Antiviral Effects And Molecular Mechanisms Of PGG Against Rabies Virus

Rabies is a deadly zoonosis infectious disease,caused by rabies virus?RABV?which mainly invades the central nervous system and causes encephalitis in mammals and humans.It has been estimated that over 60,000 humans die of rabies and more than 15 million humans worldwide receive post-exposure prophylaxis every year.Most of them occurred in the developing world,especially in Asia and Africa.Our country is a high incidence of rabies region and annually nearly thousand people die from rabies.The mortality of rabies is second in the world,only less than India.Until now,rabies is still an incurable disease and practically the only way to prevent it is pre-or post-exposure vaccination.Once clinical symptoms appear,the disease is almost always fatal.Therefore,treatment of rabies is of great significance.1,2,3,4,6-Penta-O-galloyl-b-D-glucose?PGG?is a hydrolysable phenolic tannin present in many traditional medicinal herbs.It has several biological activities,including anti-cancer,anti-inflammatory,anti-oxidation,and it has a broad spectrum of antiviral effect,whether it has anti-RABV activity has not been reported.Noting there exist some spectral inhibitors for RNA viruses,such as nucleoside analogs,interferon?IFN?,ribavirin,ketamine and isoprinosine?IPS?.IPS was reported to inhibit the growth of RABV,therefore,the study we used IPS in comparison to investigate PGG potently anti-RABV effect in vitro and in vivo,to explore the molecular mechanism of PGG against RABV.First of all,we used the SRB method to detect the cytotoxicity of PGG(half of concentration of cytoxicity,CC₅₀),to detect the inhibition rate of CVS-11 by measuring virus titers(half inhibitory concentration,IC₅₀),the result was expressed using the selectivity index(SI=CC₅₀/IC₅₀)to represent the safety range of PGG anti-RABV effect.Our results showed that the CC₅₀ and IC₅₀0 of PGG was 22.48±2.20?M and 3.90±0.82??respectively,it indicated that the SI was 5.76 and effective.Then,we demonstrated that 10?M PGG potently inhibit CVS-11 viral titers?up to 50 fold?by direct immunofluorescence test,reduce viral gene expression?up to 90%?by relative fluorescence quantitative PCR,and reduce proteins synthesis?up to 70%?by Western blotting.In addition,we found PGG not only suppress viral absorption and entry but also directly inactivate CVS-11.Furthermore,In vivo study,our result showed that intraperitoneal injection 10 mg/kg PGG could alleviate the weight loss,the clinical symptoms and reduce mortality of infected mice by 27.3%.To investigate the molecular mechanism of PGG inhibition of RABV replication,we detected the autophagy of PGG treated cells,because prior studies have shown that RABV could promote autophagy and showed that autophagy is benefit to the replication of RABV.Our results suggested that PGG suppressed the mTOR-dependent autophagy to inhibit the replication of RABV.At the same time,we found that RABV infection could change multiple miRNAs including miR-455-5p and PGG could change host proteins through the microRNA chip and liquid-phase mass spectrometry.Subsequently,by a series of experiments we confirmed that RABV could induce the down-regulation of SOCS3 protein and the activation of STAT3 to promote the replication of RABV.Combined with the results of microRNA chips,we found that PGG could reverse the up-regulated expression of miR-455-5p caused by CVS-11,while miR-455-5p can directly target SOCS3 3'-UTR and affect the expression of SOCS3,and inhibit the activation of STAT3.Therefore,we speculated that PGG could inhibit the replication of RABV by inhibiting the mi R-455-5p-SCOS3-STAT3 signaling pathway.Subsequently,we further confirmed that CVS-11 caused activation of STAT3 and suppressed the immune factor IL-10 and IFN-?expression,thus inhibiting the immune response,while PGG could reverse this process and release of STAT3 mediated IL-10 and IFN-?expression,thus activating the immune response.The above study confirmed that PGG could play an important role by reversing miR-455-5p/SOCS3/STAT3 signaling pathway caused by RABV,and releasing IL-10 and IFN-?expression,thus activating the immune response.In summary,PGG could obviously inhibit virus gene expression and viral protein synthesis of RABV,and PGG not only could inhibit viral adsorption and entry,but also directly inactivated virus.Anti-RABV effect of PGG is related to the suppression of autophagy of mTOR dependence,and PGG could reverse the mi R-455-5p/SCSO3/STAT3 signaling pathway activated by RABV to activate IL-10 and IFN-?mediated immune response to inhibit the replication of RABV.In addition,in vivo studies,the result showed that PGG could alleviate the clinical symptoms of infected mice and reduce the mortality rate by 27.3%.Our study showed PGG has a significant anti-RABV effect,indicating that this compound could be potential candidate for development of an anti-RABV drug.