| ObjectiveTo observe the effects of different light intensity and time on human retinal pigment epithelial cells,and the cell morphology,mitochondrial structure,and antioxidant effect of different concentrations of LBP on light-induced human retinal pigment epithelial cells.Pigment epithelial cell apoptosis and its effect on the PI3 K / Akt / mTOR signaling pathway.Therefore,it was explored whether lycium barbarum polysaccharide can reduce the light-induced oxidative stress damage of human retinal pigment epithelial cells and its regulatory role in PI3 K / Akt / mTOR signaling pathway.Methods MTT method was used to detect the effects of different light intensities(8500 ±200)Lux,(12500 ± 200)Lux,and(16500 ± 200)Lux on ARPE-19 cells for 24 h and 36 h on ARPE-19 cell proliferation,respectively,and screen the best Light intensity and time;meanwhile,the effect of different concentrations of LBP(10mg/L,100mg/L)on the proliferation of ARPE-19 cells 24 h was tested by MTT method.The effect of different concentrations of LBP(10mg/L,100mg/L)on the morphology of illuminated ARPE-19 cells was observed through an inverted microscope,and the morphology of different concentrations of LBP(10mg/L,100mg/L)on illuminated ARPE-19 was observed with a mitochondrial green fluorescent probe.Effects of Cell Mitochondrial Morphology and Structure.Enzyme-linked immunosorbent assay was used to detect the contents of superoxide dismutase and malondialdehyde in light-treated ARPE-19 cells at different concentrations of LBP(10mg/L,100 mg/L).Flow cytometry was used to detect the effects of different concentrations of LBP(10mg/L,100mg/L)on the content of reactive oxygen free radicals and the apoptosis ratein ARPE-19 cells.Western blotting was used to detect PI3 K,p-PI3 K,Akt,p-Akt(Thr308),mTOR,and p-mTOR proteins in different concentrations of LBP(10mg/L,100mg/L)on light ARPE-19 cells.The effect of molecular expression.Results1.MTT method was used to detect the effect of different light intensities on the survival rate of ARPE-19 cells after 24 h and 36 h induction,and the comparison between different treatment groups in the same phase: compared with the blank control group,the cell survival rate of each light-induced damage model group was significantly reduced,especially at(16500±200)Lux,blank control group and light-induced damage model group have the remarkable difference in statistics(P<0.01),and the effect of light on the survival rate of ARPE-19 cells was intensity dependent.Comparison between the same treatment groups in different phases: the cell survival rate of each treatment group decreases with time,i.e.the effect of light onthe the survival rate of ARPE-19 cells is time-dependent.combining the above,the optimal light intensity for light-induced ARPE-19 is(16500±200)Lux,and the optimal time is 24 h.2.MTT assay was used to detect the influence of different concentrations of LBP on the survival rate of ARPE-19 cells induced by light for 24 h.compared with the blank control group,the survival rate of ARPE-19 cells in the light-induced damage model group was significantly reduced,blank control group and light-induced group have the remarkable difference in statistics(P<0.01).Compared with the light-induced damage model group,the cell survival rate of LBP 10mg/L and 100mg/L groups was significantly increased,light-induced damage model group and LBP 10mg/L and 100mg/L groups have the remarkable difference in statistics(P<0.01).And with the increase of LBP concentration,the survival rate ofARPE-19 cells induced by light increased significantly.3.Effect ofLBP on morphology of light-induced ARPE-19 cells The ARPE-19 cells in blank control group showed spindle-shaped adherent growth,clear boundary,uniformdistribution of pigment particles in cytoplasm,monolayer and mosaic arrangement during cell fusion growth,and good cell state.In the light-induced damage model group,the cells showed spindle-shaped changes,partially narrowed and rounded,with unclear boundaries,uneven distribution of pigment particles in the cells,loss of mosaic arrangement characteristics,increased cell debris,and poor cell status.The cell status of LBP 10mg/L and 100mg/L groups improved significantly.4.Morphological Observation of Mitochondrial Damage Induced by LBP on ARPE-19Cells;Mitochondria in blank control group are scattered around nucleus in cytoplasm and distributed in linear,tubular reticular or branched form;Mitochondria in light-induced damage model group were swollen and showed fine granular structure.Mitochondrial morphology ofLBP 10mg/L is mainly linear and tubular reticular,which is improved compared with light-induced damage model group.Mitochondrial morphology of cells in LBP 100mg/L group is mainly tubular reticular,which is significantly improved compared with light-induced damage model group.5.Flow cytometry was used to detect the effect ofLBP on the reactive oxygen species level of ARPE-19 cells induced by light.compared with the blank control group,the ROS level of ARPE-19 cells in the light-induced damage model group was significantly increased,blank control group and light-induced group have the remarkable difference in statistics(P<0.01),indicating that light can induce the ROS level in ARPE-19 cells to increase.Compared with the light-induced damage model group,the ROS level of cells in theLBP group of 10mg/L and 100mg/L was significantly reduced,light-induced damage model group and LBP 10mg/L and 100mg/L groups have the remarkable difference in statistics(P<0.01),indicating that LBP can reduce the ROS level of ARPE-19 cells after light-induced.Compared withLBP 10 mg/L,the ROS level in ARPE-19 cells inLBP 100mg/L group was decreased,LBP 10mg/L and 100mg/L group have the remarkable difference in statistics(P<0.01),which indicated that with the increase of LBP concentration,the ROS level in light-inducedARPE-19 cells was significantly decreased,andLBP had protective effect on light-induced ARPE-19 cells,and showed concentration dependence.6.Enzyme linked immunosorbent assay(ELISA)was used to detect the effect of LBP on the total SOD activity and MDA content in ARPE-19 cells induced by light.compared with the blank control group,MDA content in ARPE-19 cells in the light-induced damage model group was significantly increased,blank control group and light-induced group have the remarkable difference in statistics(P<0.01).Compared with the light-induced damage model group,MDA content in ARPE-19 cells in LBP 10mg/L and 100mg/L groups was significantly reduced,light-induced damage model group and LBP 10mg/L and 100mg/L groups have the remarkable difference in statistics(P<0.01).Compared with LBP 10 mg/L,MDA content in ARPE-19 cells decreased in LBP 100mg/L group,LBP 10mg/L and 100mg/L group have the remarkable difference in statistics(P<0.01).The results show that light can induce the MDA content in ARPE-19 cells to increase,while LBP can reduce the MDA content in ARPE-19 cells after light-induced.With the increase of LBP concentration,the MDA content in ARPE-19 cells induced by light decreases significantly.LBP has protective effect on ARPE-19 cells induced by light,which is concentration dependent.In addition,compared with the blank control group,SOD activity in ARPE-19 cells in the light-induced damage model group was decreased,blank control group and light-induced group have the remarkable difference in statistics(P<0.01).Compared with the light-induced damage model group,SOD activity in ARPE-19 cells in LBP 10mg/L and 100mg/L groups increased significantly,light-induced damage model group and LBP 10mg/L and 100mg/L groups have the remarkable difference in statistics(P<0.01).Compared with LBP 10 mg/L,SOD activity in ARPE-19 cells in LBP 100mg/L group decreased,LBP 10mg/L and 100mg/L group have the remarkable difference in statistics(P<0.01).The results show that light can induce the decrease of total SOD activity in ARPE-19 cells,while LBP can increase the total SOD activity in ARPE-19 cells after light-induced.With the increase of LBP concentration,MDAcontent in ARPE-19 cells induced by light decreases significantly.LBP has protective effect on ARPE-19 cells induced by light,and is concentration dependent.7.Flow cytometry was used to detect the effect of LBP on light-induced apoptosis of ARPE-19 cells.compared with the blank control group,the apoptosis rate of ARPE-19 cells in the light-induced damage model group were significantly increased,blank control group and light-induced group have the remarkable difference in statistics(P<0.01).Compared with the light-induced damage model group,the apoptosis rate of ARPE-19 cells in LBP 10mg/L and 100mg/L groups were significantly reduced,light-induced damage model group and LBP10mg/L and 100mg/L groups have the remarkable difference in statistics(P<0.01).Compared with LBP 10mg/L group,the apoptosis rate of ARPE-19 cells in LBP 100mg/L group decreased,LBP 10mg/L and 100mg/L group have the remarkable difference in statistics(P<0.01),indicating that light can promote the apoptosis of ARPE-19 cells,while the apoptosis rate of ARPE-19 cells gradually decreased with the increase of LBP concentration in each treatment group,and showed concentration dependence.8.Western blot method was used to detect the effect of each group on PI3K/Akt/mTOR signaling pathway.ARPE-19 cells were induced by 24 h,(16500±200)Lux light,The levels of phosphorylation of PI3 K,Akt(Thr308)and mTOR in cells were reduced,indicating that blank control group and light-induced damage model group have the remarkable difference in statistics(P<0.01).After treatment with Lycium barbarum polysaccharides at 10mg/L and100mg/L,the levels of phosphorylation of PI3 K,Akt(Thr308)and mTOR in cells increased,demonstrating that light-induced damage model group and LBP 10mg/L and 100mg/L groups have the remarkable difference in statistics(P<0.01),indicating that LBP can enhance the activity of PI3K/Akt /mTOR signaling pathway in ARPE-19 cells and protect the ARPE-19 cells from damage induced by light.Compared with the LBP 10mg/L group,the levels of PI3 K,Akt(Thr308)and mTOR phosphorylation in ARPE-19 cells of the Lycium barbarum polysaccharide 100mg/L group were significantly increased,LBP 10mg/L and 100mg/L grouphave the remarkable difference in statistics(P<0.01),indicating a high dose LBP can enhance the activity of PI3 K / Akt / mTOR signaling pathway in ARPE-19 cells.Conclusions1.24 h and 36 h of light can significantly reduce the survival rate of ARPE-19 cells.The best light intensity of light-induced ARPE-19 is(16500 ± 200)Lux,and the optimal time is24 h.2.Light can induce changes in the morphology and mitochondrial structure of ARPE-19 cells,and can cause oxidative stress damage in ARPE-19 cells.LBP can not only improve the morphology and mitochondrial structure of ARPE-19 cells,but also can counteract light through antioxidant damage-induced ARPE-19 cells play a protective role.3.LBP can significantly reduce the apoptosis rate of light-induced ARPE-19 cells after oxidative stress injury,and the apoptosis rate decreases with increasing concentration.In addition,LBP may play a protective role against light-induced ARPE-19 cell damage by activating the PI3 K / Akt / mTOR signaling pathway. |
PDF Full Text Request