Protective Mechanism And Pharmacokinetics Of Baicalin In Rats With Estrogen-induced Intrahepatic Cholestasis

Intrahepatic cholestasis（IHC）,existing in many chronic liver diseases,result from an accumulation of bile acid in hepatocytes and impairment of bile flow excretion induced by multiple complicated factors.Without effective intervention,IHC can lead to inflammatory infiltration,hepatic injury and necrosis,and ultimately develop into hepatic fibrosis,cirrhosis and hepatocarcinogenesis.Estrogens in high level are well-known to cause IHC in susceptible women during pregnancy,administration of oral contraceptives,and postmenopausal replacement therapy.As the pathogenesis of IHC is obscure,effective drugs remain scarcity.According to the theory of traditional Chinese medicine,IHC belongs to“jaundice”in which dampness-heat in hepatobiliary system as well as spleen and stomach is the main syndrome types.Hence,protecting liver and gallbladder together with heat-clearing and damp-drying is the crucial therapeutic principle for IHC.Radix Scutellariae is adept in heat-clearing and damp-drying,and has been used for thousands years in China.Baicalin is a bioactive flavonoid isolated from Radix Scutellariae,and has many biological functions,especially with eminent hepatoprotective effects.However,the effect of baicalin for IHC,its possible mechanism and the pharmacokinetics in IHC animals have not been reported before.In the present study,we have investigated the protection of baicalin on IHC rats and the underlying pharmacological mechanism,and the pharmacokinetic characteristics of baicalin in IHC rats was also clarified,which can supply an experimental basis for its clinical application.Part Ⅰ Studies on the protection of baicalin on estrogen-induced IHC ratsObjective:To preliminarily explore the effect of baicalin on 17α-ethynylestradiol（EE）-induced IHC rats.Methods:IHC rat model was established by subcutaneously injection of EE（5mg/kg body weight）daily for five consecutive days in SD rats.Simultaneously,IHC rats were treated by baicalin at 50mg/kg,100mg/kg,200mg/kg or ursodeoxycholic acid（UDCA）at40mg/kg.We also add a control group in which normal rats were given 200mg/kg baicalin.Cholagogic action of baicalin was evaluated by examining the liver index,animal behavior and bile flow.Haematoxylin and eosin（HE）staining was performed to study the pathological injury in livers and ileums.Ultrastructure in hepatocytes was examined by transmission electron microscope（TEM）.The serum levels of total bile acid（TBA）,total bilirubin（TBil）,direct bilirubin（DBil）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）and alkaline phosphatase（AKP）were determined.Moreover,the content of superoxide dismutase（SOD）,malondialdehyde（MDA）and myeloperoxidase（MPO）in livers was also detected.Results:Baicalin significantly improved the behavioral change,the increase of liver index and decline of bile flow in IHC rats induced by EE injection.Our results showed that baicalin could dose-dependently reduce the abnormal biochemical indexes in serum including TBA,TBil,DBil,ALT,AST and AKP.HE staining and TEM observation demonstrated that the structure damage and inflammatory infiltration of livers and ileums as well as the histopathological injury on ultrastructure of hepatocytes were markedly alleviated by baicalin.Baicalin could enhance SOD activity and decrease the content of MDA and MPO in liver tissue of IHC rats.In addition,there was not any influence of baicalin at 200mg/kg on normal rats.Conclusion:Baicalin possesses the improvement effect of cholestasis on EE-induced IHC rats and alleviates the injury of livers and ileums,owing to its potential anti-oxidative damage.Among them,baicalin at 200mg/kg achieved the most outcomes and has not obvious side effect.Part Ⅱ Intervention of baicalin on bile acid homeostasis and inflammatory injury incholestatic ratsObjective:To investigate the pharmacological mechanism of baicalin on EE-induced IHC via bile acid homeostasis,inflammatory injury and farnesoid X receptor（FXR）.Methods:Normal rats and EE-induced IHC rats were orally treated with 200mg/kg baicalin respectively,to evaluate the protection on cholestasis.RT-PCR was perform to analysis the following genes:（1）enzymes for bile acid synthesis and metabolism:Cyp7a1、Cyp8b1、Cyp3a2、Sult2a1、Bal、Baat;（2）hepatic transporters for bile acid uptake and biliary secretion:Ntcp、Oatp1a1、Oatp1b2、Bsep、Mrp2、Mrp3、Mrp4、Mdr2;（3）proinflammatory factors;Tnf-α、Il-6、Il-1β;（4）hepatic Fxr and its targeted gene Shp;（5）intestinal Fxr and its targeted gene Fgf15.The protein expressions of Cyp7a1,Ntcp,Bsep,Mrp2,the proinfalmmatory cytokines,and Fxr in livers and ileums were determined by western blotting.Fxr in livers and ileums was also evaluated by immunohistochemistry.Results:Baicalin at 200mg/kg showed prominent hepatoprotective and cholagogic effect.The gene expressions of the hepatic enzymes of bile acid synthesis such as Cyp7a1 and Cyp8b1 were not affected by baicalin treatment.Baicalin could upregulate the expressions of Cyp3a2,Sult2a1,Bal and Baat.We found the gene and protein expressions of Ntcp were inhibited while those of Bsep and Mrp2 were increased.There was not any change in the gene expression of Oatp1b2,but Oatp1a1,Mrp3,Mrp4 and Mdr2 were significantly increased by baicalin.Moreover,attenuation of proinflammatory cytokines Tnf-α,Il-6,and Il-1βin livers was observed in IHC rats treated by baicalin.Results of RT-PCR,western blotting and immunohistochemistry confirmed that baicalin could upregulate the Fxr expressions in livers and ileums and promote the transcription of Fxr targeted gene Shp and Fgf15.Conclusion:Baicalin can enhance the detoxification of bile acid,accelerate the excretion of bile acid and diminish its uptake,consequently alleviate the inflammatory injury in livers and confer protection to hepatocytes.And our results show those effects are probablyrelated to Fxr activation by baicalin.Part Ⅲ Study on the pharmacokinetics of baicalin in EE-induced IHC ratsObjective:To establish an analysis method for determination of baicalin in rat plasma by liquid chromatography tandem mass spectrometry（LC-MS/MS）and study the pharmacokinetic characteristics of baicalin in normal and IHC rats.Methods:Plasma samples of rats were prepared by protein precipitation with methanol.Chromatographic condition:samples were analyzed on a C₁₈ column with mobile phase A（0.1%formic acid solution）and mobile phase B（methanol solution）at a ratio of 20:80（v/v）in 5 min,flow rate at 0.8 mL/min,10μL of injection volume and column temperature at30℃.Mass spectrometry condition:the detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source（ESI）in positive ionization mode,m/z 447.0→271.3.Single dose or five consecutive days’Baicalin at 200 mg/kg was orally given into normal or IHC rats and its blood concentration was determined by LC-MS/MS.The pharmacokinetic characteristics and parameters were calculated and recorded.Results:The method for baicalin detection was specific and showed excellent linearity over the concentration ranges of 1.01–506.00μg/mL（R²=0.9980）,and the lower limit of quantitation was 1.01μg/mL.Intra-day and inter-day precision averages were no more than10.55%.Intra-day and inter-day accuracy were between 94.94%and 109.13%,and the results of recovery test and stability test conformed to the requirement in Chinese Pharmacopoeia 2015 Edition.Pharmacokinetic study of baicalin in rats showed the concentration-time curve existed bimodal phenomenon.After multiple treatments,AUC and C_max of baicalin in rats elevated,while its CL and t_1/2 decreased.C_max and AUC of single baicalin in EE-induced IHC rats were dramatically increased,accompanying with reduced AUC,prolonged t_1/2 and slower CL.However,compared with single dose of baicalin in IHC rats,successive pretreatment of baicalin while EE modeling could lower the abnormal AUC,increase CL and shorten t_1/2,thus restoring the pharmacokinetic characteristics of baicalin in normal rats.Conclusion:Compared with single baicalin in normal rats the pharmacokinetic characteristics of single baicalin in IHC rats show elevated exposure and decreased elimination,which can be remitted by consecutive treatment of baicalin.The in-depth mechanism maybe derived from the reversion of baicalin on EE-induced the reduction of hepatic transporters and enzymes as well as intestinal injury.