| Background Hepatitis B virus(HBV)infection is still a major global public health problem.The combination of traditional Chinese medicine and Western medicine has become an important method in the treatment of chronic hepatitis B.Therefore,searching for new anti-HBV drugs with low toxicity and effectiveness from traditional Chinese herbal extracts has become an important supplementary way to discaover new anti-HBV drugs.Previous studies have shown that orientin,aloperine,vitexin,naringenin,deguelin,atractylenolide Ⅰhave certain antiviral effects.However,the effects of rabdosin on HBV have not been studied,and need to be further explored.Objective The purpose of this study is to screen whether these six Chinese herbal or natural plant extracts(orientin,aloperine,vitexin,naringenin,deguelin,atractylenolide Ⅰ)have inhibitory effect on hepatitis B virus through the cell infection model of hepatitis B virus,and to further explore the possible mechanism(s)involoved in the anti-HBV action.Methods In this research,Hep G2.2.15 cells harboring HBV genome were used as an in vitro model to screen the anti-HBV acitivity of orientin,aloperine,vitexin,naringenin,deguelin,atractylenolide Ⅰ,using HBs Ag as the preliminary screening index,and to preliminarily explore the relevant mechanism(s)of effective drugs against HBV.The experiments were divided into two parts as follows:1.The screening of anti-HBV activity of six drugs:(1)Hep G2.2.15 cells were treated with different concentrations of orientin,aloperine,vitexin,naringenin,deguelin,atractylenolide Ⅰ for 72 hours,respectively,and the control group was not treated with drugs.The effect of each drug on the cell viability of Hep G2.2.15 cells was detected by ATP method.(2)Hep G2.2.15 cells in logarithmic growth stage were seeded in 24-well plates and treated with different concentrations of orientin,aloperine,vitexin,naringenin,deguelin,atractylenolide Ⅰ for 3 and 6 days respectively.The supernatant of cell culture was collected,and the effect of each drug on HBs Ag secretion of Hep G2.2.15 cells in the supernatant(secretion from Hep G2.2.15 cells)was detected by ELISA.2.The effect of atractylenolide Ⅰ on HBV and and mechanism(s)involved in:(1)In order to ensure the reliability of the first part of the preliminary screening experiment,the inhibitory effect of atractylenolide Ⅰ on HBs Ag expression was repeatedly observed.Hep G2.2.15 cells were treated with different concentrations of atractylenolide Ⅰ for 3 days and 6 days,respectively.The supernatant of cell culture was collected,and the level of HBs Ag in supernatant was detected by ELISA.(2)After the cells were treated under the same condition,the total cellular proteins were extracted,and the HBs Ag levels in the cells that treated with different concentration of Atractylenolides Ⅰ were detected by Western blot.(3)Meanwhile,the changes of HBe Ag secretion(in the supernatant of cell culture)with different concentrations of atractylenolide Ⅰwere measured by ELISA.(4)Hep G2.2.15 cells were treated with different concentrations of atractylenolide Ⅰ for 72 hours,then the cellular RNA was extracted.The expression levels of HBV pg RNA and HBV S gene were measured by RT-PCR and q PCR.(5)In order to explore the mechanism of anti-HBV activity of Atractylenolides Ⅰ,Hep G2.2.15 cells were treated with different concentrations of Atractylenolides Ⅰ for 72 hours.The expression levels of HBx protein,an important regulatory protein of HBV,in Hep G2.2.15 cells were measured by Western blot.Meanwhile,RT-PCR and q PCR were used to screen the expression of HBV-related transcription factors.Results1.The screening of anti-HBV activity of six drugs:(1)In the concentration range of 100.00μmol/L to 0.78mol/L of orientin,the cell viability of Hep G2.2.15 was higher than 90%.There was no significant difference between the drug-treatment group and the control group(P> 0.05).Compared with the control group,the relative expression level of HBs Ag in the treatment group was67.70%-89.31%.Compared with control group,there was no significant difference in the inhibition effect(P>0.05)in treatment group.(2)In the concentration range of 100.00μmol/L to 0.78μmol/L of aloperine,the cell viability of Hep G2.2.15 cells was higher than 90%(P>0.05).Compared with the control group,the relative expression level of HBs Ag was 59.08%-79.03%,and there was no significant difference in the inhibition effect(P>0.05)in the treatment group.(3)When the concentration of Vitexin was 100.00μmol/L,the cell viability of Hep G2.2.15 cells decreased significantly(<90%),and the difference was statistically significant(P<0.05).Compared with the control group,the relative level of HBs Ag in Vitexin-treated group was higher than 70%in the concentration range without cytotoxic(<50μmol/L),and there was no significant difference in the inhibition effect(P>0.05)in the treatment group.(4)In the concentration range of 100.00μmol/L to 0.78μmol/L of naringenin,the cell viability of Hep G2.2.15 cells was higher than 90%(P>0.05).In this concentration range,naringenin had no significant cytotoxic effect on Hep G2.2.15 cells.Compared with the control group,the relative expression level of HBs Ag in naringenin-treated group was higher than 79%,and there was no significant difference in the inhibition effect(P>0.05)in the treatment group.(5)When the concentration of deguelin≥25.00μmol/L,the cell viability of Hep G2.2.15 cells decreased significantly(<90%),and the difference was statistically significant(P<0.05).Compared with the control group,the relative expression level of HBs Ag in the non-cytotoxic concentration range(<12.50μmol/L)of deguelin group was higher than 65%,and there was no obvious inhibition trend(P>0.05).(6)When the concentration of atractylenolideⅠ was 100.00μmol/L,the cell viability of Hep G2.2.15 cells was significantly reduced(<90%),the difference was statistically significant(P<0.05).Compared with the control group,in the concentration range without cytotoxic(<50.00μmol/L),the relative expression level of HBs Ag in atractylenolide Ⅰtreatment group decreased significantly(P<0.05),in a dose dependence and time dependence manner.The inhibition rate of HBs Ag in atractylenolide Ⅰtreatment group was 53.57% and 66.89% respectively after 3 and 6 days of treatment at the concentration of 50.00μmol /L.2.The effect of atractylenolide Ⅰ on HBV and and mechanism(s)involved in:(1)The inhibition rate of HBs Ag expression in cells and culture supernatant of atractylenolide Ⅰ treatment group increased with the increase of concentration and treatment time.There was a statistical difference between the control group and the treatment group(P<0.05),and the inhibition effect on HBs Ag secreted by cells(in the supernatant of cell culture)was more obvious.(2)Compared with the control group,the inhibition rate of atractylenolide Ⅰ on HBe Ag expression in cell culture supernatant increased with the increase of atractylenolide Ⅰ concentration and the prolongation of treatment time,the difference was statistically significant(P<0.05).(3)Compared with the control group,the expression level of HBV pg RNA and HBV S gene m RNA decreased with the increase of atractylenolide Ⅰ concentration,the difference was statistically significant(P<0.05).(4)With the increase of the concentration of atractylenolide Ⅰ,the expression of HBx protein in atractylenolide Ⅰtreatment group showed a downward trend compared with the control group.When the concentration of atractylenolide Ⅰ was 25.00μmol/L and 50.00μmol/L,the difference was statistically significant(P<0.05).The inhibition rate of atractylenolide Ⅰ on HBX protein in the highest concentration was about44.90%.(5)Compared with the control group,the expression level of P38 MAPK decreased significantly with the increase of atractylenolide Ⅰconcentration(P<0.05),while the level of P53 increased with the increase of atractylenolide Ⅰ concentration.Atractylenolide Ⅰ had no significant effect on the m RNA expression of ERK,NF-κB,HNF4α,JAK2 and STAT3(P>0.05).Conclusions1.Among 6 traditional Chinese herbal extracts(orientin,aloperine,vitexin,naringenin,deguelin,atractylenolide Ⅰ),atractylenolide Ⅰ exhibited obvious anti-HBV activity in the in vitro HBV-cell model.2.P38 MAPK-P53 pathway may be involved in the inhibitory effects of atractylenolide Ⅰ on HBV replication and viral expression. |
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