The Protective Effect And Mechanism Of Andrographolide Against ANIT-induced Cholestasis And Liver Injury

Cholestasis is a common symptom of liver injuries and characterized as the interruption of bile flow from hepatocytes to intestine.Cholestasis results in a dramatic increase in liver and serum bile acid levels that eventually lead to acute liver toxicity,proliferation of bile ducts,and fibrosis progressing to cirrhosis.So far,there are very few effective therapies for cholestasis.A reduction in bile flow(cholestasis)can result from gall stones,impingement from a local tumor,intrahepatic cholestasis of pregnancy,genetic deficiency in bile export proteins or in autoimmune disorders amongst other etiologies.Ursodiol,a pharmaceutical form of ursodeoxycholic acid,is the current mainline defense against cholestasis,although its efficacy is limited in some circumstances.Therefore,it is extremely important to develop new therapeutic medicines.Andrographolide,isolated from A.paniculata,has exhibited varying degrees of anti-inflammatory and anticancer activities in both in vitro and in vivo experimental models of inflammation and cancer.It has been widely used for centuries in Asian countries like China,India,Thailand and Malaysia for the treatment of sore throat,flu and upper respiratory tract infections.Recently,Andrographolide has been reported for the efficacy in liver protection based on its anti-inflammatory and antioxidant activities.However,the effect and the underlying mechanism of Andrographolide on ANIT-induced cholestasis in rats was still unknown.Part I: protective effect of Andrographolide against ANIT-induced cholestasis and liver injury.Objective: To investigate the effect of Andrographolide on ANIT-induced cholestasis in rats.Methods: Male SD rats were randomly divided into six groups.The group 1 served as normal controls and received 5% sodium carboxymethylcellulose(CMC-Na)each day and intragastrical treatment with the vehicle(olive oil)alone.Group 2 was intragastrically treated with Andrographolide(200 mg/kg per day)suspended in 5% a CMC-Na for 4 days and received vehicle(olive oil)after the second administration of Andrographolide.Group 3 received 5% CMC-Na for 4 days and was intragastrically treated with 50 mg/kg ANIT dissolved in an equal volume of olive oil after the second administration of Andrographolide.Group 4,5,and 6 received Andrographolide(50,100,200 mg/kg per day,respectively)suspended in 5% CMC-Na for 4 days and was intragastrically treated with 50 mg/kg ANIT after the second administration of Andrographolide.48 hours after the last alcohol treatment,all animals were euthanized and the liver and blood were harvested for further analysis.Liver injury was evaluated histologically by HE staining.The biochemical evaluations of serum ALT,AST,ALP,γ-GGT,total bilirubin and bile acids were performed using commercially available kits and the operation was carried out according to manufacturer’s instructions.Results: Rats pretreated with andrographolide prior to ANIT administration exhibited lower levels of serum ALT,AST,ALP,γ-GGT,total bilirubin and bile acids treated with ANIT alone.Andrographolide also decreased the incidence and extent of periductular fibrosis and bile duct proliferation.Conclusion: Andrographolide effectively attenuates ANIT-induced acute cholestasis and liver injury in rats.Part II: The protective effect of Andrographolide against ANIT-induced cholestasis and liver injury.is through Anti-inflammatory and antioxidant activityObjective: To explore the mechanism of Andrographolide on ANIT-induced cholestasis in rats.Methods: Male SD rats were randomly divided into six groups.The group 1 served as normal controls and received 5% sodium carboxymethylcellulose(CMC-Na)each day and intragastrical treatment with the vehicle(olive oil)alone.Group 2 was intragastrically treated with Andrographolide(200 mg/kg per day)suspended in 5% a CMC-Na for 4 days and received vehicle(olive oil)after the second administration of Andrographolide.Group 3 received 5% CMC-Na for 4 days and was intragastrically treated with 50 mg/kg ANIT dissolved in an equal volume of olive oil after the second administration of Andrographolide.Group 4,5,and 6 received Andrographolide(50,100,200 mg/kg per day,respectively)suspended in 5% CMC-Na for 4 days and was intragastrically treated with 50 mg/kg ANIT after the second administration of Andrographolide.48 hours after the last alcohol treatment,all animals were euthanized and the liver and blood were harvested for further analysis.Liver injury was evaluated histologically by HE staining.The biochemical evaluations of serum IL-6,TNF-α,MDA,SOD,GSH and GSH-PX were performed using commercially available kits and the operation was carried out according to manufacturer’s instructions.Western blot analysis was employed to determine the expressions of NF-κB,COX-2,SIRT1,and Nrf2.The reverse transcription-PCR analysis was employed to determine the gene expression of SIRT1.Results: Analysis of protein expression in livers from andrographolide-treated cholestatic rats revealed markedly decreased expression of NF-κB and COX-2.ANIT significantly increased TNF-α and IL-6 release and myeloperoxidase activity,decreased SOD activity and the expression of SIRT1 and Nrf2,while pretreatment with andrographolide attenuated ANITinduced oxidative stress.Conclusion: Andrographolide effectively attenuates ANIT-induced acute cholestasis and liver injury in rats,possibly through suppression of inflammatory response and oxidative stress.These findings suggest that andrographolide could be a promising therapeutic option in prevention and slowing down the progression of cholestatic liver diseases.