Qingre Xiaoyanning And Its Active Ingredient Rosmarinic Acid Reduce HSV-1 Susceptibility By Inhibiting ALOX15

HSV-1 is a neurotropic virus that usually causes damage to the surface of the body,such as cold sore and herpes simplex keratitis.HSV-1 is also one of the main pathogens of sporadic encephalitis,resulting in herpes simplex encephalitis with a high mortality rate.The occurrence of herpes infectious diseases is affected by virus and host susceptibility.Host factors play a key role in the process of viral infection,including genetic susceptibility,age,gender and immune status changes under stress.Stress causes immune dysfunction in the body and induce the development of various diseases,especially increase the susceptibility of infectious diseases.In early stage,our research team established the mouse model of restraint stress to increase the susceptibility of influenza virus,and found that the increase of CORT caused by restraint stress is the main reason for increasing the susceptibility of influenza virus.At the same time,the CORT loaded cell model of influenza virus susceptibility was established,and the effects of various Chinese medicines and natural products on reducing virus susceptibility were successfully evaluated using the model.In addition,our research team also found that CORT can cause mitochondrial morphological change and dysfunction,which may be related to increasing the susceptibility of the body virus,but the specific mechanism needs further study.Traditional Chinese medicine Qingre Xiaoyanning has a good therapeutic effect on cold sore caused by HSV-1 in clinical practice,but the pharmacological mechanism of Qingre Xiaoyaning which has been reported so far is not enough to explain its clinical efficacy.Therefore,this study established in vitro and in vivo HSV-1 susceptibility model and used the model to evaluate the protective effect of Qingre Xiaoyanning(QX)and its active ingredient rosmarinic acid(RA)on HSV-1 susceptible mice/cells that based on the previous research.From the perspective of mitochondrial damage mechanism,the mechanism of RA to reduce the susceptibility of HSV-1 induced by CORT was discussed.Through this study,we will gain a deeper understanding of the mechanism of CORT-induced susceptibility to HSV-1,further evidence of the susceptibility of emotional stress to increase disease susceptibility.The research provides the basis for pharmacology and molecular biology for drug with reduced susceptibility to HSV-1.First,this study validated the established HSV-1 susceptibility model in vitro and in vivo.The in vivo model experiments were grouped into Control,Stress,Virus,Stress+Virus and RU486+Stress+Virus groups,with 7 mice in each group.The restraint stress condition was 6 h every day for 14 days,the virus infection was intranasally infected on the 14th day(5×10~7 PFU/mL,20μL),and the RU486 group was injected subcutaneously with RU486(25 mg/kg/day)2 h before the restraint stress.Animal models are validated by health status observations and molecular indicator detection.Health status includes changes in body weight,morbidity,clinical HSE symptoms score,eye swelling score,and brain tissue pathological changes(HE staining).Molecular indicators include viral titers(plaque method),UL54 gene(qRT-PCR),UL27 gene(qRT-PCR)and ICP27 protein(Western blot)of HSV-1 in mouse brain tissue.The experimental results showed that restraint stress increased the weight loss(p<0.001),morbidity(p<0.01),clinical HSE symptoms(p<0.01)and eye swelling symptoms(p<0.001)in HSV-1 infected mice.The expression of UL54 gene(p<0.001),UL27 gene(p<0.01)and ICP27 protein of HSV-1 in mouse brain tissue was up-regulated.In vitro HSV-1 susceptibility model experiments were grouped into Control,CORT,HSV-1 and CORT+HSV-1 groups.The model group cells were infected with HSV-1 F strain(MOI=1)48 h after CORT(50μM)treatment.The results of HSV-1 protein and gene,indicated that CORT loaded SH-SY5Y cell increased the expression of UL54 gene(p<0.01),UL27 gene(p<0.01),ICP27 protein and gB protein of HSV-1 in SH-SY5Y cells.The changes of mitochondria were observed by immunofluorescence and electron microscopy.The results of immunofluorescence showed that the long columnar or reticular mitochondria were destroyed into short rods or spots after CORT treatment.The results of electron microscopy showed that the mitochondria volume became smaller and the matrix color deepened.At the same time,transcriptome sequencing analysis indicated that the differentially expressed genes were enriched in the lipid metabolism pathway,and the transcriptional expression of lipid peroxidation related gene ALOX15 was significantly increased(p<0.05).Thereby CORT induced HSV-1susceptibility may be related to mitochondrial lipid oxidative damage was presumed.The results of western blot and flow cytometry showed that CORT loaded SH-SY5Y cell increased expression of ALOX15 protein and lipid peroxidation product 4-HNE,as well as lipid peroxide accumulation liperfluo fluorescence(p<0.001).In addition,Western blot results showed that ALOX15 overexpression(ALOX15-GFP)increased the expression of HSV-1 gB and ICP27 proteins.After initial determination of ALOX15as the target to be studied,computer simulation of Qingre Xiaoyanning(Sarcandra glabra)as the source of the compound showed that rosmarinic acid and ALOX15 had the highest interaction score among the nine active components of Sarcandra glabra(Thunb.)Nakai.Secondly,this study used the HSV-1 susceptibility model to evaluate the effects of QX and RA on reducing HSV-1 susceptibility.Animal experiments were grouped into Normal,Virus,Model,QX-L(QX 0.658 g/kg/day),QX-H(QX 1.316 g/kg/day),RA-L(RA 11.7 mg/kg/day),RA-H(RA 23.4 mg/kg/day)and ACV group(acyclovir tablet0.206 g/kg/day),with 10 mice in each group,continuous observation for 14 days after viral infection.The survival rate,body weight change,clinical HSE symptoms score,eye swelling score and brain tissue pathological changes(HE staining)of each group of mice were statistically displayed.Compared with the model group,the weight loss of QX-H(p<0.001),QX-L(p<0.01),RA-H(p<0.001),and RA-L(p<0.01)groups was significantly decreased.The clinical HSE symptoms score of QX-H(p<0.001),QX-L(p<0.01),RA-H(p<0.05)and RA-L(p<0.05)groups were significantly decreased on10th day.The eye swelling score of QX-H(p<0.001),QX-L(p<0.001),RA-H(p<0.01)and RA-L(p<0.01)groups were significantly decreased on 9th day.The HE results indicated that brain tissue inflammation is reduced.The virus titers in brain tissue(plaque method)of QX-H(p<0.001),QX-L(p<0.01),RA-H(p<0.01)and RA-L(p<0.01)groups were significantly reduced.Result of western blot showed expression of HSV-1 ICP27 protein decreased.Brain tissue section positive staining for HSV-1antigen(immunohistochemistry)was also decreased.The plaque assay was used to determine the effective concentration of rosmarinic acid to reduce the increase of CORT-induced HSV-1 susceptibility.The experimental groups were Control,HSV-1,CORT+HSV-1 and RA+CORT+HSV-1 group.The results showed that RA at 100μM significantly reduced CORT-induced viral titers(p<0.05).At the same time,the results of western blot showed gB and ICP27 proteins expression of the RA+CORT+HSV-1group was down-regulated compared with the CORT+HSV-1 group.Western blot was used to detect the MAVS,p-IRF3 and IFN-βproteins in the mitochondrial antiviral signaling pathway.The results showed that expression of MAVS,p-IRF3 and IFN-βin the RA+CORT+HSV-1 group were up-regulated compared with the CORT+HSV-1group.Finally,the role of RA to reduce CORT-induced mitochondrial oxidative damage was studied.Western blot and lipoxygenase inhibitor screening kit were used to detect the effect of RA on the expression and activity of ALOX15.The results showed that the expression of ALOX15 protein in RA+CORT group had no significant change compared with CORT group while RA had inhibitory activity on ALOX15 with the inhibition rate of 92.75%.Next,the role of ALOX15 in CORT-induced lipid peroxide accumulation was studied.The experimental groups were Control,ALOX15-flag,ALOX15-flag+RA,CORT,ALOX15-flag+CORT and ALOX15-flag+RA+CORT group.The results showed that CORT-induced liperfluo fluorescence(p<0.001),4-HNE and the colocalization of liperfluo with mito red were increased under ALOX15overexpression conditions,while liperfluo fluorescence(p<0.001),4-HNE and the colocalization of liperfluo with mito red is reduced in administered to RA-treated groups.Flow cytometry and Western blot were used to detect the effect of RA on CORT-induced lipid peroxide accumulation.The results showed that the CORT-induced liperfluo fluorescence intensity(p<0.001)and 4-HNE protein expression in the RA+CORT group were reduced compared with the CORT group.Colocalization of mitochondria with lipid peroxide was detected by laser confocal microscopy.The results showed that the colocalization of liperfluo and mito red in the RA+CORT group was reduced compared with the CORT group.The mitochondrial morphology was observed by immunofluorescence as well as the network morphology was analyzed.The results showed that the mitochondrial network morphology of the cells after CORT treatment was broken,and number of the dot-like/rod-like structures(p<0.001)and network structures(p<0.001)were significantly increased.Network branch length(p<0.01)and branching degree(p<0.01)decreased,while RA could restore CORT-induced network morphological fracture.The number of point/rod structures(p<0.01)and network structures(p<0.001)were decreased,the network branch length(p<0.05)and the degree of branching(p<0.01)were increased.The mitochondrial membrane potential and ROS levels were detected by flow cytometry.The results showed that the green fluorescence of the JC-1 probe in the CORT group was enhanced,the red fluorescence was weakened,and the mitochondrial membrane potential was decreased compared with the Control group.Compared with the CORT group,RA can restore CORT-induced mitochondrial membrane potential loss(p<0.001).Compared with the Control group,DCFDA fluorescence increased(p<0.001)in the CORT group,while DCFDA fluorescence decreased(p<0.01)in the RA+CORT group compared with the CORT group.In summary,restraint stress and stress hormone CORT induce HSV-1susceptibility in vivo and in vitro.CORT load induces increased levels of mitochondrial lipid peroxidation,up-regulation of ALOX15 expression and cell mitochondrial damage.RA reduces HSV-1 susceptibility and its mechanism involves inhibition of ALOX15.RA also reduced ALOX15-mediated lipid peroxidation accumulation and CORT-induced mitochondrial damage.Expression of MAVS,p-IRF3,and IFN-βprotein in mitochondrial antiviral signaling pathways were increased that may reduce HSV-1 replication by enhancing mitochondrial antiviral immunity.