复方茵丹方对肝内胆汁淤积大鼠多药耐药相关蛋白2表达的影响
摘要
目的：观察复方茵丹方对肝内胆汁淤积大鼠模型的治疗作用,观察肝内胆汁淤积大鼠Mrp2mRNA和蛋白表达情况,探讨复方茵丹方治疗肝内胆汁淤积的作用机制
方法：选择雄性wistar大鼠45只,随机分为三组,正常对照组9只,模型组18只,中药组18只,应用α-萘异硫氰酸酯(ANIT)灌胃方法诱导大鼠肝内胆汁淤积模型。中药组于造模同时给予中药复方茵丹方灌胃,每日1次。分别于造模后24小时、48小时、72小时采集大鼠血清、肝组织标本。观察大鼠各个时相点肝功能变化；采用RT-PCR方法观察大鼠不同时相点肝细胞Mrp2mRNA表达情况；通过免疫组化方法观察大鼠Mrp2蛋白表达；通过免疫印迹法(Western-blot)检测大鼠肝组织Mrp2蛋白定量。通过SARS软件对获取数据进行统计分析,比较中药组和模型组大鼠不同时相点血清生化指标的差异、Mrp2mRNA和Mrp2蛋白表达的差异。
结果：
1血清生化指标：造模后模型组和中药组大鼠很快出现明显肝功损伤,表现为转氨酶、胆管酶、血清胆红素的迅速上升,而正常对照组大鼠肝功能在三个时间点无明显变化。与正常对照组比较,模型组24小时、48小时、72小时的血清谷丙转氨酶(ALT)、γ-谷氨酰转酞酶(GGT),碱性磷酸酶(ALP)、总胆红素(TBIL)、直接胆红素(DBIL)均显著升高P<0.01。与模型组比较,中药组在72小时的肝功能指标明显降低,P<0.05。
2肝组织病理变化：模型组和中药组24h后即可见小叶结构轻度破坏,肝细胞轻度肿胀。汇管区可见散在脱落的胆管上皮细胞,并见少量的中性粒细胞浸润。48小时损伤继续加重。24小时、48小时两组差异不明显。72小时,模型组大鼠肝小叶结构重度破坏,肝细胞肿胀,细胞核大小不一,核内染色质聚集,核仁粗大浓染,汇管区内见胆管上皮细胞增生,中性粒细胞浸润。中药组在72小时肝组织的损伤明显好于模型组。
3大鼠肝组织Mrp2mRNA RT-PCR定量结果：与同时相正常组相比,模型组大鼠Mrp2mRNA表达均有显著下降,24小时、72小时比较P<0.05,48小时比较P<0.01。与同时相模型组相比,24小时中药组Mrp2mRNA表达呈增高趋势,但统计学比较无显著差异。48小时、72小时中药组Mrp2mRNA表达显著高于模型组,P<0.05
4大鼠肝组织Mrp2转运蛋白表达结果：免疫组化结果显示,与正常对照组比较模型组各时相点Mrp2表达量均显著低于正常对照组,P<0.01。与模型组比较,24小时中药组和模型组之间无显著差异,48小时中药组Mrp2表达量逐渐增加,显著高于模型组P<0.05；72小时中药组Mrp2阳性表达量继续增加,显著高于模型组,P<0.01。Western-blot凝胶图象分析显示,与正常组比较,模型组在三个时相点Mrp2蛋白表达均显著降低,P<0.01；与模型组比较,中药组在个各时相点均显著增高,24小时P<0.05,48小时P<0.01,72小时P<0.05。
结论：
1复方茵丹方能够降低血清胆红素水平、减轻肝细胞及小胆管损伤,改善肝功能,减轻胆汁淤积,对α-萘异硫氰酸酯导致的大鼠肝内胆汁淤积具有防治作用。
2α-萘异硫氰酸酯诱导的大鼠肝内胆汁淤积及Mrp2表达的下调是复方茵丹方清热化湿、凉血活血的方证病理学基础。
3上调Mrp2mRNA和蛋白表达是复方茵丹方防治大鼠肝内胆汁淤积的关键机制之一。
Objective:To investigate the intervention effect of compound Yindan decoction in rats with acute intrahepatic cholestasis induced by alpha-naphthyl isothiocyanate (ANIT),to observe the expression of Mrp2mRNA and protein, to explore the mechanism of compound Yindan decoction in the treatment of intrahepatic cholestasis.
Methods:45 male wistar rats were randomly divided into three groups, normal control group (9), the model group (18),TCM group (18), the model of intrahepatic cholestasis can be induced by alpha-naphthyl isothiocyanate (ANIT) by gavage. The rats of TCM group were given compound Yindan decoction by gavage once a day.To collect the samples of serum and hepatic tissues after modeling 24 hours,48 hours,72 hours. To observe the changes of hepatic function at different time points; To observe the expression of Mrp2mRNA in hepatocytes at different time points by RT-PCR; To boserve protein expression of Mrp2 by Immunohistochemistry; To detect the protein quantitation of Mrp2 in the hepatic tissue by Immunoblotting (Wes tern-blot).To obtain data for statistical analysis by SARS software and compare of the differences in serum biochemical indicators, expression of Mrp2mRNA and Mrp2 protein at different time points between TCM group and model group.
Results:
1 Serum biochemical indexes:The rats of model group and TCM group appeared rapidly significant hepatic damage,manifested as the rapid rise of transaminases,biliary enzymes and serum bilirub in,while the hepatic function of normal control group had no significant change at three time points. ALT, AST, TBi1, DBi1, GGT, ALP and TBA of model group were significantly increased compared with normal control group (P<0.01).Hepatic function indexes were significantly decreased at 72 hours compared with model group (P<0.05, P<0.01).
2 Hepatic tissue pathological changes:Comparing with normal control group, the rats of model group appeared damage to the hepatic lobules strueture,hepatocyte swelling,and scattering and exfoliation of biliary epithelial cells and infiltration of neutrophils in portal area at 24 hours after modeling; hepatic tissue lesions aggravated, hepatic lobules structure destructed severely,nucleus of different sizes, nuclear chromatin gathered,nucleolus enlarged and hyperstained. At 48 hours,we can see that significant hyperplasia of biliary epithelial cells,punctate and focal necrosis of hepatic cells around biliary and a lot of infiltration of neutrophils in portal area; At 72 hours, it can be still seen the hyperplasia of biliary epithelial cells and infiltration of neutrophils in portal area.The hepatic tissue pathological changes of TCM group were not very significant at 24 hours and 48 hours compared with model group,but neutrophils infiltration significantly decreased at 72 hours in portal area.
3 RT-PCR quantitative results of Mrp2mRNA in hepatic tissue:The expression of Mrp2mRNA of rats of model group were significantly decreased compared with normal control group,at 24 hours and 72 hours, P<0.05,at 48 hours, P<0.01. The expression of Mrp2mRNA of rats of TCM group tended to increase, but there was no significant difference at 24 hours.The expression of Mrp2mRNA of rats of TCM group was ignificantly higher than model group at 48 hours and 72 hours (P<0.05)
4 Expression results of Mrp2 transporter protein in hepatic tissue: Immunohistochemical results showed that Mrp2 expression were significantly decreased compared with the normal control group at all time points,P<0.01.There is no significant difference between TCM group and model group at 24 hours, the amount of expression of Mrp2 gradually to increase, significantly increased at 48 hours (P<0.05) and at 72 hours (P<0.01). Western-b lot gel image analysis showed that the expression of
Mrp2 protein were significantly decreased at three time points compared with the normal control group(P<0.01);the expression of Mrp2 protein were significantly increased at 24 hours P<0.05,at 48 hours P<0.01, at 72 hours P<0.05 compared with the normal control group.
Conclusions:
1 Compound Yindan decoction can decrease the levels of serum bilirub in, reduce the injury of hepatic cells and bile duct, improve the hepatic function and reduce cholestasis and has a preventive effect to rat intrahepatic cholestasis caused by alpha-Naphthy1 isothiocyanate.
2 The prescriptions and syndromes pathological basis of clearing heat and removing dampness, cooling blood and activating blood circulation is the intrahepatic cholestasis in rats indeced by alpha-Naphthy1 isothiocyanate and the down-regulation of the expression of Mrp2.
3 One of the key mechanism of control intrahepatic cholestasis in rats with Compound Yindan decoction is the up-regulated expression of Mrp2mRNA and protein.
方法：选择雄性wistar大鼠45只,随机分为三组,正常对照组9只,模型组18只,中药组18只,应用α-萘异硫氰酸酯(ANIT)灌胃方法诱导大鼠肝内胆汁淤积模型。中药组于造模同时给予中药复方茵丹方灌胃,每日1次。分别于造模后24小时、48小时、72小时采集大鼠血清、肝组织标本。观察大鼠各个时相点肝功能变化；采用RT-PCR方法观察大鼠不同时相点肝细胞Mrp2mRNA表达情况；通过免疫组化方法观察大鼠Mrp2蛋白表达；通过免疫印迹法(Western-blot)检测大鼠肝组织Mrp2蛋白定量。通过SARS软件对获取数据进行统计分析,比较中药组和模型组大鼠不同时相点血清生化指标的差异、Mrp2mRNA和Mrp2蛋白表达的差异。
结果：
1血清生化指标：造模后模型组和中药组大鼠很快出现明显肝功损伤,表现为转氨酶、胆管酶、血清胆红素的迅速上升,而正常对照组大鼠肝功能在三个时间点无明显变化。与正常对照组比较,模型组24小时、48小时、72小时的血清谷丙转氨酶(ALT)、γ-谷氨酰转酞酶(GGT),碱性磷酸酶(ALP)、总胆红素(TBIL)、直接胆红素(DBIL)均显著升高P<0.01。与模型组比较,中药组在72小时的肝功能指标明显降低,P<0.05。
2肝组织病理变化：模型组和中药组24h后即可见小叶结构轻度破坏,肝细胞轻度肿胀。汇管区可见散在脱落的胆管上皮细胞,并见少量的中性粒细胞浸润。48小时损伤继续加重。24小时、48小时两组差异不明显。72小时,模型组大鼠肝小叶结构重度破坏,肝细胞肿胀,细胞核大小不一,核内染色质聚集,核仁粗大浓染,汇管区内见胆管上皮细胞增生,中性粒细胞浸润。中药组在72小时肝组织的损伤明显好于模型组。
3大鼠肝组织Mrp2mRNA RT-PCR定量结果：与同时相正常组相比,模型组大鼠Mrp2mRNA表达均有显著下降,24小时、72小时比较P<0.05,48小时比较P<0.01。与同时相模型组相比,24小时中药组Mrp2mRNA表达呈增高趋势,但统计学比较无显著差异。48小时、72小时中药组Mrp2mRNA表达显著高于模型组,P<0.05
4大鼠肝组织Mrp2转运蛋白表达结果：免疫组化结果显示,与正常对照组比较模型组各时相点Mrp2表达量均显著低于正常对照组,P<0.01。与模型组比较,24小时中药组和模型组之间无显著差异,48小时中药组Mrp2表达量逐渐增加,显著高于模型组P<0.05；72小时中药组Mrp2阳性表达量继续增加,显著高于模型组,P<0.01。Western-blot凝胶图象分析显示,与正常组比较,模型组在三个时相点Mrp2蛋白表达均显著降低,P<0.01；与模型组比较,中药组在个各时相点均显著增高,24小时P<0.05,48小时P<0.01,72小时P<0.05。
结论：
1复方茵丹方能够降低血清胆红素水平、减轻肝细胞及小胆管损伤,改善肝功能,减轻胆汁淤积,对α-萘异硫氰酸酯导致的大鼠肝内胆汁淤积具有防治作用。
2α-萘异硫氰酸酯诱导的大鼠肝内胆汁淤积及Mrp2表达的下调是复方茵丹方清热化湿、凉血活血的方证病理学基础。
3上调Mrp2mRNA和蛋白表达是复方茵丹方防治大鼠肝内胆汁淤积的关键机制之一。
Objective:To investigate the intervention effect of compound Yindan decoction in rats with acute intrahepatic cholestasis induced by alpha-naphthyl isothiocyanate (ANIT),to observe the expression of Mrp2mRNA and protein, to explore the mechanism of compound Yindan decoction in the treatment of intrahepatic cholestasis.
Methods:45 male wistar rats were randomly divided into three groups, normal control group (9), the model group (18),TCM group (18), the model of intrahepatic cholestasis can be induced by alpha-naphthyl isothiocyanate (ANIT) by gavage. The rats of TCM group were given compound Yindan decoction by gavage once a day.To collect the samples of serum and hepatic tissues after modeling 24 hours,48 hours,72 hours. To observe the changes of hepatic function at different time points; To observe the expression of Mrp2mRNA in hepatocytes at different time points by RT-PCR; To boserve protein expression of Mrp2 by Immunohistochemistry; To detect the protein quantitation of Mrp2 in the hepatic tissue by Immunoblotting (Wes tern-blot).To obtain data for statistical analysis by SARS software and compare of the differences in serum biochemical indicators, expression of Mrp2mRNA and Mrp2 protein at different time points between TCM group and model group.
Results:
1 Serum biochemical indexes:The rats of model group and TCM group appeared rapidly significant hepatic damage,manifested as the rapid rise of transaminases,biliary enzymes and serum bilirub in,while the hepatic function of normal control group had no significant change at three time points. ALT, AST, TBi1, DBi1, GGT, ALP and TBA of model group were significantly increased compared with normal control group (P<0.01).Hepatic function indexes were significantly decreased at 72 hours compared with model group (P<0.05, P<0.01).
2 Hepatic tissue pathological changes:Comparing with normal control group, the rats of model group appeared damage to the hepatic lobules strueture,hepatocyte swelling,and scattering and exfoliation of biliary epithelial cells and infiltration of neutrophils in portal area at 24 hours after modeling; hepatic tissue lesions aggravated, hepatic lobules structure destructed severely,nucleus of different sizes, nuclear chromatin gathered,nucleolus enlarged and hyperstained. At 48 hours,we can see that significant hyperplasia of biliary epithelial cells,punctate and focal necrosis of hepatic cells around biliary and a lot of infiltration of neutrophils in portal area; At 72 hours, it can be still seen the hyperplasia of biliary epithelial cells and infiltration of neutrophils in portal area.The hepatic tissue pathological changes of TCM group were not very significant at 24 hours and 48 hours compared with model group,but neutrophils infiltration significantly decreased at 72 hours in portal area.
3 RT-PCR quantitative results of Mrp2mRNA in hepatic tissue:The expression of Mrp2mRNA of rats of model group were significantly decreased compared with normal control group,at 24 hours and 72 hours, P<0.05,at 48 hours, P<0.01. The expression of Mrp2mRNA of rats of TCM group tended to increase, but there was no significant difference at 24 hours.The expression of Mrp2mRNA of rats of TCM group was ignificantly higher than model group at 48 hours and 72 hours (P<0.05)
4 Expression results of Mrp2 transporter protein in hepatic tissue: Immunohistochemical results showed that Mrp2 expression were significantly decreased compared with the normal control group at all time points,P<0.01.There is no significant difference between TCM group and model group at 24 hours, the amount of expression of Mrp2 gradually to increase, significantly increased at 48 hours (P<0.05) and at 72 hours (P<0.01). Western-b lot gel image analysis showed that the expression of
Mrp2 protein were significantly decreased at three time points compared with the normal control group(P<0.01);the expression of Mrp2 protein were significantly increased at 24 hours P<0.05,at 48 hours P<0.01, at 72 hours P<0.05 compared with the normal control group.
Conclusions:
1 Compound Yindan decoction can decrease the levels of serum bilirub in, reduce the injury of hepatic cells and bile duct, improve the hepatic function and reduce cholestasis and has a preventive effect to rat intrahepatic cholestasis caused by alpha-Naphthy1 isothiocyanate.
2 The prescriptions and syndromes pathological basis of clearing heat and removing dampness, cooling blood and activating blood circulation is the intrahepatic cholestasis in rats indeced by alpha-Naphthy1 isothiocyanate and the down-regulation of the expression of Mrp2.
3 One of the key mechanism of control intrahepatic cholestasis in rats with Compound Yindan decoction is the up-regulated expression of Mrp2mRNA and protein.
引文
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[16]王君,何炳书, 威灵仙对α-奈异硫氰酸酯所致大鼠黄疸模型的作用研究。时珍国医国药2008,19(7)：1742-1743。
[17]葛文龙,窦志华,罗摇琳,赤芍总苷对α-萘异硫氰酸酯诱导的小鼠胆汁淤积型肝损伤的保护作用。时珍国医国药2010,21(11)：2881-2882。
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[6]何鑫,吴卫华,阳国平,ABCC2转运体的研究进展。中国药学杂志2007,42(23)：1764-1766。
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