Expression Of TRPC6 In The Mouse Retina And Its Role In STZ-induced Early Retinopathy In Diabetic Mice

Backgrounds: Diabetic retinopathy(DR),also known as diabetic retinopathy,is a complication of diabetes mellitus,and hyperglycemia is the main cause of its development.The advanced stage of retinopathy can lead to severe irreversible visual impairment,and the apoptosis of retinal optic nerve cells is the root cause.Transient receptor potential(TRP)is a class of non-selective cation channels involved in a variety of physiological functions and pathological processes of the disease.However,the distribution of TRPC6 expression in the mouse retina and its role in the apoptosis of mouse diabetic retinal neurons have not been clearly investigated.Objectives: In this study,we firstly investigated the effect of TRPC6 expression in mouse retina to clarify its retinal specific expression localization,and then investigated its effect on the survival of retinal astrocytes and ganglion cells in STZ-induced diabetic mouse retina to investigate its role in STZ-induced early retinal damage in diabetic mice.Methods:(1)To detect the expression and distribution of TRPC6 in the mouse retina by Western Blot with immunofluorescence double-labeling staining.(2)We examined the proliferation of astrocytes and apoptosis of retinal ganglion cells one to three months after STZ induction using immunofluorescent labeling.(3)To establish a diabetic model by intraperitoneal injection of streptozotocin(STZ),and then to detect retinal TRPC6 protein expression by Western Blot method one to three months after STZ induction.(4)The changes in astrocyte proliferation in each group were detected by vitreous injection of TRPC6 agonist,antagonist,and by immunofluorescence method with Western Blot.(5)The ganglion cells in the TRPC6 agonist,antagonist groups were labeled using immunofluorescence method,and the apoptosis of each group of ganglion cells was detected by counting.(6)The expression of pro-BDNF and Caspase-3 in the TRPC6 agonist and antagonist groups was examined by Western Blot to investigate the cause of retinal damage caused by TRPC6.Results:(1)TRPC6 was expressed in all layers of the mouse retina,and was significantly expressed in the GCL layer.(2)The diabetic model was successfully induced by intraperitoneal injection of STZ,and the blood glucose values of the modeling group were all greater than 11.1 m M.(3)The immunostaining results showed that astrocyte proliferation appeared at one month of STZ induction and ganglion cell apoptosis appeared at three months.(4)By detecting the retinal TRPC6 protein expression from one to three months after STZ induction,TRPC6 protein expression was found to be elevated at two months of STZ induction and significantly elevated at three months.(5)By injecting TRPC6 agonist and antagonist into the vitreous and examining the proliferation of astrocytes in the antagonist and agonist groups,TRPC6 antagonist was found to decrease glial cell proliferation and agonist to increase glial cell proliferation.(6)By immunostaining and cell counting,TRPC6 antagonists were found to decrease ganglion cell apoptosis and agonists to increase ganglion cell apoptosis.(7)Finally,retinal Caspase-3 and pro-BDNF protein expression was found to be decreased in the antagonist group and increased in the agonist group by Casepase-3protein expression at three months.Conclusion:In summary,TRPC6 was expressed in the inner retinal layer,outer retinal layer and ganglion cell layer of mice;STZ-induced diabetic mouse retina showed astrocyte proliferation at one month and ganglion cell apoptosis at three months.mice in the STZ group showed elevated TRPC6 and pro-BDNF proteins at two months,and by subsequent experiments,it was concluded that TRPC6 antagonists decreased glial cell proliferation and ganglion cell apoptosis,while TRPC6 agonists increased glial cell proliferation and ganglion cell apoptosis.Finally,the effect of TRPC6 on astrocytes and ganglion cells was found to be related to pro-BDNF and Casepase-3.