消癖丸干预二甲基亚硝胺诱导大鼠肝纤维化的效应及其机制
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摘要
目的:探讨消癖丸干预二甲基亚硝胺(dimethylnitrosamine,DMN)诱导的大鼠肝纤维化的作用。方法:采用0.5%的DMN溶液2mL/kg体质量腹腔注射,每周连续注射3d,每日1次,共4周,制备大鼠肝纤维化模型。第3周开始模型大鼠随机分为模型对照组和消癖丸组,消癖丸组给予消癖丸煎剂灌胃,模型对照组给予等量的生理盐水。治疗2周后,观察大鼠肝功能、肝组织病理学改变及肝纤维化相关指标α平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)、转化生长因子β1(transforming growth factor-β1,TGF-β1)、组织金属蛋白酶抑制剂1(tissue inhibitor of metalloproteinase-1,TIMP-1)和血红素氧合酶1(heme oxygenase-1,HO-1)表达的变化。结果:(1)与正常大鼠比较,造模2和4周时模型大鼠血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、碱性磷酸酶(alkaline phosphatase,ALP)活性逐渐升高(P<0.01或P<0.05),4周时血清总胆红素(total bilirubin,TBil)含量显著增加(P<0.05);与4周模型组比较,消癖丸组血清ALT、AST、ALP、TBil水平均显著降低(P<0.01或P<0.05)。(2)模型大鼠肝组织炎症反应及胶原沉积逐渐加重,4周时部分大鼠肝组织已形成完整包绕的假小叶结构,肝组织羟脯氨酸(hydroxyproline,Hyp)含量显著增加(P<0.01);与4周模型组比较,消癖丸组肝组织炎症、胶原沉积明显减轻,肝组织Hyp含量显著降低(P<0.05)。(3)模型大鼠肝组织α-SMA蛋白表达逐渐增加,4周时显著高于2周模型组(P<0.01);聚合酶链式反应分析显示,肝组织α-SMA、TGF-β1、TIMP-1和HO-1mRNA的表达随着模型的进展逐渐升高(P<0.01);与4周模型组比较,消癖丸组肝组织α-SMA蛋白的表达及α-SMA、TGF-β1、TIMP-1、HO-1mRNA的表达均显著降低(P<0.01或P<0.05)。结论:消癖丸能够显著抑制DMN诱导大鼠肝纤维化的进展,且这一作用机制可能与抑制肝星状细胞活化有关。
OBJECTIVE: To explore the intervention effects of Xiaopi Pill (XPW), a compound traditional Chinese herbal medicine, on the development progress of dimethylnitrosamine (DMN)-induced liver fibrosis in rats. METHODS: Liver fibrosis model was established by intraperitoneal injection of 0.5% DMN 2 mL/kg thrice a week for 4 weeks. Rats were divided into control group given saline and treatment group given XPW during the 3rd week of DMN injections. Rats were sacrificed at the end of the experiment, and then liver histological changes, liver function and mRNA expression of the liver fibrosis-associated markers were observed. RESULTS: (1) At the end of the 2nd and 4th weeks of DMN injection, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) increased significantly in rats (P<0.01 or P<0.05); content of total bilirubin (TBil) increased significantly compared with the normal group until the end of the 4th week (P<0.05); compared with the model group after 4 weeks of DMN injection, the serum levels of ALT, AST, ALP and TBil were decreased remarkably in the XPW-treated group (P<0.01 or P<0.05). (2) The hepatic inflammation and collagen deposition in hepatic tissues increased by different degrees in experimental rats. Parts of pathological changes in the rat liver were found at the end of the 4th week, including a complete round structure of false flocculus round, meantime, the hydroxyproline content of hepatic tissue was increased significantly at the end of the 2nd and 4th weeks (P<0.05). Compared with the 4-week model group, the hepatic inflammation, collagen deposition and hydroxyproline content in hepatic tissues were alleviated dramatically (P<0.05). (3) Compared with the normal and 2nd week groups, protein expression of alpha-smooth muscle actin (α-SMA) was gradually increased, and that of the 4th week group were aggrandized significantly (P<0.01). Compared with the normal group, the mRNA expression of α-SMA, transforming growth factor-β1 (TGF-β1), tissue inhibitor of melalloproteinase-1 (TIMP-1), and heme oxygenase-1 (HO-1) was gradually increased. Further changes in above-mentioned abnormalities were found in the model rats at the end of the 4th week (P<0.01); while compared to the 4th week group, protein and mRNA levels of α-SMA and mRNA levels of TGF-β1, TIMP-1, and HO-1 were decreased significantly in the XPW group (P<0.01 or P<0.05). CONCLUSION: Progressive DMN-induced liver fibrosis in rats can be suppressed by XPW; the mechanism may be associated with inhibition of the activated hepatic stellate cells.
OBJECTIVE: To explore the intervention effects of Xiaopi Pill (XPW), a compound traditional Chinese herbal medicine, on the development progress of dimethylnitrosamine (DMN)-induced liver fibrosis in rats. METHODS: Liver fibrosis model was established by intraperitoneal injection of 0.5% DMN 2 mL/kg thrice a week for 4 weeks. Rats were divided into control group given saline and treatment group given XPW during the 3rd week of DMN injections. Rats were sacrificed at the end of the experiment, and then liver histological changes, liver function and mRNA expression of the liver fibrosis-associated markers were observed. RESULTS: (1) At the end of the 2nd and 4th weeks of DMN injection, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) increased significantly in rats (P<0.01 or P<0.05); content of total bilirubin (TBil) increased significantly compared with the normal group until the end of the 4th week (P<0.05); compared with the model group after 4 weeks of DMN injection, the serum levels of ALT, AST, ALP and TBil were decreased remarkably in the XPW-treated group (P<0.01 or P<0.05). (2) The hepatic inflammation and collagen deposition in hepatic tissues increased by different degrees in experimental rats. Parts of pathological changes in the rat liver were found at the end of the 4th week, including a complete round structure of false flocculus round, meantime, the hydroxyproline content of hepatic tissue was increased significantly at the end of the 2nd and 4th weeks (P<0.05). Compared with the 4-week model group, the hepatic inflammation, collagen deposition and hydroxyproline content in hepatic tissues were alleviated dramatically (P<0.05). (3) Compared with the normal and 2nd week groups, protein expression of alpha-smooth muscle actin (α-SMA) was gradually increased, and that of the 4th week group were aggrandized significantly (P<0.01). Compared with the normal group, the mRNA expression of α-SMA, transforming growth factor-β1 (TGF-β1), tissue inhibitor of melalloproteinase-1 (TIMP-1), and heme oxygenase-1 (HO-1) was gradually increased. Further changes in above-mentioned abnormalities were found in the model rats at the end of the 4th week (P<0.01); while compared to the 4th week group, protein and mRNA levels of α-SMA and mRNA levels of TGF-β1, TIMP-1, and HO-1 were decreased significantly in the XPW group (P<0.01 or P<0.05). CONCLUSION: Progressive DMN-induced liver fibrosis in rats can be suppressed by XPW; the mechanism may be associated with inhibition of the activated hepatic stellate cells.
引文
1 Ye F,Xue BY,Zhou M,Zhou ZY.Damp-heat toxinaccumulation and hepatofibrosis.Nanjing Zhong Yi YaoDa Xue Xue Bao.2005;21(6):346-349.Chinese.叶放,薛博瑜,周珉,周仲瑛.论湿热瘀毒与肝纤维化.南京中医药大学学报.2005;21(6):346-349.
2 Ala-Kokko L,Pihlajaniemi T,Myers JC,Kivirikko KI,Savolainen ER.Gene expression of typeⅠ,ⅢandⅣcollagens in hepatic fibrosis induced by dimethylnitrosaminein the rat.Biochem J.1987;244(1):75-79.
3 Jamall IS,Finelli VN,Que Hee SS.A simple methodto determine nanogram levels of 4-hydroxyproline inbiological tissues.Anal Biochem.1981;112(1):70-75.
4 Nakatani K,Seki S,Kawada N,Kitada T,Yamada T,Sakaguchi H,Kadoya H,Ikeda K,Kaneda K.Expressionof SPARC by activated hepatic stellate cells and its correlationwith the stages of fibrogenesis in human chronic hepatitis.Virchows Arch.2002;441(5):466-474.
5 Kristensen DB,Kawada N,Imamura K,Miyamoto Y,Tateno C,Seki S,Kuroki T,Yoshizato K.Proteomeanalysis of rat hepatic stellate cells.Hepatology.2000;32(2):268-277.
6 Uyama N,Shimahara Y,Okuyama H,Kawada N,Kamo S,Ikeda K,Yamaoka Y.Carbenoxolone inhibitsDNA synthesis and collagen gene expression in rat hepaticstellate cells in culture.J Hepatol.2003;39(5):749-755.
7 Jézéquel AM,Mancini R,Rinaldesi ML,Macarri G,Venturini C,Orlandi F.A morphological study of theearly stages of hepatic fibrosis induced by low doses ofdimethylnitrosamine in the rat.J Hepatol.1987;5(2):174-181.
8 Zhang B,Wang LT.Progress in experimental study oninhibitory effect of traditional Chinese medicine on liverfibrosis.J Chin Integr Med.2006;4(2):206-210.Chinese.张斌,王灵台.中医药抗肝纤维化的实验研究进展.中西医结合学报.2006;4(2):206-210.
9 Chinese Association of Integrative Medicine.Guidelinefor the diagnosis and treatment of liver fibrosis withintegrative medicine.J Chin Integr Med.2006;4(6):551-555.Chinese.中国中西医结合学会肝病专业委员会.肝纤维化中西医结合诊疗指南.中西医结合学报.2006;4(6):551-555.
10 Okada A,Garnier JM,Vicaire S,Basset P.Cloning ofthe cDNA encoding rat tissue inhibitor of metalloproteinase1(TIMP-1),amino acid comparison with other TIMPs,and gene expression in rat tissues.Gene.1994;147(2):301-302.
11 Yoshiji H,Kuriyama S,Yoshii J,Ikenaka Y,NoguchiR,Nakatani T,Tsujinoue H,Yanase K,Namisaki T,Imazu H,Fukui H.Tissue inhibitor of metalloproteinases-1attenuates spontaneous liver fibrosis resolution in thetransgenic mouse.Hepatology.2002;36(4):850-860.
12 Wang XH,Cao Q,Li J,Huang Y.The function,regulationand development of heme oxygenase-1in the liver disease.Zhongguo Yao Li Xue Tong Bao.2006;22(5):525-528.Chinese with abstract in English.王晓红,曹琦,李俊,黄艳.血红素氧化酶功能和调节及其在肝病发生中的研究进展.中国药理学通报.2006;22(5):525-528.
13 Huang XF,Chen BC,Hu ZY,Lin LN,Luo AL.Protective role of heme oxygenase-1on the liver ischemia/reperfusion injury.Gan Dan Yi Wai Ke Za Zhi.2006;18(2):95-96.Chinese with abstract in English.黄秀芳,陈必成,胡正杨,林丽娜,罗爱林.血红素加氧酶-1诱导对鼠肝缺血再灌注损伤的保护作用.肝胆胰外科杂志.2006;18(2):95-96.
14 Li YM,Zhang P,Zhang Y,Zhang QS,Wu LL,WuYJ.Alteration and role of heme oxygenase in liver duringsepsis.Chengde Yi Xue Yuan Yuan Bao.2001;18(3):202-205.Chinese with abstract in English.李玉明,张鹏,张游,张秋实,吴立玲,伍贻经.脓毒血症大鼠肝脏血红素加氧酶的变化及作用.承德医学院院报.2001;18(3):202-205.
2 Ala-Kokko L,Pihlajaniemi T,Myers JC,Kivirikko KI,Savolainen ER.Gene expression of typeⅠ,ⅢandⅣcollagens in hepatic fibrosis induced by dimethylnitrosaminein the rat.Biochem J.1987;244(1):75-79.
3 Jamall IS,Finelli VN,Que Hee SS.A simple methodto determine nanogram levels of 4-hydroxyproline inbiological tissues.Anal Biochem.1981;112(1):70-75.
4 Nakatani K,Seki S,Kawada N,Kitada T,Yamada T,Sakaguchi H,Kadoya H,Ikeda K,Kaneda K.Expressionof SPARC by activated hepatic stellate cells and its correlationwith the stages of fibrogenesis in human chronic hepatitis.Virchows Arch.2002;441(5):466-474.
5 Kristensen DB,Kawada N,Imamura K,Miyamoto Y,Tateno C,Seki S,Kuroki T,Yoshizato K.Proteomeanalysis of rat hepatic stellate cells.Hepatology.2000;32(2):268-277.
6 Uyama N,Shimahara Y,Okuyama H,Kawada N,Kamo S,Ikeda K,Yamaoka Y.Carbenoxolone inhibitsDNA synthesis and collagen gene expression in rat hepaticstellate cells in culture.J Hepatol.2003;39(5):749-755.
7 Jézéquel AM,Mancini R,Rinaldesi ML,Macarri G,Venturini C,Orlandi F.A morphological study of theearly stages of hepatic fibrosis induced by low doses ofdimethylnitrosamine in the rat.J Hepatol.1987;5(2):174-181.
8 Zhang B,Wang LT.Progress in experimental study oninhibitory effect of traditional Chinese medicine on liverfibrosis.J Chin Integr Med.2006;4(2):206-210.Chinese.张斌,王灵台.中医药抗肝纤维化的实验研究进展.中西医结合学报.2006;4(2):206-210.
9 Chinese Association of Integrative Medicine.Guidelinefor the diagnosis and treatment of liver fibrosis withintegrative medicine.J Chin Integr Med.2006;4(6):551-555.Chinese.中国中西医结合学会肝病专业委员会.肝纤维化中西医结合诊疗指南.中西医结合学报.2006;4(6):551-555.
10 Okada A,Garnier JM,Vicaire S,Basset P.Cloning ofthe cDNA encoding rat tissue inhibitor of metalloproteinase1(TIMP-1),amino acid comparison with other TIMPs,and gene expression in rat tissues.Gene.1994;147(2):301-302.
11 Yoshiji H,Kuriyama S,Yoshii J,Ikenaka Y,NoguchiR,Nakatani T,Tsujinoue H,Yanase K,Namisaki T,Imazu H,Fukui H.Tissue inhibitor of metalloproteinases-1attenuates spontaneous liver fibrosis resolution in thetransgenic mouse.Hepatology.2002;36(4):850-860.
12 Wang XH,Cao Q,Li J,Huang Y.The function,regulationand development of heme oxygenase-1in the liver disease.Zhongguo Yao Li Xue Tong Bao.2006;22(5):525-528.Chinese with abstract in English.王晓红,曹琦,李俊,黄艳.血红素氧化酶功能和调节及其在肝病发生中的研究进展.中国药理学通报.2006;22(5):525-528.
13 Huang XF,Chen BC,Hu ZY,Lin LN,Luo AL.Protective role of heme oxygenase-1on the liver ischemia/reperfusion injury.Gan Dan Yi Wai Ke Za Zhi.2006;18(2):95-96.Chinese with abstract in English.黄秀芳,陈必成,胡正杨,林丽娜,罗爱林.血红素加氧酶-1诱导对鼠肝缺血再灌注损伤的保护作用.肝胆胰外科杂志.2006;18(2):95-96.
14 Li YM,Zhang P,Zhang Y,Zhang QS,Wu LL,WuYJ.Alteration and role of heme oxygenase in liver duringsepsis.Chengde Yi Xue Yuan Yuan Bao.2001;18(3):202-205.Chinese with abstract in English.李玉明,张鹏,张游,张秋实,吴立玲,伍贻经.脓毒血症大鼠肝脏血红素加氧酶的变化及作用.承德医学院院报.2001;18(3):202-205.
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