| Purpose:Herpes simplex keratitis(HSK)is a serious blinding eye disease caused by herpes simplex virus(HSV-1)infection.It is the most common infectious corneal disease in the world today.There are about 590,000 new cases in the United States each year.The incidence rate in recent years is about 11.8/10~5;the prevalence of HSK in our population is about 11/10~4.HSK mainly manifests as corneal nerve degeneration,neovascularization,inflammatory reaction,which can lead to corneal scarring and even blindness.Eventually it will take a corneal transplant to regain sight.At present,the treatment of HSK mainly relies on nucleoside analog antiviral drugs,such as ganciclovir and acyclovir,but drugs that could effectively relieve corneal symptoms are insufficient.Pigment epithelium derived factor(PEDF)has neurotrophic effects and also has a strong inhibitory effect on neovascularization.These effects have been demonstrated in choroidal neovascularization and diabetic eye diseases.Vasoactive intestinal peptide(VIP)is an endogenous host defense protein secreted by neurons which can communicate with the immune system and participate in the regulation of inflammation.The role of PEDF and VIP in HSK have not been reported,and it have broad research prospects.In this study,the mouse HSK model was constructed to observe the corneal nerve degeneration and neovascular pathological changes in the initial infection;the expression of PEDF and VIP were detected and the effects of exogenous PEDF and VIP on the clinical symptoms of HSK were observed.The molecular mechanism of its regulation of neurodegeneration,neovascularization and inflammatory response was explored,and a new intervention strategy for the treatment of herpes simplex keratitis was provided.Methods:1.Establishment of mouse HSK model and observation of disease courseIn this study,6-8 weeks old C57BL/6 mice were used to inoculate the HSV-1 strain McKrae strain with 25G needle scribing to establish the HSK model.Slit lamp microscope was used to observe the changes of corneal infection symptoms at 3rd,7th,15th and 45th day after surgery,and the corneal transparency and neovascular growth were scored.Corneal nerve and neovascularization in HSK mice were objectively evaluated by immunofluorescence staining with?-? tublin and CD31 antibody,and corneal sensory changes were measured using a corneal sensitivity meter.The plaque assay was used to detect the amount of HSV-1 virus at various time points in the course of HSK.2.Expression changes of PEDF and VIP in mouse HSK modelThe mouse HSK model was established.The cornea was taken on the 0th,3rd,7th,15th and 45th day after operation.The expression position and quantity of PEDF and VIP in the cornea were observed by immunofluorescence staining.The mRNA and protein expression of PEDF and VIP at each time point were examined by RT-qPCR and ELISA.3.The therapeutic effect of exogenous PEDF and its derivatives and VIP on mouse HSK modelThe HSK model was established and the mouse were randomly divided into experimental group and control group.Subconjunctival injection was performed in experimental group by PEDF,PEDF derivatives and VIP on 0th and 3rd day after operation and in control group by equal volume of PBS.Slit lamp microscope was used to observe the clinical symptoms of HSK on the 3rd and 7th day after surgery and the clinical score was obtained.The corneal nerve and neovascularization of HSK mice were evaluated by?-? tublin and CD31 antibody corneal patch immunofluorescence staining.The sensitivity meter measures changes in corneal perception.4.Effects of PEDF and VIP on inflammatory cell infiltration and cytokines in mouse HSK modelThe HSK model was established and the mouse were randomly divided into experimental group and control group.Subconjunctival injection was performed in experimental group by PEDF,PEDF derivatives and VIP on 0th and 3rd day after operation and in control group by equal volume of PBS.On the 3rd and 7th day after the operation,corneal frozen section immunofluorescence staining and flow cytometry were used to detect neutrophils,macrophages,CD4~+T cells and other cells infiltration in the cornea;Expression of various cytokines in the cornea were detected by ELISA.5.Effects of PEDF and VIP on HSV-1 virus replicationThe establishment of the model is the same as above.On the 3rd and 7th day after the operation,corneal frozen section immunofluorescence staining and flow cytometry were used to detect the distribution of HSV-1 virus in cornea.The viral load of each group was detected by plaque test on the 3rd day.The expression of virus-related genes ICP0 and UL41 were detected by RT-qPCR.In vitro antiviral experiments,we used Vero cells as a vector.Different concentrations of drug were added to intervene after HSV-1infecting,the cells were collected and detected by RT-qPCR and plaque assay.Results:1.The course characteristics and the expression of PEDF and VIP of the mouse HSK model(1)The observation under the slit lamp microscope showed that the normal cornea was transparent and avascular.On the 3rd day of viral infection,the cornea got mild edema of the cornea and neovascularization.On the 7th day of infection,there was severe corneal edema with massive neovascularization.On the 15th day,the corneal edema subsided and the neovascularization receded.On the 45th day,the cornea recovered and the neovascularization disappeared completely.(2)Immunofluorescence staining of?-? tublin antibody corneal patch showed the dense subbasement nerves and nerve branches in the stroma in normal cornea.On the 3rd day after viral infection,the corneal nerve began to degenerate with a sensitivity decrease in central cornea.On the 7th day of the virus infection,the large nerve fibers,the subbasal plexus and the intraepithelial nerve endings of the sclera were almost completely resolved,and the central corneal sensitivity was minimized.On the 15th day after the virus infection,some nerves began to recover.The regenerated nerve fibers were observed in the middle and posterior stromal cells of the cornea.However,no repair of the plexus at the epithelial/matrix junction was observed,and the sensitivity in central cornea was still low.On the 45th day of virus infection,the regenerated nerves were densely branched in the corneal stroma,but the subepithelial nerve bundles were not recovered.The sensitivity in central cornea cannot restore to pre-infection levels.(3)Immunofluorescence staining of CD31 antibody corneal patch showed that the normal cornea was transparent and avascular.The angiogenesis of limbal neovascularization on the 3rd day after viral infection.On the 7th day,massive angiogenesis performed,which were vigorous and coarse,grew to the center of the cornea.On the 15th day,the neovascularization began to atrophy and recede.At 45th days,the neovascularization was almost regressed to the limbus,which was close to the normal corneal avascular state.(4)The results of plaque test showed that the viral load of the cornea infected with HSV-1was highest in the 3rd day,about 40 pfu/ml,and the virus load decreased significantly on the 7th,15th and 45th day.(5)Immunofluorescence staining showed that PEDF was mainly expressed in corneal epithelium and endothelium in normal cornea.The expression of PEDF decreased on the3rd and 7th day after HSV-1 infection,and the expression increased from the 15th day.On the 45th day,it dropped to normal level.PCR and ELISA results were consistent with immunofluorescence.(6)Immunofluorescence staining showed that VIP was mainly expressed in corneal nerve fibers in normal cornea and also expressed in corneal stromal inflammatory cells after HSV-1 infection.The results of ELISA showed that the level of VIP increased on the 3rd day after virus infection but decreased on the 7th day.On the 15th day,the expression of VIP increased again and relapse on the 45th day.2.The therapeutic effect of exogenous PEDF on mouse HSK model(1)Compared with the control group,we found that corneal edema relieved,neovascularization reduced,clinical score decreased,corneal nerve density increased,and sensitivity in central cornea increased(p<0.05)on the 3rd and 7th day in PEDF administration group.(2)Immunofluorescence staining and flow cytometry showed that the neutrophil infiltration in the cornea of the PEDF-administered group was significantly reduced on the 7th day(p<0.05),nevertheless as macrophages and CD4~+T cells(p>0.05).(3)ELISA and RT-qPCR results showed that VEGF was significantly down-regulated in the cornea of the PEDF-administered group on the 7th day after viral infection(p<0.05)and the same as IL-1?,IL-6 and TNF-?(p<0.05).(4)Immunofluorescence staining of cornea showed that the fluorescence intensity of PEDF administration group was significantly weakened on the 3rd and 7th day after virus infection.The plaque test results showed that the viral load of the PEDF-administered group was significantly reduced(p<0.05).The results of RT-qPCR showed that the expression levels of virus-related genes ICP0 and UL41 in the PEDF-administered group decreased(p<0.05).In vitro antiviral experiments found that PEDF concentration was 25ng/ml to minimize HSV-1 replication.(5)After subconjunctival injection of PEDF-derived peptides Mer44 and Mer34,there were no significantly changes of clinical symptoms between two groups on the 3rd day after viral infection.On the 7th day,there was obvious difference between two groups in neurodegeneration and neovascularization(p<0.05).The clinical symptoms of the Mer44and Mer34 administration groups were all relieved compared with the control group,the clinical score decreased(p<0.05),and the sensitivity in central cornea increased(p<0.05).However,the neuroprotective effect of the Mer44-administered group was more pronounced while the Mer34-administered group was more effective in inhibiting neovascularization.3.The therapeutic effect of exogenous VIP on mouse HSK model(1)On the 3rd and 7th day after the virus infection,the corneal edema of the VIP group was reduced,the neovascularization was reduced,the clinical score decreased,the corneal nerve density increased and the sensitivity in central cornea was improved(p<0.05).(2)Immunofluorescence staining and flow cytometry showed that the infiltration of neutrophils in the VIP group was significant lower than that in control group on the 3rd and 7th day after HSV-1 infection(p<0.05);CD4~+T cell infiltration was also significantly reduced on the 7th day(p<0.05);ELISA results showed that the activity of intramedullary myeloperoxidase MPO in the VIP group was significantly decreased on the 7th day after virus infection(p<0.05).(3)Immunofluorescence staining and ELISA results of corneal frozen sections showed that the expression of TGF-?in the cornea of the VIP group was significantly up-regulated on the 3rd and 7th day after virus infection(p<0.05).(4)ELISA results showed that on the 7th day after virus infection,the expression of IL-17and IL-17F in the cornea of the VIP administration group decreased obviously compared with the control group(p<0.05).The expression of IL-6,IL-10,IL-12,IL-23,IL-28 and IFN-?increased(p<0.05).(5)Immunofluorescence staining of corneal HSV-1 showed that there was no significant difference in HSV-1 fluorescence intensity between VIP administration group and PBS control group on the 3rd and 7th day after viral infection.The plaque test results showed that there was no significant difference in viral load between the two groups on the third day after infection(p>0.05).There was no significant difference in the expression of ICP0between HSV-1 virus-related genes in RT-qPCR(p>0.05).Conclusions:1.By establishing a mouse HSK model,the study showed the changes of clinical symptoms,corneal nerves and neovascularization during the initial infection of HSV-1.It indicated that corneal nerve regression and neovascularization were most significant on the 7th day after infection.2.The expression of PEDF in HSK is consistent with the change of clinical symptoms;exogenous PEDF play an important role on protecting nerves,inhibiting neovascularization and alleviating clinical symptoms in mouse HSK model.These effects are closely associated with the reduction of neutrophil infiltration,the decrease of VEGF and inflammatory factor expression and the inhibition of HSV-1 virus replication.3.VIP mainly expresses in the corneal nerve.It could also express in inflammatory cells in the corneal stroma after infection.Exogenous vip can protect the nerve,inhibit neovascularization and alleviate the clinical symptoms of the cornea in the mouse HSK model which are related to the reducing of infiltrated neutrophils and CD4~+T cells,decreasing the expression of pro-inflammatory factors and promoting the immunomodulatory effects of anti-inflammatory factors. |
PDF Full Text Request