Effects of Saenggantanggami-bang on Nonalcoholic Fatty Liver Disease Model Induced by Fatty Acids

지방산으로 유발된 비알코올지방간질환 실험모델에 미치는 생간탕가미방(生肝湯加味方)의 효과

  • Yoo, Jae-Yeon (Dept. of Internal Medicine, College of Oriental Medicine, Kyung-Hee University) ;
  • Lee, Jang-Hoon (Dept. of Internal Medicine, College of Oriental Medicine, Kyung-Hee University)
  • 유재연 (경희대학교 한의과대학 간계내과학교실) ;
  • 이장훈 (경희대학교 한의과대학 간계내과학교실)
  • Published : 2010.06.30

Abstract

Objectives : This study was designed to investigate the effects of Saenggantanggami-bang (SG) on nonalcoholic fatty liver disease. Methods : HepG2 cells were used in an in vitro model. HepG2 cells were divided into three groups. The Normal group was incubated with no fatty acid. The Control group was incubated with 1mM palmitic acid to introduce fat overloading. The PA-SG group was incubated with 1mM palmitic acid and various concentrations of Saenggantanggami-bang (SG). Cell viability and cytotoxicity were analyzed by MTT assay and LDH assay. Intracellular triglyceride (TG) levels, reactive oxygen species (ROS) levels, ATP amount, and GST activity were measured. Cell death pattern and protective effect of SG on cell death were studied by DNA fragmentation and caspase-3 intensity (western blot). Results : Compared with the Control group, cell viability of the PA-SG group significantly increased (P<0.01), cytotoxicity of the PA-SG group decreased (P<0.01), and intracellular TG levels and ROS levels of the PA-SG group decreased (P<0.05). In DNA fragmentation assay, necrotic pattern was observed and DNA fragment decreased in the PA-SG group. In western blot, apoptotic pattern was observed, caspase-3 intensity of the PA-SG group was reduced significantly, but there were no significant differences in intracellular ATP amount and GST activity between the control group and the PA-SG group. Conclusion : The results suggest that Saenggantanggami-bang can be a potential candidate for the clinical treatment of nonalcoholic fatty liver disease.

Keywords

References

  1. Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis : Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc. 1980;55(7):434-8.
  2. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med. 2002;346:1221-31. https://doi.org/10.1056/NEJMra011775
  3. Choi SY, Kim DH, Kang JH, Park MJ, Kim YS, Lim SH, et al. Nonalcoholic fatty liver disease as a risk factors of cardiovascular disease; relation of nonalcoholic fatty liver disease to carotid atherosclerosis. Korean J Hepatol. 2008; 14:77-88. https://doi.org/10.3350/kjhep.2008.14.1.77
  4. Park SH, Park CK. Pathogenesis and management of nonalcoholic steatohepatitis. JKMA. 2005;48: 169-78.
  5. McCullough AJ. The clinical features, diagnosis and natural history of nonalcoholic fatty liver disease. Clin Liver Dis. 2004;8:521-33. https://doi.org/10.1016/j.cld.2004.04.004
  6. Adams LA, Angulo P. Recent concepts in nonalcoholic fatty liver disease. Diabet Med. 2005;22:1129-33. https://doi.org/10.1111/j.1464-5491.2005.01748.x
  7. Marchesini G, Brizi M, Bianchi G, Cerrelli F, Lenzi M, Manini R, et al. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes. 2001;50:1844-50. https://doi.org/10.2337/diabetes.50.8.1844
  8. Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, et al. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology. 2003;37:917-23. https://doi.org/10.1053/jhep.2003.50161
  9. Feldstein AE, Werneburg NW, Canbay A, Guicciardi ME, Bronk SF, Rydzewski R, et al. Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway. Hepatology. 2004;40:185-94. https://doi.org/10.1002/hep.20283
  10. Jeffrey D Browning, Jay D Horton. Molecular mediators of hepatic steatosis and liver injury. The Journal of Clinical Investigation. 2004;114(2): 147-51.
  11. 이동석, 권기태, 조창민, 탁원영, 권영오, 김성국 외. 비알코올성 지방간질환에서 지방간염의 진단 및 간섬유화 정도의 예측인자. 대한간학회지. 2003;9(3):14-20.
  12. 김상진, 박중원, 김미경, 김형준, 홍요한, 한성혁 외. 비알코올성 지방간 환자에서 지방간염의 병리학적 심화도를 예측할 수 있는 임상적 지표들. 대한소화기학회지. 2000;36:782-92.
  13. Rallidis LS, Drakoulis CK, Parasi AS. Pravastatin in patients with nonalcoholic steatohepatitis: results of a pilot study. Atherosclerosis. 2004; 174:193-6. https://doi.org/10.1016/j.atherosclerosis.2004.01.008
  14. 유권. 비알콜성 지방간염. 대한간학회지. 2002; 8(1):66-72.
  15. 赵和平, 段晓燕. 降脂益肝冲剂治療非酒精性脂肪 肝炎的實驗硏究. Drug evaluation. 2007;4(2):114-6.
  16. 윤경수. NASH 동물모델에서 인진청간탕과 인진사령산의 효과규명. 대한한방내과학회지. 2009; 30(2):410-21.
  17. Lee KT, Kim YS, Seol IC. Effects of TongBiYeum on the Fatty Liver. Korean Journal of Oriental Medicine. 2003;24(4):11-8.
  18. 성태수, 손규목. 천궁의 열수추출액이 고지방식 이에 의한 흰쥐의 혈장 중 효소활성과 호르몬 및 간장의 지방축적에 미치는 영향. Korean Journal Food & Nutrition. 1994;7(2):108-13.
  19. 최미영. 가미생간탕이 고지방식이로 유발된 지방간에 미치는 영향. 대한한방내과학회지. 2009; 30(2):365-74.
  20. 지형준. 대한약전 및 대한약전외 한약규격주해. 서울;한국메디칼인덱스사. 1998:109-619.
  21. J.Sambrook, E.F.Fritsch, T.Maniatis. Molecular Cloning. Cold Spring Harbor Laboratory Press. 2nd ed. 1989;18:60-75.
  22. Bakon BR, Farahvash MJ, Janney CG, Neuschwander-Tetri BA. Nonalcoholic steatohepatitis: an expanded clinical entity. Gastroenterology. 1994;107:1103-9.
  23. Youssef WI, McCullough AJ. Steatohepatitis in obese indivisuals. Best Pract Res Clin Gastroenterol. 2005;45:46-52.
  24. 吴其恺, 程井军, 聂广. 脂炎消煎剂治療非酒精性 脂肪肝63例的臨床觀察. CJ GMTCM. 2007; 22(2):34-5.
  25. 전국한의과대학 간계내과학 교수 공저. 간계내과학. 서울: 동양의학연구원; 2001, P. 315-22.
  26. 이창규. 生肝湯이 $CCl_4$ 및 d-galactosamine에 의하여 유발된 흰쥐의 간장해에 미치는 영향. 경희대학교 대학원, 1986.
  27. 최서형. 生肝湯과 그 분획이 간장보호, 혈소판 응집능 및 일반 면역기능에 미치는 영향. 경희대학교 대학원; 1987.
  28. 류경하. 生肝湯이 고지혈증에 미치는 영향. 경희대학교 대학원. 1988.
  29. 승현석, 박신명, 한창우, 김영철, 이장훈, 우홍정. 加減生肝湯을 투여하여 호전된 급성 간염 2례. 대한한방내과학회. 2002;23(2):226-37.
  30. 김영철, 이장훈, 우홍정. 중풍을 동반한 초기간경변증 치험 1례. 대한한방내과학회 별책부록. 1999;19(2):21-8.
  31. 김동우, 김덕호, 우홍정, 김병운. 加減生肝湯을 투여한 간암환자에 관한 연구. 대한한의학회지. 1991;12(2):233-49.
  32. TAN De-an, FU Wei-ling, ZHOU Zhi-guang. Experimental study of effect of curcumin treatment on rat nonalcoholic fatty liver disease. 重慶醫學. 2007;36(16):1626-8.
  33. 반상석, 윤현덕, 신오철, 신유정, 박치상, 박지하 외. 고지방 식이로 유도된 비만 흰쥐에 茵蔯, 枳實, 山査가 미치는 영향. 대한본초학회지. 2006;21(3):55-67.
  34. Sun Liwei, Huang Miaozhen. The effect of Danshen on the nonalcoholic fatty liver rats MDA, SOD, TNF, Leptin. J. of Zhejiang Uni. of traditional Chinese Med. 2007;31(6):696-8.
  35. Day CP, James OF. Steatohepatitis: a tale of two "hits"? Gastroenterology. 1998;114:842-5. https://doi.org/10.1016/S0016-5085(98)70599-2
  36. Maechler P, Wolloheim CB. Mitochondrial function in normal and diabetic beta-cells. Nature. 2001 ;414:807-12. https://doi.org/10.1038/414807a
  37. St-Pierre J, Buckingham JA, Roebuck SJ, Brand MD. Topology of superoxide production from different sites in the mitochondrial electron transport chain. J. Biol chem. 2002;277:44784-90. https://doi.org/10.1074/jbc.M207217200
  38. Caldwell SH, Swerdlow RH, Khan EM, Iezzoni JC, Hespenheide EE, Parks JK, et al. Mitochondrial abnormalities in nonalcoholic steatohepatitis. J. Hepatol. 1999;31:430-4. https://doi.org/10.1016/S0168-8278(99)80033-6
  39. Chen J, Schenker S, Frosto TA, Henderson GI. Inhibition of cytochrome c oxidase activity by 4-hydroxynonenal(HNE): role of HNE adduct formation with the enzyme subunits. Biochim Biophys Acta. 1998;1380:336-44. https://doi.org/10.1016/S0304-4165(98)00002-6
  40. Poli G. Pathogenesisof liver fibrosis: role of oxidative stress. Mol Aspects Med. 2000;21: 49-98. https://doi.org/10.1016/S0098-2997(00)00004-2
  41. John D. Hayes, David J. Pulford. The Glutathione S-Transferase Supergene Family: Regulation of GST and the contribution of the Isoenzymes to Cancer Chemoprotection and Drug Resistance. Clinical Reviews in Biochemistry and Molecular Biology. 1995;30(6):445-600. https://doi.org/10.3109/10409239509083491
  42. Srinivas Kothakota, Toshifumi Azuma, Christoph Reinhard, Anke Klippel, Jay Tang, Keting Chu, et al. Caspase-3-Generated Fragment of Gelsolin: Effector of Morphological Change in Apoptosis. Science. 1997;278(10):294-7.
  43. Maria Jose Gomez-Lechon, Maria Teresa Donato, Alicia Martinez-Romero, Nuria Jimenez, Jose Vicente Castell, Jose-Enrique O'Connor. A human hepatocellular in vitro model to investigate steatosis. Chemico-Biological Interactions. 2007; 165:106-16. https://doi.org/10.1016/j.cbi.2006.11.004