Experiment Of Studying The Interventional Effect Of Babaodan On The Acute Alcoholic Liver Injury In Mice And Its Mechanism

Background and Aims:Acute alcoholic liver injury is one kind of liver disease and a worldwide public health problem with an increasing prevalence and incidence,which is caused by alcohol abuse.Oxidative stress,immune response,various cytokines and autophagy are the key drivers of alcohol-induced liver injury.Although multiple attempts have been made to improve patient outcome,the treatment of alcoholic hepatitis is still based on abstinence from alcohol and brief exposure to corticosteroids.Homeostasis is an essential factor in keeping cell growth,proliferation,function and metabolism.The occurrence and development of acute alcoholic liver injury is closely related to hepatocellular microenvironment which will be significantly disturbed by alcoholic abuse.Ethanol metabolism leads to accumulation of reactive oxygen species(ROS),mainly hydrogen peroxide(H2O2)and superoxide anion O2–,which will cause oxidative stress.Meanwhile,Alcohol can induced CYP2E1(cytochrome P450 family 2,subfamily E,polypeptide 1)which can activate toxic substances related reactions,promote ROS accumulation and participate in lipid peroxidation,thereby promoting liver cell damage.During the progress of alcoholic liver injury,metabolic products of ethanol and toxic effects of ROS will lead to apoptosis and necrosis of hepatocyte,causing changes in cell morphology in liver and damage to other tissues and organs.With the deepen study of alcoholic liver,hepatocellular microenvironment is considered to an new treatment.However,duing to the diverse biological properties and complex organizational structure of micro-environment,single gene therapy and one class-related cells is difficult to obtain the desired effect.Traditional Chinese Medicine,widely recognized by our country and the world,has pleiotropic and multi-adjustable features.Babaodan is an important TCM,mainly used in the clinical prevention and treatment of various hepatobiliary diseases.Babaodan is composed of taurine,pearl,musk,pannx and so on,containing various active ingredients,such as taurine,musk ketone,taurocholate,saponins and flavonoids.These active ingredients will create an synergistic effect on the treatment of disease.Considering that Babaodan has a clear effect in the clinical treatment of liver disease and the closed relationship between liver disease and microenvironment of hepatocytes acute alcoholic,we suspected that Babaodan could inhibit the development of acute alcoholic liver injury by improving the microenvironment of hepatocytes.In our study,we build a mice mode of acute alcoholic liver injury and gave Babaodan treatment.Then we assessed the level of ALT,AST and TG indicating the liver function,and HE staining,Oil Red staining,CCK8,DCFH-DA staining,Western blotting,Real-time PCR,si-RNA technology were also used to explore the role of Babaodan in the progress and mechanism of acute alcoholic liver injury.Methods and resultsPart 1: The drunken situation of mice orally administered Babaodan were observed by constructing mice mode of acute alcoholic liver injury,and liver function were assessed by the level of ALT and AST.The degree of liver steatosis were determined by HE staining and oil red staining,and could also be reflected in the level of TG.Cell proliferation activity of AML-12 in different groups were determined by CCK8 assay.The experimental results showed that Babaodan could significant protect mice from acute alcoholic liver injury,which was directly correlated with the concentration of Babaodan.With its increasing of concentration,Babaodan had an enhanced effect.However,the protective effect would not be enhanced after a certain concentration.Part 2: DCFH-DA staining was used to analyze the expression of ROS in hepatocellular,Real-time PCR and Western blotting were used to analyze the changes of CYP2E1,Nrf2,HO-1 and GCL in vivo and in vitro.Different kits were applied to determine levels of MDA,ADH,SOD,GSH,CAT,GPx.To observe the influence of Babaodan on acute alcoholic liver injury in vitro,the expression of Nrf2 in AML-12 were interfered by siRNA and inhibitor.The experimental results showed that Babaodan could reduce the oxidative damage on liver cells by inhibiting the secretion of CYP2E1 and reducing the synthesis of ROS which in turn decrease the generation of MDA.Babaodan could improve the protection on liver cells by activating Nrf2,which could promote the synthetic of antioxidants,such as GSH and SOD.ConclusionBabaodan have an significantly protective effect on acute alcoholic liver injury.Babaodan can inhibit the expression of CYP2E1 and active Nrf2 and its downstream target gene,further inhibiting oxidative stress and promoting the generation of antioxidants.