The Action And Mechanism Of Netrin-1 On Corneal Epithelial Wound Healing Of Diabetic Mouse

Purpose Through in vivo(STZ, streptozotocin induced diabetic mice) and in vitro(mouse corneal limbal epithelial stem / progenitor cell line, TKE2 cells) experiments, observing the function of netrin-1(axon guidance factor) in corneal epitheliun repairation, inflammatory reaction and nerve fibers regeneration of diabetic model mice, and investigating its mechanism.Metheds Mouse corneal epithelial stem / progenitor cells(TKE2 cells) and trigeminal ganglion cells were cultured in this experiment. The subjects were divided into 4 groups: normal group, mannitol group, high glucose group without and with netrin-1(50ng/ml) group. Observed the comparative expression of endogenous netrin-1 between normal group and high glucose group in TKE2 cells. After high glucose treatment, the influences of netrin-1 on migration and proliferation of TKE2 cells, on ERK and EGFR signal pathway, on oxidative stress reaction of TKE2 cells and on the growth of trigeminal ganglion cells were detected respectively. In this study, diabetic model mice induced from C57 mice using streptozotocin(STZ), and C57/BL6 mice were chosen as control subjects. The corneal epithelial wounded models of normal and diabetic mice were established and administrated with subconjunctival injection at once which divided into normal group, diabetic group, diabetic with netrin-1 group, A2 BAR antagonist group and UNC5 B neutralizing antibody group. The main contents were detected as follows, the expression differences of endogenous netrin-1 in epithelial between nomal and diabetic mice, effects of exogenous netrin-1 on diabetic mice corneal epithelial repairation and influences of netrin-1 on inflammatory reaction after epithelial injury and then to observe the function of netrin-1 for corneal nerve repairation after cornea epithelial injury.We also investigated the signaling pathway and main receptors in diabetic mouce epithelial wound healing influced by Netrin-1.Results In vitro study revealed that high glucose group showd lower expression level of endogenous netrin-1 in TKE2 cells compared with normal group, the migration area of high glucose TKE2 cells was smaller than normal and high glucose with netrin-1 groups through wound healing technology. In the cellular proliferation related study, the quanity of TKE2 cells showed a lower level in high glucose group than normal and high glucose with netrin-1 groups. In normal culture, exogenous netrin-1 could activate and up-regulate the expression of p-ERK and p-EGFR in TKE2 cells. TKE2 cells in high glucose showed increased ROS production but decreased GSH level than normal group and high glucose with Netrin-1 group. The axon length of primary cultured trigeminal ganglion cells were shorter in high glucose group than normal group, and that in high glucose with Netrin-1 group was longer than high glucose group. In vivo study revealed that the expression of endogenous Netrin-1 in diabetic mice corneal epithelium decreased significantly compared with normal mouce. The epithelium wound healing rate in diabetic group is significantely decreased compared with the normal group and the diabetic with netrin-1 group is significantly increased compared to the diabetic group. The expression of pro-inflammation factors such as COX-2, CXCL10, CXCL11 and CXCL12 in 24 hours after epithelium scraped and the number of neutrophil infiltratied in cornea were significantly higher than that in normal group, and the diabetic with netrin-1 group showed significantly lower than that data in diabetic group. As well as the same situation on the expression of i NOS and IL-12 marked by M1 macrophage and the number of M1 macrophage cells in cirnea after epithelium scraped 72 hours. Correspondingly, the expression of Arginase-1 and IL-10 marked by M2 macrophage and the number of M2 macrophage cells in diabetic group after epithelium scraped 72 hours were significantly decreased compared to the normal group and however, in diabetic with netrin-1 group it was significantly increased compared to the diabetic group. The corneal nerve length in epithelial regenerated after scraped in diabetic group were significantly slower than normal group and it was also longer in diabetic with netrin-1 group.The expression of p-ERK and p-EGFR in regenerated epithelium of diabetic group were significantly decreased compared with normal group. In diaberic with netrin-1 group, the expression of p-ERK and p-EGFR was stronger than the diabetic group. Compared to diabetic group, the protein level of p-ERK and the number of M2 macrophage were significantly decreased compared with the A2 BAR antagonist group. However the index above exist no significant difference in UNC5 B neutralizing antibody group.Conclusion Netrin-1 promotes the corneal epithelial wound healing of diabetic mouse and inhibites the early inflammatory reaction after injury and prometes transformation of M2 macrophages which could subside the inflammation. Netrin-1 can promote the regeneration and repair of corneal nerve fibers. The auxo-action to epithelium wound healing by netrin-1 related with the activation of p-ERK and p-EGFR. The receptor of netrin-1, A2 BAR, maybe take the main function to activate the ERK signal way and promotes the transconformation of M2 macrophage. In addition, netrin-1 promotes diabetic mouse corneal epithelial wound healing may be related to the function of anti-oxidative stress.