The Inhibitory Effect And Mechanism Of Thymoquinone On Hantaan Virus Replication

Background: Hantaan virus(HTNV)is the causative agent of hemorrhagic fever with renal syndrome.The current treatment of hemorrhagic fever with renal syndrome is to treat the symptoms.And there is no drug that effectively inhibits HTNV replication and has less toxic side effects.Studies have found that black grass seeds and its effective chemical composition Thymoquinone(TQ)can effectively inhibit a variety of microbial infections.This project studied the inhibitory effect of TQ on HTNV infection from both in vivo and external aspects,and further explored the effect of TQ on TLR4/NF-k B signaling pathway and inflammatory cytokine expression induced by HTNV infection.To reveal the target of TQ inhibition of Hantavirus infection,to provide experimental basis for exploring the new biological activity of TQ and as a drug against HTNV.method:(1)TQ inhibits the replication of HTNV in host cells and its mechanism.1.The cell viability of TQ against HTNV host cells was determined by MTT(tetrazolium salt)colorimetric assay for cell viability after drug treatment.2.The effects of TQ inhibition of viral replication were confirmed from four pathways: antibiotic synthesis,direct killing,anti-adsorption and prevention.3.RT-q PCR,Western Blot,enzyme-linked immunosorbent assay(ELISA)were used to detect the expression or activation of TLR4 and NF-k B in each group and detect IL-6,IP-10 and other downstream inflammatory factors are expressed inside and outside the cell.(2)Therapeutic effect of TQ on HTNV-infected animals.1.The 2-day-old Ba Lb/C suckling mice were intracranially inoculated with different titers of HTNV.The survival rate and average survival days of the suckling mice were used to determine the LD50 of HTNV-infected suckling mice.2.Using low,medium and high concentrations of TQ,intraperitoneal injection of twoday-old suckling mice,comprehensively evaluate the TQ antiviral effect by observing the survival rate,body weight change,viral nucleic acid and viral protein in the target organs.Protective effect on virus-infected suckling mice.3.The mouse tissues(brain,lung,kidney)were collected on the 3rd,5th,7th,9th and 12 th day after infection,and the tissue structure was observed by HE staining.The effect of TQ on the production of inflammatory factors in HTNV-infected animals was initially explored.result: 1.TQ in the concentration range of 25 μM,the survival rate of HUVECs in HTNV host cells is greater than 80%.The TC50 is 118.7μM.2.TQ inhibits the replication of HTNV in host cells(anti-biosynthesis)without inhibiting HTNV by direct killing,anti-adsorption and prevention.In addition,TQ inhibited HTNV in a dose-dependent and time-dependent manner.The IC50 was 8.96 μM and the TI value was 13.26.3.TQ can inhibit the activation of TLR4/NF-k B pathway induced by HTNV infection and reduce the downstream inflammatory factors IL-6,IP-10 m RNA expression and protein expression.4.TQ treatment group delayed clinical symptoms of 50LD50 HTNV-infected suckling mice compared with virus control group(weight loss,mental euphemism,rough hair,increased reactivity,arched back,tail whip-like changes,hind leg paralysis and tremor,etc.)appeared,prolonged the average survival days,the moderate concentration(10mg/kg/d)TQ effect was extended by 1.6 days,but the effect was not as good as the positive control drug ribavirin for 2.6 days.The viral load in the brain and lung tissues of suckling rats increased with the number of infection days;compared with the virus group,the viral load of brain and lung tissues in the HTNV-infected mice with low,medium and high concentrations in the TQ treatment group was significantly reduced.In the 12 th day of infection,the inhibition rate of the medium concentration TQ group in brain and lung tissues was 23.18%±6.36% and 35.72%±1.04%,respectively.6.HE-stained sections showed that TQ alleviated inflammatory damage in brain,lung and kidney tissues induced by viral infection in suckling mice.conclusion: TQ has an anti-HTNV effect in vitro.TQ exerts anti-inflammatory effects by inhibiting the TLR4/NF-k B pathway.TQ can reduce the inflammatory response of HTNV-infected suckling mice and prolong the average survival time.