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
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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
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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
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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
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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
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bodies by bioassay-guided fractionation and found that these zhankuic acids exhibited cytotoxic activity against P-388 murine leukemia cells. On cultivation of the fungus A.
cinnamomea on a medium, the bioactivity-directed fractionation displayed inhibitory nitric oxide activity by the maleimide derivatives, antrocinnamomins A [32]. Kuo et al. (2006) indicated that the molecular mechanisms during ethylacetate extract from A.
cinnamomea fruiting bodies-mediated proliferation inhibition in Hep 3B cells were due to: apoptosis induction, triggering of Ca2+/calpain pathway, disruption of mitochondrial function, and apoptotic signaling being amplified by cross-talk between the calpain/Bid/Bax and Ca2+/mitochondrial apoptotic pathways. Our data also recognized that DEN-treated livers displayed Beclin-1 mediated autophagy and TUNEL-apoptosis after 4 weeks and 8 weeks. We found that silymarin, ACW, AC co-treatment or PACW significantly inhibited DEN-enhanced autophagy and apoptosis possibly through the inhibition of ROS and NF-κB mediated inflammation.
AC may be one of the active components of A. cinnamomea fruiting bodies in inhibition of hepatic inflammation and oxidative injury. Our evidence showed that A.
cinnamomea fruiting bodies including several possible ingredients exert protective and therapeutic potential against oxidative injury and inflammation induced apoptosis, autophagy, fibrosis and tumor formation in the livers. Co-treatment of ACW displayed more efficient effects than AC or PACW to counteract liver injury and inflammation potential indicating other ingredients in ACW confer synergistic protection against DEN-induced liver injury. Our data informed that ACW was more effective than AC in the reduction of liver inflammation and damage possibly due to other important ingredients like antcin C, antcin H, dehydrosulphurenic acid, antcin B, antcin A and dehydroeburicoic acid to confer further protective effects. Therefore, we did not treat AC in post-DEN recovery experiment.
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We have compared the potential of co-treatment or therapeutic effect of A.
cinnamomea fruiting bodies ACW or AC with a positive control group of silymarin, a widely used traditional herbal medicine, by judging the degree of apoptosis and autophagy in the damaged livers of this study. Both apoptosis and autophagy evoked programmed cell death in the DEN-treated livers and possibly resulting in hepatic dysfunction. We found that the co-treatment of ACW or AC or the therapeutic use of ACW (PACW) seems to be more effective than silymarin in reduction of apoptosis and autophagy in the 8WDEN group, but not in 2WDEN and 4WDEN groups. The dosage we used in this study was ACW at 6.67 mg/kg and AC at 20 µg/kg body weight per day. However, we require to carefully screen and test the different dosage and safety use of ACW or AC and then to compare the therapeutic effect with silymarin in the next study. We explored the protective role and therapeutic potential of ACW by co-treatment with DEN injury or after 2 weeks of DEN injury. We found that different time course of ACW treatment seems to affect the final outcome by similar pathways like inhibition p85, MAPK and CYP2E1 expression and decrease of apoptosis and autophagy formation. However, the dosage used for co-treatment or therapy may be different. On the other hand, DEN induced early inflammation around 1-2 weeks, followed by 3-6 weeks of inflammation induced fibrosis and consequently led to a late stage of DEN injury with the carcinogenesis. These situations were very similar to HCC in human. Therefore, our disease model and the methods and results of ACW treatment may provide an important clue for the prevention, protection and therapeutic strategy in clinical trial in future.
Our prepared MA has been characterized by a high content of MK and total phenolic compounds and stronger anti-O2-. and anti-H2O2 (e.g., antioxidant) activities than either of its source materials (M. purpureus Went and adlay) alone and prevents
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smoke-induced lung injury [48]. The present study further indicates that daily intake of MA or MK can significantly scavenge ROS, suppress P selectin-mediated platelet activation, ADP-stimulated platelet aggregation and decrease H2O2-enhanced endothelial ICAM-1 and VCAM-1 expression in vitro. In in vivo study, MA or MK treatment FeCl3-induced oxidative stress, NF-κB p65 mediated ICAM-1 expression and adhesion molecules expression, ER stress CHOP expression and thrombosis in the rat carotid artery. MA or MK also preserved nuclear Nrf2 translocation to increase cytoprotective ability in response to FeCl3 injury. We found that the MA through its active component MK protects the vessels against FeCl3-induced thrombosis formation.
The MA products contain high nutritional potential for the higher levels of crude ash, fat, fiber, and protein than are found in uninoculated adlay [41]. In addition, MA has a bitter taste probably due to its high MK and total phenolic compound content [41,48]. Methanolic extracts from MA is more effective than adlay in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and chelating ferrous ions [42]. Our previous study [48] and the present study consistently show that MA displays a strong and effective tendency than MK in reducing O2-., H2O2 and HOCl amounts.
Trans-coniferylaldehyde, a phenolic compound in adlay, was recently found to efficiently scavenge DPPH radicals and inhibit O2-. production [53]. Generally, all these data implicate that naturally antioxidant components, such as phenolic compounds, work synergistically with MK to increase the antioxidant activity of MA.
According to our data MA and MK presenting the same effect, we further indicated that MA contains high MK, total phenolic compounds, and other nutritional factors, confers higher antioxidant activity than MK, and can be used as functional food to prevent or as therapeutic drugs to treat the cardiovascular diseases like thrombus
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formation in the future.
FeCl3 treatment would induce a H2O2-dependent Renton reaction to oxidize macromolecules like lipid and protein in the vessels. According to our data of FeCl3-treated carotid artery in vivo, the thrombus formation occurred rapidly within 10–20 min and was characterized by endothelial disruption,extensive platelet, white and red blood cell clumps, and interspersedfibrin. These data were consistent with previous findings [44,48]. Theease of thrombus formation and similarity to human thrombus makethis model appropriate for studying the influence of differentagents that can inhibit platelet and blood cell aggregation. Our evidence found that FeCl3-increased vascular ROS generation, 3-NT expression and ICAM-1 activity and expression locally in the endothelial area of the vessel wall. All the enhanced oxidative parameters observed in the artery of the FeCl3-treated rats was significantly eliminated by the dietary MA or MK pretreatment, implicating that FeCl3-increased vessel wall production of H2O2 could be scavenged by MA or MK.
In organs subjected to several kinds of oxidative injury like septic shock, smoke, hemorrhage, and ischemia/reperfusion, the excess ROS production oxidized several macromolecules, triggered pro-inflammatory signaling pathways responsible for the activation of NF-κB and AP-1 and promoted atherosclerosis and thrombosis [59-61].
Locally vascular ROS formation inhibited the bioavailability of nitric oxide (NO), impaired vascular relaxation, and increased leukocytes, platelets and fibrin adhesion to and aggregation in injured vessels [61,62]. ROS can increase fractalkine (CX3CL1) and ICAM-1 expressions on injured endothelium, attract fractalkine receptor (CX3CR1)-expression inflammatory cells to the inflamed area, and provoke atherosclerosis and vascular inflammation [63]. These activated cascades resulted in up-regulation of the ICAM-1 gene in the vascular endothelium and subsequent
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accumulation of activated neutrophils and other leukocytes in the tissue [48].
Likewise, we demonstrated that FeCl3 stimulation increased vascular ROS formation that may lead to the early activation of nuclear translocation of the p65 subunit of NF-κB and AP-1, which, in turn, promoted the expression of ICAM-1 protein and other inflammatory cytokines like CX3CL1. One previous study stated that after arterial-venous fistula surgery, increased monocyte/macrophage infiltration and pro-inflammatory cytokine-mediated adhesion molecules (i.e., ICAM-1) were highly expressed in the damaged venous wall [64]. Our recent data also demonstrated that increases in intracellular ROS, ICAM-1, and apoptosis occur in the damaged vessels easily contributing to thrombosis formation [55].
The induction of many cytoprotective enzymes in response to reactive chemical stress is regulated primarily at the transcriptional level. Activation of gene transcription is mediated primarily by Nrf2 (nuclear factor E2-related factor 2). Nrf2 controls basal expression of its genes clearly indicates that it is a constitutively and functionally active transcription factor and, notably, implies its presence in the nucleus under homeostatic conditions. Our data found that nuclear Nrf2 decreased after FeCl3 lesion, but was preserved by MA or MK treatment. We have evidenced that FeCl3 lesion enhanced nuclear p65 subunit of NF-κB expression in the carotid arteries (Figure 6). We also noted that FeCl3 lesion increased arterial 3-NT oxidative stress associated ICAM-1 and CHOP expression. We suggest that FeCl3 induced thrombus formation through the upregulation of NF-κB p65-mediated ICAM-1 and VCAM-1 expression associated oxidative stress and endoplasmic reticulum stress and downregulation of Nrf2 translocation to nucleus.
Platelet activation and aggregation is considered a crucial step in the initiation and aggravation of arterial thrombosis. ADP from activated platelets is recognized as
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major factor in thrombus formation and is a potent stimulator of ROS release from neutrophils [65]. MK affects platelet-neutrophil interactions by altering Rho-GTPase-dependent adenosine nucleotide function and consecutively inhibiting thrombin-activated platelets primed neutrophils for enhanced ROS release [65]. MK also known as simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte procoagulant protein tissue factor (TF) expression, microparticle TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels [66]. MK decreased the rise in neutrophil adhesion and ROS generation following stimulation of saphenous vein endothelial cell culture with advanced glycation end products in vitro and in vivo data from diabetic patients administered with MK showed a similar significant reduction in neutrophil adhesion and ROS generation [67]. As far we know, there is no report about MA effect on leukocytes. MK content in our MA extract was 1.57 mg/g (0.157%). All these data implied that MA may through MK inhibit platelet-neutrophil interaction and monocyte activity and reduce ROS release and inflammation.