| Objective:This study was to research the mechanism of donepezil in the retina of the photoreceptor cells apoptosis induced by MNU.Materials And Methods:Male C57BL/6J mice were intraperitoneal injected with MNU(60 mg/kg),and put to death in 1 day,3 day,5 day,7 day,then take the eye for HE staining to observe layers of retina.Mice were intravitreal injected with Hsp inhibitor I and underwent oral gavage with donepezil 10mg/kg for 3 consecutive days,then immunofluorescence was performed to observe the expression of Hsp70 and positioning.Mice were administered donepezil with different dose(1、5、10mg/kg)for 3 day by oral gavage,and different time(1day,3day,5day),then take the eye for western blot to demonstrated that donepezil induces Hsp70 expression in a time-and dose-dependent manner.In addition,these eyes were measured by western blot after mice were intravitreal injected with Hsp inhibitor I and underwent oral gavage with Bcl-2 inhibitor to detect the expression of Hsp70 and Bcl-2,at the same time by using flow cytometry to detect the situation of the photoreceptor cells apoptosis.Results:1.HE staining found that ONL significantly reduced subsequent to intraperitoneal injection of MNU,the ONL was significantly reduced in thickness by day 3 and was further reduced by day 7 the peak in the 3 day,and was further reduced by day 7,the OPL was significantly thinner on days 1,3,5 and 7,whereas the INL became significantly thinner between 5 and 7 day,however no alterations in the ganglion cell layer were observed following MNU treatment for 7 day.2.Immunofluorescence test found that the expression of Hsp70 was faintlyobserved in the retinal IS and ONL in the control group.However,strong Hsp70 immunoreactivity was observed after donepezil pretreatment for 3 day,and the expression of Hsp70 was significantly reduced subsequent to administration of HSP inhibitor I.3.western blot analysis found that the expression of Hsp70 was significantly increased at donepezil(5,10mg/kg)group,as compared with the control(0mg/kg).The peak of Hsp70 protein levels in 3 day when donepezil(3,5 day)group compared with control group(0 day).4.Western blot demonstrated that the protein expression levels of Hsp70 and Bcl-2 were increased following pretreatment with donepezil,and pretreatment with Hsp inhibitor I resulted in a significant reduction in the expression levels of Hsp70 and Bcl-2.In addition,the Bcl-2 inhibitor could only reduce the expression levels of Bcl-2,whereas the expression levels of Hsp70 were notsignificantly altered(P<0.01).5.HE staining demonstrated that the ONL was significantly thicker in donepezil plus MNU group,as compared with MNU group.However,the ONL was significantly thinner in Hsp inhibitor I plus donepezil plus MNU group.Annexin V-PE/7-AAD found that the apoptotic rate in the control group was 6.4±4.1%,MNU group was 43.2±7.4%,donepezil plus MNU group was 29.4±4.1%,Hsp inhibitor I plus donepezil plus MNU group was 40.6±2.3%,P<0.01.Conclusions : 1.Intraperitoneal treatment with MNU induces loss of photoreceptor cells.2.Donepezil induces Hsp70 expression in a time-and dose-dependent manner.In addition,donepezil can up-regulate the expression of Bcl-2.3.Donepezil prevents photoreceptor cell death caused by MNU,its protection mechanism may be through the Bcl-2,and Hsp70 may be an important factor for inhibiting apoptosis via upregulating the expression of Bcl-2. |
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