Experimental Study On Substance P Inhibiting Herpes Simplex Keratitis Recurrence In Mouse

Objective:Repeated recurrence of herpes simplex keratitis (HSK) causes corneal scar formation, neovascularization and thinning, and thus the methods inhibiting the relapse are significantly meaningful. The virus itself, the body and environmental factors are involved in the regulation of latent HSK relapse. Cornea is the most intensively innervated tissue. Meanwhile nerve degeneration and regeneration in HSK are very remarkable. Neuropeptide secreted by nerve cells, immune cells and other cells regulates the neuro-endocrine-immune system interactions. Use of antiviral host protein has unique advantages. Substance P (SP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) can regulate inflammatory processes. SP works through its receptor NK1-R. Changes of neuropeptide expression in cornea with HSK and whether or not neuropeptides are regulating pathology of HSK are not fully elucidated. So this experiment based on mouse HSK model and trigeminal ganglion (TG) cell culture model studied the expression of SP, its effects on the course of HSK and possible mechanisms, providing new targets and strategies for Precision Medicine (PM) of HSK.Method:1. HSV-1 (Mckrae strain) was produced in Vero cells. HSV-1 was injected into the corneal stroma of C57BL/6 mice to construct a mouse model of HSK (named stroma injection modeling method), and then symptoms of HSK were observed.2. ELISA method and immunofluorescence method were used for determination of concentration of neuropeptides such as SP in the cornea and TG of model at different time point. Based on latent model whose corneal score was 1, we induced recurrent HSK after subconjunctival injection of SP and L-733060 (a NK1-R receptor antagonist). Based on latently infected eyes whose corneal score was 1, we studied the effects of L-733060 on mouse recurrent HSK using inner control method. L-733060 was administrated to right eye, and normal saline (NS) was administrated to left eye as control. At recurrent 2 days (R2d), the cornea of model was photoed and scored, and HE was performed to observe the histomorphology.3. TG of C57BL/6 mouse aged 12 weeks was harvested, and ganglion cells were dissociated and cultured in vitro. Acyclovir was added in culture medium first, and then HSV-1 was added. By these methods HSV-1 latently infected TG in vitro model was successfully established. SP, L-733060 and PI3K inhibitor LY-294002 were added in the culture medium for studying the effects of SP on latency and recurrence of HSV-1 together with mechanisms. Immunofluorescence aiming at inspection of HSV-1-positive cells in vitro, fluorescence quantitative PCR aiming at detection of VP16 gene in vitro, Western Blot aiming at detection of p-Akt/Akt level in vitro and in vivo, and immunofluorescence aiming at inspection of p-Akt levels in cornea and TG at different time point were performed.Results:1. Success rate of stroma injection modeling method was above 80%. Mechanical corneal injury was mild and basically no blepharitis was observed in stroma injection modeling method. Model's corneal opacity was the severest at 7 days post infection (7 dpi) and recurrent 2 days (R2d), and the mildest at 45 dpi and R7d. The most obvious neovascularization appeared 1-2 days after the occurrence of the severest corneal opacity.2. SP concentration in HSK cornea was significantly higher than normal, and decreased at R2d. Cornea scores in SP group were significant smaller than that in the control group (Wilcoxon W=17.5, p-=0.0317). With the use of inner control method, cornea scores in L-733060 group were significantly bigger than that in the control group (t=4.908,p=0.0001). HE staining showed cornea of SP group was obviously thinner than that in control group. ELISA and immunofluorescence showed SP increased after the infection in TG as in cornea and its concentration was the highest when HSV-1 latency.Percentage of HSV-1-positive cells as well as VP16 gene expression were the lowest in SP group, and were the highest in L-733060 group. Inhibition of PI3K pathway blocked SP'effects. Levels of p-Akt increased 10 minutes and decreased 1 hour after the adding of SP to the culture medium. P-Akt level was the highest in SP group, and the lowest in L-733060 group. Western Blot and immunofluorescence showed consistent result that p-Akt level in TG increased after HSV-1 infection and was the highest when HSV-1 latency.Conclusion:The success rate and symptoms of stroma injection modeling method were similar to the scarification method, but the virus used was about 100 times fewer. Basically no blepharitis was observed and mechanical damage of corneal epithelium was mild. Expression of SP increased in model's cornea. SP can reduce the model's cornea scores at R2d, and L-733060 can increase the model's cornea scores at R2d. SP inhibited the reactivation of HSV-1 in cultured letently infected TG cell, and promoted HSV-1 latency.Antagonize SP receptor or block the PI3K/Akt signaling pathway can eliminate the effects of SP in vitro. Akt phosphorylation level and SP expression in model's corneal and TG changed in the same trend. It was suggested that HSV-1 infection induced SP expression in model's cornea and TG; Elevated SP activated PI3K/Akt signal pathway and inhibited gene expression related to HSV-1 reactivation such as VP16; SP finally prevented the recurrence and promoted the letency of HSK.