Epigallocatechin-3-gallate Inhibits Paraquat-induced Apoptosis In SK-N-SH Cells And Its Acting Mechanism

Objective:To study the epigallocatechin-3-gallate (EGCG) inhibits apoptosis of SK-N-SH cells induced by Paraquat(PQ) and its acting mechanism.Methods:SK-N-SH cells were cultured and were added with PQ (400μmol·L-1) for 72 hours to induce the model of cell apoptosis.The cultured cells were randomly divided into 6 groups:blank control group, paraquat control group(400μmol·L-1),3 EGCG (10,5 and 1μmol·L-1)+PQ (400μmol·L-1) groups respectively and vitamin E (10μmol·L-1)+PQ (400μmol·L-1) group. After treatment of SK-N-SH cells with VE and different concentration of EGCG for 2 hours respectively, paraquat (400μmol·L-1) was added to the cultures for 72 hours. Then MTT was used to determine the conditions of cells viability, and these cultured cells supernatant was taken to determine the leakage of LDH. The morphological change and rate of cell apoptosis were detected by Hoechst 33258 staining and flow cytometry, respectively. Then, mitochondrial membrane potential (MMP) was detected by Rhodamine 123 staining and expression of bcl-2 mRNA and bax mRNA were detected by RT-PCR. Furthermore, the activity of SOD, the contents of MDA and ROS were assayed separately.Results:1. Compared with blank group, different concentration of PQ(200,400,800, 1200,1600μmol·L-1) were added to cultured cells for 24hours,48hours and 72hours showed the decline significantly in dose-amd time-dependence.2. PQ(400μmol·L-1) were added to cultured cells for 72hours, the cell viability was declined significantly to 55.20% of normal group. Vitamin E (10μmol·L-1) group and 3 EGCG (10,5 and 1μmol·L-1) groups could elevate the cell viability to 60.17±2.82%,74.08±5.32%,70.22±2.27%,59.22±6.91%(P＜0.05, P<0.01)3. SK-N-SH cells were treated with PQ(400μmol·L-1) and the leakage of LDH was increased significantly (P＜0.01) compared with that of normal group. Following treatments of different concentration of EGCG(10,5 and 1μmol·L-1), the leakage of LDH was reduced (P＜0.05, P＜0.01) significantly.4. After Hoechst 33258 staining, the nuclei of normal cells appeared regular contours, round and large in size. After PQ (400μmol·L-1) for 72 hours, many cells showed an asymmetric and bright blue fluorescence.Cell membrane appeared breakage and vacuole, and induced apoptosis with a rate of occurrence up to 34.90%(P＜0.01, Compared with normal group). Treated with vitamin E(10μmol·L-1) and 3 EGCG (10,5 and 1μmol·L-1), the morphology of apoptosic nuclei was improvement and apoptosis rate was decreased (P<0.01) significantly.5. Flow cytometry showed that cells apoptosic rate of PQ treatment was higher than that of normal cells(P<0.01). Compared with PQ group, vitamin E (10μmol·L-1 and 3 EGCG (10,5 and 1μmol·L-1) could reduce the apoptotic rate(P<0.01).6. The collapse of MMP of SK-N-SH cells was observed with the probe rhodamine 123. Treated with PQ(400μmol·L-1) for 72 hours, the fluorescent intensity was reduced significantly(P<0.01). Vitamin E (10μmol·L-1) and 3 EGCG (10,5 and 1μmol·L-1) could reduced the decline of MMP caused by PQ significantly(P<0.05, P＜0.01).7. Compared with normal group, the expression of bcl-2 mRNA appeared weak and bax mRNA appeared stronger, and the volum of bcl-2 mRNA/bax mRNA was lower than that of normal group(P＜0.01). Vitamin E (10μmol·L-1) and 3 EGCG (10,5 and 1μmol·L-1) added to the cultured cells, the expression of bcl-2 mRNA and bax mRNA returned to normal levels.8. The activity of SOD was decreased and the contents of MDA and ROS were increased significantly(P＜0.01) treated with PQ. Vitamin E(10μmol·L-1)and 3 EGCG (10,5 and 1μmol·L-1) could elevated the activity of SOD and reduced the contents of MDA and ROS respectively(P＜0.01).Conclusions:1. The cultured SK-N-SH cells treated with different concentration of PQ (200 to 1600μmol·L1) for 72h can induce the damage and apoptosis change in dose-and time-dependent manner. 2. For the cells treated by PQ (400μmol-L1), EGCG can enhance the cell viability significantly, increse the MMP of cells and improve the cells condition in morphology, reduce the leakage of LDH and apoptosis rate, increase the activity of SOD and decrease the contents of MDA and ROS.3. EGCG can reduce the SK-N-SH cells apoptosic rate induced by PQ and enhance the mitochondrial's MMP, increase the expression of bcl-2 mRNA and reduce the expression of bax mRNA.These effects of EGCG may be concerned with enhancing the antioxidative ability, protecting the mitochondrial function and structure in normal condition, inhibiting the over expresstion of apoptosic gene in mitochondria.