Study On Screening And Mechanism Of Anti-HBV Drugs In Vitro

Objective:Hepatitis B virus(HBV)is a hepatotropic DNA virus.HBV infection can cause acute or chronic viral hepatitis,which can further cause liver fibrosis,cirrhosis and even hepatocellular carcinoma.Hepatitis B virus causes Hepatitis B,and Hepatitis B has been a serious threat to human health.At present,anti-HBV drugs widely used clinically include immunomodulators and nucleoside analogues.Anti-virus is currently an important treatment for chronic hepatitis B.The main drugs are interferons and nucleoside analogues(NAs).Interferon achieves the effect of treating CHB through dual mechanisms of antiviral and immune regulation,and has the advantages of high sustained response rate,long-lasting and stable curative effect,relatively stable treatment course,and no drug resistance.However,the therapeutic effect of interferon is normal.In the early stage,flu-like symptoms often accompanied by a certain period of time can cause adverse reactions such as myelosuppression and hepatic damage,and have serious consequences for patients whose liver function is nearly decompensated or has already occurred.In addition,the use of interferon is inconvenient.It is usually injected subcutaneously and is not suitable for patients with compromised immune function.Therefore,the clinical application of interferon is greatly limited.Oral administration of nucleoside analogues facilitates high bioavailability and strong antiviral effects,does not depend on the patient's immune response to HBV,and therefore has less adverse reactions and milder side effects.It can be used in patients with decompensated liver function,so it is widely used in clinical practice.However,nucleoside analogues cannot completely eliminate the virus in vivo and need to be used for a long period of time,which is easy to produce drug resistance.The purpose of this project is to study the anti-HBV activity and its mechanism of action of 10 TCM compounds in vitro,and lay a foundation for the search for new anti-HBV drugs.Method:In this study,Hep G2.2.15 cell line and Hep GRL1 cell line were used as models to screen the anti-HBV effect of 10 compounds in vitro and the preliminary study on the HBV X gene related mechanism.The experiment was divided into two parts.The content was as follows:The first part of the in vitro screening of 10 compounds against HBV1.Cytotoxicity assay of drug: Hep G2 2.2.15 and Hep GRL1 cells were cultured in 96-well cell culture plates for 24 hours,and then added with different concentrations of drug-containing culture medium,and continued to culture for 3 days.On the third day,the compounds were tested for their toxicity to cells.2.Detection of HBV e and s antigen inhibition by drugs: Hep G2 2.2.15 and Hep GRL1 cells were cultured in 96-well cell culture plates for 24 hours,and then added with different concentrations of drug-containing culture broth to continue cultivation for 6 days.The liquid was changed every 3 days.The supernatant was collected and the antigen expression levels of HBV s and e were detected by ELISA.3.Drug HBV DNA Inhibition Assay: Hep G2 2.2.15 and Hep GRL1 cells were cultured in 24-well cell culture plates for 24 hours,and then added with different concentrations of drug-containing culture fluids,and cultured for 6 days(changed once every 3 days).The supernatant and cells were collected,and the effect of the compound on the intracellular and extracellular HBV DNA was detected by PCR.The second part of the preliminary study of 10 compounds against HBV X gene related mechanismsThe HBV X gene expression of HepG2.2.15 cells and HepGRL1 cells was detected by RT-PCR.After Hep G2 2.2.15 and Hep GRL1 cells were cultured in 12-well cell culture plates for 24 hours,cultures containing compounds at different concentrations were added.Positive control group used 3-TC and ETV.Blank group only added medium and continued cultivation.Days(changed once every 3 days),cells were collected and reversed for RNA,and the effect of compound on intracellular HBV X gene expression was detected by RT-PCR.Result:The first part of the in vitro screening of 10 compounds against HBV1.10 different compounds acted on HepG2.2.15 cells and HepGRL1 cells,and the TC50 was calculated by MTT assay for the toxicity of the compound to both cells.2.Elisa assay of compounds on the two cell HBV e antigen and s antigen expression found that yxy1741,yxy1483 e,yxy1483c,yxy1452 a,yxy1403b on Hep G2.2.15 cells HBV e antigen and s antigen has a strong inhibitory effect,in addition yxy1741 Yxy1483 e,yxy1483c,yxy1452 a,yxy1403b had strong inhibitory effect on HBV e antigen of Hep GRL1 cells.3.The effects of 10 compounds on Hep G2.2.15 cells and Hep GRL1 cell supernatants and intracellular HBV DNA secretion were detected by Q-PCR,and it was found that yxy1741,yxy1483 e,and yxy1483 c significantly inhibited HBV DNA replication and inhibited compared with the blank group.The rate exceeds 50%,and the compound significantly inhibits HBe Ag,HBs Ag levels.The second part of the preliminary study of 10 compounds against HBV X gene related mechanisms4.The RT-PCR results showed that yxy1483 e not only inhibited the HBV e antigen,HBV s antigen and HBV DNA in the two cells,but also inhibited the expression of HBV-X RNA in the cellsConclusionThrough this study,yxy1483 e can significantly inhibit the replication of HBV DNA in Hep G2.2.15 cells and Hep GRL1 cell culture experiments,and can also have a strong inhibitory effect on the expression of HBs Ag and HBe Ag,and less cytotoxicity to HBV-X.The inhibition of RNA expression is expected to be a new class of anti-HBV drugs.