本文探讨哈蟆油含药血清对H2O2致大鼠卵巢颗粒细胞氧化应激损伤的保护作用与分子机制.将大鼠原代卵巢颗粒细胞分为6组:正常对照组、H2O2模型组、阳性药对照组、哈蟆油含药血清低、中、高剂量组.CCK-8法检测哈蟆油含药血清对H2O2致颗粒细胞氧化应激损伤细胞活力的影响;实时荧光RT-PCR法检测PI3K、AKT3 mRNA的表达水平;Western blot检测p-Akt(Ser473)、NF-κB信号通路相关蛋白的表达.结果发现与正常对照组相比,H2O2刺激后细胞活性明显下降,哈蟆油含药血清能够提高颗粒细胞存活率.机制研究发现,哈蟆油含药血清通过恢复H2O2诱导细胞增殖相关PI3K、AKT3 mRNA水平,上调p-Akt(Ser473)、IKKβ、NF-κB(p65)蛋白的表达,下调IκBα蛋白的表达.以上结果表明,哈蟆油对H2O2诱导卵巢颗粒细胞氧化应激损伤具有保护作用,其作用机制可能与调控PI3K/Akt/NF-κB信号通路密切相关.这可能是其抗氧化的作用机制之一.%The aim of this study was to investigate the function and mechanism of Oviductus Ranae (OR) on protecting rat ovarian granulosa cells from H2O2-induced oxidative damage.Granulosa cells were divided into six groups:control,model,positive control,as well as low,middle and high doses of OR-containing serum group.The granulosa cells were exposed to H2O2 for 2 h,and then treated with the OR-containing serum for 48 h.After that,the viability of granulosa cells was detected by CCK-8 assay.mRNA expressions of PI3K and AKT3 were assayed by real-time qPCR.The related proteins,such as NF-κB (p65),IκBα,IKKβ,and p-Akt (Ser473),were detected by Western blotting.The results showed that the viability of granulosa cells was significantly decreased after stimulating H2O2.OR-containing serum can significantly increase the viability of granulosa cells.Furthermore,OR-containing serum can rescue the descending expression of PI3K and AKT3 mRNA induced by H2O2,and up-regulate the protein levels of p-Akt (Ser473),IKKβ and NF-κB (p65),and down-regulate the expression of IκBα.These results indicated that OR protected granulosa cells from oxidative stress-induced injury by regulating the PI3K/Akt/NF-κB signal pathway,which may be one of the antioxidant mechanism of OR.
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