The Study Of Effect And Mechanism Of Substance P In Mice With Herpes Simplex Keratitis

Part Ⅰ Effect of substance P deletion on pathogenesis of herpes simplex keratitis in mice Objective: Studies have shown that substance P(SP)is highly expressed in the initial stage of herpes simplex keratitis and plays an immunoregulatory role in up-regulating early inflammatory cytokines and promoting cytopathic changes,but the effect of SP gene knockout on the course and mechanism of herpes simplex keratitis in mice remains unclear.To investigate the pathogenesis of herpes simplex keratitis in mice model after knockout of neuropeptide SP and detect the viral load in cornea,exploring the characteristic changes in the disease process.And giving C57BL/6 mice with intraperitoneal injection of SP receptor inhibitor to explore whether this could imitating the course of HSK in the SP gene knockout mouse.Methods:1.The SP knockout mice were identified by the mouse tail identification method,and compare the normal SP knockout mice with C57BL/6 mice in terms of slit lamp photography,corneal HE staining,corneal nerve staining,corneal sensitivity and basic tear secretion.2.At different time points after HSV-1 infection in C57BL/6 mice,changes of SP expression in the cornea were detected by ELISA.3.Herpes simplex keratitis model was constructed in SP knockout mice and C57BL/6 mice,and the changes of corneal opacity,neovascularization and sensitivity in the 3,7,11,28 days after infection and 2 days after recurrence were observed and scored.Immunofluorescence was used to detect the expression of HSV-1 in corneal tissue at different time points.The virus titer in corneal tissue was measured by plaque assay at 3 and 7 days after infection.Furthermore,absolute quantitative RT-PCR was used to detect the changes of ICP0,ICP27,TK,VP16,UL41 and LAT in cornea and trigeminal ganglia at different time points.4.We explore intraperitoneal injection of L-733060 in C57BL/6 mice to simulate SP knockout mice.C57BL/6 mice were randomly divided into the SP group,L-733060 group and control group.After inoculating the three group with HSV-1,the clinical signs of different groups were observed,and corneal lesion,viral titer and index related to virus infection were tested to verify whether the intraperitoneal injection of L-733060 could successfully simulate the SP knockout mice.Results:1.Compared SP knockout mice with normal C57BL/6 mice in terms of corneal morphology,corneal innervation,corneal sensitivity and tear secretion,there was no significant difference between the two groups(P>0.05).2.In the model of herpes simplex keratitis in C57BL/6 mice,the expression of SP in the cornea increased at the third day of initial infection stage,then gradually decreased as the corneal symptoms subsided and the course of keratitis entered into latent stage.Then the expression of SP increased again at the second day of recurrence.3.There was no significant difference in corneal lesion morphology,corneal opacity,neovascularization and sensitivity between C57BL/6 and SP knockout mice at different time points after HSV-1 infection(P>0.05).Immunofluorescence detection of HSV-1 in corneal tissue at different time points showed that the expression of HSV-1 in the two groups was mainly located in the epithelium.But on the 3 days after infection and the 2 days after recurrence,the expression of HSV-1 in the SP knockout mice were significantly more than C57BL/6 mice,and the staining of HSV-1 in the two groups was not obvious on the 7th day.The titer detected by plaque assay is consistent with above results.Further detection related index expression of virus in the cornea and trigeminal nerve tissue by using absolute quantitative RT-PCR.The result suggested that virus related indicators in corneal tissue such as ICP27,TK,UL41 in SP knockout mice were significantly more than C57BL/6 mice at third day after infection(P < 0.001).The expression of ICP0,ICP27,VP16,UL41,except TK,in trigeminal ganglia,SP knockout mice group were significantly more than C57BL/6 mice on the 3 days after infection(P < 0.05).4.There were no significant differences in lesions of HSK among in the three intraperitoneal injection groups of SP,L-733060 and control,same as corneal opacity,neovascularization and sensitivity among the groups at different time points(P>0.05).The expression of HSV-1 in the cornea on 3rd day after infection was detected by immunofluorescence,results showed that the L-733060 group was significantly higher than that of the other two groups.Plaque assay showed that the viral titer of L-733060 group was higher than that of the other two groups at the 3rd day.Using absolute quantitative RT-PCR to detect the indexes related HSV-1 infection among three group in corneal tissue and trigeminal ganglia at different time points.The results suggested that ICP0,ICP27,TK,VP16,UL41 in L-733060 groups were significantly more than the other two groups(P < 0.05)in cornea at the 3rd after infection,the expression of indexes of virus in trigeminal ganglia were consistent with those in cornea,indexes related virus infection in L-733060 groups were significantly more than the other two groups(P < 0.05).Conclusion: SP gene knockout had no significant effect on corneal morphology,innervation,corneal sensitivity and basal tear secretion in mice.We first time found that the absence of SP increased the virus expression in the cornea and trigeminal ganglia of herpes simplex keratitis in mice.The HSK model constructed by intraperitoneal injection of SP receptor inhibitor L-733060 could simulate with the HSK model of SP gene knockout mice,and intraperitoneal injection L-733060 in mice could be used for further experiments.Part Ⅱ The mechanism of substance P gene knockout affecting virus replication and inflammatory infiltration in mouse HSK modelObjective: Preliminary experimental results found that the corneal viral titer was increased in SP gene knockout mice with herpes simplex keratitis,but no difference in the symptoms comparing with the wild type.Further researches were needed in HSV-1 replication-related factors and inflammatory cell infiltration,in order to discuss the effects and mechanism of the absence of substance P in a mouse model with herpes simplex keratitis.Methods: 1.Immunofluorescence was used to detect the distribution of neutrophils,macrophages,CD4 and CD8 labeled immune cells in the corneal tissues of C57BL/6 and SP knockout mice at 3 and 7 days after infection and 2 days after recurrence.C57BL/6 mice were randomly divided into the SP group,L-733060 group and the control group,and HSK model was constructed in the three groups.The infiltration of neutrophils and macrophages in the cornea among each group was detected by immunofluorescence on the third day after HSV-1 infection.2.RT-PCR was used to detect the expressions of neutrophils related chemokines CXCL2,CCL3,MCP-1 and IL-17 m RNA in the corneal tissues of C57BL/6 and SP knockout mice at 3rd day post infection.3.Immunofluorescence and RT-PCR were used to detect the expression of heparanase,a kind of receptor related virus invasion,in the cornea of C57BL/6 and SP knockout mice on the third day after infection.The expression of HVEM,nectin-1,NEAT1 and tetherin,as well as type I interferon will be tested by RT-PCR in the cornea of C57BL/6 and SP knockout mice.4.The expressions of factors related virus invasion and type I interferon m RNA in the cornea were detected by RT-PCR among the SP group,L-733060 group and control group.Results: 1.Immunofluorescence staining showed that F4/80 labeled macrophages infiltrated into the epithelium and stroma layer of the cornea in SP knockout mice and C57BL/6 mice on the third and seventh days after infection.The Ly6 G labeled neutrophils in the two groups was mainly located in the epithelium and stroma,and the number of Ly6 G labeled neutrophils was significantly reduced compared with the control group.At the second day of recurrence,both groups had significant infiltration of macrophages and neutrophils.On the third day after infection,the CD4 labeled cells were mainly located in the corneal epithelium and stromal layer,there was no significant difference between the two groups.CD8 a labeled cells were expressed in the corneal epithelium and stromal layer of C57BL/6 mice,but not in the cornea of the SP knockout mice.2.RT-PCR results indicated that the expressions of neutrophil related chemokines CXCL2,CCL3 and IL-17 B in the cornea of SP-KO mice on day 3 after infection were lower than that in C57BL/6 mice(P<0.05),and the expression level of MCP-1 m RNA in SP knockout mice was higher than that in C57BL/6 mice(P<0.01).3.At 3rd day postinfection,the RT-PCR and immunofluorescence results showed that there was no significant difference in the expression of heparanase between SP knockout mice and wild type mice(P>0.05),but the expression of the other receptors related virus invasion,nectin-1 and NEAT1,were increased in SP knockout mice(P < 0.001),the expression of the virus restrictive factor tetherin was lower in SP knockout mice cornea(P < 0.001).4.The expression of IFN-α and IFN-β m RNA in SP konckout mice were lower than those in C57BL/6 mice on the third day after infection(P<0.05).The expression levels of IFN-γ m RNA in the HSK model of SP konckout mice were significantly higher than those in the control group on the 28 th day after infection(P<0.01).The expression of IFN-α and IFN-β m RNA were lower in the L-733060 group than the other two groups(P<0.05),consistent with that of SP knockout mice.Conclusion: After SP deletion,the chemotaxis of neutrophils in the corneal tissue was reduced on 3rd day of HSK,while the increase of virus titer in the cornea might be related to the increase of cell surface receptors related virus recognition,the decrease of antiviral factors IFN-α and IFN-β,and the decrease of immune cells infiltrating into the cornea.The above indicated that SP was involved in regulating the expression of genes related HSV-1 invasion and replication,thus playing a certain control role on the virus.Part Ⅲ Substance P regulates HSV-1 infection through STING/TBK1/IRF3 signaling pathway in human corneal epithelial cellsObjective: Corneal epithelium is the first defense line against virus invasion,but how SP regulate HSV-1 infection in epithelial cells remained unknown.Through establishment of HSV-1 infected model in human corneal epithelial cells(HCEC)in vitro,observing the effects on virus replication while dealing with SP and different concentrations of L-733060,and further exploring the mechanism related to virus infection after the deletion of SP.Methods: Human corneal epithelial cell lines were cultured in vitro,and CCK-8 was used to detect the effect of SP and L-733060 treatment on cell viability.The cells were divided into control group,HSV-1 group,HSV-1+SP 10μmol/L group,HSV-1+L-733060 0.1μmol/L group,HSV-1+L-733060 1μmol/L group,and HSV-1+ L-733060 10μmol/L group.The morphological changes of cells were observed after pretreatment with drugs for 12 hours and infection with HSV-1 for 24 hours.And then western blot was used to test the expression of proteins and corresponding phosphorylated proteins in STING/TBK1/IRF3 signaling pathway among above groups.Results: 1.CCK-8 assay was used to detect the changes in cell viability of HCECs treated with different types and concentrations of drugs,and the results showed that the cell viability of HCECs after 12 hours of intervention with L-733060 20 μmol/L was significantly lower than the other groups(P<0.05),while there was no significant difference among other groups(P>0.05).2.To observe the effect of SP and different concentrations of L-733060 on the cytopathic effect of HCECs infected with HSV-1,the control group of HCECs could be paved over the bottom of the culture plate,and different degrees of cytopathic changes could be observed in the other groups infected with HSV-1.Compared with other groups,the degree of cytopathic changes was more obvious in the group treated with L-733060 10 μmol/L.3.Western-blot analysis of proteins and their phosphorylated proteins expressions in STING/TBK1/IRF3 signaling pathway after HCECs treated by SP and different concentrations of L-733060 and HSV-1.The results showed that the expression levels of p-IRF3 and p-TBK1 gradually decreased with the increasing of the intervention concentration of L-733060,and the most significant decrease was observed in the L-733060 10 μmol/L group(P<0.05).There was no significant difference in STING protein expression between groups(P>0.05).Compared with HSV-1 group,the expression of tetherin significantly decreased in the L-733060 10 μmol/L group(P<0.001).Conclusion: Exogenous L-733060 can regulate the infection process of HSV-1 in HCECs through the STING/TBK1/IRF3 signaling pathway.L-733060 may down-regulating the expression of TBK1 and IRF3 to increase cell viral titer and interfere with the transduction of antiviral signaling.