| Objective:To investigate the mechanism of ultraviolet B(UVB)-induced apoptosis in human lens epithelial cells(HLECs)and the protective effect of green tea extract epigallocatechin gallate(EGCG),and to elucidate the mechanism of mitogen-activated protein kinases(p38MAPK)signaling pathway,oxidative damage,endoplasmic reticulum stress(ERS)in UVB-induced apoptosis of HLEC cells and the protective effect of EGCG.Methods:This experiment is divided into three parts.Part I:HLEC cells were cultured with 1640 medium supplemented with 10%fetal bovine serum.To screen a half lethal dose(LD50)of UVB radiation and a safe and effective concentration of EGCG and to verify the toxicity of 10 μM p38 inhibitor SB203580 on HLEC cells.The human lens epithelial cell line HLE B-3 was used as the research model,and the UVB irradation with a central irradiation intensity of 0.2 mW/cm2 was used in the present study.The irradiation doses were 10,20,40 and 60 mJ/cm2,respectively.The HLEB-3 cells were irradiated and cultured after UVB irradiation.Different EGCG concentrations(0,10,20,40,60,80,90,100,120 and 150 μg/ml,respectively)were used.Cells were cultured in 1640 medium containing 10%fetal bovine serum for 24h and 48h,respectively.Meanwhile,the cells were cultured with 10 μM of SB203580 solution which dissolved in 1640 medium supplemented with 10%fetal bovine serum for 24 hours and 48 hours,respectively.Morphological changes of cells after UVB irradiation were observed under inverted phase contrast fluorescence microscope.CCK-8 was used to detect the cell viability after different treatments including UVB irradiation,different concentrations of EGCG,and 10μM of SB203580 solution.Part II:To explore the mechanism of UVB-induced apoptosis in human lens epithelial cells based on p38MAPK signaling pathway.The experiment was divided into normal control group,SB203580 group,UVB group and UVB+SB203580 group.Among them,the normal control group and SB203580 group had no UVB irradiation,while SB203580 group and UVB+SB203580 were pretreated with 10%fetal bovine serum 1640 medium with 10 μM of SB203580 for 2 h.For UVB group and UVB+SB203580 group,the UVB irradiation dose was 40 mJ/cm2,and further cultured for 6h,24h after UVB irradiation.After that,cells were collected for relevant analysis.CCK-8 was used to detect the cell viability of each experimental group at 6h and 24h after UVB irradiation.Flow cytometry was used to detect the changes of reactive oxygen species(ROS),mitochondrial membrane potential(ΔΨm)and apoptosis after UVB irradiation.Moreover,the changes of intracellular p38MAPK,phospho-p38,caspase-3,C/EBP homologous protein(CHOP),glucose regulated protein 78(GRP78),calreticulin(CRT)after UVB irradiation were further investigated by real-time fluorescence quantitative PCR(Q-PCR)and Western Blot.Part III:Study on the mechanism of EGCG inhibiting UVB-induced apoptosis of HLECs by blocking P38MAPK signaling pathway.The experiment was divided into normal control group,EGCG low concentration group,EGCG high concentration group,UVB group,UVB+EGCG low concentration group and UVB+EGCG high concentration group.The low concentration group of EGCG and the low concentration group of UVB+EGCG were first pretreated with 20 μg/ml EGCG dissolved in 1640 medium containing 10%fetal bovine serumfor 2 hours;the high concentration group of EGCG and the high concentration group of UVB+EGCG were pretreated with 1640 medium containing 80 μg/ml EGCG for 2 hours before UVB irradiation;the normal control group,the low concentration group of EGCG and the high concentration group of EGCG were not exposed to UVB irradiation.Ultraviolet B irradiation(40 mJ/cm2)was used to irradiate other groups.Low concentration group plus UVB and the high concentration group plus UVB+EGCG were exposed to UVB irradiation.After irradiation,the cells were cultured and collected at designated time points for different analysis.Cell morphological changes were observed and recorded under inverted phase contrast fluorescence microscopy;CCK-8 was used to detect the cell viability for each experimental group after UVB irradiation;ROS level,mitochondrial membrane potential(ΔΨm)changes and cell apoptosis were detected by flow cytometry after UVB irradiation.Apoptosis;Q-PCR and Western Blot analyses were used to detect the changes of mRNA and protein expression of p38,p-p38,Caspase-3,CHOP,GRP78 and CRT molecules in HLE B-3 cells after UVB irradiation.Results:The first part1.The morphology and quantity of HLEB-3 cells changed significantly after UVB irradiation,and this change gradually became obvious with the increase of irradiation dose.2.After normal HLEB-3 cells were irradiated by UVB,the cell survival rate decreased,and there was dose-time dependent(P<0.01).The cell survival rate was about 55%when the irradiation dose was 40mJ/cm2 for 6h,and 40mJ/cm2 was used as the experimental irradiation dose in our experiment.3.EGCG between 0-80 μg/ml had no obvious toxic effect on HLEB-3 cell viability.4.10μM SB203580 had no significant toxicity on HLEB-3 cell viability.The second part1.Changes of cell survival rate and apoptosis:UVB irradiation can reduce the survival rate of HLEB-3 cells(P<0.01).The proportion of early apoptotic cells and late apoptotic and/or necrotic cells increased under UVB irradiation(P<0.01),After treatment with p38 blocker SB203580,the survival rate of cells and the percentage of apoptotic cells were increased(P<0.01).2.Intracellular ROS production:Compared with the normal control group,ROS level increased significantly after UVB irradiation(P<0.01),whereas ROS production within HLEB-3 cells decreased after SB203580 intervention(P<0.01).3.Changes of mitochondrial membrane potential(ΔΨm):UVB irradiation resulted in damage of mitochondrial function in HLEB-3 cells,and ΔΨm decreased sharply(P<0.01);p38 blocker SB203580 could significantly reduce mitochondrial function damage and restored the ΔΨm(P<0.01).4.Caspase-3 expression:Caspase-3 expression increased sharply after UVB irradiation,and EGCG intervention could significantly reduce the Caspase-3 expression level(P<0.01).These results suggest that the elevated ROS in HLEB-3 cells induced apoptosis after UVB irradiation,which is one of the main mechanisms of cataract induced by UVB.Inhibiting p38MAPK signaling pathway can significantly reduce the apoptosis HLECS cells induced by ROS under UVB,thus decreasing Caspase-3 can protect HLEB-3 cells from cell damage induced by UVB irradiation.5.The expression of p38 signaling pathway related molecules:UVB irradiation can activate p38 signaling pathway in HLEB-3 cells,increase the expressions of both p38,and phosphorylated p38 protein(both P<0.01).SB203580 could inhibit the phosphorylation of p38 protein.These results showed that UVB irradiation could activate p38 signaling pathway and induce apoptosis of HLEB-3 cells,indicating the important role of p38 activation in UVB-induced apoptosis.6.Expression of endoplasmic reticulum stress markers:UVB irradiation activates endoplasmic reticulum stress response in HLEB-3 cells and induces apoptosis.The main markers of endoplasmic reticulum stress were high expression of GRP78,CHOP and CRT(P<0.01).SB203580 could reduce the expression of GRP78,CHOP and CRT(P<0.01),inhibit the endoplasmic reticulum stress induced by UVB,and retard cell apoptosis.The results also showed that the activation of P38 signaling pathway occurred in the upstream of endoplasmic reticulum stress.The third part:1.Cell morphological changes:After UVB irradiation,HLEB-3 cells showed abnormal changes in morphology and number.EGCG intervention could effectively improve the abnormal changes in morphology and number of HLEB-3 cells,and maintain the original characteristics of epithelial cells in a concentration-dependent manner.These results suggest that EGCG has protective effect on HLECS cells against UVB irradiation.2.Changes in cell survival rate:EGCG increased the survival rate of HLEB-3 cells after UVB irradiation in a dose-dependent manner(P<0.05),suggesting that EGCG intervention could restore the viability of HLEB-3 cells after UVB irradiation.3.Apoptosis:Compared with the normal control group,the proportion of early apoptotic cells and late apoptotic and/or necrotic cells increased in HLEB-3 cells after UVB irradiation(P=0.001,0.000).EGCG intervention could significantly reduce the apoptosis of HLEB-3 cells induced by UVB(P<0.01).Both high concentration and low concentration of EGCG could reduce the proportion of early apoptotic cells,and there was no significant difference between them(P=0.094).4.Intracellular ROS production:Compared with the normal control group,ROS level increased significantly after UVB irradiation(P<0.01).EGCG intervention could significantly alleviate the increase of ROS in HLEB-3 cells after UVB irradiation(P<0.01),and there was a concentration-dependent manner.5.Changes of mitochondrial membrane potential(ΔΨm):Compared with the normal control group,the level of ΔΨm in HLEB-3 cells decreased significantly after UVB irradiation(P<0.01).EGCG intervention could reduce mitochondrial dysfunction and decrease the level of ΔΨm(P<0.01),was accompanied by a concentration-dependent manner.These results indicate that EGCG has strong ROS-quenching ability,blocking the initial and direct cell events in UVB-mediated oxidative damage,and alleviating the apoptosis induced by UVB-mediated oxidative damage.6.Changes of Caspase-3 expression in cells:The expression of Caspase-3 increased sharply after UVB irradiation,and the expression of Caspase-3 decreased significantly after EGCG intervention(P<0.01).7.Expression of related molecules in p38 signaling pathway:Compared with the normal control group,UVB irradiation activated the P38 pathway,increased the phosphorylation level of p38,and enhanced the expression of p38 and p-p38 in cells(P<0.01).EGCG intervention blocked the activation of p38 in HLEB-3 cells induced by UV-B,and decreased the phosphorylation level of p38(P<0.01).These results indicated that the activation of p38 signaling pathway activated under UV-B irradiation.EGCG could block the activation of p38 signaling pathway.8.Expression of endoplasmic reticulum stress markers:ROS induced by UVB irradiation can lead to endoplasmic reticulum stress and consequent apoptosis.Compared with normal control group,UVB irradiation activated endoplasmic reticulum stress in HLEB-3 cells,increased GRP78 and CHOP expression,whereas EGCG intervention could reduce GRP78 and CHOP expression levels(P<0.01).These results suggest that EGCG can alleviate endoplasmic reticulum stress in HLEB-3 cells under UVB irradiation,suggesting that both p38 pathway and the activation of endoplasmic reticulum stress activated after UVB irradiation.EGCG can inhibit the activation of p38 pathway and endoplasmic reticulum stress,thus inhibiting the apoptosis induced by UVB.Conclusions:The first part1.After UVB irradiation,the morphology and number of HLEB-3 cells changed abnormally,the survival rate of HLEB-3 cells decreased and it was dose-dependent with UVB irradiation,which is one of the main mechanisms of cataract under UVB irradiation.2.EGCG concentration between 0 and 80μg/ml had no apparent cytotoxic effect on HLE B-3 cells.The second part1.Excessive UVB irradiation can lead to the decrease of cell viability and apoptosis of HLEB-3 cells.2.ROS produced by UVB irradiation mediates p38 activation,mitochondrial damage,endoplasmic reticulum stress,leading to apoptosis of HLEB-3 cells.3.Inhibition of p38MAPK signaling pathway can reduce ROS,mitochondrial damage and HLECS cell apoptosis induced by UVB,and protect HLEB-3 cells from UVB-induced cell damage and apoptosis.4.The activation of p38MAPK signaling pathway induced by UVB irradiation on HLEB-3 cells occurs in upstream of endoplasmic reticulum stress.Blocking p38MAPK signaling pathway can inhibit endoplasmic reticulum stress.5.p38 MAPK plays an important role in UVB-induced apoptosis in HLECS and may be an important target of cataract treatment.The third part1.EGCG exhibits a very significant ROS quenching activity,which can block mitochondrial damage,p38MAPK pathway and endoplasmic reticulum stress,thereby inhibiting UVB-induced apoptosis of HLECs cells,thus inhibiting the apoptosis induced by UVB.2.EGCG plays an important role in protecting HLEB-3 cells against oxidative stress and apoptosis induced by UVB.These findings may provide a theoretical basis for EGCG to become an effective drug for the prevention and treatment of cataract protection in clinical practice. |
