To assess whether A. camphorata constituents can inhibit LPS-induced nitric oxide production in macrophages, mouse macrophage RAW 264.7 cells were cultured with LPS (20μg/ml) and A. camphorata constituents for 16h. When compare with control group, the production of nitric oxide in LPS-induced RAW 264.7 cells was suppressed by A. camphorata constituents (Figure 8). Among them, both AC-10 and AC-12 constituents showed the inhibition activity at higher concentration, while AC-3 has better inhibition effect with all concentrations. Thus, these results demonstrated that AC-3 isolate might inhibit LPS-induced nitric oxide production in mouse
macrophage RAW 264.7 cells.
Western blot of inhibition of iNOS production
To investigate the effects of A. camphorata constituents on the cytosolic protein levels of iNOS, RAW 264.7 cells were treated with or without LPS and various concentrations of A. camphorata constituents for 16h and the protein levels of iNOS were analyzed by Western blot. As shown in Figure 9, AC-3 and AC-10 constituents show inhibition effects of iNOS protein expression. Only AC-3 isolate shows higher inhibition effect at treated with maximus concentration (0.02mM). This result indicated that A. camphorata constituents might inhibit LPS-induced nitric oxide production in macrophages.
Discussion and conclusion
There are many kinds of A. camphorata products in market, and in this study we use pure chemical constituents of A. camphorata. Our results showed that A.
camphorata constituents have their effects on the inhibition of H. pylori-induced
inflammation. The effects of A. camphorata constituents on anti-bacteria activity were similar to the standard drug metronidazole (MTZ). In addition, the A. camphorata constituents showed no cytotoxic effect on both gastric epithelial cells and
macrophage RAW 264.7 cells.
Among lots of A. camphorata commodities in the world, most of them are used for liver protection, for instance, in vitro or in vivo treatment of ethanol-induced hepatis or hepatitis B virus infection in a murine model system (Lee et al., 2002; Lu et al., 2007). Using A. camphorata compounds for treatment to bacteria is unusual, however, it seems to be a good direction for eradication of bacterial infection. In this study, our results show that constituents of AC-3 and AC-10 have better inhibition effects on H. pylori-induced inflammation of gastric epithelial cells. This was consistent with previous study of anti-inflammatory and anti-cancer properties of A.
camphorata (Rao et al., 2007).
In a macrophage RAW 264.7 cell system, A. camphorata constituents show their inhibitory effects by decreasing the production of nitric oxide. Although all the A.
camphorata constituents have the same trends, AC-3 shows the best effect than others.
Our data also showed that iNOS expression in LPS-induced macrophage was
inhibited by AC-3. In addition, MTT assay shows that all A. camphorata constituents have no cytotoxic in macrophage RAW 264.7 cells. These results maybe indicated that A. camphorata constituents would be a good candidate for therapy of
inflammatory diseases.
In this study, we demonstrated that A. camphorata constituents displayed potential anti-H. pylori activity. The constituents of AC-3 and AC-10 inhibit the H.
pylori adhesion and invasion to AGS cells. In addition, these constituents also inhibit
the IL-8 protein secretion and NF-κB signal activation in H. pylori-infected gastric epithelial cells. This inhibition of NF-κB activation and IL-8 secretion by AC-3 and AC-10 might contribute to the potential role of A. camphorata as an
anti-inflammatory action in H. pylori induced gastric epithelial cells damage. Future studies are needed to clarify the molecular mechanisms by which A. camphorata inhibits H. pylori-mediated activation of NF-κB expression in AGS cells and to identify additional targets in gene regulation.
For the future, first we can do T cell experiment like test spleen cells by ex vivo mouse model system to examine if AC constituents can inhibit mitogen
(conA)-induce T cell activation or not. Otherwise detect AC constituents inhibit TCR
(T cell receptor) induced by Jurkat cells activation in using Flow cytometry and cell cycle assay
References
Ao, Z.H., Xu, Z.H., Lu, Z.M., Xu, H.Y., Zhang, X.M. and Dou, W.F., 2009.
Niuchangchih (Antrodia camphorata) and its potential in treating liver diseases.
J Ethnopharmacol 121, 194-212.
Bochenek, W.J., Peters, S., Fraga, P.D., Wang, W., Mack, M.E., Osato, M.S., El-Zimaity, H.M., Davis, K.D. and Graham, D.Y., 2003. Eradication of
Helicobacter pylori by 7-day triple-therapy regimens combining pantoprazole
with clarithromycin, metronidazole, or amoxicillin in patients with peptic ulcer disease: results of two double-blind, randomized studies. Helicobacter 8, 626-642.
Brandt, S., Kwok, T., Hartig, R., Konig, W. and Backert, S., 2005. NF-kappaB activation and potentiation of proinflammatory responses by the Helicobacter
pylori CagA protein. Proc Natl Acad Sci U S A 102, 9300-9305.
Chan, Y. and Chan, C.H., 2003. Antibiotic resistance of pathogenic bacteria from odontogenic infections in Taiwan. J Microbiol Immunol Infect 36, 105-110.
Chen, K.C., Peng, C.C., Peng, R.Y., Su, C.H., Chiang, H.S., Yan, J.H. and Hsieh-Li, H.M., 2007. Unique formosan mushroom Antrodia camphorata differentially inhibits androgen-responsive LNCaP and -independent PC-3 prostate cancer cells. Nutr Cancer 57, 111-121.
Chien, S.C., Chen, M.L., Kuo, H.T., Tsai, Y.C., Lin, B.F. and Kuo, Y.H., 2008.
Anti-inflammatory activities of new succinic and maleic derivatives from the fruiting body of Antrodia camphorata. J Agric Food Chem 56, 7017-7022.
Chourasia, D., Misra, A., Pandey, R. and Ghoshal, U.C., 2008. Gastric atrophy and intestinal metaplasia in a patient on long-term proton pump inhibitor therapy.
Trop Gastroenterol 29, 172-174.
de Boer, W.A., Driessen, W.M., Jansz, A.R. and Tytgat, G.N., 1995. Quadruple therapy compared with dual therapy for eradication of Helicobacter pylori in ulcer patients: results of a randomized prospective single-centre study. Eur J Gastroenterol Hepatol 7, 1189-1194.
de Boer, W.A., Driessen, W.M., Potters, V.P. and Tytgat, G.N., 1994. Randomized study comparing 1 with 2 weeks of quadruple therapy for eradicating
Helicobacter pylori. Am J Gastroenterol 89, 1993-1997.
Filipec Kanizaj, T., Katicic, M., Skurla, B., Ticak, M., Plecko, V. and Kalenic, S., 2009. Helicobacter pylori eradication therapy success regarding different treatment period based on clarithromycin or metronidazole triple-therapy regimens. Helicobacter 14, 29-35.
Fong, Y.C., Maa, M.C., Tsai, F.J., Chen, W.C., Lin, J.G., Jeng, L.B., Yang, R.S., Fu, W.M. and Tang, C.H., 2008. Osteoblast-derived TGF-beta1 stimulates IL-8 release through AP-1 and NF-kappaB in human cancer cells. J Bone Miner Res 23, 961-970.
Gobert, A.P., McGee, D.J., Akhtar, M., Mendz, G.L., Newton, J.C., Cheng, Y., Mobley, H.L. and Wilson, K.T., 2001. Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: a strategy for bacterial survival. Proc Natl Acad Sci U S A 98, 13844-13849.
Graham, D.Y., Lu, H. and Yamaoka, Y., 2009. African, Asian or Indian enigma, the East Asian Helicobacter pylori: facts or medical myths. J Dig Dis 10, 77-84.
Gu, Q., Xia, H.H., Wang, J.D., Wong, W.M., Chan, A.O., Lai, K.C., Chan, C.K., Yuen, M.F., Fung, F.M., Wong, K.W., Lam, S.K. and Wong, B.C., 2006. Update on clarithromycin resistance in Helicobacter pylori in Hong Kong and its effect on clarithromycin-based triple therapy. Digestion 73, 101-106.
Hacin, B., Rogelj, I. and Matijasic, B.B., 2008. Lactobacillus isolates from weaned piglets' mucosa with inhibitory activity against common porcine pathogens.
Folia Microbiol (Praha) 53, 569-576.
Harris, P.R., Smythies, L.E., Smith, P.D. and Dubois, A., 2000. Inflammatory cytokine mRNA expression during early and persistent Helicobacter pylori infection in nonhuman primates. J Infect Dis 181, 783-786.
Hentschel, E., Brandstatter, G., Dragosics, B., Hirschl, A.M., Nemec, H., Schutze, K., Taufer, M. and Wurzer, H., 1993. Effect of ranitidine and amoxicillin plus metronidazole on the eradication of Helicobacter pylori and the recurrence of duodenal ulcer. N Engl J Med 328, 308-312.
Hseu, Y.C., Chen, S.C., Yech, Y.J., Wang, L. and Yang, H.L., 2008. Antioxidant
activity of Antrodia camphorata on free radical-induced endothelial cell damage.
J Ethnopharmacol 118, 237-245.
Hsiao, G., Shen, M.Y., Lin, K.H., Lan, M.H., Wu, L.Y., Chou, D.S., Lin, C.H., Su, C.H. and Sheu, J.R., 2003. Antioxidative and hepatoprotective effects of
Antrodia camphorata extract. J Agric Food Chem 51, 3302-3308.
Hsu, P.I., Lai, K.H., Lin, C.K., Chen, W.C., Yu, H.C., Cheng, J.S., Tsay, F.W., Wu, C.J., Lo, C.C., Tseng, H.H., Yamaoka, Y., Chen, J.L. and Lo, G.H., 2005. A prospective randomized trial of esomeprazole- versus pantoprazole-based triple therapy for Helicobacter pylori eradication. Am J Gastroenterol 100, 2387-2392.
Jodlowski, T.Z., Lam, S. and Ashby, C.R., Jr., 2008. Emerging therapies for the treatment of Helicobacter pylori infections. Ann Pharmacother 42, 1621-1639.
Kim, S.Y., Lee, Y.C., Kim, H.K. and Blaser, M.J., 2006. Helicobacter pylori CagA transfection of gastric epithelial cells induces interleukin-8. Cell Microbiol 8, 97-106.
Lai, C.H., Chang, Y.C., Du, S.Y., Wang, H.J., Kuo, C.H., Fang, S.H., Fu, H.W., Lin, H.H., Chiang, A.S. and Wang, W.C., 2008a. Cholesterol depletion reduces
Helicobacter pylori CagA translocation and CagA-induced responses in AGS cells. Infect Immun 76, 3293-3303.
Lai, C.H., Fang, S.H., Rao, Y.K., Geethangili, M., Tang, C.H., Lin, Y.J., Hung, C.H.,
Wang, W.C. and Tzeng, Y.M., 2008b. Inhibition of Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells by Phyllanthus urinaria extracts. J Ethnopharmacol 118, 522-526.
Lai, C.H., Kuo, C.H., Chen, P.Y., Poon, S.K., Chang, C.S. and Wang, W.C., 2006.
Association of antibiotic resistance and higher internalization activity in resistant Helicobacter pylori isolates. J Antimicrob Chemother 57, 466-471.
Lam, S.K. and Talley, N.J., 1998. Report of the 1997 Asia Pacific Consensus
Conference on the management of Helicobacter pylori infection. J Gastroenterol Hepatol 13, 1-12.
Lee, I.H., Huang, R.L., Chen, C.T., Chen, H.C., Hsu, W.C. and Lu, M.K., 2002.
Antrodia camphorata polysaccharides exhibit anti-hepatitis B virus effects.
FEMS Microbiol Lett 209, 63-67.
Logan, R.P., Gummett, P.A., Misiewicz, J.J., Karim, Q.N., Walker, M.M. and Baron, J.H., 1991. One week eradication regimen for Helicobacter pylori. Lancet 338, 1249-1252.
Lu, Z.M., Tao, W.Y., Zou, X.L., Fu, H.Z. and Ao, Z.H., 2007. Protective effects of mycelia of Antrodia camphorata and Armillariella tabescens in submerged culture against ethanol-induced hepatic toxicity in rats. J Ethnopharmacol 110, 160-164.
Marshall, B., 2002. Helicobacter pylori: 20 years on. Clin Med 2, 147-152.
McMahon, B.J., Hennessy, T.W., Bensler, J.M., Bruden, D.L., Parkinson, A.J., Morris, J.M., Reasonover, A.L., Hurlburt, D.A., Bruce, M.G., Sacco, F. and Butler, J.C., 2003. The relationship among previous antimicrobial use, antimicrobial
resistance, and treatment outcomes for Helicobacter pylori infections. Ann Intern Med 139, 463-469.
Megraud, F. and Lamouliatte, H., 2003. Review article: the treatment of refractory
Helicobacter pylori infection. Aliment Pharmacol Ther 17, 1333-1343.
Mirbagheri, S.A., Hasibi, M., Abouzari, M. and Rashidi, A., 2006. Triple, standard quadruple and ampicillin-sulbactam-based quadruple therapies for H. pylori eradication: a comparative three-armed randomized clinical trial. World J Gastroenterol 12, 4888-4891.
Nozawa, Y., Nishihara, K., Peek, R.M., Nakano, M., Uji, T., Ajioka, H., Matsuura, N.
and Miyake, H., 2002. Identification of a signaling cascade for interleukin-8 production by Helicobacter pylori in human gastric epithelial cells. Biochem Pharmacol 64, 21-30.
Parsonnet, J., 1998. Helicobacter pylori. Infect Dis Clin North Am 12, 185-197.
Peek, R.M., 2004. Helicobacter pylori and Gastroesophageal Reflux Disease. Curr Treat Options Gastroenterol 7, 59-70.
Poon, S.K., Chang, C.S., Su, J., Lai, C.H., Yang, C.C., Chen, G.H. and Wang, W.C., 2002. Primary resistance to antibiotics and its clinical impact on the efficacy of
Helicobacter pylori lansoprazole-based triple therapies. Aliment Pharmacol
Ther 16, 291-296.
Rao, Y.K., Fang, S.H. and Tzeng, Y.M., 2007. Evaluation of the anti-inflammatory and anti-proliferation tumoral cells activities of Antrodia camphorata,
Cordyceps sinensis, and Cinnamomum osmophloeum bark extracts. J
Ethnopharmacol 114, 78-85.
Salcedo, J.A. and Al-Kawas, F., 1998. Treatment of Helicobacter pylori infection.
Arch Intern Med 158, 842-851.
Sartor, R.B., 1994. Cytokines in intestinal inflammation: pathophysiological and clinical considerations. Gastroenterology 106, 533-539.
Shen, C.C., Kuo, Y.C. and Huang, R.L., 2003. New ergostane and lanostane from
Antrodia camphorata. J Chin Med 14, 247-258.
Suerbaum, S. and Michetti, P., 2002. Helicobacter pylori infection. N Engl J Med 347, 1175-1186.
Tang, C.H., Yang, R.S., Chen, Y.F. and Fu, W.M., 2007. Basic fibroblast growth factor stimulates fibronectin expression through phospholipase C gamma, protein kinase C alpha, c-Src, NF-kappaB, and p300 pathway in osteoblasts. J Cell Physiol 211, 45-55.
Tankovic, J., Lamarque, D., Lascols, C., Soussy, C.J. and Delchier, J.C., 2001a.
Clarithromycin resistance of Helicobacter pylori has a major impact on the
efficacy of the omeprazole-amoxicillin-clarithromycin therapy. Pathol Biol (Paris) 49, 528-533.
Tankovic, J., Lamarque, D., Lascols, C., Soussy, C.J. and Delchier, J.C., 2001b.
Impact of Helicobacter pylori resistance to clarithromycin on the efficacy of the omeprazole-amoxicillin-clarithromycin therapy. Aliment Pharmacol Ther 15, 707-713.
Walsh, C., 2000. Molecular mechanisms that confer antibacterial drug resistance.
Nature 406, 775-781.
Walsh, J.H. and Peterson, W.L., 1995. The treatment of Helicobacter pylori infection in the management of peptic ulcer disease. N Engl J Med 333, 984-991.
Warren, J.R., 1983. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet i, 1273.
Wong, W.M., Wong, B.C., Lu, H., Gu, Q., Yin, Y., Wang, W.H., Fung, F.M., Lai, K.C., Xia, H.H., Xiao, S.D. and Lam, S.K., 2002. One-week omeprazole, furazolidone and amoxicillin rescue therapy after failure of Helicobacter pylori eradication with standard triple therapies. Aliment Pharmacol Ther 16, 793-798.
Yamaoka, Y., Kita, M., Kodama, T., Sawai, N. and Imanishi, J., 1996. Helicobacter
pylori cagA gene and expression of cytokine messenger RNA in gastric mucosa.
Gastroenterology 110, 1744-1752.
Yang, H.L., Chen, C.S., Chang, W.H., Lu, F.J., Lai, Y.C., Chen, C.C., Hseu, T.H., Kuo,
C.T. and Hseu, Y.C., 2006. Growth inhibition and induction of apoptosis in MCF-7 breast cancer cells by Antrodia camphorata. Cancer Lett 231, 215-227.
Zullo, A., Gatta, L., De Francesco, V., Hassan, C., Ricci, C., Bernabucci, V., Cavina, M., Ierardi, E., Morini, S. and Vaira, D., 2005. High rate of Helicobacter pylori eradication with sequential therapy in elderly patients with peptic ulcer: a prospective controlled study. Aliment Pharmacol Ther 21, 1419-1424.
Table 1 Molecular weight of A. camphorata constituents
Compound Molecular weight (M.W)
AC-1 482
AC-2 468
AC-3 526
AC-4 470
AC-5 468
AC-6 484
AC-7 486
AC-8 466
AC-9 454
AC-10 470
Table 2 Inhibitory effects of A. camphorata constituents Compounds Inhibition zone (mm)
AC-1 -
† DMSO was used as negative control, and the antibiotics amoxicillin (AMX,
0.05 mg/ml), clarithromycin (CLR, 0.05 mg/ml), and metronidazole (MTZ, 0.8 mg/ml) were used as positive control.
Fig. 1. Chemiacl structures of A. camphorata constituents 1-11
Fig. 1. Chemiacl structures of A. camphorata constituents 1-11 (continuous)
COOH
3 6
25 17
Antcin B (or) Zhankuic acid A (5)
O O
COOCH3
Fig. 1. Chemiacl structures of A. camphorata constituents 1-11 (continuous)
COOH
Fig. 2. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) and
DMSO control on gastric epithelial cell viability. The AGS cells were exposed to
various concentrations of the constituents during a 20 h incubation period and treated with no constituents for control. Cell viability was assessed by MTT assay. Results represent mean values ± SD from at least triplicate independent experiments.
Fig. 3. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) on H.
pylori adhesion of gastric epithelial cells. The bacteria to AGS cells that were
untreated with A. camphorata constituents for DMSO control and others treated with
A. camphorata constituents, followed by infection of H. pylori at a MOI of 50 for 6 h.
Each experiment was shown represent mean values ± SD of at least six independent experiments. The significant difference was set at * P < 0.05; ** P < 0.01.
Fig. 4. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) on H.
pylori invasion of gastric epithelial cells. The bacteria to AGS cells that were
untreated with A. camphorata constituents for DMSO control and others treated with
A. camphorata constituents, followed by infection of Helicobacter pylori at a MOI of
50 for 6 h. Each experiment was shown represent mean values ± SD of at least six independent experiments. The significant difference was set at * P < 0.05; ** P <
0.01.
Fig. 5. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) on H.
pylori-induced NF-κB activation in gastric epithelial cells. The luciferase activity
was determined as described in materials and methods. Cells treated without any A.
camphorata constituents for control. Results are shown by mean values ± SD from at least three independent experiments. The significant difference was set at * P < 0.05;
** P < 0.01.
Fig. 6. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) on H.
pylori-induced IL-8–luciferase activity in gastric epithelial cells. The luciferase
activity was determined as described in materials and methods. Cells treated without any A. camphorata constituents for control. Results are shown by mean values ± SD from at least three independent experiments. The significant difference was set at * P
< 0.05; ** P < 0.01.
Fig. 7. Effect of A. camphorata constituents (AC-3, AC-10, and AC-12) and
DMSO control in RAW 264.7 cells. The RAW 264.7 cells were treated with or
without LPS (20μg/ml) and others treated with both LPS and A. camphorata constituents or DMSO control during a 20 h incubation period. Cell viability was assessed by MTT assay. Results represent mean values ± SD from at least triplicate independent experiments.
Fig. 8. Inhibition by A. camphorata constituents (AC-3, AC-10, and AC-12) and
DMSO control of LPS-induced NO production in RAW 264.7 cells. Cells were
treated with or without LPS (20μg/ml) and others treated with both LPS and A.
camphorata constituents or DMSO control. After incubation over 20 h, the culture
supernatant of RAW 264.7 cell line was then collected for the assay of nitric oxide production, respectively. Each experiment was shown represent mean values ± SD of at least six independent experiments. The significant difference was set at * P < 0.05;
** P < 0.01.
Fig. 9. Effects of A. camphorata constituents (AC-3 and AC-10) on the iNOS
protein expression in RAW 264.7 cells. Cells were treated with or without LPS
(20μg/ml) and others treated with both LPS and A. camphorata constituents. After 20 h, cells were washed with PBS and homogenized. Each lane contained protein 20μg constituents. Electrophoresis was performed using 10% SDS-PAGE and detected with anti-iNOS and anti-β-actin antibodies, respectively.