodies with high content of active triterpenoids like Antcin K (AC) and Antcin C [20]. In this study, we determined the potential effects and mechanisms of water extract (ACW) and its novel and active component AC from the fruiting bodies of A. cinnamomea on DEN-induced inflammation, fibrosis and carcinogenesis in the rat livers. Traditional Chinese herbal medicine has been widely used in modern medicine for health promotion, disease prevention and treatment. These include products made from fungi, such as Monascus spp., and grass crops, such as adlay (Cois lachrymal-jobi L. var. ma-yuen Stapf; also known as Chinese pearl barley and soft-shelled Job’s tears). Products of fermented Monascus spp. (e.g., anka and red koji) were first mentioned in the ancient Chinese pharmacopoeia, Pen-Chow-Kang-Mu (Systematic Pharmacopoeia ) by Li, S. C. in 1596, and are widely used as a Chinese cuisine [38-42] for its metabolic products, including monacolin K, γ-aminobutyric acid, dimerumic acid, and polyketide pigments and so on. The major metabolic product from fermented Monascus species is monacolin K (MK), which is known to 7.

(9) have. hypocholesterolemic,. anti-fibrosis,. anti-inflammatory,. antioxidant,. and. anti-apoptosis properties [43]. Adlay is widely and popularly planted in Taiwan, China, and Japan. It has a high nutritional value and is effective in the treatment of rheumatism and neuralgia as well as anti-inflammatory, antioxidant, anti-tumeric, and anti-helmintic properties [44,45]. Excess production of reactive oxygen species (ROS) may contribute to abnormal signal transduction or cellular dysfunction, initiating an oxidative stress cascade that leads to ICAM-1 mediated endothelial dysfunction and thrombosis formation [46,47]. Thrombus formation is a highly dynamic process that is mediated by proteins of the coagulation cascade, the subendothelial matrix as well as surface adhesion proteins on platelets, leukocytes and endothelial cells. In the pathological condition of thrombosis, oxidative stress or increased ROS is associated with impaired nitric oxide (NO) bioavailability and endothelial dysfunction, and contributes to the pathogenesis of acute vascular syndromes by predisposing to plaque rupture and intravascular thrombosis [46,47]. A dominant mechanisms of impaired vascular NO bioavailability relates to its oxidative inactivation by O2−•. A central feature of impaired endothelial function is the presence of ROS manifested by oxidized low-density lipoprotein in hypercholesterolemia, glycoxidation products in hyperglycemia, redox-active compounds in tobacco smoke, and lipid peroxides in hyperhomocysteinemia. [46].. Oxidized. lipids. can. also. be. generated. by. metal-dependent Fenton oxidation; enzyme-catalyzed oxidation by lipoxygenase or reaction with HOCl generated by myeloperoxidase; cell-dependent oxidation via a diversity of O2 − • and H2O2-generating oxidases; and oxidation by NO-derived reactive nitrogen species such as •NO2, nitryl chloride (NO2Cl), and peroxynitrite [46,47]. The improvement of endothelial function by decreasing oxidative stress and 8.

(10) inflammation would attenuate endothelial progenitor cell apoptosis and thrombosis formation [43]. We have demonstrated that inoculating cooked adlay with Monascus spp. produces monascus adlay (MA) containing both functional and active components [48]. MA extracts including rich MK and total phenol contents display higher antioxidant activity, reducing power, scavenging and chelating abilities than uninoculated adlay products [42,48]. MA and MK can depress oxidative stress evoked endoplasmic reticulum stress, apoptosis, autophagy and pyroptosis [48]. MK or lovastatin can inhibit urotensin enhanced expression of VCAM-1 and ICAM-1 by modulating the Rho activation, and NF-kB inhibitors [49]. In addition, phenolic components from adlay inhibit the release and secretion of inflammatory mediators/cytokines [50,51] and decrease O2-. production/generation [52]. However, the anti-thrombotic effect of MA has never been explored. We therefore explored whether dietary MA through its active component, MK, enhances antioxidant, anti-inflammatory, anti-endoplasmic reticulum stress and anti-adhesive mechanisms to counteracting oxidative stress-induced cardiovascular diseases, like thrombosis, in our FeCl3-induced thrombosis.. 3. Materials and Methods 3.1 Chemicals The powdered fruiting bodies of A. cinnamomea (POZO-4, BCRC930103, DNA sequencing listed in NCBI data bank, 18S ribosomal gene sequencing recorded number EU077558~61 and ITS ribosomal gene recorded number EU077562~65) are purchased from Po-Zone Enterprises Co., LTD (New Taipei City, Taiwan). The powdered fruiting bodies of A. cinnamomea (100 g) were boiled in the 1000 mL of distilled water for 3 hr. The water extract of fruiting bodies of A. cinnamomea (ACW) 9.

(11) filtered with 0.45 µm was analyzed by high-performance liquid chromatography (HPLC, Hewlett Packard 1100 series). The HPLC fingerprint of ACW was performed by Medical and Pharmaceutical Industry Technology and Development Center (Taipei, Taiwan). In the animal experiment, the ACW was given orally to rats at a dose of 6.67 mg/kg body weight/day for 8 weeks as described previously [23]. Control rats were given access to water alone. The reagents of H2O2, and N-nitrosodiethylamine (DEN) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). AC (Figure 2) was isolated from the fruiting bodies of A. cinnamomea as described previously [24] and provided by Po-Zone Enterprises Co., LTD (New Taipei City, Taiwan).. 3.2 Experimental model of liver injury Total 108 male Wistar rats (200-250 g) were housed at the Experimental Animal Center, National Taiwan Normal University, at a constant temperature and with a consistent light cycle (light from 07:00 to 18:00 O'clock). Food and water were provided ad libitum. All surgical and experimental procedures were approved by National Taiwan Normal University Animal Care and Use Committee and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997). The grouping and experimental design were indicated in Figure 1. To evaluate the co-treating effect of ACW or AC on DEN-induced liver injury, 72 animals were randomly divided into ten groups, group 1: control group (Con, n=18); group 2 with 2 weeks of DEN treatment (2WDEN, n=6); group 3 with 4 weeks of DEN treatment (4WDEN, n=6); group 4 with 8 weeks of DEN treatment (8WDEN, n=6); group 5 with 2 weeks of ACW and DEN treatment (2WDEN+ACW, n=6); group 6 with 4 weeks of ACW and DEN treatment (4WDEN+ACW, n=6), group 7 with 8 weeks of ACW and DEN treatment (8WDEN+ACW, n=6), group 8 with 2WDEN treatment plus antcin K (AC, 20 µg/kg body weight) co-treatment 10.

(12) (2WDEN+AC, n=6), group 9 with 4WDEN treatment plus AC co-treatment (4WDEN+AC, n=6) and group 10 with 8WDEN treatment plus AC co-treatment (8WDEN+AC, n=6). For exploration of the therapeutic potential of ACW, we also included the effect of post-treatment of ACW after 2 weeks of DEN injury in the rats subjected to 2 weeks (2WDEN+PACW, n=6), 4 weeks (4WDEN+PACW, n=6) and 8 weeks of DEN injury (8WDEN+PACW, n=6). For reducing the animal number in this study, the control group was used the group 1 as described above. In another 18 rats, an effective silymarin treatment (200 mg/kg) on DEN-treated hepatic injury was performed in the 2WDEN+silymarin (n=6), 4WDEN+silymarin (n=6) and 8WDEN+silymarin (n=6) groups. DEN was given as an initiator of liver injury at 500 ppm in the drinking water throughout the entire experiment (8 weeks). ACW at a dosage of 6.67 mg/kg body weight was given by daily gavage twice for 8 weeks.. 3.3 In vivo and in vitro chemiluminescence recording for ROS activity The in vivo ROS generation in response to DEN injury was measured from the liver. surface. by. intravenous. infusion. of. 2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo-. a. superoxide. anion. probe,. [1,2-a]-pyrazin-. 3-one-hydrochloride (MCLA) (0.2 mg/ml/h, TCI-Ace, Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan) and by the use of a Chemiluminescence Analyzing System (CLD-110, Tohoku Electronic In. Co., Sendai, Japan) [9]. MCLA, lucigenin and luminol are very sensitive chemiluminescence probes used to detect ROS and reactive nitrogen species formation. Briefly, the rat was maintained on a respirator and a circulating water pad at 37°C in a dark box with a shielded plate. Only the liver window was left unshielded and was positioned under a reflector, which reflects the photons from the exposed liver surface by the MCLA-amplified chemiluminescence onto the detector area. The 11.

(13) real-time displayed chemiluminescence signal was indicated as O2-. ROS level from the liver surface. The measurement of bile ROS or in vitro experiment was also detected by a lucigenin-amplification for O2-. determination [9] and luminol-amplification for H2O2 measurement [34]. In brief, the bile samples were immediately wrapped in aluminum foil and kept on ice until chemiluminescence measurement, usually done within 2 h [54]. Immediately before ROS measurement, 0.1 mL of phosphate-buffered saline (pH 7.4) was added to 0.2 ml of bile sample. The chemiluminescence was measured in a completely dark chamber of the Chemiluminescence Analyzing System (CLD-110). After 100-s background level determination, 1.0 mL of 0.1 mM lucigenin [54] or 1.0 mL of 0.2 mM luminol in phosphate-buffered saline (pH 7.4) was injected into the sample. The ROS signal was monitored continuously for an additional 300 s. The total amount of O2-. or H2O2 chemiluminescence was calculated by integrating of the area under the curve and subtracting it from the background level. Three major ROS like O2-., hydrogen peroxide (H2O2) and nitric oxide (NO) can initiate inflammation [9,35]. Therefore, we determined these three ROS in cell-free system in vitro, respectively. Superoxide generation was measured in a cell-free enzyme system by lucigenin-enhanced chemiluminescence. Assay solutions consisting of lucigenin (0.1 mM in 1.0 mL) and xanthine (10−5 M in 100 µl) were prepared in Krebs-HEPES buffer (composition (mM): NaCl 99.0, KCl 4.7, KH2PO4 1.0, MgSO4·7H2O 1.2, D-glucose 11.0, NaHCO3 25.0, CaCl2·2H2O 2.5, Na-HEPES 20.0) [36]. Aliquots (0.2 mL) of the assay solutions containing distilled water, different dosage of ACW or AC were placed into the CLD-110 chemiluminescence analyzer to measure background photon emission over 30 sec. After background counting was completed, xanthine oxidase (5 × 10−6 M, final concentration 10 U/mL) 12.

(14) was added to the mixture and photon emission, as a measure of O2− production, was counted for 300 sec. For H2O2 determination, chemiluminescent signals emitted from the test mixture containing PBS, different dosage of ACW or AC and 50 µL of H2O2 (0.03 %) were amplified by 1.0 mL of 0.2 mM luminol (Sigma) was measured with a ultrasensitive chemiluminescent analyzer (CLD-110) and the same protocol as O2-. detection [34]. For NO measurement, the NO chemiluminescence counts were examined in the test mixture containing the chemiluminescent probe 0.1 mM luminol, 10 mM H2O2, desferrioxamine 0.15 mM and K2CO3 2 mM. A NO probe in sodium phosphate buffer (0.1 M; pH 7.4) was prepared from zwitterionic polyamine/NO adducts for releasing NO including 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-l-triazene (NOC5) and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl- 1-triazene (NOC7) (Dojin Chemicals Co., Ltd., Kumamoto, Japan) [37]. The different dosage of ACW or AC was administered in the NO probe. After measuring the background of chemiluminescent. solution. mixture,. the. NO. probe. was. added. into. the. chemiluminescent solution and the chemiluminescent counts were detected by a chemiluminescence analyzer (CLD-110). All the assays were performed in six times for each sample, and total chemiluminescence counts in 300 s were calculated by integrating the area under the curve.. 3.4 Inflammation, ED-1, 3-NT, 4-HNE, apoptosis and autophagy in the liver The histologic features were assessed and scored for inflammation (portal and lobular) by hematoxylin and eosin and fibrosis by Masson trichrome as following. The grade of lobular inflammation was scored as 0 = no foci, 1 = < 2 foci/200xfield, 2 = 2-4 foci/200xfield, and 3 = > 4 foci/200xfield. Fibrosis was scored as 0 = none, 1 = zone 3 perisinusoidal fibrosis or portal/periportal, 2 = perisinusoidal and 13.

(15) portal/periportal fibrosis, 3 = bridging fibrosis, and 4 = cirrhosis [25]. We suggested that the high levels of ROS might promote hepatic accumulation of. leukocytes,. nitrated. 3-nityrotyrosine. (3-NT),. protein. and. lipid. 4-hydroxynonenal. peroxides. (4-HNE),. We and. immunostained ED-1. in. the. paraffin-embedded sections of liver tissues [9]. For these oxidative injury measurement, the rats (n=6 in each group) were sacrificed at the end of experiment. Hepatic sections were deparaffinized, rehydrated, and stained immunohistochemically for presence of in vivo markers of lipid peroxidation, 4-HNE-protein adducts, by incubation with a polyclonal antibody (Alpha Diagnostic International; San Antonio, TX, USA) and with rabbit polyclonal anti-3-NT antibody (Alpha Diagnostic International; San Antonio, TX, USA) diluted at 1:50. The percentage of 3-NT, or 4-HNE expression was calculated as 3-NT-, or 4-HNE-stained area/total area × 100% and analyzed by Adobe Photoshop 7.0.1 image software analysis. For hepatic macrophage (ED-1) staining, the tissue sections were incubated overnight at 4°C with a mouse anti-rat antibody to ED-1 (CD68, 1:200, Serotec, Sydney, NSW, Australia). A biotinylated secondary antibody (Dako, Botany, NSW, Australia) was then applied followed by streptavidin conjugated to HRP (Dako). The chromogen used was Dako Liquid diaminobenzene (DAB). Twenty high-power (×400) fields were randomly selected for each liver section, and the value of ED-1 positive cells was counted. We performed Beclin-1-related autophagy and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) apoptosis method [10] to investigate the presence and extent of two types of programmed cell death in DEN-induced hepatic injury. The hepatic sections (5 µm) were prepared, deparaffinized, and stained by the hematoxylin & eosin, Beclin-1staing, and TUNEL-avidin-biotin-complex 14.

(16) methods. A biotinylated secondary antibody (Dako, Botany, NSW, Australia) was then applied followed by streptavidin conjugated to HRP (Dako). The chromogen used was Dako Liquid diaminobenzene (DAB). Twenty high-power (×400) fields were randomly selected for each section, and the value of each oxidative stress was analyzed using a Sonix Image Setup (Sonix Technology Co., Ltd) containing image analyzing software Carl Zeiss AxioVision Rel.4.8.2 (Future Optics & Tech. Co. Ltd., Hangzhou, China).. 3.5 Western Blotting for p85, MAPK and CYP2E1 in the livers The liver protein concentration was determined by a BioRad Protein Assay (BioRad Laboratories, Hercules, CA, USA). Ten µg of protein was electrophoresed as described previously [9]. The expression of phosphorylated p85, phosphorylated MAPK, and CYP2E1 in liver tissue was evaluated by Western immunoblotting and densitometry. Briefly, the total proteins were homogenized with a prechilled mortar and pestle in extraction buffer, which consisted of 10 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 2% β-mercaptoethanol, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin. The mixtures were homogenized completely by vortexing and kept at 4°C for 30 min. The homogenate was centrifuged at 12,000 g for 12 min at 4°C, the supernatant was collected, and the protein concentrations were determined by BioRad Protein Assay (BioRad Laboratories). Antibodies raised against cytochrome P450 CYP2E1 (Chemicon International Inc., Temecular, CA, USA), phosphorylated p85 (Anti-PI3K p85, phosphor Y607, Abcam, Cambridge, MS, USA), phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204, Cell Signaling, Danver, MA, USA) and monoclonal mouse antimouse β-actin (Sigma, Saint Louis, MI, USA) were used at 1:400. All of these antibodies cross-react with the respective rat antigens. Proteins on SDS-PAGE gels 15.

(17) were transferred to nitrocellulose filters and stained as described. The density of the band with the appropriate molecular mass was determined semi-quantitatively by densitometry using an image analyzing system (Alpha Innotech, San Leandro, CA).. 3.6 Preparation of Monascus adlay (MA) Monascus fermented products were prepared on an adlay substrate, using a solid-state culture method as described previously [41,48]. In brief, adlay (obtained from the Erhlin Farmers’ Association, Changhua County, Taiwan) was immersed in deionized water for 2 h, dried and autoclaved at 121°C for 20 min. We inoculated M. purpureus Went (CCRC 31498; obtained from the Culture Collection and Research Center, Food Industry Research and Development Institute, Hsinchu City, Taiwan) onto malt extract agar (Difco agar, Voigt Global Distribution Inc, Kansas City, Missouri, USA) at 25°C for 72 h. We inoculated the mycelium into potato dextrose broth (Difco) and incubated it at 25°C for 7 d. The culture was homogenized in a blender and inoculated into autoclaved adlay with 10% M. purpureus Went at an inoculation rate of 5%. MA products were harvested after the fungal mycelia had colonized for 7 d at 25°C. MA was autoclaved at 121°C for 20 min and air-dried in an oven at 40°C. The dried MA was ground into a coarse powder (20 mesh) using a Restsch Ultra Centrifugal Mill and Sieving Machine (Haan, Germany). Our previous report had indicated the MK content in MA extract by a high-performance liquid chromatography (HPLC) [48]. Briefly, 1 g of powdered MA was extracted with 5 mL ethyl acetate at 70°C for 1.5 h. After 5 min of centrifugation at 1800 rpm and filtration through a 0.45-µm membrane, the filtrate was dried under a vacuum. One milliliter of acetonitrile was added to the resulting mixture, which was then filtered with a 0.45-µm pore size filter and analyzed by an HPLC system (Model L-6200, Hitachi, Japan). MK content in our prepared MA extract was 1.57 mg/g (0.157%). 16.

(18) 3.7 Animals and MA Treatments The onset of hemolysis and subsequent thrombosis and tissue infarction is faster in female than in male rats [53]. Therefore, we explored the medical efficacy of an MA diet and MK on 24 female Wistar rats (200–220 g; mean body weight, 206 ± 10 g) housed at the Experimental Animal Center of National Taiwan Normal University. The standard rat chow diet contained 58% carbohydrates, 28.5% proteins, and 13.5% fat (Laboratory Rodent diet 5001; Young Li Trading Company Ltd., Sijhih City, New Taipei City, Taiwan). We mixed powdered rat chow diet and powdered MA at a ratio of 99:1 by combining them through a 20-mesh sieve (aperture = 0.94 mm) [48]. We used 2% corn starch and 3% soybean oil to re-form the product into the MA lump diet. The same methods were used to crush and re-form the control, standard rat chow diet into food lumps. Animals were provided with food and tap water ad libitum.. 3.8 FeCl3-induced carotid arterial time to occlusion (TTO) All the rats were anesthetized by subcutaneous injection of 1.2 g/kg urethane (Sigma-Aldrich Inc., St. Louis, MO, USA). After arterial isolation, transonic flow probes (Probe# 0.5VBB517, Transonic Systems Inc., Ithaca, NY, USA) for carotid arterial blood flow measurement were applied and displayed on a small animal blood flow meter (Model 206, Transonic Systems Inc., Ithaca, NY, USA). All the blood flow signals were continuously recorded with an ADI System (PowerLab/16S, ADI Instruments, Pty Ltd, Castle Hill, Australia). The carotid arteries were injured as previously described11 with a slight modification. In brief, a filter paper (1 mm × 2 mm), soaked with 30% FeCl3 solution (Ferric chloride, Sigma, St. Louis, MO, USA), was applied to the artery for 3 minutes and the cavity was filled with saline immediately. The flow rate was continuously recorded, and the time to occlusion (TTO, arterial blood flow decreases to zero) was determined. For some animals, the arterial rings were fixed in 10% formalin after completing the thrombosis protocol, as 17.

(19) described previously [48]. The injured arterial segments were excised, embedded in paraffin, sectioned, and subjected to hematoxylin and eosin staining.. 3.9 Grouping Rats were randomly divided into the following groups: control diet without TTO (n=6), control diet with TTO (n=6), MA diet with TTO (n=6), and MK pretreatment with TTO (n=6). Both MA and MK treatments lasted for 2 weeks. MK (lovastatin) (A.G. Scientific, CA, USA) was dissolved in deionized water at a concentration of 1.0 mg/100 mL. We found that rats in the MA group ingested 30±3 g apiece, or approximately 0.47 mg of MK per rat [48]. Animals in the MK group drank approximately 40-50 mL of treated water apiece, for a mean dosage of MK around 0.47 mg/rat. These doses were based on Boyd's formula for body surface area, such that daily doses of MA and MK were equivalent to the daily recommended supplemental doses for adult humans (~2 g of Monascus-fermented products and ~20 mg of MK). All surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of National Taiwan Normal University and are in accordance with the guidelines of the National Science Council of the Republic of China (NSC 1997). All possible efforts were made to reduce the numbers of animals used and to minimize animal suffering during the experiment.. 3.10 Lucigenin-Enhanced Chemiluminescence (CL) Counts We considered that MA or MK may affect the degree and process of oxidative stress in the carotid arteries. We selected 900 sec of FeCl3 lesion to the carotid artery to determine the degree of ROS amount before its complete occlusion. We used lucigenin- and luminol-amplified chemiluminescence (CL) methods detect O2-., H2O2 and HOCl amounts in MA (65 mg/mL containing 0.1 mg MK), and MK (0.1 mg/mL). These data were compared to the control values measured in distilled water [54]. The 18.

(20) lucigenin-enhanced CL method has been confirmed to be a reliable assay for oxidative stress in damaged tissue [54]. We compared the differences in CL counts from carotid arteries in the rat subjected FeCl3 injury as well as the MA or MK treatment. The carotid artery was removed after each treatment and homogenized with saline in a 0.1 mL volume. ROS levels in the homogenized arteries were determined by a CL analyzer (CLD-110; Tohoku Electronic Industrial, Japan) after administration of 1.0 ml of 0.1 mM lucigenin in phosphate-buffered saline (pH 7.4) into the tested samples. The assay was performed in duplicate for each sample, and total CL counts in 600 s were calculated by integrating the area under the curve.. 3.11 Soluble form of Vascular ICAM-1 and ROS assay To obtain the quantified data of oxidative stress, in some rats after FeCl3 lesion for 900 s, the homogenates of arterial rings were used for measurement of soluble form of ICAM-1 (sICAM-1) by an ELISA kit (rat ICAM-1/CD54 Quantikine ELISA Kit) and H2O2–ROS amount by a luminol-amplified CL assay as described above. sICAM-1 is an important biomarker and the main cause for neutrophil adhesion to endothelium then to release ROS and to trigger thrombosis in the vessel wall [48,51].. 3.12 In situ demonstration of ROS production and amount in the carotid artery High levels of ROS might promote the expression of 3-nitrotyrosine (3-NT) and ICAM-1 in the endothelium to trigger thrombotic cascades. We considered that MA or MK may affect the degree and process of oxidative stress in the carotid arteries. We selected 900 s of FeCl3 lesion to the carotid artery to determine the degree of oxidative. stress. 3-NT and. ICAM-1. by immunocytochemical. stains.. For. immunocytochemical stains, the rats (n=3 in each group) were sacrificed at the end of experiment. 3-NT and the ICAM-1 [55] expression in the paraffin-embedded sections of the vascular rings were immunostained. The 5-µm cross-sections were stained with 19.

(21) anti-3-NT antibody (Alpha Diagnostic International; San Antonio, TX) and with ICAM-1 antibody (R&D Systems, Minneapolis, MN). The 3-NT and ICAM-1 stains were photographed on a Leica microscope (Leica Microsystems Wetzlar, Wetzlar, Germany). The percentage of staining in the vascular rings was calculated by the formula: % staining = stained curved length/total curved length. The thrombus size was determined by Adobe Photoshop 7.0.1 imaging software using the following formula: % thrombus size = thrombus area of intravascular area/total intravascular area × 100. 3.13 Immunoblot analysis for NFκB, 3-NT, ICAM-1, CHOP, and Nrf2 To explore proteins expression, we selected 4 h of FeCl3 lesion to the carotid artery to determine the degree of proinflammation transcription factor, NF-κB, oxidative stress biomarkers, 3-NT and ICAM-1, endoplasmic reticulum stress biomarker, CCAAT/-enhancer-binding protein homologous protein (CHOP), by western blotting. The immunoblotting method for western blotting was performed as described previously [51]. We determined the expression of 3-NT, ICAM-1, CHOP and β-actin in the total homogenates of carotid arterial tissues subjected to 30% FeCl3 lesion. We evaluated NF-κB p65 and Nrf2 in the nuclear proteins. The isolated arteries were placed in ice-cold isolation buffer containing 0.5 M sacarose, 10 mM Tris-HCl, 1.5 mM MgCl2, 10 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 2 mg/mL aprotinin, 4 mg/mL leupeptin, 2 mg/mL chymostatin, 2 mg/mL pepstatin, and 100 mg/mL 4-(2 aminoethyl)- benzenesulfonyl fluoride at pH 7.4 and were homogenized by using a tissue grinder. Then, the supernatant was resuspended in isolation buffer and the aliquots (nuclear fractions) were stored at -70°C. Antibodies 20.

(22) raised against NFκB (R&D Systems, Minneapolis, MN), LaminA/C, Nrf2, CHOP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 3-NT (Alpha Diagnostic International, San Antonio, TX), ICAM-1 (R&D Systems, Minneapolis, MN, USA) and β-actin (catalog no. A5316, clone AC-74, Sigma) were used. The immunoreactive bands were detected by incubation with each respective antibody; the secondary antibody. alkaline. phosphatase;. and,. finally,. nitroblue. tetrazolium,. 5-bromo-4-chloro-3-indolyl phosphate, and a toluidine salt (Roche Diagnostic, Mannheim, Germany) stock solution for 30 minutes at room temperature.. 3.14 Preparation of Platelet Suspensions for Platelet activation and aggregation Rat platelet suspensions were prepared as described previously [56]. In brief, blood (5 mL) was drawn from the carotid artery into plastic tubes containing 1 mL of 3.8% sodium citrate buffer (blood : sodium citrate = 9 : 1). Platelet-rich plasma was obtained by low-speed centrifugation (1500 rpm for 10 min) and further centrifuged at 15,000 rpm for 10 min to obtain a platelet pellet. The platelets were suspended in of 190 µL aliquots of Ca2+-free Tyrode's solution (pH 7.35) and were incubated with the indicated concentrations of MA extract, MK or vehicle (DMSO) for 30 min at 37 °C. MA or MK was dissolved in DMSO as a stock solution and stored at − 20 °C. The platelet number was adjusted to 3 × 108/mL before use. A turbidimetric method was adapted to measure platelet aggregation with a lumi-aggregometer (Payton Scientific, Scarborough, ON, Canada). Specifically, 10 µL of 2 mM ADP was administered to the platelet suspension (0.4 mL) for 5 min and the response of aggregation was expressed in light-transmission units. For platelet activation, the washed platelets, pretreated with MA or MK were stimulated with collagen (2.5 µg/mL) and incubated for 5 min at 37°C. A detailed procedure was described below. The reaction was terminated, and the platelets were 21.

(23) centrifuged followed by resuspension in ice-cold phosphate buffere saline (PBS) containing 10% fetal calf serum (FCS) and 1% sodium azide. The platelets were incubated with CD62P primary antibody (P-selectin, Thermo Fisher Scientific Taiwan, Co., Ltd., Taipei, Taiwan) in 3% bovine serum albumin (BSA)/PBS for 30 min at 4 °C in the dark, then fixed and washed three times by centrifugation at 400×g for 5 min. The platelets were resuspended in ice-cold PBS followed by FITC-conjugated secondary antibody (Santa Cruz Biotechnology) incubation in 3% BSA/PBS for 30 min at 4 °C in the dark. Platelets were again washed three times by centrifugation at 400×g for 5 min and resuspended in ice-cold PBS, 3% BSA and 1% sodium azide. The samples were analyzed with a FACSCalibur flow cytometer using CellQuest software (BD Biosciences, San Jose, CA, USA).. 3.15 Expression of ICAM-1 and VCAM-1 on ROS-treated endothelial cells To investigate the effect of MA or MK on ROS-treated cells, human umbilical vein endothelial cell (HUVEC), HUVEC were exposed to H2O2 (100 nmol/L) in the presence of MA extract (255 µg/mL), MK (400 ng/mL) or isovolumetic solvent control (0.5% DMSO) for 24 h. HUVECs (ATCC CRL-2873) were cultured in EGM2 medium supplemented with 2% (v/v) fetal calf serum as described [56]. Cells were used at passages 3–4, and all cells exhibited the specific characteristics of endothelial cells. HUVECs were seeded at 1 × 105 cells/mL on 0.8% gelatin-coated 24-well plates. After 24 h, the medium was replaced with serum-free medium, and cells were incubated overnight. After 1 h, the medium was replaced with normal growth medium or medium containing MA, MK or vehcle (1–50 µg/mL). Cell plates were examined by fluorescence microscopy (Nikon TS100, Tokyo, Japan). The ICAM-1 and VCAM-1 expression level in HUVEC were determined by immunofluorescence staining [57,58]. For ICAM-1 and VCAM-1 expression studies, cells were stained by 22.

(24) a rat anti-human/mouse ICAM-1 or VCAM-1 antibody (Santa Cruz Biotechnology) at 4 °C overnight. The secondary antibody, FITC-labeled goat anti-rat (Santa Cruz Biotechnology), was applied at 37 °C for 30 min followed by staining of the nuclei with 4,6-diamidino-2-phenylindole (DAPI; Sigma).. 3.16 Statistical analyses All data of Antrodia cinnamomea were expressed as mean ± standard error mean. Differences within groups were evaluated by a paired t test. Two-way ANOVA was used to establish differences among groups. Intergroup comparisons were made by Duncan’s multiple-range test. Differences were regarded as significant if P < 0.05 was attained. All values of Monascus adlay were expressed as mean ± standard error mean (SEM). Differences within groups were evaluated by paired t-test. One-way analysis of variance was used for establishing differences among groups. Intergroup comparisons were made by Duncan's multiple-range test. Differences were regarded as significant if P < 0.05 was adapted.. 4. Final Results 4.1 Ingredients of A. cinnamomea fruiting body We determined the possible ingredients existing in the water extract of A. cinnamomea fruiting body. As shown in Figure 1, HPLC fingerprint identified seven major components and the respective structure of antcin K (ST14, 1680.5 mg), antcin C (ST4, 248.2 mg), antcin H (ST12, 105.2 mg), dehydrosulphurenic acid (TR15, 2.3 mg), antcin B (ST3, 250.3 mg), antcin A (1.3 mg) and dehydroeburicoic acid (TR2, 2.1 mg) from 100 g of A. cinnamomea fruiting body. Our data found that the highest content of ergostane-type triterpenoids is antcin K (1.68 %) existing in the fruiting body of A. cinnamomea. 23.

(25) 4.2 Scavenging O2-., H2O2 and HOCl ability by ACW and AC. We explored the dosage response of ACW and AC on scavenging O2-., H2O2. and NO activity in vitro. As shown in Figure 3, we found that ACW (Figure 3A) at the concentration larger than 0.25 mg/ml and AC (Figure 3B) at the concentration larger than 1 µg/ml significantly and dose-dependently scavenged O2-., H2O2 and NO activity in vitro. Higher concentration of ACW and AC directly and further reduced O2-., H2O2 and NO activity. These data implicate AC and ACW can scavenge these ROS directly and in a dose-dependent manner.. 4.3 Exploring Anti-inflammatory effect of AC and ACW by 3-NT and 4-HNE stain and ED-1 infiltration We explored the effect of co-administration of AC and ACW on inflammation by H&E stain and oxidative stress by 3-NT and 4-HNE stain and ED-1 infiltration after 2 weeks of DEN-induced liver injury. There are significantly increases in inflammation (Figure 4B), 3-NT (Figure 4F), 4-HNE (Figure 4J) and ED-1 stain (Figure 4N) in 2WDEN group as compared to Con group. Co-treatment of AC (2WDEN+AC) or ACW (2WDEN+ACW) significantly decreased these inflammatory and. oxidative. parameters. (Figures. 4I-4L).. We. explored. the. effect. of. co-administration of AC and ACW on inflammation by H&E stain, oxidative stress by 3-NT and 4 HNE stain and ED-1 infiltration after 4 weeks of DEN-induced liver injury (Figure 5). 4WDEN markedly increased in inflammation (Figure 5B), 3-NT (Figure 5F), 4-HNE stain (Figure 5J) and ED-1 number (Figure 5N) in the livers compared to Con group. Co-treatment of AC or ACW for 4 weeks significantly reduced these parameters in the 4WDEN+AC and 4WDEN+ACW groups (Figures 5I-5L). We determined the effect of co-treatment of AC and ACW on tumor formation by H&E stain and oxidative stress by 3-NT and 4 HNE stain and ED-1 infiltration 24.

(26) after 8 weeks of DEN-induced liver injury (Figure 6). Eight weeks of DEN injury induced HCC formation by the increased ratio in liver weight/body weight (Figure 6Q) and significantly increased 3-NT (Figure 6R), 4-HNE (Figure 6S) and ED-1 infiltration (Figure 6T) in the 8WDEN group as compared to Con group. Co-treatment of AC or ACW for 8 weeks significantly reduced these parameters in the 8WDEN+ACW and 8WDEN+AC groups. We therefore determined the possible therapeutic effect of post-treatment of ACW on liver function and oxidative parameters after 2 weeks of DEN injury in the 2WDEN+PACW, 4WDEN+PACW and 8WDEN+PACW groups (Figure 7). After 2 weeks of DEN injury, ACW was started to administer to the rats and at this moment, all the parameters were not depressed in 2WDEN+PACW group as compared to 2WDEN group. We also found that the degree of inflammation, 3-NT, 4 HNE stains and ED-1 number was significantly reduced in 4WDEN+PACW and 8WDEN+PACW groups, not in 2WDEN+PACW (Figure 7) as compared respective time-matched DEN group.. 4.4 Anti-fibrosis effect of AC and ACW We evaluated the effect of co-treatment of AC and ACW on DEN-induced hepatic fibrosis by Masson stain and collagen content of the damaged livers (Figure 8). There are significantly blue Masson stains and higher liver collagen contents in 2WDEN, 4WDEN and 8WDEN livers.. Co-treatment of AC or ACW significantly. reduced these fibrosis parameters in the 2WDEN+AC, 2WDEN+ACW, 4WDEN+AC, 4WDEN+ACW, 8WDEN+AC and 8WDEN+ACW livers.. 4.5 ACW or AC treatment inhibiting NF-κB mediated inflammation We explored the effect of co-administration of AC or ACW on DEN-induced NF-κB mediated inflammation by EMSA assay (Figure 9). Gel shift of nuclear transcription factor NF-κB by EMSA found that NF-κB transcription factor increased 25.

(27) in the nucleus fraction of 2WDEN, 4WDEN and 8WDEN livers. ACW or AC treatment greatly inhibited DEN-induced NF-κB transcription factor shift in 2WDEN+ACW, 4WDEN+ACW, 8WDEN+ACW and 8WDEN+AC livers.. 4.6 inhibition of phosphorylated p85, phosphorylated MAPK and CYP2E1 expression by AC or ACW We determined the response of oxidative stress protein and upstream molecules for regulating NF-κB transcription factor in the AC or ACW treated livers subjected to DEN injury. The effect of concomitant administration of AC or ACW, and post-treatment ACW (PACW) on DEN-induced phosphorylated p85, phosphorylated MAPK and CYP2E1 was indicated in Figure 10. DEN treatment time-dependently and significantly enhanced phosphorylated p85 and MAPK expression and CYP2E1 protein expression in the 2WDEN, 4WDEN and 8WDEN livers compared to Con group. Co-treatment of ACW in 2DEN+ACW, 4DEN+ACW and 8WDEN+ACW groups significantly depressed DEN-enhanced hepatic p85, MAPK and CYP2E1 expression compared to respective DEN group. Co-treatment of AC in 2DEN+AC, 4DEN+AC and 8WDEN+AC groups also significantly decreased DEN-enhanced hepatic phosphorylated p85, phosphorylated MAPK and CYP2E1 expression compared to respective DEN group. After 2 weeks of DEN induction, PACW treatment significantly depressed DEN-enhanced hepatic phosphorylated p85, phosphorylated. MAPK. and. CYP2E1. expression. compared. to. respective. 4WDEN+PACW and 8WDEN+PACW groups, but not in 2WDEN+PACW group. According to our data, the protective potential in inhibition of phosphorylated p85, phosphorylated MAPK and CYP2E1 expression seems to be displayed in an order of ACW > AC >PACW.. 4.7 Reducing DEN-evoked apoptosis and autophagy by AC and ACW in the livers 26.

(28) We compared several treatments on DEN-induced apoptosis and autophagy in the livers. Figure 11 indicated the effect of silymarin, AC, ACW co-treatment or post-treatment PACW on DEN-induced Beclin-1-mediated autophagy (Figure 11A) and apoptosis-TUNEL responses (Figure 11B) by immunohistochemistry. These data showed that DEN toxicity significantly increased Beclin-1-positive stain and TUNEL-positive stain in 4WDEN and 8WDEN livers. Co-treatment of silymarin, AC, ACW or PACW significantly depressed DEN-enhanced Beclin-1 and TUNEL positive stain after 4 or 8 weeks of DEN injury when compared to their respective 4WDEN or 8WDEN livers. 8WDEN+AC, 8WDEN+ACW and 8WDEN+PACW significantly reduced DEN-evoked apoptosis and autophagy when compared to 8WDEN group.. 27.

(29) Figure 1. The grouping and experimental design.of Antrodia cinnamomea. 28.

(30) Figure 2. HPLC analysis of several components of extracts from the fruiting bodies of A. cinnamomea. Within 110 min, seven components are identified by the HPLC 29.

(31) fingerprint analysis and the respective structure of antcin K (AC, ST14), Antcin C (ST4), Antcin H (ST12), Dehydrosulphurenic acid (TR15), Antcin B (ST3), Antcin A and Dehydroeburicoic acid (TR2) is indicated. Within 80 min, five major components are identified. Note that the highest content is Antcin K.. Figure 3. Different concentration of water extract (ACW) (A) and Antctin K (AC) (B) of A. cinnamomea on scavenging O2-., H2O2 and NO activity in vitro. These data show that the concentration from ACW of A. cinnamomea at a dose larger than 0.25 mg/mL significantly reduced the oxidative stress in O2-., H2O2 and NO. In addition, AC at a dose larger than 1 µg/mL displayed a similar response in significant inhibition of O2-., H2O2 and NO activity like ACW. All the experiments were repeated six times. * P < 0.05 when compared to control value. 30.

(32) Figure 4. Effect of concomitant administration of Antcin K (AC) and water extract (ACW) of Antrodia cinnamomea on inflammation by H&E stain (A-D), oxidative stress by 3-NT (E-H) and 4-HNE (I-L) stain and ED-1 infiltration (M-P) after 2 weeks of DEN-induced liver injury (2WDEN). There are significantly increases in inflammation (Q), 3-NT (R), 4-HNE (S) and ED-1 stain (T) in 2WDEN livers as compared to Con rats. Co-treatment of AC and ACW significantly reduced these parameters in 2WDEN+AC and 2WDEN+ACW rats. The scale bar is 50 µm. * P < 0.05 when compared to Con value. # P <0.05 when compared to 2WDEN group. 31.

(33) Figure 5. Effect of concomitant administration of Antcin K (AC) and water extract (ACW) of Antrodia cinnamomea on inflammation by H&E stain (A-D), oxidative stress by 3-NT (E-H) and 4 HNE (I-L) stain and ED-1 infiltration (M-P) after 4 weeks of DEN-induced liver injury (4WDEN). There are significantly increases in inflammation (Q), 3-NT (R), 4-HNE (S) and ED-1 stain (T) in 4WDEN livers. Co-treatment of AC and ACW significantly reduced these parameters in 4WDEN+AC and 4WDEN+ACW rats. The scale bar is 50 µm. * P < 0.05 when compared to Con value. # P <0.05 when compared to 4WDEN group.. 32.

(34) Figure 6. Effect of concomitant administration of Antcin K (AC) and water extract (ACW) of Antrodia cinnamomea on inflammation and tumor formation by H&E stain (A-D), oxidative stress by 3-NT (E-H) and 4 HNE (I-L) stain and ED-1 infiltration (M-P) after 8 weeks of DEN-induced liver injury (8WDEN). There are significantly increases in ratio of liver weight/body weight (Q), 3-NT (R), 4-HNE (S) and ED-1 stain (T) in 8WDEN livers. Co-treatment of AC and ACW significantly reduced these parameters in 8WDEN+AC and 8WDEN+ACW rats. The scale bar is 50 µm. * P < 0.05 when compared to Con value. # P <0.05 when compared to 8WDEN group.. 33.

(35) Figure 7. Effect of post-treatment of water extract (PACW) of Antrodia cinnamomea on inflammation by H&E stain (A-D), oxidative stress by 3-NT (E-H) and 4 HNE (I-L) stain and ED-1 infiltration (M-P) in the 2WDEN+PACW, 4WDEN+PACW and 8WDEN+PACW livers. The degree of inflammation (Q), 3-NT (R), 4-HNE (S), ED-1 stain (T) and plasma level of γ-GT (U) is indicated respectively in these DEN-treated livers. The scale bar is 50 µm. * P < 0.05 when compared to Con value. # P <0.05 when compared to 2WDEN+PACW group.. 34.

(36) Figure 8. Effect of co-treatment of Antcin K (AC) and water extract (ACW) of A. cinnamomea on fibrosis by Masson stain (A, B) and collagen content (C) in the DEN-treated livers. There are significantly blue Masson stains and hepatic collagen contents in DEN-treated livers in 2WDEN, 4WDEN and 8WDEN groups. 35.

(37) Co-treatment of AC or ACW significantly reduced these parameters in 2WDEN+ACW, 2WDEN+AC, 4WDEN+ACW, 4WDEN+AC, 8WDEN+ACW and 8WDEN+AC livers. The scale bar is 50 µm. * P < 0.05 when compared to Control value. a P < 0.05 when compared to 2WDEN. b P < 0.05 when compared to 4WDEN. c P < 0.05 when compared to 8WDEN. The level of collagen was determined with the Sircol collagen kit according to the manufacture’s instruction.. 36.

(38) Figure 9. Effect of concomitant administration of water extract of A. cinnamomea (ACW) and AC on DEN-induced inflammation by EMSA assay. Gel shift of nuclear transcription factor NF-κB by EMSA found NF-κB transcription factor increased by DEN stimulation for 2-8 weeks (A). ACW or AC treatment markedly reduced DEN-induced NF-κB transcription factor shift. NC, negative control. The statistic data from triple experiments are demonstrated in (B). * P < 0.05 compared to Con group. # P < 0.05 compared to respectively time-matched DEN group.. 37.

(39) Figure 10. Effect of concomitant administration of Antcin K (AC) or water extract of A. cinnamomea (ACW), and post-treatment ACW (PACW) on phosphorelated p85, phosphorylated MAPK and CYP2E1 expression by western blot (A). DEN injury significantly enhanced p85 (B), MAPK (C) and CYP2E1 protein expression (D) in the 38.

(40) 2WDEN, 4WDEN and 8WDEN livers compared to Con group. Co-treatment of ACW in 2DEN+ACW, 4DEN+ACW and 8WDEN+ACW groups significantly depressed DEN-enhanced hepatic phosphorylated p85, phosphorylated MAPK and CYP2E1 expression compared to respective DEN group. Co-treatment of AC in 2DEN+AC, 4DEN+AC and 8WDEN+AC groups also significantly decreased DEN-enhanced hepatic p85, MAPK and CYP2E1 expression compared to respective DEN group. After 2 weeks of DEN induction, PACW treatment significantly depressed DEN-enhanced hepatic p85, MAPK and CYP2E1 expression compared to respective 4WDEN+PACW and 8WDEN+PACW groups. * P < 0.05 compared to Con group. # P < 0.05 compared to respectively time-matched DEN group. a P < 0.05 compared to respective DEN+ACW group. b P < 0.05 compared to respective DEN+AC group.. 39.

(41) Figure 11. Effect of silymarin, antcin K (AC), co-treatment of water extract of A. cinnamomea pretreatment (ACW) or post-treatment of water extract of A. 40.

(42) cinnamomea (PACW) 2 weeks after DEN injury on Beclin-1-mediated autophagy (A) and apoptosis-TUNEL (B) responses by immunohistochemistry. DEN injury significantly enhanced Beclin-1-positive stain and TUNEL-positive stain at 4WDEN and 8WDEN livers. Co-treatment of silymarin, AC, ACW or PACW significantly depressed DEN-enhanced Beclin-1 and TUNEL positive stains compared to respective 4WDEN and 8WDEN livers. 8WDEN+AC and 8WDEN+ACW displayed a stronger effect in decreasing Beclin-1-autophagy and TUNEL-apoptosis than the positive group of 8WDEN+silymarin. * P < 0.05 compared to 2WDEN group. # P < 0.05 compared to 4WDEN group. a P < 0.05 compared to respectively time-matched DEN group. b P < 0.05 compared to time-matched silymarin group. c P < 0.05 compared to time-matched AC group. d P < 0.05 compared to time-matched ACW group.. 41.

(43) 4.8 MA or MK pretreatment delays FeCl3-induced TTO As shown in Figure 12A, the definition of TTO was indicated in one control, MA- and MK-treated rat subjected to FeCl3 lesion, respectively. Topical application of 30% FeCl3 efficiently induced carotid arterial blood flow cessation, which was designated to TTO within 10-20 min. The use of MA or MK pretreatment for two weeks significantly (P < 0.05) elongated FeCl3-induced TTO levels (Figure 12B) when compared to TTO group. The value of TTO between MA+TTO and MK+TTO groups was not significantly different.. Figure 12. Effects of MA and MK treatment on FeCl3-reduced carotid arterial blood 42.

(44) flow (A) and the statistical data of TTO levels (B) in three groups of control, MA or MK treated rats (n=6 each). MA or MA treatment significantly delayed FeCl3-induced carotid arterial blood flow occlusion to zero. *, P < 0.05 versus the Control group.. 4.9 MA or MK reduces FeCl3-induced arterial thrombosis and endothelial 3-NT and ICAM-1 As shown in Figures 13B-13D, we found that FeCl3-induced carotid arterial thrombosis by the appearance of thrombus in the carotid artery when compared to non-treated control rat (Figure 13A). A less expression of endothelial 3-NT (Figure 13E) and ICAM-1 (Figure 13I) was found in the control carotid arteries. FeCl3 stimulation markedly enhanced endothelial 3-NT (Figures 13F-13H) and ICAM-1 (Figures 13J-13L) expression in the carotid arteries. MA or MK pretreatment effectively reduced carotid arterial thrombotic area by the inhibition of endothelial 3-NT and ICAM-1 accumulation indicated by black arrows in Figure 13.. 43.

(45) Figure. 13 Effects of MA or MK treatment on FeCl3-induced carotid arterial thrombosis (A-D) by H&E stain and oxidative stress (E-H) analyzed by 3-NT stain and I-CAM-1 (I-L) in the carotid arteries from Control, TTO, TTO+MA and TTO+MK groups. FeCl3 markedly induced thrombosis and increased 3-NT and ICAM-1 stains in the endothelium indicated by black arrows. MA or MK treatment efficiently reduced thrombosis and 3-NT and ICAM-1 stains in the endothelial area.. 44.

(46) 4.10 MK or MK inhibited platelet activation and aggregation and endothelial ICAM-1 and VCAM-1 expression MA or MK significantly decreased collagen-enhanced platelet activation by the downregulation of P selectin expression in the washed platelets (Figure 14A). In addition, MA or MK significantly inhibited platelet aggregation stimulated by ADP (Figure 14B). The statistic data showed that MA seems to have a high tendency but not significant in inhibition of platelet activation and aggregation compared with MK. H2O2 significantly increased fluorescent ICAM-1 expression (Figure 14C) and fluorescent VCAM-1 expression (Figure 14D) in the cultured HUVECs. MA or MK significantly depressed these ICAM-1 and VCAM-1 expressions with a similar effect between MA and MK.. 45.

(47) B *. 25 #. 20. #. 15. *. *. 10 5 0. Co. C Fluorescent ICAM-1 (%). 100. *. 80 60. #. 40. *. #. *. 20 0. n Co DMSO n+MA n+MK e e en+ ollag ollag ll ag C C. n Co DMSO P+MA P+MK AD AD P+ AD. D 30. *. 25 20. #. 15 #. 10. *. *. 5 0. n Co. K A O2 H2 2+M O2+M O H2 H2. Fluorescent VCAM-1 (%). P-selectin (%). 30. Platelet aggregation (%). A. 40 30 # 20. #. *. 10 0. *. n Co. H2. K A O2 +M +M O2 O2 2 2 H H. Figure 14. Effect of MA or MK on platelet activation and aggregation in washed platelets and H2O2-induced endothelial ICAM-1 and VCAM-1 expression in endothelial cells. A: Collagen activated the platelets by the increase of P selectin expression. MA or MK significantly inhibited platelet activation by downregulating P selectin expression. B: ADP enhanced platelets aggregation, whereas MA or MK significantly inhibited ADP-enhanced platelet aggregation. H2O2 significantly increased fluorescent endothelial ICAM-1 (C) and VCAM-1 expression (D) in endothelial cells. MA or MK treatment significantly depressed H2O2-enhanced. 46.

(48) endothelial ICAM-1 and VCAM-1 expression in endothelial cells. *P <0.05 versus DMSO-control (Con) group; #P < 0.05 versus H2O2 group.. 4.11 Scavenging O2-., H2O2 and HOCl ability by MA and MK. As shown in Figure 15, MA or MK significantly (P < 0.05) reduced O2-., H2O2 and HOCl counts implicating that their wide-range antioxidant ability in MA or MK. This data also informed that MA via its active component, MK, can inhibit O2-., H2O2 and HOCl activity. Our data also evidence that MA is more effective than MK in reducing O2-., H2O2 and HOCl counts, implicating the phenolic compounds from adlay work synergistically with MK to increase the antioxidant ability of MA.. 47.

(49) O2-. CL (counts/10 s) H2O2 CL (counts/10 s) HOCl CL (counts/10 s). 6000 4500 3000. #. 1500 0. * Con. MA. * MK. 8000 6000 4000. # 2000 0. * Con. MA. * MK. 4000 3000 2000. #. 1000. *. *. 0. Con. MA. MK. Figure 15. O2-.-, H2O2- and HOCl scavenging activity of the control (distilled water; Con), MA, and monacolin K (MK). MA is better than MK in scavenging O2-.-, H2O2and HOCl activity. *, P < 0.05 in comparison with Con; #, P < 0.05 in comparisons with MA.. 48.

(50) 4.12 MA or MK treatment on FeCl3-enhanced arterial ROS and sICAM-1 levels To further confirm FeCl3-induced oxidative stress in the carotid arteries, we determined the level of ROS and sICAM-1 by biochemical assay in the carotid arterial homogenates in the control, MA and MK groups. Figure 16A demonstrated that FeCl3 significantly (P < 0.05) increased ROS CL counts in the control, MA and MK group by luminol-amplified CL methods when compared to non-treated control arteries. MA or MK treatment significantly (P < 0.05) decreased FeCl3-enhanced ROS CL counts compared to TTO group. Figure 4B also showed that FeCl3 significantly (P < 0.05) elevated sICAM-1 levels in the control, MA or MK group by an ELISA assay compared to non-treated control group. Similarly, MA or MK treatment significantly (P < 0.05) depressed FeCl3-enhanced arterial sICAM-1 levels compared with TTO group. There is no significance between MA and MK groups.. 49.

(51) ROS CL (counts/10 sec). A. sICAM-1 (pg/mg protein). B. 2000. * 1500. #. #. *. 1000. *. 500 0. rol Cont. TTO TO+MA TO+MK T T. 2000. * #. 1500. #. *. *. 1000 500 0. rol Cont. TTO TO+MA TO+MK T T. Figure 16. Effect of MA or MK treatment on FeCl3-enhanced ROS (A) and sICAM-1 (B) levels in the FeCl3-treated carotid arteries in Control, TTO, TTO+MA or TTO+MK. group.. A:. TTO. significantly. increased. ROS. CL counts. by. luminol-amplified CL methods when compared to Control arteries. MA or MK treatment significantly attenuated TTO-enhanced ROS CL counts compared with TTO group. B: TTO significantly elevated sICAM-1 levels by ELISA assay compared to Control group. MA or MK treatment significantly decreased FeCl3-enhanced arterial sICAM-1 levels compared with TTO group. * P < 0.05 when compared to Control group. # P < 0.05 when compared to TTO group. 50.

(52) 4.13 MA or MK on 3-NT, ICAM-1, CHOP and n-Nrf2 expression in FeCl3-treated arteries With western blot analysis, FeCl3 significantly enhanced 3-NT (Figure 17A), ICAM-1 (Figure 17B), and CHOP expression (Figure 17D) and decreased n-Nrf2 expression (Figure 17C) in the damaged carotid arteries of the TTO group when compared to Control group. MA or MK treatment significantly attenuated FeCl3-enhanced 3-NT, ICAM-1 and CHOP expression and partly preserved n-Nrf2 expression compared with TTO group.. Figure 17. Effect of MA or MK treatment on FeCl3-enhanced nuclear NF-κB translocation (A), 3-NT (B), ICAM-1 (C), CHOP (D) and nuclear Nrf2 (n-Nrf2) (E) 51.

(53) expression in the FeCl3-treated carotid arteries in Control, TTO (T), TTO+MA (T+MA) or TTO+MK (T+MK) group. FeCl3 significantly enhanced nuclear NF-κB translocation, increased arterial 3-NT, ICAM-1 and CHOP expression and significantly decreased n-Nrf2 expression when compared to Control arteries. MA or MK treatment significantly attenuated FeCl3-enhanced nuclear NFκB translocation, 3-NT, ICAM-1 and CHOP expression and partly preserved n-Nrf2 expression compared with TTO group. * P < 0.05 when compared to Con group. # P < 0.05 when compared to T group.. 5. Discussion. According to our findings, DEN treatment increased oxidative stress. including the increased level in liver ROS, bile O2-. and H2O2, ED-1 stain, 3-NT and 4-HNE expression. After 4 weeks or 8 weeks of DEN, hepatic fibrosis and HCC occurred in the livers. ACW and its active component AC displayed dose-dependent and efficient ability to scavenge O2-., H2O2, and NO. Co-treatment of ACW or AC and post-treatment of ACW effectively ameliorated DEN-induced inflammation, fibrosis and carcinogenesis in the damaged livers. ACW or AC protected the livers against DEN-induced injury through the inhibition of oxidative stress protein CYP2E1 expression and the inhibition of DEN-enhanced NF-κB translocation by downregulation of the upstream signaling of phosphorylated p85/PI3K and phosphorylated MAPK and CYP2E1. We found that excess ROS production contributed to DEN-induced liver inflammation, fibrosis and carcinogenesis in a time-dependent manner in this study. We have developed an enhanced chemiluminescence method to measure ROS, including O2.- , H2O2, and NO production in the liver, blood and bile [5]; [9] and consistently observed excess ROS production in bile and liver in vivo with previous 52.

(54) finding [9]. We showed that the level of amplified chemiluminescence detected from the liver surface, bile secretion and plasma increased after DEN treatment. By employing an in situ immunocytochemistry technique, we showed that the cellular source of ROS, especially O2 .- synthesis, was located in Kupffer cells of the insulted liver. Formation of ROS occurs in a variety of forms of liver injury [9]; [11]. In our study, there is an accumulation of neutrophils and Kupffer cells in the liver, an elevation of hepatic and bile ROS and increased 3-NT and 4-HNE oxidative product accumulation in the DEN-treated liver. We found that overt ROS and NO production could be due to the high CYP2E1 and iNOS protein expression in the DEN-treated livers. Similar to the iNOS inflammatory response, the apoptosis and autophagy signaling pathway was also enhanced after DEN stimulation. DEN activated Bax expression, but decreased Bcl-2, MnSOD and catalase expression in the livers [5]. The overproduced ROS may contribute to hepatic apoptotic cell death, which was confirmed by the increased Bax and decreased Bcl-2 expression and the increased Bax/Bcl-2 ratio and PARP expression. Our data further confirmed that the DEN also enhanced Beclin-1 protein expression as described previously [5]. Based on our data, the formation of apoptosis and autophagy contributed to liver inflammation, fibrosis and carcinogenesis and the inhibition of apoptosis and autophagy may be a therapeutic target on reducing liver injury. A. cinnamomea with several potentially active ingredients in the mycelia or fruiting body have been used for treatment of various cancers and liver diseases. Yang et al. (2009) found that anti-angiogenesis activity of polysaccharides from A. cinnamomea mycelia with molecular weight > 100 kDa significantly and concentration-dependently decreased the secretion of vascular endothelial growth factor in human leukemia cells, inhibited the matrigel tube formation in human 53.

(55) umbilical vein endothelial cells through the increased levels of interleukin-12 and interferon-gamma gamma. Anti-hepatoma activity of 4-acetylantroquinonol B from the purified A. cinnamomea mycelium produced by submerged fermentation [27]. Chu et al. (2010) found cytochrome P450 and glutathione-S-transferase were expressed 3.66- and 2.75-fold in fruiting body of A. cinnamomea compared with mycelium, and perxoiredoxin and manganese superoxide dismutase were displayed similar expressions in fruiting body and mycelium indicating the fruiting body with higher antioxidant activity. A neutral polysaccharide named ACN2a separated from the water extract of mycelia of A. cinnamomea (0.4, 0.8 g/kg/day, p.o.) significantly prevented increases in serum AST and ALT activities in mice treated with Propionibacterium acnes and lipopolysaccharide [29]. Yu et al. (2009) found a triterpenoid methyl antcinate K isolated from A. cinnamomea promoted mouse bone marrow-derived dendritic cells maturation through the enhancement in the expression of MHC class II and CD86, and secretion of TNF-α, MCP-1, and MIP-1β and primed Th2 responses in immunotherapy. A. cinnamomea is also used for treatment of long-term infection of hepatitis C virus induced liver cancer through the maleic and succinic acid constituents antrodin A and antrodin C to inhibit hepatitis C virus protease [31]. Crude and fractionated polysaccharides of A. cinnamomea inhibited angiogenic-related gene expression, decreased VEGF receptor 2 phosphorylation on tyrosine 1054/1059, cyclin D1 promotor activity, and protein expression induced by VEGF [16]. In our experiment, HPLC fingerprint identified seven major components including antcin K (AC), antcin C, antcin H, dehydrosulphurenic acid, antcin B, antcin A and dehydroeburicoic acid from basswood-cultivated A. cinnamomea fruiting bodies. The highest content of antcin K (AC) has been explored for its inhibition of 54.

(56) metastasis via suppression of integrin-mediated adhesion, migration, and invasion in human hepatoma cells [20]. Antcin K effectively inhibited adhesion, migration, and invasion of Hep 3B cells were within 24 h of treatment by the actions of reducing the protein expression and activity of MMP-2 and MMP-9 and of down-regulating vimentin and up-regulated E-cadherin [20]. In addition, antcin K (AC) reduced the protein expression of integrin β1, β3, α5, and αv and suppressed phosphorylation of FAK, Src, PI3K, AKT, MEK, ERK, and JNK implicating antcin K (AC) to inhibit the metastasis of human hepatoma cells through suppression of integrin-mediated adhesion, migration, and invasion. Our in vitro results displayed that ACW and AC dose-dependently reduced O2-., H2O2 and NO amount in the cell-free system. ACW and AC significantly inhibited DEN-induced hepatic inflammation, fibrosis and carcinoma by improved pathology, decreased bile and liver ROS amounts, 3-nitrotyrosine and 4-hydroxynonenal expression, Kupffer cell infiltration, plasma γ-glutamyl transpeptidase level, and collagen content. The levels of tyrosine phosphorylated form of p85 subunit of PI3K and phosphorylated MAPKs of the upstream regulators for the translocation of NF-κB [12] were higher in 2WDEN, 4WDEN and 8WDEN livers compared to Con livers (Figure 11). ACW and AC suppressed DEN-enhanced NF-κB translocation through the inhibition of the upstream signaling molecules like phosphorylated p85/PI3K and MAPK suggesting their anti-inflammatory signaling pathways. On the other hand, our results show that DEN activated hepatocytes in CYP2E1 expression and subsequently released diffusible mediators including ROS, which can activate hepatic stellate cells [8]. Thus, besides perturbing the homeostasis of hepatocytes, CYP2E1-derived diffusible oxidants may also interact with stellate cells and contribute to hepatic fibrosis [8]. Our data found that ACW. and AC decreased DEN-enhanced oxidative stress and fibrosis possibly through the 55.

(57) inhibition of the CYP2E1 signaling implicating their antioxidant and anti-fibrotic signaling pathways. We have no evidence for the antioxidant and anti-inflammatory effects of ACW or AC dose-dependently in the DEN-treated livers in the rats. It requires further. experiments to explore whether ACW and AC evoked anti-inflammation and antioxidant effect is also dose-dependently in in vivo system. Using adenosine deaminase or adenosine A2A receptor antagonist to delete adenosine signaling, Lu et al. (2006) reported that the protective effect of A. cinnamomea is owed to its active component, adenosine, which acts through activation of adenosine A2A receptor to prevent serum deprivation-induced PC12 cell apoptosis. They further found that serum deprivation resulted in decreased phosphorylation of ERK and increased phosphorylations of JNK and p38 of MAPKs; however, A. cinnamomea reversed these phenomena through adenosine/adenosine A2A receptor-mediated protein kinase A-dependent pathway and by suppression of JNK and p38 activities [19]. Anti-inflammatory herbal medicine blocked the activation and translocation of NF-κB and AP-1 by suppressing the upstream kinases including IκBα, IκBα kinase, Akt, phosphoinositide-dependent kinase 1, p85/PI3K, MAPK/ERK [12]. Our data also evidenced that ACW and AC decreased NF-κB translocation. and. inhibited. CYP2E1,. phosphorylated. p85,. phosphorylated. MAPK/ERK expression in DEN-treated livers. Hsu et al. (2007) found that the anti-invasive effect of ethylacetate extract from A. cinnamomea fruiting bodies inhibited TNF-α-activated NF-κB-dependent reporter gene expression of MMP-9 and VEGF associated with a concomitant decrease in the level and activity of VEGF, MMP-2, MMP-9 and MT1-MMP, and an increase in the expression of TIMP-1 and TIMP-2 in the human liver cancer cell line PLC/PRF/5. Chen et al. (1995) identified three types of steroids, zhankuic acids A, B, and C from the A. cinnamomea fruiting 56.