Study On The Inhibitory Effect And Mechanism Of Five Sophora Flavescens Alkaloids On Human Respiratory Syncytial Virus In Vitro

Objective:The aims of this project were to compare the anti-viral effects of Oxymatrine,Matrine,Sophocarpine oxide,Sophocarpine,and Sophoridine on human Respiratory Syncytial Virus（hRSV）in vitro,and to explore the mechanism of anti-hRSV infection of Oxymatrine,Matrine,Sophocarpine oxide,Sophocarpine,and Sophoridine by inhibiting the apoptosis of host cells.Such project would provide a novel mechanism for the study of anti-virus mechanism of ethnic medicine in Guizhou Province and a new candidate drug for the clinical application of ethnic medicine in Guizhou Province.Methods:（1）Using MTT experiment to determine the cytotoxicity of Oxymatrine,Matrine,Sophocarpine oxide,Sophocarpine,and Sophoridine,and to calculate the TC₅₀ of each matrine alkaloid;（2）Using four modes of administration,including inhibition of virus biosynthesis,inhibition of virus adhesion to cells,inhibition of virus penetration into cells,and preventive treatment of cells in advance,to explore the anti-hRSV mechanism.The anti-viral effect of five kinds of Sophora flavescens alkaloid were compared by TCID₅₀ and real-time fluorescent quantitative PCR technology respectively.Anti-hRSV effect of five alkaloids and positive control ribavirin under different administration methods:TCID₅₀ test was used to detect five matrine alkaloids and positive control ribavirin after acting on hRSV-infected cells in the above-mentioned different ways.The virus content in the supernatant was calculated to measure the IC₅₀ and TI of each matrine alkaloid and the positive control ribavirin;real-time fluorescent quantitative PCR assay was used to detect the five matrine alkaloids and the positive control ribavirin with absolute quantitative method.The above-mentioned different methods acted on the virus copy number in the cells after hRSV infection,calculate the IC₅₀ and TI of each matrine alkaloid and the positive control ribavirin;（3）Use flow cytometry assay to detect five matrine alkaloids caused cell apoptosis after hRSV inoculation;（4）Western blot assay was conducted to detect five kinds of matrine alkaloids and positive control ribavirin under the intervention of hRSV-infected cells with NF-κB and the expression changes of BCL-2,TRAF₂,c-FLIP,and c-IAP₂.Results:（1）MTT experiment:According to the results of cytotoxicity,each alkaloid was absolutely safe to HEp-2 cells at or under a concentration of 150μg/ml and below 72 h after administration,and they are totally no cytotoxicity;they are calculated using the probability regression equation.TC₅₀ of each alkaloid(Oxymatrine TC₅₀=1,765μg/ml,Matrine TC₅₀=1,805μg/ml,Sophocarpine oxide TC₅₀=4,853μg/ml,sophocarpine TC₅₀=1,068μg/ml,Sophoridine TC₅₀=1,142μg/ml).（2）TCID₅₀ and real-time fluorescent quantitative PCR experimental results:1)Under the administration mode of inhibiting virus synthesis,the five matrine alkaloids have significant inhibitory effects on the hRSV virus;2)Under the administration mode of inhibiting virus adsorption cells,After 72 hours of incubation,the results of high-concentration doses showed that the sophocarpine and sophoridine groups showed a stronger inhibitory effect,which was better than the positive control ribavirin group.Compared with the virus control,p<0.05,which was statistically significant;3)Under the administration method of inhibiting virus penetration into cells,after 72 hours of culture,the results of high-concentration doses showed that the five matrine alkaloids have a significant effect on inhibiting virus penetration into cells.Compared with the virus control,p<0.05,With statistical significance;4)Under the pretreatment of the five kinds of alkaloids of matrine to the cells,the sophocarpine and sophoridine groups have a better inhibitory effect on the virus than the positive control ribavirin,and compared with the virus By comparison,p<0.01,which is statistically significant.（3）The results of cell apoptosis detected by flow cytometry:24 h post infection,no apoptosis was observed in the five matrine alkaloid intervention group,virus infection group and normal cell group;48 h post infection,the apoptosis rate of the matrine alkaloid intervention group was lower than that of the virus-infected group,and there was no significant difference from the normal cell group;72 h post infection,the five matrine alkaloid intervention group had a lower apoptosis rate than the virus-infected group.（4）Western blot results:The expression trend of NF-κB in the virus group and the drug group was similar at 24h post infection,and the expression of NF-κB was up-regulated;48 h post infection,the virus group began to inhibit the translocation of NF-κB from the cytoplasm to the nucleus.The translocation of five matrine alkaloids NF-κB from the cytoplasm to the nucleus was continuously up-regulated.Compared with the normal cell group and the virus-infected group,p<0.05,which was statistically significant;anti-apoptosis at 48 h and 72 h post infection.The expression levels of anti-apoptosis proteins BCL-2,TRAF₂,c-FLIP,and c-IAP₂ of the five matrine alkaloid intervention groups were all up-regulated compared with the virus infection group.Conclusions:The five alkaloids have shown obvious anti-hRSV effects under four administration ways including inhibition of virus biosynthesis,preventive treatment of cells in advance,and inhibition of virus adhesion and penetration.Among them,Sophocarpine oxide has the most prominent anti-infection effect,especially in inhibiting the adhesion and penetration of the virus.It is speculated that the five matrine alkaloids may activate the NF-κB signaling pathway and increase the expression of its downstream anti-apoptotic factors such as BCL-2,TRAF₂,c-FLIP,and c-IAP₂ to achieve anti-apoptosis and then inhibit virus infection.