Effect of Triptolide on Apoptosis and Oxidative Stress of Ovarian and Granulosa Cells in Rats
Objective:To investigate the effects of different concentrations of triptolide(TP)on oxidative stress and apoptosis in rat ovary and granulosa cells.Methods:A total of 32 female SD rats of SPF grade were randomly divided into 4 groups,including control group,TP low-dose group,TP medium-dose group and TP high-dose group,8 rats in each group.Rats were given TP intraperitoneal injection of 60,120,and 240μg/kg respectively in TP low,medium and high-dose groups,for 14 consecutive days.The body mass and ovarian wet weight were weighed.Ovarian index was calculated.Serum estradiol(E2),progesterone(P),follicle-stimulating hormone(FSH)and luteinizing hormone(LH)levels were measured by enzyme-linked immunosorbent assay(ELISA).Hematoxylin-eosin(HE)staining was used to observe the morphology of ovarian tissues,and biochemical kits were used to detect the levels of superoxide dismutase(SOD),catalase(CAT)and malondialde-hyde(MDA)in ovarian tissues.Dihydroethidium(DHE)staining was used to detect the levels of reactive oxygen species(ROS)in ovarian tissues,and Western blotting was used to detect the levels of aromatase(CYP19A1),B lymphocytoma-2(Bcl-2),Bcl-2-related X protein(Bax),active cysteine aspartate protease-3(Cle-caspase-3),nuclear factor erythroid lineage 2-associated factor 2(Nrf2)and heme oxygenase 1(HO-1)protein expression.KGN cells were divided into a control group,TP low-dose group,TP medium-dose group and TP high-dose group.KGN cells in TP low,medium,and high dose-groups were treated with TP of 20,40,and 60 nmol/L,respectively.Cell viability was detected by CCK8 assay,and cell supernatant E2 was detected by ELISA.Cell morphology was observed by microscopy,and DNA breakage was detected by TUNEL staining.Apoptosis rate was detected by flow cytometry,and cellular ROS level was detected by DCFH-DA probe.Western blotting was used to detect CYP19A1,Bcl-2,Bax,Cle-caspase-3,Nrf2 and HO-1 protein ex-pression.Results:In animal experiment,compared with the control group,the body mass,ovarian index,serum E2 and P levels were reduced in the TP medium and high-dose groups,while FSH and LH levels were increased.The atretic follicles increased and developing follicles decreased in the TP medium and high-dose groups.The SOD and CAT enzyme activity of the ovarian tissues was decreased,while the MDA content and ROS levels of the ovarian tissues increased in the TP medium and high-dose groups.The expression of CYP19A1,Bcl-2,Nrf2 and HO-1 expression decreased,while Bax and Cle-caspase-3 expression increased in the TP medium and high-dose groups.The differences were statistically significant(P＜0.05).In cell experiments,compared with the control group,the viability of KGN cells in the TP-treated groups was significantly decreased,and the supernatant E2 content of cells was reduced in the TP medium and high-dose groups.The cells were obviously wrinkled,rounded and smaller in size in the TP medium and high-dose group,and the number of TUNEL-positively expressing cells was increased in the TP medium and high-dose group.The rate of apoptosis and cellular ROS level was increased in the TP medium and high-dose group.The expression of CYP19A1,Bcl-2,Nrf2 and HO-1 was reduced,while the expression of Bax and Cle-caspase-3 increased in the TP medium and high-dose group.The differences were statistically significant(P＜0.05).Conclusion:TP can significantly cause ovarian toxicity,and the mechanism of action may be related to oxidative stress and apoptosis.
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