The Function And Mechanism Study On How DNase ? Improves Corneal Epithelium And Nerve Regeneration In Diabetic Mice

Purpose China is the country with the largest number of diabetes patients in the world.The latest data shows that the population of diabetes in China aged 20-79 has reached 114 million.Primary keratopathy occurs in 47% to 64% of patients with diabetes,and the clinical manifestations cover delayed epithelial healing,corneal sensation and neurotrophic corneal ulcer.The prolongation of corneal wounds has been a difficulty in clinical treatment.Diabetes patients carry a higher risk of injury and infection,and cataracts and fundus surgery can give corneal patients a chance of corneal damage.Neutrophils are inflammatory cells that play a major role at the early stages of wound healing.It is delayed in the process of tissue damage in diabetic individuals,and a special type of death called NETosis occurs,releasing histones and DNA in the nucleus,forming neutrophil extracellular traps?NETs?,bringing about tissue damage.The PAD4enzyme is a key enzyme that catalyzes NETosis.Extracellular DNA?eDNA?and histones are key components of NETs,which induce a strong immune response in the host,leading to tissue damage.Targeted NETs treatment can promote diabetic skin healing significantly.Removal of DNase ? from eDNA and PAD4 inhibitors that inhibit the formation of NETs are two major treatments for targeting NETs.However,there is no research on the role and mechanism of NETs formation in the regeneration of diabetic corneal epithelium.The study of NETs and nerve regeneration has not been reported either.This study first observed and compared the effects of DNase ? and PAD4 inhibitors on diabetic corneal epithelium and nerve regeneration,then explored the mechanism of action of NETs in diabetic corneal epithelium and nerve regeneration.Methods 1.Observation on the effect of anti-NETs treatment of corneal epithelial injury and the effect of DNase ? on corneal epithelial regeneration in diabetic mice C57BL/6J male mice were selected and a type 1 diabetes model induced by multiple intraperitoneal injections of streptozotocin?STZ?was used,and a corneal epithelial injury model was established.The administration method of anti-NETs treatment?DNase ? and PAD4 inhibitor?for treating corneal epithelial damage in type 1 diabetic mice was determined by preliminary experiments.The effects of anti-NETs?DNase ? eye drops and systemic application of PAD4 inhibitors?on corneal epithelial damage in normal and diabetic mice were observed.The effects of 1 mg/ml DNase ? eye drops on the expression of corneal epithelial regeneration-related signaling pathways?pAkt,IGF-1R and Sirt1?,oxidative stress-related signaling pathways?ROS,NOX2 and NOX4?,and HIF-1?in corneal epithelium of diabetic mice were examined by immunofluorescence and western blot.2.Effect of DNase ? on inflammatory response during corneal epithelial regeneration in diabetic mice The expression sites and levels of NETs marker H3Cit and neutrophil marker Ly6G were detected by immunofluorescence and western blot at different time points after corneal epithelial injury in normal and diabetic mice.The effects of DNase ? eye drops on the expression of neutrophil marker?Ly6G?,NETosis markers?H3Cit,neutrophil elastase and myeloperoxidase?in diabetic injured cornea were detected by immunofluorescence,western blot and Elisa.The effects of DNase ? eye drops on the expression of macrophage marker F4/80 and M2 macrophage marker CD206 in diabetic injured cornea were detected by immunofluorescence.The effect of DNase ? on the expression of macrophage associated mRNA in the injured cornea of diabetic mice was examined by qPCR.3.Effect of DNase ? on corneal nerve regeneration in diabetic mice The effects of DNase ? eye drops on corneal nerve regeneration in diabetic mice were observed by corneal nerve staining and corneal sensitivity measurement.Results 1.Anti-NETs treatment promoted corneal epithelial regeneration in normal and diabetic mice DNase ? eye drops promoted corneal epithelial regeneration in normal mice?P<0.01?,and also in diabetic mice?P<0.01?.The therapeutic effect was exhibited at 24h.Systemic application of Cl-amidine had no effect on the rate of corneal epithelial regeneration in diabetic mice at 24h?P>0.05?,but showed a significant promotion in corneal epithelial regeneration at 48h?P<0.01?.2.DNase ? promoted corneal epithelial regeneration in diabetic mice?1?DNase ? activated corneal epithelial regeneration-related signaling pathway in diabetic mice Expression of pAkt,IGF-1R,and Sirt1 was concentrated in the corneal epithelial layer.The expression of pAkt,IGF-1R and Sirt1 in diabetic cornea was weaker than that in normal mice?P<0.01?.DNase ? treatment enhanced the expression of pAkt,IGF-1R and Sirt1 in diabetic cornea?P<0.01?.?2?DNase ? inhibited corneal oxidative stress-related signaling pathway in diabetic mice The corneal epithelial ROS accumulated in diabetic mice,and the fluorescence intensity was higher than that of normal mice?P<0.01?.DNase ? reduced the accumulation of ROS in the epithelial layer of diabetic mice?P<0.01?.The expression of NOX2 and NOX4 was concentrated in the corneal epithelial layer.The protein expression levels of NOX2 and NOX4 in the cornea of diabetic mice were higher than the same in normal mice?P<0.05?.DNase ? treatment reduced the accumulation of NOX2 and NOX4 in the diabetic cornea?P<0.05?.?3?DNase ? regulated the expression of HIF-1?in the cornea of diabetic mice The expression of HIF-1?was mainly located in the corneal epithelial layer.The protein expression level of HIF-1?in the cornea of diabetic mice was higher than that in normal mice?P<0.01?.DNase ? treatment reduced the accumulation of HIF-1?in the cornea of diabetic mice?P<0.01?.In the process of injury repair,the expression level of HIF-1?in the cornea of diabetic mice was lower than that in normal mice?P<0.01?.DNase ? treatment increased the expression of HIF-1?in the diabetic cornea during injury repair?P<0.01?.3.DNase ? reduced the inflammatory response of in diabetic cornea ?1?Increased neutrophil infiltration and NETs formation after corneal epithelial injury in diabetic mice At 24h after scraping corneal epithelium,Ly6G appeared in the superficial corneal stroma of normal mice;at 48h,the expression of Ly6G decreased.The expression of H3Cit was always located in the cornea of the neutrophil infiltration site,and the immunofluorescence intensity at 24h was higher than the same at 48h.At 24h after scraping corneal epithelium,the immunofluorescence intensity of Ly6G in diabetic mice was weaker than that in normal mice;it increased significantly at48h,and the immunofluorescence intensity was stronger than that in normal mice.The expression of H3Cit was always located in the cornea ofthe neutrophil infiltration site,and the immunofluorescence intensity at 48h was higher than the same at 24h.There was no significant change in the total corneal H3Cit/H3 at all time points in the normal group?P>0.05?,but it increased in the diabetic group at48h.There was statistical difference compared with the normal group?P<0.01?.?2?DNase ? inhibits inflammatory response of neutrophils during corneal epithelial regeneration in diabetic mice At 48h after corneal epithelial scraping,the citrullination rate of H3Cit/H3 and the expression level of Ly6G of diabetic cornea,were higher than those in normal cornea?P<0.01?.After DNase ? treatment,H3Cit/H3 and Ly6G expression decreased to normal mouse levels?P>0.05?.At the same time,the expression levels of myeloperoxidase and neutrophil elastase in the diabetic cornea were higher than those in normal cornea?P<0.01?.After 48h of DNase ? treatment,the expression levels of myeloperoxidase and neutrophil elastase in the diabetic cornea decreased?P<0.05?.?3?DNase ? inhibits inflammatory response of macrophages during corneal epithelial regeneration in diabetic mice At 48h after corneal epithelial injury,the expression of F4/80 in the cornea of diabetic mice increased compared with normal mice.After treatment with DNase ?,not only the number of infiltrating macrophages in the corneal stroma was significantly reduced,but also CD206 as a surface marker of M2 macrophages,and its immunofluorescence intensity was also enhanced at the same time.QPCR results showed that the mRNA levels of iNOS,CD86,TNF-?,MCP-1 and IL-12 associated with M1 macrophages in the cornea of diabetic mice were higher than those in normal mice?P<0.001?.The mRNA levels of IL-10,Arg-1,and CD206 associated with M2 macrophages were lower than those in normal mice?P<0.01?.After treatment with DNase ?,the mRNA levels associated with M1 macrophages in the diabetic mice cornea were lower than those in untreated diabetic mice?P<0.05?.DNase ? treatment increased the expression of mRNA associated with M2macrophages in the diabetic cornea?P<0.01?.4.DNase ? promoted corneal nerve regeneration in diabetic mice?1?DNase ? promoted nerve fiber regeneration in the subepithelial plexus of the cornea On the 21^th day after corneal epithelial injury,the density of the subepithelial plexus nerve fiber densities of normal mice,diabetic mice,and DNase ?-treated diabetic mice were 18.3±2.7%,5.3±1.9%,and 8.7±3.7%,respectively,in the central region of the cornea.The nerve fiber density in diabetic mice were lower than that in normal mice?P<0.001?,though increased after treatment with DNase ??P<0.01?,but still lower than in normal mice?P<0.01?.In the peripheral region of the cornea,the nerve fiber density of normal mice,diabetic mice,and DNase ?-treated diabetic mice was 14.6±2.7%,5.0±2.1%,and 8.0±2.8%,respectively.The nerve fiber density in diabetic mice were lower than that in normal mice?P<0.05?,though increased after treatment with DNase ??P<0.05?,but still lower than in normal mice?P<0.05?.?2?DNase ? promoted corneal sensitivity recovery After scraping the corneal epithelium,the corneal sensitivity of the mice decreased significantly.On the 7^th day of corneal epithelial injury,corneal sensitivity in normal mice nearly recovered to uninjured levels.On the 3^th,7^th,14^th,and 21^th days after corneal epithelial injury,the corneal sensitivity of diabetic mice decreased compared with normal mice?P<0.01?.The DNase ? eye drops significantly increased the corneal sensitivity of diabetic mice?P<0.05?.The corneal sensitivity of diabetic mice recovered to the recovery level of normal mice starting from the 3^th day?P>0.05?.Conclusions 1.Anti-NETs treatment promotes corneal epithelial regeneration in both normal and diabetic mice.The therapeutic effect of DNase ? to clear eDNA is superior to that of PAD4 inhibitors that inhibit the formation of NETs.DNase ? promotes diabetic corneal epithelial regeneration by activating signal pathways involved in corneal epithelial regeneration,inhibiting corneal oxidative stress-related signaling pathways,and regulating HIF-1?activation.2.DNase ? promotes corneal epithelial regeneration in diabetic mice by inhibiting inflammation caused by neutrophils and macrophages after corneal injury,suggesting that eDNA is an important cause of excessive inflammation after diabetic corneal injury.3.DNase ? promotes the regeneration of corneal epithelial plexus nerve fibers in diabetic mice and promotes the recovery of corneal sensitivity,suggesting that the increase of eDNA production and the delay of elimination are important reasons for putting off the regeneration of diabetic corneal nerve.Innovation and significance:For the first time,this study demonstrates that anti-NETs therapy can be used to promote the repair of diabetic corneal injury.It is also the first comparison study in the therapeutic effects of DNase ? and PAD4 inhibitors.It is found that the efficacy of DNase ? in clearing eDNA is superior to a PAD4 inhibitor that inhibits the formation of NETs applied to the treatment of diabetic corneal injury.This study provides that the mechanism of DNase ?'s action in clearing eDNA during corneal epithelial regeneration involves not only inhibition of inflammatory response during corneal injury repair,but also promotion of corneal epithelial regeneration and regulation of oxidative stress.What's more,it is the first report on eDNA clearing's ability to promote corneal nerve fiber regeneration.Given the multi-faceted mechanism in the treatment of diabetic corneal injury,and economically accessible property,DNase ? has a sound clinical application prospect.