Effect And Mechanism Of Huangqi Injection Combined With Entecavir Against Hepatitis B Virus Infection

ObjectivesTo study the effect and mechanism of Huangqi injection combined with Entecavir against hepatitis B virus infection in vivo and vitro.Materials and methods1.Huang Qi injection combined with Enticavir against Duck Heptitis B virus in vivo experiment. 48 one day age ducklings(weighting 50 g each)infected DHBV were screened out,and randomly divided into 6 experimental groups, including virus control group,Enticavir treatment group(0.1mg/kg),high,medium and low doses(20ml/kg,10ml/kg,5ml/kg) Huang Qi injection combined with Enticavir groups and high dose Huang Qi injection(20ml/kg) 6 experimental groups,with 8 animals in each group.Viral load was measured at prior treatment(T0),7 days treatment(T7),14 days treatment(T14),21 days treatment(T21),5 days withdrawl(P5),10 days withdrawl(P10),20 days withdrawl(P20)and compared the data.2.Huang Qi injection combined with Enticavir against Heptitis B virus in vitro Hep G2.2.15 cells experiment.Cultivating Hep G2.2.15 cells were divided into 8 experimental groups,including virus control group,Enticavir treatment group(30nmol/L),high,medium and low doses Huang Qi injection combined with Enticavir groups and high,medium and low doses( 1000ng/ml,500ng/ml and 250ng/ml) Huang Qi injection groups,with 9 wells in each group.The cells proliferation of drug treatment groups were detected,on the third day of using drugs(T3)and the third day of withdrawaling(P3). HBVDNA was detected in cell supernatant and intracellular with PCR fluorescence probe method.3.Machanism of Huang Qi injection combined with Enticavir against Heptitis B virus infection in vitro Hep G2 and Hep G2.2.15 cells.Cultivating Hep G2 cells and Hep G2.2.15 cells. Untransfected HBV Hep G2 cells were used as blank control group,and transfected HBV Hep G2.2.15 cells were used as drug groups and virus control groups.Cells were devided into 9 groups:blank group,Enticavir treatment group(30nmol/L),high,medium and low doses Huang Qi injection combined with Enticavir groups,high,medium and low doses(1000ng/ml,500ng/ml and 250ng/ml)Huang Qi injection groups and virus control group,with 6 wells in each group. using drugs 3 days and withdrawaling drugs 3 days,at the fifth day,digesting and splitting the cells of 3 wells,applicated fluorescence quantitative assay detected the drugs’ effection on JAK-STAT signal pathway m RNAs.the other 3 wells of cells were digested and splited for detection of NF-кB、OAS、PKR、Caspase3 proteins intracellular by western blotting assay.4.HuangQi injection combined with Enticavir treated NIH mouse immunological liver injury study.Puchased 72 male NIH mice were randomly divided into 9 experimental groups:normal control group,Enticavir treatment group(45μg/kg), high,medium and low doses Huang Qi injection combined with Enticavir groups, high,medium and low doses(1.5,1,0.5ml/10g)Huang Qi injection groups,model control group,with 8 animals in each group.In addition to the normal control group,the rest of the mice received tails intravenous injection of Con A 20mg/kg.Then intragastric drugs in the morning and at the afternoon of the first day and at the afternoon of the second day after modeling.Model control groups and drugs treatment groups intravenous injected Con A 20mg/kg at the last 4 hours of the final treatment.Food was forbided,and water was nonforbided,Blood was collected by Extracting mice’s eyeballs,and plasma was separated by centrifugal.ALT,IFN-γ,TNF-α,SOD, MDA and NO were detected in mice serum in 8h.Randomly taking 100 mg the tissues of the livers,except the left leaf,of the 3 mice every group,grided and splited tissues of 100 mg the rest livers,and applicated Western Blotting assay to observe changes of NF-кB,OAS,PKR, Caspase3 proteins expression of the liver cells.Results1.Huang Qi injection combined with Enticavir against Duck Heptitis B vir us in vivo experiment.In this experiment,virus control animal serum DHBV DNA level was stable.Huangqi injection combined with Enticavir three dose s groups and Enticavir group in the treatment of 7 days,the duckling seru m DHBV DNA was declined.Compared with the serum of the virus control grou p at the same time was significant(P<0.01).The viral load of Huangqi in jection combined with Entecavir three doses groups rose again in 20 days after drug withdrawal.There was no obvious bounce of the virus load in 5 days,10 days drugs discontinuation,and compared with the virus control g roup at the same time and the time before drug delivering was significant(P<0.01),the virus appeared obviously bouncing in drugs discontinuation for 20 days.This phenomenon illustrated DHBV DNA quantities of Huangqi in jection combined with Enticavir three dose groups rebounding were slowed.The medium dose of Huangqi injection combined with Enticavir was most obv ious.And the load of virus in Enticavir group appeared obvious bouncing i n 5 days after drug discontinuation.Alone with Huangqi injection high dos e group in the treatment of 21 days duck serum DHBV DNA had no decline.Co mpared with the virus control group,at the same time,there wasn’t signifi cance(P>0.05).This illustrated Huangqi injection itself antiviral effect was weak.2.Huangqi injection combined with Enticavir against Hepatitis B infection in vitro Hep G2.2.15 cells experimental study. According to the results of preliminary experiment,the drug concentration of Enticavir was 30nmol/L,and Huangqi injection high,medium and low three drug concentration were 1000ng/ml,500ng/ml,250ng/ml respectively. Medication for 3 days, Enticavir could inhibit the secretion of extra and intracellular HBVDNA(P<0.05),quantitative compared with the virus control group had significant difference(P<0.01).Drugs discontinue for 3 days, the extra and intracells HBVDNA quantities were rebounded.Having no difference with virus control group(P>0.05).Medication for 3 days, every Huangqi injection group had no effect on HBVDNA extra and intracellular,compared with virus control group had no difference(P>0.05).Medication for 3 days, High and medium dose Huangqi injection combined with Enticavir could inhibit extra and intracellular HBVDNA,the HBVDNA quantities in extracellular compared with virus control group had difference(P <0.05),the HBVDNA quantities intracellular compared with virus control group had obvious difference(P<0.01). But the HBVDNA quantities of extra and intracells were rebounded in 3 days.Except the HBVDNA quantities of high dose Huangqi injection combined with Enticavir of extracellular was less than virus control group(P<0.05),other treatment groups Having no differences with the virus control group(P > 0.05).This means,High dose of Huangqi injection combined with Enticavir had effect of delaying the rebounce of HBVDNA in extracellular.3. Machanism of Huang Qi injection combined with Enticavir against Heptitis B virus infection in vitro Hep G2 and Hep G2.2.15 cells.Compared with blank control group,the expression of ISGF3 m RNA of JAK-STAT path of virus control group was declined(P<0.05),other m RNAs had no change.Compared with virus control group,Enticavir could make the expression of STAT2 m RNA to rose(P<0.05),other drug groups had no effects on it.Enticavir and Huangqi injection medium and low doses combined with Enticavir maked the expression of ISGF3 m RNA rose(P<0.05),All drug groups had no effect on STAT1,OAS,PKR m RNAs(P>0.05).All in all,The ISGF3 m RNA expression of JAK- STAT signal pathway was diminished by HBV infection,Huangqi injection medium and low dose combined with Enticavir could make it back to normal(compared with blank control group, P>0.05),Although Enticavir could Enhanced its expression,effect of it was weaker than the combination groups(compared with blank control group,P<0.05).Effects of Huangqi injection combined with Enticavir on proteins of inflammatory reaction of Hep G2.2.15 cells.Compared with blank control group,there was no significant difference in Caspase 3 protein expression in virus control group(P>0.05).While others rose obviously(P<0.01).Compared with virus control group,NF-кB of Huangqi injection low dose combined with Enticavir treatment group was declined(P<0.05),OAS of Enticavir treatment group,Huangqi injection medium dose combined with Enticavir treatment group,Huangqi injection medium and low dose treatment group were declined too(P<0.05 or P<0.01),PKR of Huangqi injection low dose combined with Enticavir treatment group,Huangqi injection high dose treatment group were declined(P<0.05),the rest medication group had no effect on NF-кB,OAS,PKR(P>0.05).There were no effects on Caspase3 expression in all treatment groups(P>0.05).4.The immune liver injury in NIH mice experiment,compared with normal control group,ALT of model group rose.compared with model group,Except Huangqi injection low dose treatment group,ALT of the other groups were declined(P<0.05).Compared with Enticavir treatment group,The declining effection of ALT on Huangqi injection high dose combined with Enticavir treatment group and Enticavir treatment group were equal(P>0.05).Other groups were less effective(P<0.01).Compared with normal control group,SOD,MDA,and NO in model control group rose(P<0.01).Compared with model control group,the other medication groups reduced SOD(P<0.01).Except Huangqi injection in low dose group,the other treatment groups declined MDA(P<0.01).Each groups declined NO(P<0.05 or P<0.01).Compared with Enticavir group,each group declined SOD less than Enticavir(P<0.05 or P<0.01),the effect declining MDA of Huangqi injection high dose combination group was equal to the Enticavir group(P>0.05),the other treatment groups effect on it were less than the Enticavir group(P<0.01);The effects of declining NO of each combination groups and Enticavir group were equal,while the other treatment groups were less than the Enticavir group(P<0.05 or P<0.01).Compared with normal control group,TNF-αand INF-γ of model control group rose obviously(P<0.01).Compared with model control group,TNF-α of Enticavir medication group, Huangqi injection high and medium doses combined with Enticavir groups were declined obviously(P<0.05 or P<0.01),the other groups had no effect on TNF-α.Compared with the Enticavir group,Huangqi injection high and medium doses combined with Enticavir groups’ effects on it were equal(P > 0.05).Each treatment groups had obviously reducing effect on INF-γ(P<0.01),Huangqi injection high dose effect was equal to Enticavir treatment group(P>0.05),the other treatment groups were worse(P<0.05 or P<0.01). Compared with normal control group,NF-кβ,OAS,PKR,Caspase3 of model control group rose(P<0.01).Compared with model control group,NF-кB of the Enticavir group rose(P<0.05),the other groups had no effect on it(P>0.05);Except Huangqi injection low and medium dose combined with Enticavir had no effect of OAS,OAS of the other treatment groups were declined(P<0.05 or P<0.01). PKR of Huangqi injection medium and low dose combined with Enticavir groups rose(P<0.05 or P<0.01),PKR of Huangqi high dose combined with Enticavir group and Huangqi high dose injection group were declined(P<0.05),PKR of the other medication groups had no change(P>0.05);Caspase3 of Each groups had no change(P>0.05).Liver index,compared with normal control group,the liver index of model group was rose obviously.Compared with model group,Enticavir and Huangqi injection low dose group rose obviously(P<0.01),Huangqi injection medium and low dose combined with Enticavir had no change(P>0.05),Huangqi injection high and medium doses combined with Enticavir groups,Huangqi injection high dose group had obviously reducing effect(P < 0.01).Compared with normal group,Huangqi injection high dose combined with Enticavir treatment group had little change(P>0.05).Spleen index,compared with normal control group,the spleen index of model control group rose obviously(P<0.01).Compared with model control group, Enticavir and low dose Huangqi injection group rose obviously(P<0.01),medium and low dose Huangqi injection combined with Enticavir had no change(P>0.05),Huangqi injection high and medium doses combined with Enticavir and high dose of Huangqi injecton had obviously reducing effect(P<0.05 or P<0.01). Thymus index,compared with normal control group,model group declined obviously(P<0.01).compared with model group, Enticavir and low dose Huangqi injection groups declined obviously(P<0.01),Huangqi injection medium dose group had no change(P>0.05),combination groups and Huangqi injection high dose group rose obviously(P<0.01). the pathological slices of liver,each treatment group compared with normal control group,had tissue injuries(P<0.01),each medication group compared with model control group,injury of the tissues was reduced(P<0.01).ConclusionHuangqi injection combined with Enticavir had the very good effect against Heptitis B virus infection,and could delay the hepatitis B virus rebound after drugs discontinuation.The mechanism of the action concluded adjusting JAK-STAT pathway,antiviral cytokines and proteins on the immune response.The function of descending glutamic-pyruvic transaminase of Huangqi injecttion combined with Enticavir,inflammatory reaction factor and protecting the liver cells,in general, the total therapeutic effects of antiviral,protecting liver,adjusting immune response are better than using these two drugs alone.Huangqi injection combined with Enticavir or used alone,the curative effect of antiviral and immune adjustment are dose dependent.On the whole, experimental results indicated that the antiviral function of high and medium doses of Huangqi injection combined with Enticavir were outstanding,but the function of Huang Qi injection low dose promoted immune reaction was stick out.The experiments indicated clinical treatment of Huangqi for Chronic hepatitis B must according to the needs of the patient,using concentration flexibly.The combination of two medicines application could be more safe and effective,while the function needs verifications of clinical trails.