The Study On The Anti-influenza Activity Of Eleutheroside B1 And Its Mechanism

Influenza virus can cause serious lung disease and has multi-host.Its genes are easy to be mutable,recombination which help these virus spread between human and humans,animals and humans,and cause a pandemic.Genetic recombination and adaptability to the environment increased the difficulty to prevent and control influenza virus,which causes a serious threat to human health.Prevention and treatment of influenza virus with Chinese medicine has a long history with clinical effects.Chinese medicine,Sarcandra glabra has been used to treat respiratory tract infection for a long time.Modern pharmacological studies have shown that Sarcandra glabra and its extraction have antibacterial antiviral activity,and can control the respiratory tract infection with alleviating clinical symptoms.However,the active compounds and the mechanism of Sarcandra glabra for anti-influenza virus is unclear.Based on this,the 14 main components in Sarcandra glabra were screened for anti-influenza virus activity.One of the components,eleutheroside B1 was found to have antiviral activity.Therefore,in this study,eleutheroside B1 was selected as the research target and systematically studied for the anti-influenza mechanism,which provided a scientific basis for clinical use.Part Ⅰ The study on the anti-influenza virus activity of main components from Sarcandra glabraObjective1.14 main components in Sarcandra glabra were screened for anti-infl uenza virus activity,and the component with anti-influenza activity will be selected for the research target.2.Determine the pharmacodynamic model,time-course,and point of the research target to inhibit influenza virus replication.3.Study on the pharmacodynamic mechanism of research target against the influenza virus.Methods1.Using the drug cytotoxicity test(MTT method)detect 14 main comp onents of Sarcandra glabra(astilbin,neoastilbin,Isofraxidin,eleuthe roside B1,protocatechuic acid,caffeic acid,rosmarinic acid,chloranthal actone E,5-0-caffeoylshikimic acid,Sarcaboside A,Fraxin,Esculetin,raxeti n,Scoparone)on MDCK cells(Madin Darby Canine Kidney,MDCK cell line)for the half toxic concentration(the TC50).This concentration can be used for subsequent experiments.Cytopathic effect inhibition assay(CPE)detectthe activity of these components to inhibit PR/8/34 strain in MDCK cellsmodel,which can be calculated for semi-inhibitory concentration(IC50)and selecti on index SI(SI=TC50/IC50)by Reed-Mench method.According to these results,the component with antiviral activity will be selected as a research targ et.Then,research target will be prepared by high-performance liquid chr omatography for subsequent experiments,and its structure will be identif ied by nuclear magnetic resonance technique.2.CPE method was used to detect the inhibitory effect of research target for A/Guangzhou/GIRD07/09(H1N1),A/Aihi/68(H3N2),A/Duck/Guangdong/2009,(H6N2),A/Duck/Guangdong/1994(H7N3)and A/Chicken/Guangdong/1996(H9N2).In prevention,treatment and direct action model,research target was investigated for its anti-influenza virus.Time-course experiment and immuno-fluorescence test were used to detect action point of research target in the single replication cycle of influenza A/PR/8/34 virus on MDCK cells.In the two experiments,at the time point(-2h,Oh,2h,4h,6h,and 8h)after the infection of influenza A/PR/8/34 virus,eleutheroside B1 was given for treatment on MDCK cells,then these cells were collected for the experiments.3.Real-time quantitative PCR(Quantitative Real-time PCR)was used to detect the mRNA expression levels of influenza virus gene fragments in each group.The experimental groups were divided into the virus infection model group,the group of eleutheroside B1(25μ g/mL,50μg/mL,100μg/mL)treatment on A549 cells with virus infection,eleutheroside B1 control group.An experiment was performed as follows,A549 cells were infected with influenzaA virus A/PR/8/34 strain,then treated with eleutheroside Bl.24 hours after infection,the cells were collected for RNA extraction.The luciferase gene and Influenza virus polymerase(RNP)related gene were transfected into 293T cells(human kidney epithelial cell line)with liposome.After 24h,cell supernatants were collected for luciferase activity detection with a Luciferase reporter gene assay kit.Real-time PCR detected mRNA expression levels of cytokine including TNF,IL27,IL1B,NFκ B1,IL-6,CXCL8,CCL-2 and IFNG.Results1.MTT results were showed as follows,the half toxic concentration(the TC 50)of 14 compounds on MDCK cells were 0.455 mg/mL,0.783mg/mL,0.252mg/mL,4.127mg/mL,0.196 mg/mL,0.56mg/mL,0.126 mg/mL,2.004mg/mL,0.128mg/mL,>2.300mg/mL,>lmg/mL,>0.015mg/mL,>0.063mg/mL and>0.25mg/mL.In thisstudy,subsequent experiments will use this concentration as the maximum concentration.The results of CPE experiments showed that astilbin and eleutheroside Blhave anti-influenza activity.Their IC 50 is 0.455 mg/mL and 4.127 mg/mL respectively,and the SI index is 1.74 and 11.79 respectively.However,astilbin is with low-efficient,and eleutheroside B1 is low toxicity with strong antiviral activity.The other 12 compounds are without antiviral activity,and their SI index is less than 1.Therefore,eleutheroside B1 was selected as the research target for subsequent studies.Eleutheroside Blwas extracted by HPLC and identified as molecular weight(384),molecular formula(C17H2409),structure type(coumarin).2.Eleutheroside Bl have an inhibitory effect on A/Guangzhou/GIRD07/09(H1N1),A/Aichi/68(H3N2)influenza virus strains.The IC 50 were 0.064 mg/mL and 0.125 mg/mL respectively,and the SI index were 3.91 and 3.Ele utheroside B1 had no inhibitory effect on A/Duck/Guangdong/2009(H6N2),A/D uck/Guangdong/1994(H7N3),A/Chicken/Guangdong/1996(H9N2),and the SI index was less than 1.In the treatment mode,eleutheroside B1 inhibited the influenza A virus A/PR/8/34 strain with IC 50 of 0.04 mg/mL and SI index of 3.625.In the direct mode and prevention model,eleutherosides B1 didn’t show anti-influenza virus activity with SI index<1.From the results,we can see that eleutheroside B1 had no effect on the influenza virus particles,and its anti-influenza virus activity was achieved by regulating the host cells.Therefore,in the future,the mechanism of eleutheroside B1 against the influenza virus on the host cells in anti-influenza virus will be explored.In the results of the time-course assay,compared with viral infection group,the virus titer was significantly decreased after the treatment of eleutherosides B1(100ug/mL)at 0-6 hours in the viral replication cycle(P<0.05).While eleutherosides B1(100 ug/mL)was added at-2hour or 8hour,virus titers did not change(P>0.05).This suggests that eleutherosides B1 primarily acts in the early stages of influenza virus replication.In the immunofluorescence results,eleutherosides B1(100 ug/mL)was added at Oh,2h,4h,6h,8h dpi,and the viral NP proteins synthesis was decreased,but NP proteins were not limited in the nucleus,still observed in the cytoplasm.While eleutherosides B1(100 μg/mL)was added at-2h dpi,the NP proteins synthesis did not change and was not restricted to the nucleus.3.In the real-time PCR results,compared to the virus-infected model group,eleutherosides B1(100μg/mL)can reduce the expression of viral genes including PB1,PB2,PA,HA,NA,NP and NS mRNA(P<0.05),and had no effect on the expression of viral gene M mRNA(P>0.05).In the result of influenza virus RNA polymerase(RNP)activity inhibition test,eleutherosides B1(200 p g/mL)inhibited RNP activity compared with the RNP control group(P<0.05).In the real-time PCR results,eleutherosides B1(100μg/mL)significantly reduced the mRNA expression levels of host cell inflammatory factors induced by the influenza virus,including TNF,IL27,IL1B,NFκ B1,IL-6,CXCL8 CCL-2,and IFNG,compared with the virus-infected model group.(P<0.05).ConclusionEleutherosides B1 was identified with antiviral activity.In the first time,eleutherosides B1 was proved to mainly work in the early stage of influenza virus replication,and down-regulated the expression level of the influenza virus NP proteins.It also reduced the mRNA expression level of viral gene and host cytokine and inhibited influenza virus RNA polymerase(RNP)activity.Part Ⅱ The study on the differential gene expression regulated by eleutherosides B1 in the anti-influenza virus processObjective1.Study on the differential gene expression regulated by eleutherosides B1 in the host cells for its anti-influenza virus activity2.Valid the differential expressed gene induced by eleutheroside B1,and screen potential targets.Methods1.RNA high-throughput sequencing and analysis:The experimental groups were set as follows.A549 cells as control cells(A549)group,influenza virus A/PR/8/34 infected A549 cells was virus-infected group(PR8),and the infected A549 cells with eleutheroside B1 treatment(100 μg/mL)was eleutherosides B1 intervention group(PR8+eleu).The cells in each group were given the corresponding intervention.24h later,the cells were collected for RNA extraction and high throughput sequencing.After obtaining sequencing data,the R language software was used to analyze the differential expression of RNA(mRNA)and non-coding RNA(ncRNA)levels between the experimental groups.Then the program combined RNA sequence data with the database for functional enrichment and cluster analysis.In this study,the follows database were used,gene ontology(GO)and the Kyoto Encyclopedia of Genes and genomes book(KEGG).2.Analyze the significant differential expressed mRNAs/ncRNAs between PR8+eleu/PR8 and PR8/A549,and obtain the same differential expressed mRNAs/ncRNAs between them.These mRNAs/ncRNAs were performed for functional enrichment analysis.Significant differential expressed mRNAs/ncRNAs involved in pathways associated with influenza virus infection were selected for further validation.Real-time PCR technology was used to validate significant differential expressed mRNAs/ncRNAs the screening of gene detection in the high-throughput RNA sequencing of genes.These mRNAs/ncRNAs with statistically significant and consistent with RNA sequencing result would be chosen for molecular docking.Using ChemDraw ultra 8.0 and SYBYL-X2.1.1 software analyzed the space interaction between eleutherosides B1 and gene.Results1.In PR8+ eleu/PR8 groups,there are 1871 significant differentially expressed mRNA(Fold Change>2,P-value<0.05),of which 958 were up-regulated and 913 down-regulated.The same significantly differentially expressed mRNAs between PR8+eleu/PR8 groups and the PR8/A549 groups are 846.For GO analysis,these mRNAs were enriched in Various types of N-glycan biosynthesis,chemokine signaling pathways,cytokine-cytokine receptor interactions,and RNA polymerase pathways.In PR8 + eleu/PR8 groups,there are 8 significant differentially expressed ncRNAs(Fold Change>2,P-value<0.05),of which 5 were up-regulated and 3 down-regulated.The same significantly differentially expressed ncRNAs between PR8+Eleu/PR8 groups and the PR8/A549 groups was only NEAT1.For GO analysis,this ncRNA s were enriched in oxidative phosphorylation,influenza A,allograft rejection,autoimmune thyroid disease,type Ⅰ diabetes,cytokine-cytokine receptor interaction and other channels.2.For validation,we selected 46 same significantly differentials expressed mRNAs between PR8+eleu/PR8 groups and the PR8/A549 groups,which involved in Various types of N-glycan biosynthesis,chemokine signaling pathways,cytokine-cytokine receptor interactions,and RNA polymerase pathways.The most significant differential expressed gene,POLR2A(downregulated 16 384-fold)was included.Significant differential expression ncRNAs(NEAT1)was also added for validation.Then,47 significant differentially expressed mRNAs/ncRNAs were validated by real-time PCR.In the 47 significant differentially expressed mRNAs/ncRNAs,compared to virus-infected group,eleutherosides B1(100ug/mL)treatment down-regulated POLR2A,ALG1,ALG9 and IL1RAP expression(P<0.05)in real-time quantitative PCR,which was consistent with the results of high-throughput sequencing RNA.For the other 43 genes,other screened gene expression between the two groups,there is no significant difference between eleutherosides B1(100ug/mL)treatment and virus-infected group(P>0.05).In the molecular docking results,for the host genes,such as POLR2A,ALG1,ALG9 and IL1RAP,eleutherosides B1 show highest match with the structure of POLR2A.Their evaluation score is 9.0133.POLR2A was selected for subsequent experimental studies.ConclusionEleutheroside B1 regulates the expression of multiple genes and pathways in the host cell for its anti-influenza virus activity.POLR2A gene may play an important role in the eleutherosides B1 function to inhibit the replication of the influenza virus.Part Ⅲ The regulation of eleutheros ides B1 on POLR2A for its anti-influenza virus activityObjective1.Verify the key role of POLR2A in anti-influenza virus activity of eleutherosides B12.Explore the regulation mechanism of eleutherosides B1 on POLR2A.Methods1.The lentiviral particles with POLR2A gene infected MDCK cells.48 hours,puromycin was added for screening monoclonal cell.After the establishment of the monoclonal cell,cells were expanded for enough amount.Then POLR2A over-expression cell model was constructed.CRISPR-Cas9 technology was used for the construction of POLR2A knockout cell model.Firstly,Cas9 plasmid was constructed and prepared for high-quality.Then,these plasmids were transfected into MDCK cells.To validate the POLR2A Overexpression/knockdown stable cells,western blot test was used to detect POLR2A expression levels in the cell samples.For the construction of influenza virus with a green fluorescent protein(GFP),a non-structural protein(NS)recombinant plasmid labeled with GFP was constructed,and co-transfected with 7 plasmids of PB2,PB1,PA,HA,NP,NA and M into 293T cells,which produce recombinant influenza virus with GFP.Then,this recombinant influenza virus was amplified in the MDCK cells and chicken embryo.Through the observation of the fluorescence microscope,the virus hemagglutination test and TEM technology,recombinant influenza virus with GFP were identified as successfully construction.Recombinant influenza virus with GFP infected POLR2A gene overexpression/knockdown stable cell.Then,The difference of GFP expression in the NS gene was observed by fluorescence microscopy between eleutherosides B1(100ug/mL)treatment group and virus-infected group.The cytopathic effect inhibition test was used to detect the inhibitory effect of eleutherosides B1 in the POLR2A Overexpression/knockdown stable cells for influenza virus a/PR/8/34 strain.2.The promoter activity inhibition assay was used to detect the effect of eleutherosides B1 on the activity of POLR2A promoter.A luciferase-tagged POLR2A promoter stable cell line was constructed firstly and infected with influenza virus.Then eleutherosides B1 was added for intervention.After this process,the supernatants of these cells were collected and detected with the luciferase assay kit.The experimental groups were divided as into the virus infection model group,the group of eleutheroside B1(25μg/mL,50μg/mL,100μg/mL)treatment on A549 cells with virus infection,eleutheroside B1 control group.Bisulfite sequencing method was used to detect the methylation levels in CpG island of POLR2A.Western blot experiments detected the POLR2A protein expression levels in each experimental group.Results1.Western blot results show that the expression levels of POLR2A protein in the POLR2A overexpression stable cells were higher than normal MDCK cells group(P<0.05),which represent the successful construction of POLR2A overexpression stable cell.While in POLR2A gene knockout cells,protein expression of POLR2A was same with normal MDCK cells(P>0.05),which mean that the construction of POLR2A gene knockout cells model was not a success.Therefore,the POLR2A overexpression cell model was selected for subsequent studies.Under a fluorescence microscope,recombinant influenza virus with a green fluorescent protein(GFP)was able to express GFP in the infected MDCK cells.Viral hemagglutination experimental results also confirmed that the influenza virus with GFP has hemagglutination activity.Influenza virus particles were observed by an electron microscope in the sample.Under fluorescence microscopy,there was no GFP expression difference in the POLR2A Overexpression stable cells between eleutherosides B1(100ug/mL)treatment group and virus-infected group.The result of cytopathic effect inhibition test shows that eleutherosides B1 did not inhibit influenza virus a/PR/8/34 strain in the POLR2A Overexpression stable cells(SI index<1).2.The promoter activity assay results showed that,compared to virus infection model group,eleutherosides B1 with the concentration of 100μg/mL inhibited POLR2A promoter activity(P<0.05),while at the concentration of 25μg/mL or 50μg/mL,eleutherosides B1 did not show inhibition effect for POLR2A promoter(P>0.05).Bisulfite sequencing(BSP)results that,compared to virus infection model group,the ratio of POLR2A CpG methylation levels in the area of-222-72 bp,was increased(P<0.05),while in the area of-606-268 bp or-183 is-476bp,there was no change for methylation levels(P>0.05)in the eleutherosides B1(100 ug/mL)intervention group.Western blot results showed that,compared with virus infection model group,the expression of the POLR2A protein had no difference in the group of eleutheroside B1(25μg/mL,50μg/mL,100μg/mL)treatment on A549 cells with virus infection(P>0.05).ConclusionDecreasing the expression of the POLR2A gene is an important mechanism for eleutheroside B1 against the influenza virus.Eleutheroside B1 regulates the expression of the POLR2A gene by affecting the activity of the POLR2A promoter and the CpG methylation level.