發(fā)布時間：2018-05-08 21:00

  本文選題：肝內膽汁淤積 + 體外培育牛黃��； 參考：《華中科技大學》2014年博士論文

【摘要】：肝內膽汁淤積(intrahepatic cholestasis, IC),又稱淤膽型肝炎,是由各種病因引起的肝細胞內膽汁分泌器結構與功能障礙,導致肝內膽汁淤滯和血液中膽汁成分增多,引起的以部分或完全性膽汁流阻滯為特征的綜合癥候群,臨床表現(xiàn)為重度黃疸、皮膚瘙癢、大便顏色淺、心動過緩,伴血清生化指標升高等系列癥狀。其主要臨床表現(xiàn)與肝外阻塞性黃疸相似,但患者并無膽道機械性阻塞。IC也是多種肝病所共有的基礎性病變,其進行性發(fā)展最終可致肝硬化、肝衰竭而威脅生命。IC的常見病因包括感染、代謝、免疫以及遺傳等,發(fā)病機制較為復雜,迄今尚未完全清楚,一般認為主要是由致病因子導致的肝細胞細胞器和毛細膽管損傷,造成膽汁排泌障礙,或者毛細膽管內膽栓形成。目前用于治療肝內膽汁淤積癥的可選藥物不多,亦無特效藥物,故研究新穎的治療肝內膽汁淤積的藥物并闡明其作用機制具有重要價值和意義。 牛黃(Calculus Bovis, Niuhuang)是我國傳統(tǒng)名貴中藥,具有較好的保肝護肝作用。牛黃天然來源于�？苿游锱５哪懩摇⒛懝芑蚋喂芙Y石,稀有緊缺,近年來國內開始廣泛應用體外培育牛黃(Calculus Bovis Sativus, CBS)作為天然牛黃的理想代用品,其藥理作用與牛黃近似。本文主要就體外培育牛黃對肝內膽汁淤積大鼠的保護作用及機制進行了系統(tǒng)研究。 第一部分考察CBS對ANIT誘導肝內膽汁淤積大鼠的作用。雄性Wistar大鼠30只隨機分為5組,每組6只：正常對照組,模型組,ANIT+CBS50mg/kg組,ANIT+CBS100mg/kg組,ANIT+CBS200mg/kg組。正常對照組每日灌胃給予生理鹽水,其余各組每日灌胃給予相應劑量的藥物,于第5日,正常對照組一次性灌胃給予橄欖油,其余各組一次性灌胃給予ANIT橄欖油溶液(100mg/kg)。造模后繼續(xù)給藥,于造模后48h,麻醉固定大鼠,行膽管插管引流術,收集2h內膽汁,計算膽汁流量；采血分離血清測定血清生化指標；取部分肝臟組織固定后行HE染色及透射電鏡觀察,部分肝臟組織勻漿后測定SOD活性及MDA含量。結果發(fā)現(xiàn)：1.CBS顯著逆轉ANIT導致的大鼠膽汁流量下降；2.CBS下調ANIT誘導肝內膽汁淤積大鼠血清ALT、AST、ALP、TBIL值；3.CBS減輕ANIT誘導肝內膽汁淤積大鼠肝組織病理損傷；4.CBS升高ANIT誘導肝內膽汁淤積大鼠肝組織SOD活性,減少肝組織MDA含量。結果顯示,CBS(50,100or200mg/kg)對ANIT誘導的肝內膽汁淤積大鼠具有明顯保護作用,可顯著減輕ANIT引起的肝臟損傷,機制可能與減少炎性細胞浸潤,減輕肝臟氧化損傷有關。 第二部分考察CBS對EE誘導肝內膽汁淤積大鼠的作用。雄性大鼠36只隨機分為6組,每組6只：正常對照組,模型組,EE+CBS50mg/kg組,EE+CBS150mg/kg組,CBS50mg/kg組,CBS150mg/kg組。正常對照組每日給予丙二醇皮下注射,同時給予NS灌胃,模型組每日給予EE(5mg/kg)皮下注射,同時給予NS灌胃,EE+CBS50mg/kg組每日給予EE(5mg/kg)皮下注射,同時給予CBS (50mg/kg)灌胃,EE+CBS150mg/kg組每日給予EE(5mg/kg)皮下注射,同時給予CBS (150mg/kg)灌胃；CBS50mg/kg組每日給予丙二醇皮下注射,同時給予CBS (50mg/kg)灌胃,CBS150mg/kg組每日給予丙二醇皮下注射,同時給予CBS (150mg/kg)灌胃。連續(xù)給藥5天后,各組大鼠麻醉固定,行膽管插管引流術,收集2h內膽汁,計算膽汁流量；采血分離血清測定血清生化指標；取部分肝臟組織固定后行HE染色,部分肝臟組織勻漿后測定SOD活性及MDA含量。結果發(fā)現(xiàn)：1.CBS顯著逆轉EE導致的大鼠膽汁流量下降；2.CBS下調EE誘導肝內膽汁淤積大鼠血清ALT、AST、ALP、TBIL值；3.CBS減輕EE誘導肝內膽汁淤積大鼠肝組織病理損傷；4.CBS升高EE誘導肝內膽汁淤積大鼠肝組織SOD活性,減少肝組織MDA含量。結果顯示,CBS對EE誘導的肝內膽汁淤積大鼠具有保護作用,可顯著減輕肝臟損傷,機制可能與減輕肝臟氧化損傷有關。 第三部分考察CBS對EE誘導肝內膽汁淤積大鼠肝臟Mrp2、Bcrp表達的影響,對肝臟雌激素受體ERa、轉運體調節(jié)蛋白PDZK1表達的影響。雄性大鼠36只隨機分為6組,每組6只：正常對照組,模型組,EE+CBS50mg/kg組,EE+CBS150mg/kg組,CBS50mg/kg組,CBS150mg/kg組。正常對照組每日給予丙二醇皮下注射,同時給予NS灌胃,模型組每日給予EE(5mg/kg)皮下注射,同時給予NS灌胃,EE+CBS50mg/kg組每日給予EE(5mg/kg)皮下注射,同時給予CBS(50mg/kg)灌胃,EE+CBS150mg/kg組每日給予EE(5mg/kg)皮下注射,同時給予CBS(150mg/kg)灌胃；CBS50mg/kg組每日給予丙二醇皮下注射,同時給予CBS(50mg/kg)灌胃,CBS150mg/kg組每日給予丙二醇皮下注射,同時給予CBS(150mg/kg)灌胃。連續(xù)給藥5天后,處死各組大鼠,取肝臟組織,采用RT-PCR和Western blotting方法檢測Mrp2、Bcrp、ERα、PDZK1表達。結果發(fā)現(xiàn)：1.EE顯著下調大鼠肝臟組織Mrp2、Bcrp的蛋白表達,顯著降低BcrpmRNA表達；2.EE顯著下調大鼠肝臟組織PDZK1的mRNA及蛋白表達,上調大鼠肝臟組織ERa的蛋白表達；3.與模型組相比,CBS顯著上調Mrp2、Bcrp mRNA及蛋白表達,上調PDZK1mRNA及蛋白表達,下調ERα蛋白表達。結果顯示：CBS對EE誘導肝內膽汁淤積大鼠肝臟轉運體Mrp2、Bcrp表達具有上調作用；CBS可能通過恢復肝臟轉運體的表達,增加膽汁流量,發(fā)揮保護作用；同時,CBS對EE誘導肝內膽汁淤積大鼠肝臟組織轉運體調節(jié)蛋白PDZK1表達具有明顯上調作用,對ERa蛋白表達具有下調作用；因此,CBS可能通過影響雌激素信號通路及相關蛋白發(fā)揮對肝臟轉運體的上調作用。 第四部分考察CBS對肝內膽汁淤積大鼠Mrp2、Bcrp轉運功能的影響。雄性Wistar大鼠24只隨機分為4組,每組6只：正常對照組,模型組,ANIT+CBS50mg/kg組,ANIT+CBS150mg/kg組。正常對照組、模型組每日灌胃給予生理鹽水,ANIT+CBS組每日灌胃給予相應劑量的藥物；于第5日,正常對照組一次性灌胃給予等量的橄欖油,其余各組一次性灌胃給予ANIT橄欖油溶液(100mg/kg)。造模后繼續(xù)給藥,于造模后48h,禁食過夜,麻醉固定大鼠,行膽管插管引流術；尾靜脈注射一次性劑量的黃芩苷溶液(20mg/kg),按照預定時間點收集膽汁,凍存待測；雄性Wistar大鼠24只隨機分為4組,每組6只：正常對照組,模型組,EE+CBS50mg/kg組,EE+CBS150mg/kg組。正常對照組每日給予丙二醇皮下注射,同時給予NS灌胃；模型組每日給予EE(5mg/kg)皮下注射,同時給予NS灌胃；EE+CBS組每日給予EE(5mg/kg)皮下注射,同時給予CBS灌胃。連續(xù)給藥5天后,禁食過夜,麻醉固定大鼠,行膽管插管引流術；尾靜脈注射一次性劑量的米托葸醌溶液(2mg/kg),按照預設時間點收集膽汁樣品,凍存待測。結果發(fā)現(xiàn)：1.ANIT導致肝內膽汁淤積大鼠靜脈注射黃芩苷經膽汁排泄速率降低,累積排泄量減少；2.CBS可部分對抗這種抑制效應；與模型組相比,CBS明顯增加黃芩苷經膽汁排泄量；3.EE導致肝內膽汁淤積大鼠靜脈注射米托蒽醌經膽汁排泄速率降低,累積排泄量減少；4.CBS可部分對抗這種抑制效應；與模型組相比,CBS明顯增加米托葸醌經膽汁排泄量。結果顯示：CBS可以增加ANIT誘導肝內膽汁淤積大鼠黃芩苷的膽汁排泄量,可能與CBS增強Mrp2轉運功能有關；CBS可以增加EE誘導肝內膽汁淤積大鼠米托葸醌的膽汁排泄量,可能與CBS增強Bcrp轉運功能有關。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R285.5

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1 劉圣p，

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