Screening And Antiviral Assessment Of Steroidal Pyridine Compounds Against Respiratory Syncytial Virus

Respiratory syncytial virus can cause severe lower respiratory tract infections,and almost all children will be infected before the age of three,placing a huge burden on the world economy.Ribavirin,a drug used in clinical for respiratory syncytial virus,is only used in critical patients because of its side effects.Although vaccines are being developed,there are still no vaccines available clinical.Therefore,it is particularly important to find new drugs that are effective against respiratory syncytial virus.The purpose of this project is to screen a series of butene lactone and steroidal pyridine compounds,find safe and effective compounds,evaluate their antiviral effects,and explore their antiviral mechanism.Fristly,the MTT method and CPE method were used to detect the anti-rsv activity of 36 butene lactone and 26 steroidal pyridine compounds at the cellular level.The half inhibition concentration(CC50)value was measured.The antiviral activity was measured by 100 TCID50 and the half effective concentration(EC50)was measured,the selectivity therapeutic index(SI)is the ratio of CC50 and EC50.The compound 4L was selected because of its low toxicity,high activity,and the highest therapeutic index,and then its mechanism of action prevention,direct blocking,and therapeutic effect are explored.The inhibitory effect of the added compound on the virus at different times were tested,the compound was added at 0 h,2 h,4 h,6 h,8 h,10 h,12 h,and 24 h after respiratory syncytial virus inoculation.After adding compound 4L for 24 h,qRT-PCR was used to detect the changes of TLR-3,RIG-I,IL-6 and IFN-?,which are the key factors of TLR-3 and RIG-I pathway.Secondly,an animal infection model was established to explore the mechanism of action of compound in vivo.We first divided 126 mice into 6 groups and 18 groups.The mice in the experimental group were nasally infected with the virus,and the mice in the normal control group were infected with the same amount of medium.After 24 hours,they were administered for five days.Mice were dissected on day 2,4,and 6 after virus infection to collect lung tissue.HE staining was used to detect inflammatory lesions in mouse lung tissue,IHC was used to detect virus expression in mouse lung tissue,qRT-PCR was used to detect the expression of M gene,cytokines TLR-3,RIG-I,IL-6,IFN-?,IL-10.The experimental results showed,compound 4L exhibited low cytotoxicity,high antiviral activity,and the highest index.During the exploration of the antiviral mechanism,it was found that compound 4L mainly plays a therapeutic role.Experiments with compound added at different times showed that compound 4L is added 2 hours after virus infection had the best effect,the inhibition rate of the virus remained at 70%in added 4-12 hours after infection.The compound 4L was added 24 hours after the virus infection,and it exhibited almost no antiviral effect.qRT-PCR results showed the expression of the key factors TLR-3,RIG-I,IL-6,and IFN-? were reduced after dosing.Compound 4L exerted antiviral effects by suppressing virus replication and inhibiting the major inflammatory factors of TRL-3,RIG-I pathway at the cellular level.In mice model,the symptoms such as weight loss and malaise in mice were improved after compound 4L administration.The lung index,inflammation and pathological injury in lung were relieved by compound 4L.The expression of viral antigens and the replication of the viral M gene were decreased after compound 4L administration.The expression of TLR-3,RIG-I,IL-6,IFN-? and IL-10 were down-regulated in 4L-treated mice groups,and TLR-3,RIG-I,IL-6,IFN-? are the main factors of TLR-3 and RIG-I signaling pathway.The above results indicated that 4L relieves lung injury by suppressing inflammatory and maintaining immune balance.In summary,steroidal pyridine compound 4L exhibites a good anti-respiratory syncytial virus effect both in vitro and in vivo.