TIPE2 Regulates Diabetic Corneal Epithelial And Nerve Regeneration Through Rac1/AKT/NF-κB P50 Signalling Pathway

Background:Diabetic keratopathy（DK）is one of the main complications of diabetic eyes.The main clinical features of DK are corneal epithelial exfoliation,delayed epithelial healing,decreased corneal nerve density,decreased corneal perception,etc.Severe cases may have corneal ulcer or even perforation.Healthy innervation is essential to maintain corneal structure and function.Diabetic patients are prone to peripheral neuropathy,and the damaged sensory innervation or function may damage the homeostasis of corneal epithelium.In addition,corneal epithelial injury repair is closely related to epithelial cell apoptosis.At present,the treatment of DK mainly focuses on corneal re-epithelialization and nerve regeneration.It has been reported that the expression of tumor necrosis factor-α-induced protein 8-like 2（TIPE2）in glomeruli of diabetic rats induced by streptozotocin（STZ）and kidney in diabetics is increased,suggesting that TIPE2 may be involved in the occurrence and development of diabetic complications.TIPE2 has a death effect domain like structure,which can promote many types of cells apoptosis.It has been shown that the mechanism of TIPE2 promoting apoptosis is at least partly due to its inhibition of Protein kinase B（AKT）signaling.As the target of AKT,nuclear factor（NF）-κB is a strong anti apoptosis signal in most cells.NF-κB p50 is one of the most common subunits of NF-κB.Wang et al.Confirmed that the corneal epithelial healing of NF-κB p50 knockout mice was significantly delayed;some scholars also reported that NF-κB p50 gene deficient mice can spontaneously develop into optic neuropathy.It has also been found that the molecular mechanism of the antiproliferative effect of TIPE2 lies in its negative regulation of Ras-related C3 botulinum toxin substrate（Rac）signaling pathway.However,there is no study on the specific function and mechanism of TIPE2 in DK,which needs to be systematically and deeply explored.In this study,normal C57 mice,TIPE2 knockout(TIPE2^-/-)mice and STZ induced type I diabetic mice were used to establish corneal epithelial injury repair models.The purpose of this study was to investigate whether TIPE2 affects corneal epithelial and nerve regeneration in diabetic mice at the molecular level,and to explore its molecular mechanism by studying its regulation of NF-κB p50/Akt/Rac1 signaling pathway.Part one:TIPE2 knockdown promotes diabetic corneal epithelial wound repair and nerve regenerationObjective:To investigate the TIPE2 expression in diabetic corneal during wound healing,and to explore its role in re-epithelialization and nerve regeneration.Methods:1.The central corneal epithelium in normal（NL）and diabetic（DM）mice was scraped to establish a corneal epithelial injury model.The expression of TIPE2 m RNA and protein in corneal tissue was detected before and after corneal epithelial injury in this two groups of mice.2.Immunofluorescence staining was emploied to detect the TIPE2 expression in cornea of normal people and diabetic patients.3.Diabetic mice were injected subconjunctivally with TIPE2-si RNA or nonspecific control（NC）,WT(TIPE2^+/+)TIPE2^+/+and TIPE2^-/-mice as the control group,namely TIPE2^+/+group,TIPE2^-/-group,DM+NC group and DM+si RNA group,compared the epithelial defect rate.4.The density of subbasal nerve plexus and corneal sensitivity of the above four groups mice were detected on the 5^th day after epithelial injury.Results:1.Compared with normal mice,the TIPE2 m RNA in diabetic cornea increased（P<0.05）,the TIPE2 m RNA in normal（P<0.001）and diabetic mice（P<0.001）was increased significantly at 24 h after corneal epithelial curettage,the latter is higher than the former（P<0.001）;The protein level of TIPE2 was detected by western blotting,and the trend was consistent with PCR.2.The fluorescence staining in diabetic patients is stronger,and mainly showed in epithelial layer.3.The corneal epithelium of TIPE2^-/-mice was repaired quickly,and repaired completely 24 h post wounding,while that of TIPE2^+/+mice was repaired completely48 h after injury;Epithelial repair was also accelerated in diabetic mice injected subconjunctivally with TIPE2-si RNA,the percentage of epithelial defect in DM+si RNA group was 0.97±1.34%at 48 h after injury,which was significant smaller than DM+NC group（P<0.001,10.00±3.00%）.4.The density of subbasal nerve plexus in TIPE2^-/-mice was significantly higher than that in TIPE2^+/+mice（P<0.001）on the 5th day of epithelial curettage;The density in DM+si RNA group was significantly higher than that in DM+NC group（P<0.001）;On the 7th day after injury,the corneal sensitivity of TIPE2^-/-group was significantly higher than that of TIPE2^+/+group（P<0.01）,and that of DM+si RNA group was significantly higher than DM+NC group（P<0.001）.Conclusion:The TIPE2 expression was up-regulated in diabetic cornea,TIPE2knockdown promotes corneal epithelial wound repair and nerve regeneration in normal and diabetic cornea.Part two:TIPE2 knockdown promote diabetic corneal epithelial wound repair and nerve regeneration through Rac1/AKT/NF-κB p50 signal pathwayObjective:To investigate the regulatory effect of TIPE2 on the signal pathways of Rac1,Akt and NF-κB P50 in the process of corneal epithelial injury and repair.Methods:1.The NF-κB p50 and p-NF-κB p65 expression in TIPE2^+/+and TIPE2^-/-mice were detected by Immunofluorescence and western blotting before and after the corneal epithelium injury;TIPE2^+/+and TIPE2^-/-mice were injected subconjunctivally with NF-κB p50 inhibitor（NF-κB p50i）and control protein（con）,compared the epithelial defect rate and the density of subbasal nerve plexus.2.The p-AKT1 expression in TIPE2^+/+and TIPE2^-/-mice were detected 24 h after corneal epithelial injury;TIPE2^+/+and TIPE2^-/-mice were injected subconjunctivally with AKT inhibitor（AKTi）or PBS（con）,then the protein level of NF-κB p50 was detected and the corneal epithelial injury repair and nerve regeneration were observed.3.The NF-κB p50 and p-NF-κB p65 expression in normal and diabetic mice were detected before and after corneal epithelial injury;Local knockdown of TIPE2and NF-κB p50 or AKT inhibitor were injected subconjunctivally,diabetic mice were divided into DM+NC group,DM+si RNA group,DM+si RNA+NF-κB p50i group and DM+si RNA+AKTi group,to compare the epithelial defect rate and the density of subbasal nerve plexus,NF-κB p50 expression was detected after 24h epithelial curettage.4.mouse corneal epithelial stem/progenitor cell（TKE2）were cultured in vitro.The experimental groups were:normal group,mannitol control group,high glucose group,high glucose+TIPE2-si RNA group,high glucose+TIPE2-si RNA+NF-κB p50i group,high glucose+TIPE2-si RNA+Akti group.The GTP-Rac1 was detected by pull down technique.5.TIPE2^+/+mice,TIPE2^-/-mice and diabetic mice treated with TIPE2-si RNA were injected subconjunctivally with Rac1 inhibitor（Rac1i）or PBS（con）,the corneal epithelial repair and nerve regeneration were observed,and the protein levels of p-AKT1 and NF-κB p50 were detected after 24h epithelial curettage.Results:1.Compared with normal TIPE2^+/+mice,the NF-κB p50 expression in corneal epithelium of TIPE2^-/-mice was significantly increased（P<0.001）,the results of immunofluorescence were consistent;The p-NF-κB p65 expression was decreased significantly（P<0.05）;The epithelial repair in TIPE2^-/-+NF-κB p50i group was slower than in TIPE2^-/-group,the latter was completely repaired at 24 h after epithelial curettage,while the former was completely repaired at 48 hours after injury;NF-κB p50 inhibitor significantly inhibited the promotion of nerve regeneration in TIPE2^-/-mice（P<0.001）.2.The protein level of p-AKT1 in TIPE2^-/-group was significantly higher than that in TIPE2^+/+group（P<0.05）at 24h after the corneal epithelium scraping;The protein level of NF-κB p50 in TIPE2^-/-+AKTi group was significantly lower than that in TIPE2^-/-group（P<0.01）;The epithelial wound healing in TIPE2^-/-+AKTi group was significantly slower than that in TIPE2^-/-group,the epithelial in the latter repaired almost completely at 24h after injury,the former was 24.16±3.59%;Epithelial defect in the TIPE2^-/-+AKTi group healing faster than that in TIPE2^+/++AKTi group（P<0.001）at 48h after injury;AKT inhibitor significantly inhibited the promoting effect of TIPE2^-/-mice on nerve regeneration（P<0.001）.3.The NF-κB p50 protein level in diabetic corneal epithelium was significantly lower than that in normal mice,no matter in unwound（P<0.05）or wound epithelium（P<0.001）,the immunofluorescence results was consistent;However,the p-NF-κB p65 level in diabetic corneal epithelium was significantly increased before and after injury（P<0.05）,and NF-κB p50 inhibitor significantly inhibited the effect of TIPE2-si RNA on corneal epithelial healing and nerve regeneration in diabetic mice（P<0.001）.AKT inhibitors significantly inhibited the effects of TIPE2-si RNA on NF-κB p50 expression,epithelial repair and nerve regeneration in diabetic mice corneal epithelium（P<0.001）.4.GTP-Rac1 pull down showed that the Rac1 activation of TKE2 cells（P<0.01）;while TIPE2-si RNA restored the inhibition of high glucose on Rac1 activation（P<0.001）,while inhibition of NF-κB p50（P<0.001）and AKT（P<0.001）significantly inhibited the activation of Rac1 by TIPE2-si RNA.5.Rac1 inhibitor can significantly inhibit regeneration of corneal epithelium（P<0.001,24h;P<0.01,36h）and nerve（P<0.001）and the expression of p-AKT1（P<0.05）and NF-κB p50（P<0.01）in TIPE2^-/-mice;Rac1 inhibitor can also inhibit the promoting effect of TIPE2-si RNA on corneal epithelium regeneration（P<0.001）,nerve（P<0.01）and the expression of p-AKT1（P<0.001）and NF-κB p50（P<0.01）in diabetic mice.Conclusion:TIPE2 knockdown promotes corneal epithelial wound repair and nerve regeneration by activating Rac1/AKT/NF-κB p50 signaling pathway.Part three:TIPE2 promotes diabetic epithelial cell apoptosis by inhibiting NF-κB p50 during wound repairObjective:To investigate the apoptosis mechanism of TIPE2 on diabetic corneal epithelium during wound repair.Methods:1.TUNEL staining and wes were used to detect the expression of Bax,cleaved caspase-3,and Bcl-2 in the regenerated corneal epithelium of normal and diabetic mice at 24 h after injury.2.To compare apoptosis of corneal epithelial cells,Bax,cleaved caspase-3,and Bcl-2 expression in TIPE2^+/+and TIPE2^-/-mice.3.Diabetic mice were injected subconjunctivally with TIPE2-si RNA or NC,compared the Bax,cleaved caspase-3 and Bcl-2 expression in corneal epithelium of NL group,DM+NC group and DM+si RNA group.4.TIPE2^+/+and TIPE2^-/-mice were injected subconjunctivally with NF-κB p50inhibitor or control protein,and to detect apoptosis cells,Bax,cleaved caspase-3 and Bcl-2 expression in corneal epithelium.Results:1.Apoptotic cells,Bax（P<0.05）and cleaved caspase-3（P<0.05）expression in DM group were increased,and Bcl-2 level（P<0.05）were decreased at24 h after injury.2.Apoptotic cells in corneal epithelial of TIPE2^-/-group decreased,the Bax（P<0.05）and cleaved caspase-3（P<0.01）expression were also decreased significantly in TIPE2^-/-group,and the Bcl-2 expression increased（P<0.01）.3.TIPE2-si RNA can significantly reduce apoptosis cells in diabetic corneal epithelium,and the expression of Bax（P<0.05）and cleaved caspase-3（P<0.05）were significantly decreased,and Bcl-2 was significantly increased（P<0.05）.4.Apoptotic cells in corneal epithelium of TIPE2^+/+and TIPE2^-/-mice were increased after NF-κB p50 inhibition,the expression of Bax（P<0.01;P<0.05）and cleaved caspase-3（P<0.01;P<0.05）were increased significantly,and Bcl-2 was decreased（P<0.05;P<0.05）.Conclusion:TIPE2 can regulate the apoptosis of corneal epithelial cells in normal and diabetic mice through NF-κB p50.