体外培育牛黄对肝内胆汁淤积大鼠的作用及部分机制研究

发布时间：2018-05-08 21:00

本文选题：肝内胆汁淤积 + 体外培育牛黄　；参考：《华中科技大学》2014年博士论文

【摘要】：肝内胆汁淤积(intrahepatic cholestasis, IC),又称淤胆型肝炎,是由各种病因引起的肝细胞内胆汁分泌器结构与功能障碍,导致肝内胆汁淤滞和血液中胆汁成分增多,引起的以部分或完全性胆汁流阻滞为特征的综合症候群,临床表现为重度黄疸、皮肤瘙痒、大便颜色浅、心动过缓,伴血清生化指标升高等系列症状。其主要临床表现与肝外阻塞性黄疸相似,但患者并无胆道机械性阻塞。IC也是多种肝病所共有的基础性病变,其进行性发展最终可致肝硬化、肝衰竭而威胁生命。IC的常见病因包括感染、代谢、免疫以及遗传等,发病机制较为复杂,迄今尚未完全清楚,一般认为主要是由致病因子导致的肝细胞细胞器和毛细胆管损伤,造成胆汁排泌障碍,或者毛细胆管内胆栓形成。目前用于治疗肝内胆汁淤积症的可选药物不多,亦无特效药物,故研究新颖的治疗肝内胆汁淤积的药物并阐明其作用机制具有重要价值和意义。牛黄(Calculus Bovis, Niuhuang)是我国传统名贵中药,具有较好的保肝护肝作用。牛黄天然来源于牛科动物牛的胆囊、胆管或肝管结石,稀有紧缺,近年来国内开始广泛应用体外培育牛黄(Calculus Bovis Sativus, CBS)作为天然牛黄的理想代用品,其药理作用与牛黄近似。本文主要就体外培育牛黄对肝内胆汁淤积大鼠的保护作用及机制进行了系统研究。第一部分考察CBS对ANIT诱导肝内胆汁淤积大鼠的作用。雄性Wistar大鼠30只随机分为5组,每组6只：正常对照组,模型组,ANIT+CBS50mg/kg组,ANIT+CBS100mg/kg组,ANIT+CBS200mg/kg组。正常对照组每日灌胃给予生理盐水,其余各组每日灌胃给予相应剂量的药物,于第5日,正常对照组一次性灌胃给予橄榄油,其余各组一次性灌胃给予ANIT橄榄油溶液(100mg/kg)。造模后继续给药,于造模后48h,麻醉固定大鼠,行胆管插管引流术,收集2h内胆汁,计算胆汁流量；采血分离血清测定血清生化指标；取部分肝脏组织固定后行HE染色及透射电镜观察,部分肝脏组织匀浆后测定SOD活性及MDA含量。结果发现：1.CBS显著逆转ANIT导致的大鼠胆汁流量下降；2.CBS下调ANIT诱导肝内胆汁淤积大鼠血清ALT、AST、ALP、TBIL值；3.CBS减轻ANIT诱导肝内胆汁淤积大鼠肝组织病理损伤；4.CBS升高ANIT诱导肝内胆汁淤积大鼠肝组织SOD活性,减少肝组织MDA含量。结果显示,CBS(50,100or200mg/kg)对ANIT诱导的肝内胆汁淤积大鼠具有明显保护作用,可显著减轻ANIT引起的肝脏损伤,机制可能与减少炎性细胞浸润,减轻肝脏氧化损伤有关。第二部分考察CBS对EE诱导肝内胆汁淤积大鼠的作用。雄性大鼠36只随机分为6组,每组6只：正常对照组,模型组,EE+CBS50mg/kg组,EE+CBS150mg/kg组,CBS50mg/kg组,CBS150mg/kg组。正常对照组每日给予丙二醇皮下注射,同时给予NS灌胃,模型组每日给予EE(5mg/kg)皮下注射,同时给予NS灌胃,EE+CBS50mg/kg组每日给予EE(5mg/kg)皮下注射,同时给予CBS (50mg/kg)灌胃,EE+CBS150mg/kg组每日给予EE(5mg/kg)皮下注射,同时给予CBS (150mg/kg)灌胃；CBS50mg/kg组每日给予丙二醇皮下注射,同时给予CBS (50mg/kg)灌胃,CBS150mg/kg组每日给予丙二醇皮下注射,同时给予CBS (150mg/kg)灌胃。连续给药5天后,各组大鼠麻醉固定,行胆管插管引流术,收集2h内胆汁,计算胆汁流量；采血分离血清测定血清生化指标；取部分肝脏组织固定后行HE染色,部分肝脏组织匀浆后测定SOD活性及MDA含量。结果发现：1.CBS显著逆转EE导致的大鼠胆汁流量下降；2.CBS下调EE诱导肝内胆汁淤积大鼠血清ALT、AST、ALP、TBIL值；3.CBS减轻EE诱导肝内胆汁淤积大鼠肝组织病理损伤；4.CBS升高EE诱导肝内胆汁淤积大鼠肝组织SOD活性,减少肝组织MDA含量。结果显示,CBS对EE诱导的肝内胆汁淤积大鼠具有保护作用,可显著减轻肝脏损伤,机制可能与减轻肝脏氧化损伤有关。第三部分考察CBS对EE诱导肝内胆汁淤积大鼠肝脏Mrp2、Bcrp表达的影响,对肝脏雌激素受体ERa、转运体调节蛋白PDZK1表达的影响。雄性大鼠36只随机分为6组,每组6只：正常对照组,模型组,EE+CBS50mg/kg组,EE+CBS150mg/kg组,CBS50mg/kg组,CBS150mg/kg组。正常对照组每日给予丙二醇皮下注射,同时给予NS灌胃,模型组每日给予EE(5mg/kg)皮下注射,同时给予NS灌胃,EE+CBS50mg/kg组每日给予EE(5mg/kg)皮下注射,同时给予CBS(50mg/kg)灌胃,EE+CBS150mg/kg组每日给予EE(5mg/kg)皮下注射,同时给予CBS(150mg/kg)灌胃；CBS50mg/kg组每日给予丙二醇皮下注射,同时给予CBS(50mg/kg)灌胃,CBS150mg/kg组每日给予丙二醇皮下注射,同时给予CBS(150mg/kg)灌胃。连续给药5天后,处死各组大鼠,取肝脏组织,采用RT-PCR和Western blotting方法检测Mrp2、Bcrp、ERα、PDZK1表达。结果发现：1.EE显著下调大鼠肝脏组织Mrp2、Bcrp的蛋白表达,显著降低BcrpmRNA表达；2.EE显著下调大鼠肝脏组织PDZK1的mRNA及蛋白表达,上调大鼠肝脏组织ERa的蛋白表达；3.与模型组相比,CBS显著上调Mrp2、Bcrp mRNA及蛋白表达,上调PDZK1mRNA及蛋白表达,下调ERα蛋白表达。结果显示：CBS对EE诱导肝内胆汁淤积大鼠肝脏转运体Mrp2、Bcrp表达具有上调作用；CBS可能通过恢复肝脏转运体的表达,增加胆汁流量,发挥保护作用；同时,CBS对EE诱导肝内胆汁淤积大鼠肝脏组织转运体调节蛋白PDZK1表达具有明显上调作用,对ERa蛋白表达具有下调作用；因此,CBS可能通过影响雌激素信号通路及相关蛋白发挥对肝脏转运体的上调作用。第四部分考察CBS对肝内胆汁淤积大鼠Mrp2、Bcrp转运功能的影响。雄性Wistar大鼠24只随机分为4组,每组6只：正常对照组,模型组,ANIT+CBS50mg/kg组,ANIT+CBS150mg/kg组。正常对照组、模型组每日灌胃给予生理盐水,ANIT+CBS组每日灌胃给予相应剂量的药物；于第5日,正常对照组一次性灌胃给予等量的橄榄油,其余各组一次性灌胃给予ANIT橄榄油溶液(100mg/kg)。造模后继续给药,于造模后48h,禁食过夜,麻醉固定大鼠,行胆管插管引流术；尾静脉注射一次性剂量的黄芩苷溶液(20mg/kg),按照预定时间点收集胆汁,冻存待测；雄性Wistar大鼠24只随机分为4组,每组6只：正常对照组,模型组,EE+CBS50mg/kg组,EE+CBS150mg/kg组。正常对照组每日给予丙二醇皮下注射,同时给予NS灌胃；模型组每日给予EE(5mg/kg)皮下注射,同时给予NS灌胃；EE+CBS组每日给予EE(5mg/kg)皮下注射,同时给予CBS灌胃。连续给药5天后,禁食过夜,麻醉固定大鼠,行胆管插管引流术；尾静脉注射一次性剂量的米托葸醌溶液(2mg/kg),按照预设时间点收集胆汁样品,冻存待测。结果发现：1.ANIT导致肝内胆汁淤积大鼠静脉注射黄芩苷经胆汁排泄速率降低,累积排泄量减少；2.CBS可部分对抗这种抑制效应；与模型组相比,CBS明显增加黄芩苷经胆汁排泄量；3.EE导致肝内胆汁淤积大鼠静脉注射米托蒽醌经胆汁排泄速率降低,累积排泄量减少；4.CBS可部分对抗这种抑制效应；与模型组相比,CBS明显增加米托葸醌经胆汁排泄量。结果显示：CBS可以增加ANIT诱导肝内胆汁淤积大鼠黄芩苷的胆汁排泄量,可能与CBS增强Mrp2转运功能有关；CBS可以增加EE诱导肝内胆汁淤积大鼠米托葸醌的胆汁排泄量,可能与CBS增强Bcrp转运功能有关。
[Abstract]:Intrahepatic cholestasis (intrahepatic cholestasis, IC), also known as cholestatic hepatitis, is a comprehensive syndrome characterized by intrahepatic cholestasis and increased bile composition in the liver, which is caused by various causes, and is characterized by partial or complete bile flow block. The clinical manifestation is severe yellow. Jaundice, skin pruritus, shallow stool color, bradycardia, and serum biochemical indicators. Its main clinical manifestations are similar to extrahepatic obstructive jaundice, but there is no mechanical obstruction of biliary tract in patients with.IC, a basic disease common to multiple liver diseases, and its progressive development can eventually lead to cirrhosis, and liver failure threatens the life of.IC. The etiology includes infection, metabolism, immunity and heredity. The pathogenesis is more complex and has not yet been fully understood. It is generally considered to be mainly caused by pathogenic factors of hepatocyte organelles and capillary bile duct injury, causing bile excretion obstruction, or capillary bile duct formation. The optional drugs used for the treatment of intrahepatic cholestasis are currently used. Therefore, it is of great value and significance to study new drugs for intrahepatic cholestasis and elucidate its mechanism.
Calculus Bovis (Niuhuang) is a traditional Chinese traditional Chinese medicine. It has good liver protection and liver protection. It is natural from the gallbladder, bile duct or hepatic duct stone of cattle from cattle. It is rare and scarce. In recent years, it began to widely use Calculus Bovis Sativus (CBS) in vitro as an ideal substitute for natural cattle. The protective effect and mechanism of in vitro cultured calculus bovis on intrahepatic cholestasis in rats were systematically studied.
The first part investigated the effect of CBS on ANIT induced intrahepatic cholestasis in rats. 30 male Wistar rats were randomly divided into 5 groups, 6 rats in each group: normal control group, model group, ANIT+CBS50mg/kg group, ANIT+CBS100mg/kg group and ANIT+CBS200mg/kg group. The normal control group was given the normal saline daily, the other groups were given the corresponding dose of the stomach every day. On the fifth day, the normal control group was given the olive oil for the normal control group. The other groups were given the ANIT olive oil solution (100mg/kg) at one time. After making the model, the normal control group was given the medicine. After the model was built, the rats were anesthetized and fixed, and the bile duct intubation and drainage was performed. The bile of the 2H was collected and the bile flow was calculated. The serum biochemical indexes were measured by the blood extraction separation serum; take the part of the serum; take the part of the serum. After the liver tissue was fixed, HE staining and transmission electron microscopy were performed, and some liver tissue homogenate was used to determine the activity of SOD and the content of MDA. The results showed that 1.CBS significantly reversed the decrease of bile flow in rats induced by ANIT, and 2.CBS down regulated the serum ALT, AST, ALP, TBIL in the serum of the intrahepatic cholestasis induced by ANIT; 3.CBS alleviated the intrahepatic cholestasis induced by 3.CBS. The pathological damage of rat liver tissue, 4.CBS increased ANIT induced SOD activity in hepatic cholestasis of rats and reduced the MDA content of liver tissue. The results showed that CBS (50100or200mg/kg) had obvious protective effect on intrahepatic cholestasis induced by ANIT, which could significantly reduce the liver damage caused by ANIT, and the mechanism may reduce inflammatory cell infiltration and decrease the infiltration of inflammatory cells. The oxidative damage of light liver is related.
The second part investigated the effect of CBS on EE induced intrahepatic cholestasis in rats. 36 male rats were randomly divided into 6 groups, with 6 rats in each group: normal control group, model group, EE+CBS50mg/kg group, EE+CBS150mg/kg group, CBS50mg/kg group, CBS150mg/kg group. The normal control group was given subcutaneous injection of propanediol daily, and NS was given to the stomach, and EE (5m) was given daily in the model group. G/kg) subcutaneous injection and NS gavage at the same time. Group EE+CBS50mg/kg was given EE (5mg/kg) subcutaneous injection every day, and CBS (50mg/kg) was given to the stomach. EE+CBS150mg/kg group was given EE (5mg/kg) subcutaneous injection every day, and CBS (150mg/kg) was given to the stomach. Group a daily administration of propanediol was given subcutaneous injection, and CBS (150mg/kg) was given to the stomach. After 5 days of continuous administration, the rats in each group were anesthetized and fixed. Bile duct intubation and drainage were performed. Bile flow in 2H was collected and bile flow was calculated. Serum biochemical indexes were measured by blood extraction separation sera. After partial liver tissue was fixed, HE staining was performed and partial liver tissue homogenate was measured. SOD activity and MDA content. The results showed that 1.CBS significantly reversed the decrease of bile flow in rats induced by EE; 2.CBS reduced EE induced the serum ALT, AST, ALP, TBIL values in the serum of rat intrahepatic cholestasis; 3.CBS alleviated the pathological damage of liver tissue in rats with intrahepatic cholestasis induced by EE, and decreased the activity of liver tissue in rats with intrahepatic cholestasis. MDA content of liver tissue. The results showed that CBS had protective effect on EE induced intrahepatic cholestasis in rats, and could significantly reduce the liver damage. The mechanism may be related to the reduction of liver oxidative damage.
The third part investigated the effect of CBS on the expression of Mrp2 and Bcrp in the liver of rats with intrahepatic cholestasis induced by EE, the effect on the liver estrogen receptor ERa and the expression of the transporter regulatory protein PDZK1. The male rats were randomly divided into 6 groups, 6 rats in each group: the normal control group, the model group, the EE+CBS50mg/kg group, EE+CBS150mg/kg group, CBS50mg/kg group, CBS150mg/kg group. The control group was given hypodermic injection of propanediol daily and given NS gavage at the same time. The model group was given EE (5mg/kg) subcutaneous injection every day, and NS was given to the stomach. EE+CBS50mg/kg group was given EE (5mg/kg) subcutaneous injection every day, and CBS (50mg/kg) was given to the stomach. The EE+CBS150mg/kg group was given EE (5mg/kg) subcutaneous injection every day. Group CBS50mg/kg was given subcutaneous injection of propanediol daily, CBS (50mg/kg) was given to the stomach, group CBS150mg/kg was given subcutaneous injection of propanediol, and CBS (150mg/kg) was given to the stomach at the same time. After 5 days of continuous administration, the rats were killed and the liver tissues were taken. RT-PCR and Western blotting methods were used to detect Mrp2, Bcrp, ER alpha and expression. The results were found. 1.EE significantly lowered the protein expression of Mrp2 and Bcrp in rat liver tissue and significantly reduced the expression of BcrpmRNA; 2.EE significantly lowered the mRNA and protein expression of PDZK1 in rat liver tissue, up regulation of the protein expression of ERa in rat liver tissue; 3. The expression of ER alpha protein was adjusted. The results showed that CBS could increase the expression of Mrp2 and Bcrp expression in hepatic cholestasis induced by EE in rats. CBS may increase the expression of hepatic transporter, increase the flow of bile, and play a protective role. At the same time, CBS induces the PDZK1 expression of the regulatory protein of the hepatic tissue transporter in the rat hepatic cholestasis induced by EE. It has an obvious up-regulation effect on the expression of ERa protein, and therefore, CBS may play an up-regulated role in the liver transporters by affecting the estrogen signaling pathway and related proteins.
The fourth part investigated the effect of CBS on Mrp2 and Bcrp transport in rats with intrahepatic cholestasis. 24 male rats were randomly divided into 4 groups, with 6 rats in each group: the normal control group, the model group, the ANIT+CBS50mg/kg group, the ANIT+CBS150mg/kg group and the normal control group. The model group was given the saline daily by gavage, and the ANIT+CBS group was given the corresponding dose of the stomach every day. On the fifth day, the normal control group was given the same amount of olive oil for the normal control group, and the other groups were given ANIT olive oil solution (100mg/kg) at one time. After making the model, the normal control group continued to give the medicine. After the model, 48h, the fasting for the night, the anesthesia fixed rats, the bile duct intubation drainage, and the tail vein injection of baicalin solution (20mg/kg) with the dose of one-time dose (20mg/kg). 24 male Wistar rats were randomly divided into 4 groups, 6 rats in each group, 6 in each group: normal control group, model group, EE+CBS50mg/kg group, EE+CBS150mg/kg group. The normal control group was given propanediol subcutaneous injection every day and given NS gavage at the same time; the model group was given EE (5mg/kg) subcutaneous injection at the same time, and NS gavage was given at the same time; EE+ Group CBS was given a daily subcutaneous injection of EE (5mg/kg) and given to the gavage of CBS at the same time. After 5 days of continuous administration, the fasting was overnight, the anesthetized rats were fixed, the bile duct intubation drainage was performed, and the tail vein was injected with a one-time dose of mitoxone quinone solution (2mg/kg). The bile samples were collected at the preset time point and were frozen to be tested. The results found that 1.ANIT led to intrahepatic cholestasis. The cumulative excretion of baicalin was reduced and the cumulative excretion of baicalin was reduced by intravenous injection of baicalin, and 2.CBS could partially antagonism the inhibitory effect. Compared with the model group, CBS significantly increased the excretion of baicalin through bile; 3.EE resulted in a decrease in the excretion rate of mitoxantrone by intravenous injection of mitoxantrone in the intrahepatic cholestasis of rats and a decrease in the cumulative excretion of the bile. 4. CBS can partially antagonism this inhibitory effect; compared with the model group, CBS significantly increased the excretion of mitoquinone through bile. The results showed that CBS could increase the bile excretion of Baicalin in rat liver cholestasis induced by ANIT, which may be related to the enhanced Mrp2 transport function of CBS; CBS can increase the induced intrahepatic cholestasis of the rat Mito quinone by adding EE. The amount of bile excretion may be related to the enhanced Bcrp transport function of CBS.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R285.5

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