| Objective 1.To explore the effect of autophagy on ARPE-19 cells in the retinal photo-induced injuries model mediated by different light durations,and the effect of 3MA intervention on photo-induced autophagy in ARPE-19 cells.2.Based on the PI3K/AKT/mTOR signaling pathway,to explore the interventional effect and mechanism of LBP on autophagy.Methods The ARPE-19 cells cultured in vitro were randomly divided into: control group,model group,3MA group,LBP low-dose group and LBP high-dose group.The ARPE-19 cells were exposed to 16500(±500)lux light for 12 h,18h and24 h.Transmission electron microscope was used to observe the changes of cell ultrastructure before and after photo-induced.The activity and apoptosis of ARPE-19 cells in each group were detected by CCK8 and flow cytometry(annexinv/pi double staining).The morphology and fluorescence intensity of autophagic lysosomes were analyzed qualitatively and quantitatively by laser scanning confocal microscope combined with inverted fluorescence microscope.Immunofluorescence method was used to detect the staining degree of LC3Ⅱ,P62 before and after photo-induced,and after intervention of different concentrations of LBP.The expression of Beclin1,LC3,p62,PI3 K,p-mTOR,mTOR,p-Akt and Akt were detected by Westernblot method.Take PI3 K inhibitor(3MA or 3methyladenine,autophagy inhibitor)combined with Western blot method to detect the expression of pathway-related proteins and the level of autophagy.The autophagy activity of ARPE-19 cells was further studied under different light conditions.Based on the signal pathway of PI3K/AKT/mTOR,the effects of LBP on autophagy of ARPE-19 cells were studied.Results 1.CCK8 method was used to detect the changes of ARPE-19 cell survival rate induced by different time long light.The illumination time was 12 h,18h and 24 h,compare each time period,the cell survival rate of the model group after photo-induced injuries was significantly lower than that of the control group(P<0.01).The cell survival rate of LBP low and high dose groups after photo-induced injuries was higher than that of the model group(P<0.01).The cell survival rate of the high-dose LBP group was higher than that of the low-dose LBP group(P<0.01).The illumination time was 12 h,18h and 24 h,compared different times,the cell survival rate of the control group did not change significantly(P>0.05),the cell survival rate of the model group,LBP low and high dose groups after light damage decreased with the prolonged light time(P <0.05).These results indicated that LBP could protect ARPE-19 cells against light damage,and the cell survival rate was positively correlated with LBP concentration.ARPE-19 cells were damaged by different length of time illumination,and the cell survival rate was negatively correlated with the duration of illumination.2.Flow cytometry was used to detect the changes of apoptosis rate of ARPE-19 cells induced by different duration of illumination.Experiment 1: The illumination time was12 h,18h and 24 h,compare each time period,the apoptosis rate of model group was significantly higher than that of control group(P < 0.01).The apoptosis rate of3 MA group was higher than that of model group after 12 hours of photo-induced injuries(P < 0.01),and lower than that of model group after 18 hours and 24 hours of photo-induced injuries.The illumination time was 12 h,18h and 24 h,compared different times.There was no significant difference in the apoptosis rate of the control group with the prolongation of light time(P > 0.05).The apoptosis rate of the model group increased with the prolongation of light time(P < 0.05),and the apoptosis rate of the 3MA group decreased with the prolongation of light time(P< 0.05).Experiment 2:The illumination time was 12 h,18h and 24 h,compare each time period,the cell apoptosis rate of model group was significantly higher than that of LBP low-dose group,LBP high-dose group and control group(P < 0.01),and the cell apoptosis rate of LBP high-dose group was lower than that of LBP low-dose group(P < 0.01).The illumination time was 12 h,18h and 24 h,compared different times.There was no significant difference in apoptosis rate between control groups(P>0.05),and the apoptosis rate of LBP low-dose group,high-dose group and model group increased with the prolongation of light time(except for the comparison of 12 h and 18 h in low dose group,P>0.05,comparison between the remaining groups P<0.01).The above results show that,(1)3MA could promote the apoptosis of ARPE-19 cells after 12 h illumination,while 3MA could inhibit the apoptosis of ARPE-19 cells after 18 h and 24 h illumination.(2)LBP could inhibit the apoptosis of ARPE-19 cells induced by light,and its ability to inhibit apoptosis was positively correlated with the concentration of LBP the apoptosis rate of each group(except the control group)increased with the extension of light time.3.The ultrastructure of ARPE-19 cells was observed by transmission electron microscope.In the model group,typical double-membrane autophagosomes,single membranous autophagy lysosomes and autophagy characteristics were found.In the control group,autophagosomes and autophagy lysosomes were rare,the organelles were clear,the mitochondrial crests were continuous,the endoplasmic reticulum had not been expanded,and the Golgi apparatus was visible.The above results indicate that 16500(±500)lux visible light irradiation can induce autophagy in ARPE-19 cells.4.Observation of autophagy lysosome morphology with laser scanning confocal microscope.The illumination time was 12 h,18h and 24 h,compare each time period,the cells of the control group were fusiform or long fusiform,some of the cells of the 3MA group were elliptical and the intercellular space was widened,and the cell morphology of the model group was round or ellipse and the autophagy-lysosome fluorescence brightness increased.The illumination time was12 h,18h and 24 h,compared different times,the number of red autophagy lysosomal particles in the model group increased with the prolonged light time,and there was no significant difference between the control group and the 3MA group.The above results indicate that 16500(±500)lux visible light irradiation can induce autophagy in ARPE-19 cells,and the autophagy activity increases with the prolonging of light.5.Analyze the fluorescence intensity of autophagy-lysosomes using an inverted fluorescence microscope.The illumination time was 12 h,18h and24 h,compare each time period,the autophagic lysosomal fluorescence intensity of ARPE-19 cells in the model group was significantly higher than that in the control group(P<0.01),and the fluorescence intensity of the cells in the 3MA group was between the control group and the model group(P< 0.01).The illumination time was 12 h,18h and 24 h,compared different times,there was no significant difference in the fluorescence intensity of the control group(P>0.05),and the fluorescence intensity of the model group increased with the prolonged illumination time(P < 0.01).The fluorescence brightness of the 3MA group increased with the prolonged illumination time(except for the comparison of 12 h and 18 h,P>0.05,comparison between the remaining groups P<0.01).The above results show that ARPE-19 cells have enhanced autophagy activity under light induction,and 3MA may inhibit the production of autophagic lysosomes in A-RPE19 cells under light induction.6.Use cell immunofluorescence to detect the staining of LC3Ⅱ and P62.Staining of LC3Ⅱ.The illumination time was 12 h,18h and 24 h,compare each time period,the degree of LC3Ⅱ staining in the model group was deeper than that of the control group and the low and high dose groups of LBP(except for the low and high dose groups of LBP at 18 h and 24 h,P>0.05,comparison between the remaining groups P<0.01).The illumination time was 12 h,18h and 24 h,compared different times,there was no significant difference in the degree of LC3Ⅱ staining in the control group(P>0.05).There was no significant difference in the degree of LC3Ⅱ staining in the model group after 12 h and 18 h of light(P>0.05).The degree of LC3Ⅱ staining among the other groups in the model group deepened with the prolonging of the light(P<0.01).The staining degree of LC3Ⅱ of LBP low and high-dose groups increased with the prolonging of light time(P<0.01).(2)P62 staining condition.The illumination time was 12 h,18h and24 h,compare each time period,the P62 staining degree of LBP low-dose and high-dose group and control group was deeper than that of the model group(P<0.01),and the P62 staining degree of LBP high-dose group was deeper than that of LBP low-dose group(P<0.01).The illumination time was 12 h,18h and24 h,compared different times,there was no significant change in the degree of P62 staining in the control group(P>0.05).The degree of P62 staining in the model group,low-dose and high-dose LBP groups all became lighter with the prolonged light time(the comparison of 12 h and 18 h in low-dose LBP group and 18 h and24h in high-dose LBP group P > 0.05,comparison between the remaining groups P<0.01).The results showed that long-term illumination could induce excessive autophagy of ARPE-19 cells,and LBP could inhibit the autophagy of ARPE-19 cells after illuminationt induction to help the cells cope with photo-induced injuries,and its ability to inhibit cell autophagy is positively correlated with the concentration of LBP.7.Western blot method was used to detect the expression of autophagy-related proteins and the ratio of LC3Ⅱ/LC3Ⅰ.Experiment 1: The illumination time was 12 h,18h and 24 h,compare each time period,the protein expression of Beclin1 and LC3Ⅱ and the ratio of LC3Ⅱ/LC3Ⅰ in the model group were higher than those in the control groups(P<0.01).The expression of P62 protein in the model group was lower than that in the control group(P<0.01).The expression of Beclin1 protein and the ratio of LC3Ⅱ/LC3Ⅰ in the 3MA group were lower than those in the model group(P<0.01).The expression of P62 protein in 3MA group was higher than that in model group(P < 0.01).The illumination time was 12 h,18h and 24 h,compared different times,the expression of Beclin1 protein and the ratio of LC3Ⅱ/LC3Ⅰ,in control group there was no significant difference.In model group and 3MA group,the expression of Beclin1 protein and the ratio of LC3Ⅱ/LC3Ⅰ increased with the prolonged light time(P<0.05),and the expression of P62 protein decreased with the prolonged light time(P < 0.05).The results showed that the autophagy activity of the cells was enhanced with the time of illumination,and the autophagy activity was decreased after 3MA intervention.Experiment 2 : The illumination time was 12 h,18h and24 h,compare each time period,the expression of Beclin1,LC3 Ⅱ and LC3 Ⅱ /LC3 Ⅰ ratio in model group were higher than those in control group(P <0.01).The expression of P62 protein in model group was significantly lower than that of the control group(P<0.01).The protein expression of Beclin1(except for the low-dose group of LBP compared with the model group,at 24 h,P>0.05,comparison between the remaining groups P<0.01)and LC3 Ⅱ and the ratio of LC3 Ⅱ / LC3 Ⅰ in LBP low-dose and high-dose groups were lower than those in model group(P<0.01).The illumination time was 12 h,18h and 24 h,compared different times,In the model group,the expression of Beclin1 protein(the exception of 18 h and 24 h P>0.05,the other groups P<0.05)and the ratio of LC3 Ⅱ/ LC3 Ⅰ(P<0.01)increased with the extension of light time,the expression of p62 protein decreased with the prolongation of illumination time(P<0.01).In LBP low-dose and high-dose groups,the expression of Beclin1 protein and the ratio of LC3 Ⅱ / LC3 Ⅰ increased with the prolongation of light time(P< 0.05),and the expression of p62 protein decreased with the prolongation of illumination time(the comparison of 18 h and 24 h in the LBP low-dose group,P>0.05,the comparison among the other groups P<0.05).There was no significant difference in Beclin1,P62 protein expression and LC3Ⅱ/LC3Ⅰ ratio in the control group(P>0.05).The above results indicate that 16500(±500)lux visible light irradiation can induce autophagy and increase autophagy flux in ARPE-19 cells,LBP can inhibit photo-induced autophagy in ARPE-19 cells,and its ability to inhibit autophagy is positively correlated with the concentration of LBP.8.Western blot method was used to detect the influence of each group on the PI3K/Akt/mTOR signal pathway.The illumination time was 12 h,18h and24 h,compare each time period,the expression of p-mTOR,p-Akt,PI3 K and the ratio of p-mTOR / mTOR,p-Akt / Akt in the model group were lower than those in the control group(P < 0.01),the expression of PI3 K,p-mTOR,p-Akt and the ratio of p-mTOR / mTOR,p-Akt / Akt in LBP low and high dose groups were higher than those in model group(P < 0.05).These results suggest that LBP may promote the phosphorylation of mTOR and Akt proteins in a concentration dependent manner,thus activating PI3 K / Akt / mTOR signaling pathway,inhibiting the expression of Beclin1 and LC3 Ⅱ proteins,and reducing autophagy level of ARPE-19 cells.Conclusion At 16500(±500)lux light intensity can induce autophagy in ARPE-19 cells,and the autophagy activity increases with the prolonged light time.The autophagy level of ARPE-19 cells after 12 h of light can resist light damage and reduce the rate of apoptosis,while the rate of apoptosis increased after the intervention of 3MA.The autophagy level of ARPE-19 cells under 18 h of light can resist light damage and reduce the rate of apoptosis.The rate of apoptosis is slightly lower after the intervention of 3MA.After 24 h of light,ARPE-19 cell autophagy activity exceeded its normal regulation range,and the apoptosis rate increased,The use of autophagy inhibitor 3MA can inhibit light-induced excessive autophagy of ARPE-19 cells,reduce the rate of apoptosis,and protect cells against light damage. |
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