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目的:探讨黄芩素对雷公藤甲素诱导的大鼠卵巢颗粒细胞凋亡的保护作用。方法:收集大鼠卵巢颗粒细胞,经原代培养24 h后,用雷公藤甲素损伤大鼠卵巢颗粒细胞;干预组添加终浓度分别为50、100、200μmol/L的黄芩素,对照组添加相同体积的二甲基亚砜(DMSO)溶液,继续培养24 h(或以上)。采用MTT法测定卵巢颗粒细胞活力,流式细胞术Annexin V/PI双染色检测细胞凋亡,Western blot法检测与细胞凋亡相关蛋白的表达情况;测定各组卵巢颗粒细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)含量,评价抗氧化能力。结果:雷公藤甲素可显著降低大鼠卵巢颗粒细胞活力,诱导细胞凋亡,并使SOD和GSH-Px活性降低,MDA含量增加;雷公藤甲素处理细胞的同时添加黄芩素干预后,卵巢颗粒细胞相对活力增加,细胞凋亡减弱,胞内SOD和GSH-Px活性升高,MDA含量降低。Western blot结果显示,经过雷公藤甲素处理,卵巢颗粒细胞中促凋亡蛋白Bax表达量显著上升,抗凋亡蛋白Bcl-2表达量明显降低;黄芩素干预后可以显著下调Bax表达量,上调Bcl-2表达量。结论:黄芩素可明显改变卵巢颗粒细胞的抗凋亡及抗氧化能力,对雷公藤甲素诱导的细胞凋亡起保护作用。
Objective: To investigate the protective effect of baicalein on apoptosis of rat ovarian granulosa cells induced by triptolide. Methods: Ovarian granulosa cells were harvested from rat ovaries. After primary culture for 24 h, rat ovarial granulosa cells were injured by triptolide. The intervention group was given baicalein at the final concentrations of 50, 100 and 200 μmol / L, and the control group The same volume of dimethyl sulfoxide (DMSO) solution was added, and the culture was continued for 24 h (or above). The viability of ovarian granulosa cells was determined by MTT assay. The apoptosis of ovarian granulosa cells was detected by flow cytometry with Annexin V / PI double staining. Western blot was used to detect the expression of apoptosis related proteins. The levels of superoxide dismutase SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured to evaluate the antioxidant capacity. Results: Triptolide significantly decreased the vitality of ovarian granulosa cells, induced apoptosis, decreased the activities of SOD and GSH-Px, and increased the content of MDA. After treatment with triptolide and baicalein, ovarian Granulosa cells increased relative activity, decreased apoptosis, intracellular SOD and GSH-Px activity, MDA content decreased. Western blot results showed that after triptolide treatment, the expression of Bax protein in ovarian granulosa cells increased significantly and the expression of anti-apoptotic protein Bcl-2 decreased significantly; baicalein significantly down-regulated the expression of Bax and up-regulated Bcl-2 expression. CONCLUSION: Baicalein can obviously change the anti-apoptosis and anti-oxidation ability of ovarian granulosa cells and protect the apoptosis induced by triptolide.