Role And Mechanism Study Of Vasoactive Intestinal Polypeptide In Corneal Epithelium And Nerve Regeneration In Diabetic Mice

Diabetes mellitus is a systemic disease in the world and the incidence of which has increased significantly in recent decades.According to some data,the global prevalence of diabetes is estimated at 415 million(8.8%),which is predicted to rise to 642 million in next 25 years.There are about 114 million diabetic patients in China,and the prevalence rate is 10.4%,which accounts for 27% of the world’s diabetes patients,and has become the largest number of diabetic patients in the world.Diabetic complications in ocular are the main cause of blindness in adults of working age.Diabetic neuropathy characterized by progressive loss of nerve fibers is a common complication and affecting about 50% of diabetics.Although diabetic retinopathy is the main and most severe ocular complication of diabetes,other parts of the eye also have diabetic complications,such as cornea,iris,lens and optic nerve.Diabetic neuropathy is one of the main complications of diabetics.Cornea is the most innervated tissue and its nerve density is 300 to 600 times that of skin.In diabetic keratopathy,most of the sensory nerve fibers were damaged,nerve fiber density and corneal sensitivity were decreased.Corneal innervation is a necessary factor to maintain corneal sensitivity,blink reflex,ocular surface homeostasis and to regulate corneal wound healing progress.Corneal neuropathy is one of the causes of diabetic keratopathy,the main manifestations of which include superficial punctate keratosis and persistent corneal epithelial erosion,and delayed healing of epithelial wounds,which may develop into corneal ulcers and have no sensitive to general clinical treatment.Sensory nerve fibers play an important role in maintaining the homeostasis of corneal epithelium.There are a variety of bioactive peptides expressed in corneal nerve fibers,for example,vasoactive intestinal polypeptide,calcitonin gene-related peptide,substance P,neuropeptide Y,growth hormone peptide,et al.Lack of enough neuropeptides,such as CGRP,SP and VIP,is one of the pathogenesis of diabetic neurokeratosis.VIP is an endogenous multidirectional neuropeptide that plays a biological role in a variety of tissues and organs through two G protein-coupled receptors,VIPR1 and VIPR2,in which VIPR1 receptors have preferential affinity.VIP plays an important role in the development of the nervous system,which not only promotes the differentiation of neurons and the growth of neuronal processes,but also plays an important role in the development of the nervous system.VIP can also protect the peripheral nervous system and promote the regeneration of sciatic nerve in rats and plays a protective role in the neurogenic reaction of skin.Endogenous VIP can inhibit the sustained inflammation and immune response,promote the extinction of inflammation,and maintain and regulate the immune homeostasis.Published results showed that VIP was expressed in mouse corneal nerve fibers and had the function of anti-Pseudomonas aeruginosa infection.In diabetic mice,VIP can improve the structure and cell structure of pancreatic islets and insulin secretion activity and has antioxidant stress and anti-inflammatory effects.Although the research on VIP is extensive,but there is no research on diabetic keratopathy.Therefore,our study focused on the neuroprotective and anti-inflammatory functions of VIP and to investigate whether VIP could affect the diabetic corneal wound repair and to investigate the mechanism at molecular level.The purpose of this study is to observe the role of VIP in corneal epithelial wound healing,inflammatory response and nerve regeneration in diabetic mice in vitro and in vivo,and further to explore the mechanism.PART 1Purpose To build corneal nerve degenerated mouse model.To detect the effect of VIP,α-CGRP and SP on the wound healing progress and inflammatory response of corneal nerve degenerated mouse.Methods To prepare resiniferatoxin eye drops and administrated to C57/BL6 mice to establish cornea nerve denervation model.Normal mice were divided into six groups: normal group(NL),RTX group(RTX),normal wound group(NW),RTX wound group(RTXW),RTXW with α-CGRP group,RTXW with SP group,RTXW with VIP group.The corneal sensitivity of mice in NL group and RTX group was measured by Aesthesiometer,and corneas were stained with Bengal red and fluorescein sodium solution to detect the integrity.Mouse corneal nerve density was measured by corneal whole-mount immunofluorescence staining with β-tubulin III antibody.To establish a model of corneal epithelial injury in normal and RTX treated mice.To observe the difference of corneal epithelial healing rate post wound in NW group,RTXW group,RTXW with α-CGRP group,RTXW with SP group,RTXW with VIP group.To detect the gene level expression of IL1β,IL1 RN,CXCL2,IL10,F4/80,NOS2 and ARG1 in normal and RTX treated mice by RT-PCR.To prepare resiniferatoxin eye drops and administrated to C57/BL6 mice to establish cornea nerve denervation model.Normal mice were divided into six groups: normal group(NL),RTX group(RTX),normal wound group(NW),RTX wound group(RTXW),RTXW with α-CGRP group,RTXW with SP group and RTXW with VIP group.The corneal sensitivity,corneal epithelial integrity and corneal nerve density were measured in normal and RTX mice respectively.To establish a model of corneal epithelial injury in normal and RTX treated mice.To observe the difference of corneal epithelial healing rate post wound in NW group,RTXW group,RTXW with α-CGRP group,RTXW with SP group and RTXW with VIP group.To detect the gene level expression of IL1β,IL1 RN,CXCL2,IL10,F4/80,NOS2 and ARG1 in normal and RTX treated mice by RT-PCR.Results C57/BL6 corneal nerve regeneration mouse model was successfully established.The corneal sensitivity and nerve density were decreased in RTX group compared to NL group.The corneal epithelial integrity had no difference in normal and RTX treated mice.Based on the mouse wound healing model,we found that the healing rate in RTXW group was significantly decreased compared to NW group.However,the healing rate in RTXW with VIP,RTXW with α-CGRP and RTXW with SP group were significantly increased compared to RTXW group.Based on the RT-PCR results,we found that,the expression of IL1 RN,F4/80,NOS2 and ARG1 were increased obviously in RTX group compared to NL group.The expression of IL1β,IL1 RN,CXCL2,IL10,F4/80,NOS2 and ARG1 were increased obviously in NW group compared to NL group.The expression of IL1β,CXCL2,IL10 and NOS2 in RTXW group was significantly higher than that in RTX group.Compared with RTXW group,the expression of IL1β,CXCL2 and NOS2 decreased significantly in RTXW with α-CGRP group.The expression of CXCL2、IL10 and NOS2 were decreased significantly in RTXW with SP group compared to RTXW group.In RTXW with VIP group,the expression of IL1β,CXCL2,F4/80 and NOS2 were decreased and IL10 was increased compared to RTXW group.Conclusion RTX eye drops is a safe and effective method to establish C57/BL6 mouse corneal denervation model.The function or neuropeptide VIP in promoting wound healing and inhibit corneal inflammatory response during the repair progress after corneal injury in corneal nerve denervation mice is much stronger than CGRP and SP.PART2Purpose To detect the effect of exogenous VIP1 receptor antagonist and VIP on the proliferation and migration of normal glucose and high glucose cultured mouse corneal epithelial stem / progenitor cells(TKE2 cells)after injury in vitro respectively.To detect the effect of VIP on murine bone marrow derived macrophages in vitro.To detect the effect of recombinant mouse Sonic Hedgehog(rm SHH)on the repair of porcine corneal epithelial injury cultured in high glucose in vitro.Methods TKE2 cells were cultured in vitro and divided into normal group,mannitol group,high glucose group and high glucose with VIP group.TKE2 cells were cultured in normal glucose medium to detect the effects of VIP on ERK and SHH signaling pathway with different concentration and at different time point.To detect the effect of VIP1 receptor antagonist and VIP on the healing progress of TKE2 cells after injury and the effect on ERK and SHH signal pathway activity after TKE2 cell injury in normal and high glucose cultured TKE2 cells respectively.To culture macrophages derived from normal and diabetic mouse respectively in vitro.The experimental groups were divided into eight groups: normal group,high glucose group,normal with VIP group,high glucose with VIP group,normal with IL1β group,high glucose with IL1β group,normal with IL1β and VIP group and high glucose with IL1β and VIP group.Porcine cornea was cultured in vitro.The experimental groups were mannitol group,mannitol with SHH receptor antagonist group(SHH Ra),high glucose group and high glucose with rm SHH group.To observe the effect of rm SHH and SHH Ra on the healing progress of porcine corneal epithelium after injury.Results Through the study on TKE2 cells in vivo,we found that the activation of ERK and the expression of SHH in TKE2 cells were significantly enhanced when the concentration of VIP was higher than that of 250ng/ml.After VIP administrated for 6 hours,ERK and SHH were activated obviously.The healing area of TKE2 cells post-wound in normal glucose with VIP1 Ra group was significantly lower than that in normal glucose group.The healing area of TKE2 cells post-wound in high glucose group was significantly lower than that in high glucose with VIP group.The expression of p-ERK and SHH in TKE2 cells in normal glucose with VIP1 Ra group and high glucose group was significantly lower than that in normal glucose group,and the expression of p-ERK and SHH in high glucose with VIP group was significantly higher than that in high glucose group.Based on the study of macrophages in vitro,we found that the expression of IL1β,CXCL2 and NOS2 in normal with IL1β group and high glucose with IL1β group were higher than those in normal and high glucose group,IL1 RN in normal with IL1β group was higher than that in normal with IL1β group.IL10 and ARG1 levels in normal with IL1β group had no change.The IL10 expression in high glucose with IL1β group was higher than that of normal group,but IL1 RN and ARG1 had no change.The expression of IL1β,CXCL2 and NOS2 in the normal with IL1β and VIP groups were significantly lower than that in the normal with IL1β group,ARG1 was significantly higher than that in the normal with IL1β group.IL1 RN and IL10 had no change.The expression of NOS2 in high glucose with IL1β and VIP groups were significantly lower than that in normal with IL1β group and IL1 RN,IL10 and ARG1 were significantly increased,IL1β and CXCL2 had no change.Based on the study of porcine cornea in vitro,we found that the defect area of corneal epithelium in mannitol with SHH Ra group was significantly higher than that in mannitol group.The defect area in high glucose group was significantly higher than that in mannitol group.Conclusion VIP promotes the healing progress of TKE2 cells in high glucose medium by enhancing the activation of ERK and SHH through VIPR1 signal pathway.VIP inhibited the expression of pro-inflammatory cytokines in macrophages and promoted the expression of anti-inflammatory cytokines.rm SHH promoted the healing progress of corneal epithelial after injury in high glucose cultured medium by binding with receptors.PART 3Purpose To induce type I diabetic mice by streptozotocin.To observe the effect of VIP on corneal epithelial healing progress,corneal inflammatory response and nerve repair in diabetic mice and to explore the mechanisms of them.Methods Type I diabetic mice induced by streptozotocin and normal C57/BL6 mice were selected.The model of corneal epithelium injury in mice was established and subconjunctival injection was used.Normal and diabetic mice were divided in to nine groups: normal group(NL),diabetic group(DM),normal wound group(NW),diabetic wound group(DMW),NW with VIP group,NW with VIP1 receptor antagonist(VIP1Ra)group,DMW with VIP group,NW with SHH Ra group,DMW with rm SHH group and DMW with VIP and SHH Ra group.The expression of endogenous VIP and its receptors in corneal epithelium and trigeminal nerve ganglion of normal and diabetic mice were detected by RT-PCR,Western-blotting and immunofluorescence staining.To build mouse corneal epithelia wound model and detect the difference of corneal epithelial healing rate and nerve density in NW group,DMW group,NW with VIP group,NW with VIP1 Ra group,DMW with VIP group,NW with SHH Ra group,DMW with rm SHH group and DMW with VIP and SHH Ra group mice respectively.To detect the nerve growth factors and inflammatory factors in NL group,DM group,NW group,NW with VIP1 Ra group and DMW with VIP group by RT-PCR.To detect the difference of corneal infiltrated neutrophil and macrophage numbers in NW group,NW with VIP1 Ra group and DMW with VIP group by whole-mount immunofluorescence staining.To detect the expression level of p-ERK and SHH in NL group,DM group,NW group,NW with VIP1 Ra group and DMW with VIP group by western-blotting and immunofluorescence staining.Results Based on the study of normal and diabetic mice,we found that the expression of VIP and VIPR1 in corneal epithelium were increased in NW group compared to NL group,and they were decreased in DMW group compared to NW group.The results of immunofluorescence staining showed that VIPR1 was mainly expressed in corneal limbal region and peripheral epithelium.The expression of VIPR1 in corneal epithelium of DM group and DMW group were significantly lower than that of NL group and NW group respectively.The results of microscope showed that,compared to NW group,the corneal healing rate was significantly increased in NW with VIP group,and decreased in DMW group,NW+VIP1Ra group and NW+SHH Ra group.Compared to DMW group,the corneal healing rate was significantly increased in DMW with VIP group and DMW with rm SHH group,and significantly decreased in DMW with VIP and SHH Ra group.The variation tendency of corneal nerve density in these groups was almost corresponding to the trend of healing rate.The expression of NGF and CNTF in NW group was significantly higher than that in NL group and compared to NW group,those were decreased significantly in NW with VIP1 Ra group and DMW group.Compared to DMW group,the expression of NGF and CNTF in DMW with VIP group were increased significantly.Compared to NW group,the expression level of IL1β,CXCL2 and NOS2 in NW with VIP1 Ra group and DMW group were significantly increased and IL1 RN,IL10 and ARG1 were significantly decreased.Compared to DMW group,the pro-inflammatory factors were decreased,and anti-inflammatory factors increased in DMW with VIP group.Corneal whole-mount immunofluorescence staining results showed that corneal infiltrated neutrophil and macrophage numbers in NW with VIP1 Ra group and DMW group were much more than NW group.Compared to DMW group,those were decreased significantly in DMW with VIP group.The results of western-blotting showed that the expression of p-ERK and SHH protein increased significantly after corneal epithelial injury in normal mice.However,the expression of p-ERK and SHH were obviously decreased in NW with VIP1 Ra group and DMW group compared to NW group and those expression in DMW with VIP were increased significantly compared to DMW group.The results of microscope and confocal microscopy showed that the corneal healing rate and nerve density in NW with SHH Ra group were significantly lower than those in NW group.Compared to DMW group,the corneal healing rate and nerve density were significantly increased.Conclusion Exogenous VIP promotes corneal epithelial injury repair and nerve regeneration in diabetic mice through its receptor VIPR1.The mechanism of VIP promoting corneal healing and nerve regeneration by upregulating the expression of p-ERK and SHH and the expression of NGF and CNTF.Exogenous SHH promotes corneal epithelial repair and nerve regeneration in diabetic mice.