Clinical and Molecular Hepatology (대한간학회지)

The Korean Association for the Study of the Liver (대한간학회)
권호 표지

Suppressive Effects of Antioxidant DA-9601 on Hepatic Fibrosis in Rats

항산화제 DA-9601에 의한 백서 간섬유화 억제 효과

Cheong, Jae-Youn;Oh, Tae-Young;Lee, Ki-Myung;Kim, Do-Hyun;Ahn, Byoung-Ok;Kim, Won-Bae;Kim, Young-Bae;Yoo, Byung-Moo;Hahm, Ki-Baik;Kim, Jin-Hong;Cho, Sung-Won
정재연;오태영;이기명;김도현;안병옥;김원배;김영배;유병무;함기백;김진홍;조성원

  • Published : 20020000
⟨ Previous Next ⟩

Abstract

Background/Aims : Oxidative stress is one of the important underlying mechanisms of hepatic fibrosis. DA-9601, the ethanol extracts of Artemisia asiatica, has been reported to possess strong antioxidative and cytoprotective actions. We tried to evaluate whether antioxidant can ameliorate dimethylnitrosamine (DMN)-induced hepatic fibrosis. Methods: Rat hepatic fibrosis was induced by intraperitoneal administrations of 10 mg DMN six times. Additionally, rats of one group were started daily with DA-9601 30 mg/kg containing diets and another group was fed a pellet diet containing DA-9601 100 mg/kg. The immunohistochemical studies for collagen, $\alpha$-smooth muscle actin ($\alpha$-SMA), and fibronectin, the measurements of hepatic malondialdehyde (MDA) and collagens, and the changes of liver function profiles were performed. Hepatic stellate cells (HSC) were isolated and in vitro effects of DA-9601 on HSC activations were measured. Results: DA-9601 significantly attenuated the loss of body weights (p<0.05), the reduction of liver wet weights (p<0.05), and the elevation of liver enzymes provoked by DMN administrations. DMN injections caused the severe fibrosis of portal tract, hepatic inflammation, and significant oxidative damages, but DA-9601 treatment significantly reduced the mean scores of hepatic fibrosis, the amounts of hepatic collagens, and hepatic MDA levels. The prominent decreases in the expressions of collagens type I & III, $\alpha$-SMA, and fibronectin or hepatic inflammations were observed in DA-9601-treated groups dose-dependently and similar efficacy was also proven in in vitro HSC experiment. Conclusions: DA-9601 effectively protected rat liver tissues against the DMN-induced hepatic fibrosis. Antioxidant could be considered as a supplementary therapeutic for alleviating the hepatic fibrosis.

목적: 산화적 스트레스와 이에 따른 지질 과산화는 간섬유화의 중요한 기전이며, 항산화제는 이를 억제함으로써 간섬유화를 예방할 수 있다. DA-9601은 쑥의 일종인 Artemisia asiatica의 추출물에서 합성한 약물로 항산화작용이 있는 것으로 알려져 있다. 본 연구는 항산화제인 DA-9601이 dimethylnitrosamine (DMN)에 의한 간섬유화를 억제하는지를 알아보고자 하였다. 대상과 방법: 숫 Sprague-Dawley계 백서를 10마리씩 4군으로 분류하여 2군, 3군, 4군에는 DMN 10 mg/kg을 1주일에 3번씩 2주간 복강내로 주사하였고, 1군은 생리 식염수를 같은 방법으로 주사하였다. DMN 주사 1주전 부터 DMN 주사기간 동안, 3군은 DA-9601 30 mg/kg, 4군은 DA-9601 100 mg/kg을 동시에 투여하였다. 적출된 간조직의 파라핀 블록에서 H-E염색과 collagen, $\alpha$-smooth muscle actin ($\alpha$-SMA), fibronectin에 대한 면역조직화학적 염색을 실시하였고, collagen 및 malondialdehyde (MDA)를 정량 하였고, 간기능검사의 변화를 살펴 간섬유화에 대한 항산화제의 효과를 알아보았다. 또한, 간성상세포 (hepatic stellate cell, HSC)를 분리하여, in vitro상에서 HSC 활성화에 대한 DA-9601의 효과를 측정 하였다. 결과: 1) DA-9601은 DMN 주사에 의한 백서의 체중감소를 둔화시켰고(2군: $245.5{\pm}7.5$g, 4 군: $266.8{\pm}4.1$g, p<0.05), 간섬유화에 의한 간무게감소를 줄였으며(1군: $15.2{\pm}1.8$g, 2군: $7.7{\pm}1.9$g, 3군$:12.6{\pm}2.0$g, 4군: $14.1{\pm}2.2$g, p<0.05), 간효소치 상승을 감소시켰다(ALT 2군 $177.2{\pm}40.3$ IU/L, 4군 $96.0{\pm}21.6$IU/L, p<0.05; AST 2군 $236.5{\pm}33.7$IU/L, 4군 $108.8{\pm}23.2$ IU/L, p<0.05). 2) DMN 주사에 의해 간문맥계의 섬유화, 간세포 염증괴사, 산화에 의한 심한 간세포 손상을 보였으나, DA-9601 투여는 DMN 주사에 의한 간섬유화, collagen의 증가, 간 MDA의 증가를 감소시켰다. 3) DA-9601 투여군에서 collagen I형과 III형, a-SMA, fibronectin 이 감소하였고, 간세포 염증이 약물 용량에 비례하여 감소하였으며, HSC에 대한 in vitro 실험에서도 유사한 양상을 보였다. 결론: DA-9601은 DMN에 의한 간섬유화를 효과적으로 억제하였다. 따라서 항산화제는 간섬유화 및 간세포 염증반응을 줄일 수 있는 치료제로 고려할 수 있을 것으로 생각된다.

Keywords

References

  1. Jezequel AM, Ballardini G, Mancini R, Paolucci F, Bianchi FB, Orlandi F. Modulation of extracellular matrix components during dimethylnitrosamine-induced cirrhosis. J Hepatol 1990;11:206-14. https://doi.org/10.1016/0168-8278(90)90115-8
  2. Jenkins SA, Grandison A, Baxter JN, Day DW, Taylor I, Shields R. A dimethylnitrosamine-induced model of cirrhosis and portal hypertension in the rat. J Hepatol 1985;1:489-499. https://doi.org/10.1016/S0168-8278(85)80747-9
  3. Jezequel AM, Mancini R, Rinaldesi ML, Macarri G, Venturini C, Orlandi F. A morphological study of the early stages of hepatic fibrosis induced by low doses of dimethylnitrosamine in the rat. J Hepatol 1987;5:174-181. https://doi.org/10.1016/S0168-8278(87)80570-6
  4. Baroni GS, Dambrosio L, Ferretti G et al. Fibrogenic effect of oxidative stress on rat hepatic stellate cells. Hepatology 1998;27:720-726. https://doi.org/10.1002/hep.510270313
  5. Nieto N, Friedman SL, Greenwel P, Cederbaum AI. CYP2E1-mediated oxidative stress induced collagen type I expression in rat hepatic stellate cells. Hepatology 1999; 30:987-996. https://doi.org/10.1002/hep.510300433
  6. Olaso E, Friedman SL. Molecular regulation of hepatic fibrogenesis. J Hepatol 1998;29:836-847. https://doi.org/10.1016/S0168-8278(98)80269-9
  7. Gressner AM. Cytokines and cellular crosstalk involved in the activation of fat storing cells. J Hepatol 1995;22:28-36. https://doi.org/10.1016/0270-9139(95)94092-8
  8. Xu Y, Rojkind M, Czaja MJ. Regulation of monocyte chemoattractant protein 1 by cytokines and oxygen free radicals in rat hepatic fat storing cells. Gastroenterology 1996;110: 1870-1877. https://doi.org/10.1053/gast.1996.v110.pm8964413
  9. Vogl S, Petermann H, Dargel R. Oxygen radical formation, proliferative activity and phagocytic capacity of cultivated macrophages from cirrhotic rat livers. Liver 1996;16:313-320.
  10. Casini A, Ceni E, Salzano R, et al. Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells: Role of nitric oxide. Hepatology 1997;25:361-367. https://doi.org/10.1002/hep.510250218
  11. Swart PJ, Hirano T, Kuipers ME, et al. Targeting of superoxide dismutase to the liver results in antiinflammatory effects in rats with fibrotic livers. J Hepatol 1999;31:1034-1043. https://doi.org/10.1016/S0168-8278(99)80316-X
  12. Sokol RJ. Antioxidant defenses in metal-induced liver damage. Semin Liver Dis 1996;16:39-46. https://doi.org/10.1055/s-2007-1007217
  13. Miyamura M, Ono M, Kyotani S, Nishioka Y. Effects of Sho-saiko-to extract on fibrosis and regeneration of the liver in rats. J Pharm Pharmacol 1998;50:97-105.
  14. Kayano K, Sakaida I, Uchida K, Okita K. Inhibitory effects of the herbal medicine Sho-saiko-to (TJ-9) and cell proliferation and procollagen gene expressions in cultured rat hepatic stellate cells. J Hepatol 1998;29: 642-649. https://doi.org/10.1016/S0168-8278(98)80161-X
  15. Oh TY, Ryu BK, Ko JI, et al. Protective effect of DA-9601, an extract of Artemisia herba, against naproxen-induced gastric damage in arthritic rats. Arch Pharm Res 1997;20: 414-419. https://doi.org/10.1007/BF02973932
  16. Hahm KB, Kim JH, Yoo BM et al. Induction of apoptosis with an extract of Artemisia asiatica attenuates the severity of cerulein-induced pancreatitis in rats. Pancreas 1998;17:153-157. https://doi.org/10.1097/00006676-199808000-00007
  17. Ahn BO, Ryu BK, Ko JI, et al. Beneficial effect of DA-9601, an Extract of Artemisia herba, on animal models of inflammatory bowel disease. J Appl Pharm 1997;5: 165-173.
  18. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275.
  19. Apte MV, Haber PS, Applegate TL, et al. Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture. Gut 1998;43:128-133. https://doi.org/10.1136/gut.43.1.128
  20. Shimizu I, Yasuda M, Mizobuchi Y, et al. Suppressive effect of oestradiol on chemical hepatocarcinogenesis in rats. Gut 1998;42:112-119. https://doi.org/10.1136/gut.42.1.112
  21. Yasuda M, Shimizu I, Shiba M, Ito S. Suppressive effects of estradiol on dimethylnitrosamine-induced fibrosis of the liver in rats. Hepatology 1999;29:719-727. https://doi.org/10.1002/hep.510290307
  22. Matsuda Y, Matsumoto K, Yamada A, et al. Preventive and therapeutic effects in rats of hepatocytes growth factor infusion on liver fibrosis/cirrhosis. Hepatology 1997; 26:81-89. https://doi.org/10.1002/hep.510260111
  23. Yasuda H, Imai E, Shiota A, Fujise N, Morinaga T, Higashio K. Antifibrogenic effect of a deletion variant of hepatocytes growth factor on liver fibrosis in rats. Hepatology 1996;24:636-642. https://doi.org/10.1002/hep.510240328
  24. 서강석, 이창훈, 설미영, 이선경. Dimethylnitrosamine에 의 한 백서의 간섬유화 과정에서 Pentoxifylline과 Ciprofloxacine의 영향. 대한간학회지 2001;7:34-46.
  25. Mancini R, Benedetti A, Jezequel AM. An interleukin-1 receptor antagonist decreases fibrosis induced by dimethylnitrosamine in rat liver. Virchows Arch 1994;424: 25-31.
  26. George J, Roulot D, Koteliansky VE, Bissell DM. In vitro inhibition of rat stellate cell activation by soluble transforming growth factor type II receptor: A potential new therapy for hepatic fibrosis. Proc Natl Acad Sci USA 1999;96:12719-12724. https://doi.org/10.1073/pnas.96.22.12719
  27. Baroni GS, Ambrosio LD, Curto P, et al. Interferon gamma decreases hepatic stellate cell activation and extracellular matrix deposition in rat liver fibrosis. Hepatology 1996; 23:1189-1199 https://doi.org/10.1002/hep.510230538
  28. Pines M, Knopov V, Genina O, Lavelin I, Nagler A. Halofuginone, a specific inhibitor of collagen type I synthesis, prevents dimethylnitrosamine-induced liver cirrhosis. J Hepatol 1997;27:391-398. https://doi.org/10.1016/S0168-8278(97)80186-9
  29. 이정일, 이관식, 정준표 등. 사염화탄소로 급성 간손상이 유발된 흰쥐에서 혈청 및 간조직의 Transforming Growth Factor-$\beta$-1의 변화와 비타민 E의 효과. 대한간학회지 2000: 6;147-155
  30. Gualdi R, Casalgrandi G, Montosi G, Ventura E, Pietrangelo A. Excess iron into hepatocytes is required for activation of collagen type I gene during experimental sclerosis. Gastroenterology 1994;107:1118-1124. https://doi.org/10.1016/0016-5085(94)90237-2
  31. Britton RS, Bacon BR. Role of free radicals in liver diseases and hepatic fibrosis. Hepatogastroenterology 1994;41:343-348.
  32. Montosi G, Garuti C, Iannone A, Pietrangelo A. Spatial and remporal dynamics of hepatic stellate cell activation during oxidant-stress-induced fibrogenesis. Am J Pathol 1998;152:1319-1326.
  33. Paolucci F, Mancini R, Marucci L, Benedetti A, Jezequel AM, Orlandi F. Immunohistochemical identification of proliferating cells following dimethylnitrosamine-induced liver injury. Liver 1990;10:278-281.
  34. Mancini R, Jezequel AM, Benedetti A, Paolucci F, Trozzi L, Orlandi F. Quantitative analysis of proliferating sinusoidal cells in dimethylnitrosamine-induced cirrhosis-An immunohistochemical study. J Hepatol 1992;15:361-366. https://doi.org/10.1016/0168-8278(92)90069-2
  35. Shiba M, Shimizu I, Yasuda M, Ii K, Ito S. Expression of type I and type III collagens during the course of dimethylnitrosamine-induced hepatic fibrisis in rats. Liver 1998;18:196-204.
  36. Yamamoto Y, Yamashita S, Fujisawa A. Oxidative stress in patients with hepatitis, cirrhosis and hepatoma evaluated by plasma antioxidants. Biochem Biophys Res Commun 1998;247:166-170. https://doi.org/10.1006/bbrc.1998.8752
  37. Iredale JP, Benyon RC, Pickering J et al. Mechanisms of spontaneous resolution of rat liver fibrosis. J Clin Invest 1998;102: 538-549. https://doi.org/10.1172/JCI1018
  38. Mitsuyoshi H, Nakashima T, Sumida Y et al. Ursodeoxycholic acid protects hepatocytes against oxidative injury via induction of antioxidants. Biochem Biophys Res Commun 1999;263: 537-542. https://doi.org/10.1006/bbrc.1999.1403
  39. Shimizu I, Ma YR, Mizobuchi Y, et al. Effects of Shosaiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats. Hepatology 1999;29: 149-160. https://doi.org/10.1002/hep.510290108
  40. Kovacs EJ. Fibrogenic cytokines. The role of immune mediators in the development of scar tissue. Immunol Today 1991;12 :17-23. https://doi.org/10.1016/0167-5699(91)90107-5
  41. Brown KE, Poulos JE, Li L, et al. Effect of vitamine E supplementation on hepatic fibrogenesis in chronic dietary iron overload. Am J Physiol 1997;272: G116-123.
  42. Ham AJ, Liebler DC. Antioxidant reactions of vitamin E in the perfused rat liver: product distribution and effect of dietary vitamin E supplementation. Arch Biochem Biophys 1997;339:157-164. https://doi.org/10.1006/abbi.1996.9856
  43. Pastor A, Collado PS, Almar M, Gonzalez-Gallego J. Antioxidant enzyme status in biliary obstracted rats: effects of N-acetylcystein. J Hepatol 1997;27:363-370. https://doi.org/10.1016/S0168-8278(97)80183-3