| Objective:In order to observe that ginsenoside Rbl to inhibit the ability of Marc-145cells infected with PRRSV in vitro and the mode of action, and investigate whether the ginsenoside Rbl by inhibiting the virus N protein expression of the rise to suppress the virus replicaton.Methods:The assay determined50%cytotoxic concentration and maximum non-cytotoxic concentration of ginsenoside Rbl against PRRSV which was estimated using visualization of cytopathologic effect assay and MTT test on Marc-145cell in vitro. Within maximum non-cytotoxic concentration to determine the maximum suppression ratio and50%Effect concentration of ginsenoside Rbl inhibited PRRSV. SI index was calculated to illustrate the ginsenoside Rbl inhibitory effect of PRRSV infection in Marc-145cells, then explored the mechanism by time-of-addition assay, adsorption inhibition assay and virucidal assay. In order to explain the impact of ginsenoside Rbl to N protein through confirming the number of the N protein gene by FQ-PCR, surving N protein content changes via IFA method which combined with DAPI staining method. Qualitative index was the results of IFA which to observe N protein positive cells number changes in the inverted fluorescence microscope. Quantitative index was the results of Western Blot which to detect N protein content in infected cells.Results:1. The inhibition ratio of ginsenoside Rbl against PRRSV up to88.2%in the maximum non-cytotoxic concentration (MNTC). CC50of ginsenoside Rbl is larger than4000μg/mL. EC50respectively were equal to342.9±37.56μg/mL, SI was greater than11.67.2. The inhibition ratios more than50%of the virucidal assay in which medicine poisonous mixing process for30to150min, the maximum inhibition rate of ginsenoside Rb1was up to54.65%and ratios did not change significantly with time (P>0.05).3. The maximum inhibition rate of ginsenoside Rbl in the time-of-addition assay was up to59.38%and all ratios within first12h more than50%which showed ginsenoside Rbl has the ability to interfering with viral replication. But inhibition rate in2.5h to12h had no significant change (P>0.05),1h to12h dosing group is significantly higher than14h drug group (P<0.05).4. The ratios lower than20%and had no significant change (P>0.05) in the adsorption inhibition assay by means of ginsenoside Rbl mixed with cells incubated with different time, then join the virus. It showed that ginsenoside Rbl did not inhibite virus attach Marc-145cell.5. In the assay within12to72h test time of Ginsenoside Rbl effects on N protein gene, copy number of N protein gene of positive drug group and4mg/mL,2mg/mL of ginsenoside Rb1group significantly lower than the virus control group (P<0.05), which showed that positive drug and4mg/mL,2mg/mL group of ginsenoside Rbl could reduced the amount of the virus gene. The gene number of the2mg/mL ginsenoside Rb1group lower than4mg/mL ginsenoside Rbl group at12h\24h and not significant at36h\60h\72h(P>0.05). the copy number of1mg/mL ginsenoside Rbl group was not significantly different with virus control at12h\36h\60h(P>0.05), but significant less than virus group at24h\72h(P<0.05). This two information stated the effect of ginsenoside Rb1reduced the N gene synthesis ability has the dose difference.6. The test which added virus before append medicine then training48h to study ginsenoside Rbl effect on N protein content, through the IFA method that combined with DAPI staining observing cell attached N protein numbers and using Western Blot to observe N protein content after training60h. The results stated that4mg/mL ginsenoside Rbl group and positive control group compared with the virus control, the positive cells number and N protein content in the least were obvious difference, which showed that4mg/mL ginsenoside Rbl and positive drugs can reduce virus N protein in the cell. The positive cells number and.N protein content of1mg/mL,2mg/mL ginsenoside Rbl group were slightly more than positive control group and4mg/mL group,with2mg/mL group lower than1mg/mL group, which showed that1mg/mL,2mg/mL ginsenoside Rbl could reduced N protein loads and the ability would drop along with the lessen of drug density.Conclusion:The experimental results demonstrated that ginsenoside Rbl could reduce the damage of PRRSV to Marc-145cell effectively. The maximum inhibition rate of ginsenoside Rb1was up to80%in vitro. Its mechanism of antiviral actions could be due to inhibiting the virus the early or middle stage of replication or/and inactivating the virus directly. Further, ginsenoside Rbl make purpose to inhibiting viral replication by decrease the virus N protein expression. It suggested that ginsenoside Rbl could be used for developing a new drug or precursor in treating and preventing PRRSV. |
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