| HCV(Hepatitis C Virus)is the main pathogen leading to non-A non-B hepatitis after blood transfusion.Chronic HCV infection leads to chronic liver inflammation and necrosis,fibrosis and hepatocellular carcinoma.According to the latest report from WHO,there is about 180 million people infected with HCV in world wide,with the annual 3～4 million new infected cases.Having the high or middle HCV epidemic,it is estimated that about 41 million people,were infected with HCV.Highly gene variation is the main characteristic of HCV.According to the nucleic acid sequence homology,HCV can be divided into 7 genotypes and various subtypes.Different genotype of HCV has the distinctly geographic distribution.In China and further in Yunnan,genotypes 1,3 and 6 is the predominant HCV genotype.Interferon combined with RBV is the most used therapy for hepatitis C.However,the curative effects of this therapy are far difference for different genotypes.And,its side effects are also obvious.Recently,HCV NS3/4A protease inhibitors(Telaprevir,Boceprevir and simeprevir)and HCVNS5B polymerase inhibitors(Sofosbuvir)is in the development as the new anti-HCV drugs.However,side effects and the expensive price are still existed.It is urgent to develop new anti-HCV drugs.Cyclophilin A could bind with HCV NS5A effectively and boost the peptide bond isomerization of Proline.The inhibitor against Cyclophilin A could interrupt HCV replication by interacting with NS5A.Cyclophilin inhibitors can inhibit the replication of the virus by destroying cyclophilin inter action with NS5A.In current study,based on J6-JFH/Huh 7.5.1 HCV culture system,IFN and RBV was taken as positive control to establish the anti-HCV compounds screening technique.Using this technique,the compounds of Cyclosporine A and NEW were candidated to have the testing on anti-HCV action.It was shown that the concentration for 50%of maximal effect(EC50)of IFNa-2b is 0.194 IU/mL;the half maximal inhibitory concentration of(IC50)RBV is 22.87 μg/mL,EC50 is 2.445 μg/mL;the select index value is calculated to be 9.354.According to the primary screening on other candidate compounds,there is no obvious cytotoxicity and high anti-HCV activity for and 7 compounds in concentration range of 0.010-3.00 μmol/L.The value of EC50 is 0.118 μmol/L for CsA,0.052μmol/L for 4-OH-CsA,0.010 μmol/L for NIM811,0.146 μmol/L for SCY635,0.088 μmol/L for NEW-80,0.125 μmol/L for NEW-U and 0.053 μmol/L for NEW-C respectively.Among them,no obvious cell toxicity was detected for 7 compounds.Particularly,the compound named New having the lower EC50 value is considered to have the good potential for industrial development.To further describe the influence of serum on compound effect,anti-HCV activity of NEW and CsA in different concentrations serum condition were determined.The cytotoxicity of CsA was decreased with the serum concentrations from 5%,10%to 20%.But there was no significant change in the antiviral activity with EC50 values of 0.026,0.053 and 0.038 μmol/L for these three serum concentrations respectively.The cytotoxicity of NEW was decreased slightly with the serum concentrations from 5%,10%to 20%.The antiviral activity with EC50 values of 0.018,0.178 and 0.177 μmol/L for these three serum concentrations respectively.The antiviral activity was decreased with increase of serum concentrations.But the trend is not obvious in the high concentration of serum.EC90 value is the evaluation index of anti-HCV activity of NEW and CsA in different time points.The obvious inhibitory effect was detected for CsA in the first 12 hours,and the inhibition has reached 80%in the 24 hours.Western blot to detect the expression level of NS5A proteinin EC00,EC20,EC50,EC80 concentration of drug treatment.The expression of NS5A protein was decreased with the increase of drug concentration.No obvious cell toxicity was detected for NEW.So we improve the drug concentration to 31.60 μmol/L in the cell toxicity experiment.The results showed that there are still no significant cytotoxicity.For different batches of NEW and CsA in the screening of anti HCV activity,the value of EC50 is 0.034 μmol/L±0.009 for NEW,was better than 0.071 μmol/L±0.028 for CsA.In China and further in Yunnan,genotypes 1 is mainly papular HCV genotype.It is necessary to construct the HCV lb subgenomic replicon cell model for HCV study.Luciferase fusion gene fragment was replaced with the secreted luciferase gene fragment in APP76(Con1 SG-Neo(Ⅰ)hRluc2aUb)plasmid.HCV subgenomic replicon RNA was obtained by in vitro transcription,and then transfer into Huh-7.5.1 cell by liposome.After screening the cells in the medium containing G418 about 3 weeks,we got the cell colonies with HCV lb subgenomic replicon.Then insert the colony to expand culture,to get a large number of HCV lb subgenomic replicon cell.We detected that the HCV replicon fragments include NS5B and Sluc gene from cell total RNAby RT-PCR.In current study,based on J6-JFH/Huh7.5.1 HCV culture system,we successfully screening out the NEW compounds of Cyclosporine A which has the good potential for industrial development.And we also have successfully established a cell model for HCV lb subgenomic replicon in vitro.The cell model is useful tool for the study on the development of antiviral drugs. |
