| Objective To explore the effects of 3-Methyladenine(3-MA)and starvation on the autophagy in,,as well as matrix metalloproteinase(MMP-2)expression by,human glaucomatous trabecular meshwork cells.Methods1.Human glaucomatous trabecular meshwork cells culture and identification:the trabecular meshwork tissues were cultured in vitro and passaged for 3 times.Then they were identified by microscope and immunocytochemistry.2.Cells were divided into three groups:group control,group 3-MA and group starvation.Group 3-MA were treated with different concentrations of 3-MA(2.5,5,10,20mmol/L),group starvation were grown in Earle ? s Balanced Salt Solution(EBSS)instead of normal media for different time(6h,12 h,18h,24 h,36h),the other were grown in normal media as control group.The inhibition rate of 3-MA and starvation for POAG trabecular meshwork cells were measured by MTT method.3.Monodansylcadaverine fluorescent staining was performed to detect the occurrence of autophagy.4.Cells were divided into three groups:group control,group 3-MA and group starvation.Group 3-MA were treated with different concentrations of 3-MA(1.25,2.5,5,10mmol/L),group starvation were intervened for different hours(3h,6h,9h,12h),extract RNA and protein respectively.RT-PCR was used to examine the difference of MMP-2,LC3-(40)(40)and Beclin1 m RNA expression,Western blot was used to examine the difference of MMP-2,LC3-(40)(40)and Beclin1 protein expression.Results1.Glaucomatous trabecular meshwork cells were successfully cultured and identified in vitro.2.3-MA and starvation both inhibited the proliferation of glaucomatous trabecular cells,the inhibition rate of cells was founded to have increased in a time-dependent and dose-dependent manner.The inhibition rate mount up obviously when the drug concentration was 20mmol/L in group 3-MA or starved for 18 h in group starvation.3.Monodansylcadaverine(MDC)fluorescent staining showed that compared to group control,the numbers of MDC-labeled cells in starvation group was increased,while decreased in group 3-MA,which implies that the autophagy formation was inhibited in 3-MA group and promoted in starvation group.4.Group 3-MA:Compared to group control,with the increase of the concentration,the expression of LC3-II and Beclin1 were declined gradually,while the expression of MMP-2 was upregulated.The expression of LC3-II m RNA and protein were declined especially when 3-MA at 5 mmol/L(P1=0.000,P2=0.046,P<0.05),the expression of Beclin1 m RNA and protein were declined especially when 3-MA at 5mmol/L(P1=0.000,P2=0.009,P<0.05),the expression of MMP-2 m RNA and protein were upregulated especially when 3-MA at 5 mmol/L(P1=0.037,P2=0.034,P<0.05).5.Group starvation:Compared to group control,with the prolongation of starvation time,the expression of LC3-II and Beclin1 were upregulated,while the expression of MMP-2 was decreased in group stravation.The expression of LC3-II m RNA and protein were upregulated especially when starve at 6h and 9h respectively(P1=0.045,P2=0.042,P<0.05),the expression of Beclin1 m RNA and protein were upregulated when starve at 6h(P1=0.021,P2=0.018,P<0.05),the expression of MMP-2 m RNA and protein were declined especially when starve at9 h and 6h respectively(P1=0.009,P2=0.001,P<0.05).Conclusions3-MA could inhibit the autophagy of glaucomatous trabecular meshwork cells,while increase the expression of MMP-2.Starvation exerted a promotive effect on the autophagy of glaucomatous trabecular meshwork cells,accompanied with the downregulation of MMP-2.It is speculated that autophagy is involved in the pathogenesis of POAG,and it might regulate the deposition of extracellular matrix and the outflow resistance through MMP-2. |
PDF Full Text Request