| Objective By means of cultured rabbit retinal pigment epithelium(retinal pigment epithelium,RPE)cells at different times of light stimulus,screening out the best illumination time points of rabbit RPE cells light damage model.To study the expression of apoptosis factor in rabbit RPE cells after light injury and the effect of intervention by Lycium barbarum polysaccharides(LBP)on the expression of apoptotic factors.To investigate the protective mechanism of LBP on light injury of rabbit RPE cells and its possible signal transduction pathways,and provide theoretical basis for the clinical application of LBP in the treatment of age-related macular degeneration and retinal light injury.Methods1.Rabbit RPE cells cultured in vitro as the research object,using three primary colors LED cold light lamp as light source,light intensity(16500±200)Lux,for different time(11h,16 h,24h)light stimulation,after the light were determined,MTT assay was used to detect the effect of different light stimulation time on the activity of rabbit RPE cells,flow cytometry was used to detect the effect of different stimulation time on the apoptosis of rabbit RPE cells.2.Preparation of high,medium and low safe concentrations of LBP medium,in the preventive observation experiment,LBP medium with different safe concentrations was pretreated 8h before irradiation,at the end of the light treatment with 0h,6h,12 h,24h for each corresponding index detection respectively;The therapeutic observation experiment was carried out by adding high,medium and low concentration of LBP medium after the end of light,and continue to culture in the incubator 6h,12 h,18h and 24 h to detect thecorresponding indexes.Preventive and therapeutic observation experiments were set up five groups,namely: normal group,light injury group,LBP low concentration group,LBP medium concentration group,LBP high concentration group.3.The microstructure changes of rabbit RPE cells after light injury were observed by transmission electron microscopy;Flow cytometry was used to detect the changes of intracellular free Ca2+ concentration in rabbit RPE cells after light injury and LBP prevention and treatment;The expression of Caspase-12 protein in rabbit RPE cells was detected by Western blot(Western blot)after light injury and LBP prevention and treatment.To investigate the molecular mechanism of LBP on light injury protection of rabbit RPE cells and the possible signal pathway.Results1.MTT assay results showed that: compared with no light group,rabbit RPE cells after11 h,16h,24 h light stimulation,OD values decreased,and the difference was statistically significant(F=80.691,P=0.000);Also,the difference between the different lighting groups was satistically significant(P<0.05),the longer the duration of the light,the more damage the rabbit RPE cells had,and the more pronounced the decrease in OD.2.Annexin V-FITC/PI double staining combined with flow cytometry results showed that:after continuous illumination of 11 h,16h and 24 h in rabbit RPE cells,the main form of cell damage was apoptosis,and the rate of apoptosis of rabbit RPE cells increased gradually with the extension of light stimulation time.Compared with no light group,the apoptosis rate increased in the light group 11 h,but the difference was not statistically significant(P>0.05),The rate of apoptosis in the light,16 h and 24 h groups increased significantly,and the difference was statistically significant(P<0.05).There was statistical significance between the groups of illumination(P<0.05).3.Transmission electron microscopy showed that: no light group of rabbit RPE cell surface has many microvilli,cell membrane integrity,abundant cytoplasmic organelles,visible to the mitochondria,rough endoplasmic reticulum,ribosome,nuclear chromatin distribution.The rabbit RPE cells in the 16 h illumination group showed typical morphological changes of apoptosis: cell volume reduction,Surface microvilli decreased or disappeared,the cell membrane structure is intact without breakage,cytosolic organelles decreased or disappeared,there were a large number of vacuoles,condensed cytoplasm,nuclear chromatin gathered together.Margination and dispersed,or broken into pieces.4.Flow cytometry was used to detect the changes of intracellular free Ca2+concentration in rabbit RPE cells,the results showed that:(1)Preventive observation showed that:(1)Comparison between different treatment groups at the same time:Compared with the normal group,the fluorescence intensity of Ca2+was significantly increased in the injured group(P<0.01).Compared with the light injury group,the fluorescence intensity of Ca2+ in the low,medium and high concentration group of LBP in 0h and 12 h decreased significantly,especially in the LBP high concentration group(P<0.01),6h,24 h phase only LBP high concentration group significantly inhibited the fluorescence intensity of Ca2+(P<0.05).LBP was compared between two groups with low,medium and high concentration:There was no significant difference between LBP low concentration group and LBP medium concentration group in each phase(P>0.05);The difference between the LBP low concentration group and the LBP high concentration group: 0h,6h and 12 h was statistically significant(P<0.05),and there was no significant difference in 24 h phase(P>0.05);The difference between LBP medium concentration group and LBP high concentration group: only 6h,12 h phase difference was statistically significant(P<0.05).(2)Comparison between different phases in the same treatment group:Each treatment group except 6h and 12 h changes in intracellular Ca2+fluorescence intensity is not obvious,others are as follows: with the time prolonging,the fluorescence intensity of Ca2+ showed a downward trend,especially in LBP high concentration group,24 h decreased most significantly(P<0.05).(2)Therapeutic observation showed that:(1)Comparison between different treatment groups at the same time:Compared with the normalgroup,the fluorescence intensity of Ca2+in the injured group was significantly increased(P<0.01).Compared with the light injury group,12 h,18h,24 h in LBP low,middle and high concentration group of intracellular Ca2+ fluorescence intensity decreased significantly,especially in the high concentration of LBP group was reduced significantly(P<0.01),6h Ca2+fluorescence intensity phase only LBP high concentration group cells significantly inhibited(P<0.05).LBP was compared between two groups with low,medium and high concentration:There was no significant difference in 6h concentration between LBP low concentration group and LBP medium group(P>0.05),12 h,18h and 24 h were statistically significant(P<0.05);The difference between LBP low concentration group and LBP high concentration group: each phase difference was statistically significant(P<0.05);The difference between LBP medium concentration group and LBP high concentration group:only 6h the phase was statistically significant(P<0.05).(2)Comparison between different phases in the same treatment group:In the light injury group,LBP low concentration group and LBP high concentration group:with the time prolonging,the fluorescence intensity of Ca2+ showed a downward trend,in which there was no significant difference between 18 h and 24h(P>0.05);LBP medium concentration group: compared with 6h,the fluorescence intensity of Ca2+ in 12 h,18h and 24 h were significantly decreased,the difference was statistically significant(P<0.05),but there was no significant difference between the 12 h,18h and 24 h groups(P>0.05).5.Western-blot method was used to detect the expression of Caspase-12 protein,the results showed that:(1)Preventive observation showed that:(1)Comparison between different treatment groups at the same time:Compared with the normal group,the expression of Caspase-12 protein was significantly increased in each phase of light injury group(P<0.01);Compared with the light injury group,the expression of LBP in both medium and high concentration group of Caspase-12 protein was significantly inhibited,especially in the high LBP concentration inhibited the most significant(P<0.01),12 h,24h phase LBP in thelow concentration group which effectively inhibited the expression of Caspase-12 protein(P<0.05);LBP was compared between two groups with low,medium and high concentration:The difference between LBP low concentration group and LBP medium concentration group: 6h was statistically significant(P<0.05),and there was no significant difference between 0h,12 h and 24h(P>0.05);The difference between LBP low concentration group and LBP high concentration group: each phase difference was statistically significant(P<0.05);The difference between LBP medium concentration group and LBP high concentration group: only 12 h,24h phase difference was statistically significant(P<0.05).(2)Comparison between different phases in the same treatment group:with the time prolonging,the expression of Caspase-12 protein in rabbit RPE cells showed a downward trend,especially in LBP high concentration group,24 h decreased most significantly(P<0.05).(2)Therapeutic observation showed that:(1)Comparison between different treatment groups at the same time:Compared with the normal group,the expression of Caspase-12 protein was significantly increased in each phase of light injury group(P<0.01);Compared with the light injury group,the expression of Caspase-12 protein in the low,medium and high concentration groups of LBP was significantly inhibited in 6h,18 h and 24 h,especially in the LBP high concentration inhibited the most significant(P<0.01),12 h phase only LBP high concentration group decreased significantly(P<0.05);LBP was compared between two groups with low,medium and high concentration:The difference between LBP low concentration group and LBP medium concentration group: there was significant difference in 12h(P<0.05),there were no significant difference in 6h,18 h and 24h(P>0.05);The difference between LBP low concentration group and LBP high concentration group: each phase difference was statistically significant(P<0.05);The difference between LBP concentration group and LBP high concentration group: 18 h and 24 h was statistically significant(P<0.05),and there was no significant difference between 6h and 12h(P>0.05).(2)Comparison between different phases in the same treatment group: in addition to light injury group 18 h and group 24 h,LBP lowconcentration 6h and 12 h had no significant difference(P>0.05),the other showed that,with the time prolonging,the expression of Caspase-12 protein in rabbit RPE cells showed a downward trend,especially in LBP high concentration group,24 h decreased most significantly(P<0.05).Conclusions1.Illumination intensity(16500±200)lux,illumination time 11 h,16h,24 h have different degrees of cell injury and apoptosis on rabbit RPE cells cultured in vitro.2.Illumination intensity(16500 ± 200)lux,and illumination time 16 h were the best conditions for the construction of the best light damage model.3.The microstructure of RPE cells cultured in vitro was changed after light stimulation.4.Light injury promoted the increase of intracellular free Ca2+ concentration and the expression of Caspase-12 protein factor.5.Lycium barbarum polysaccharides can protect rabbit RPE cells by inhibiting the concentration of free Ca2+ and the expression of Caspase-12 protein in rabbit RPE cells.6.The protective effect of LBP on light induced apoptosis in rabbit RPE cells may be achieved by inhibiting the endoplasmic reticulum signaling pathway. |
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