基于PTEN/mTOR/HIF-1α通路的血筒素调控氧糖剥夺PC12细胞自噬机制研究  

Mechanism of schisanlactone E in regulating oxygen-glucose deprivation of autophagy in PC12 cells based on the PTEN/mTOR/HIF-1αpathway

作　　者：陈笑一 彭南波 田梦芝 龙佳欣 杜可[1] CHEN Xiao-yi;PENG Nan-bo;TIAN Meng-zhi;LONG Jia-xin;DU Ke(Hunan University of Traditional Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学,长沙410208

出　　处：《天然产物研究与开发》2025年第3期430-437,共8页Natural Product Research and Development

基　　金：湖南省自然科学基金(2020JJ9050);湖南省普通高等学校教学改革研究项目(HNJG-2022-0134);研究生创新创业训练计划(CX20230828);大学生创新创业训练计划(S202310541068)。

摘　　要：本研究旨在探讨血筒素(schisanlactone E,SE)通过磷酸酯酶与张力蛋白同源物(phosphatase and tensin homolog,PTEN)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycinm,mTOR)/缺氧诱导因子1亚基α(hypoxia-inducible factor 1α,HIF-1α)通路调节PC12细胞自噬抗氧糖剥夺(oxygen-glucose deprivation,OGD)的作用机制。构建OGD-PC12细胞体外模型,分为正常组(Con)、OGD组、SE组、PTEN抑制剂(bisperoxovanadium,Bpv)组、Bpv+SE组。通过噻唑蓝染色(MTT)法、流式细胞术、透射电镜法、单丹磺酰尸胺荧光染色(monodansylcadaverine,MDC)法、Western blot法检测相关指标。结果表明,与OGD组相比,SE组细胞存活率提高(P<0.01),凋亡率降低(P<0.01),SE组和Bpv组细胞自噬小体和溶酶体数量均减少(P<0.01),自噬小体的荧光强度均减弱(P<0.01),螯合体1(sequestosome1,p62)、磷酸化雷帕霉素靶蛋白(p-mTOR)/mTOR蛋白表达量均升高(P<0.01或P<0.05),微管相关蛋白轻链3-II/I(microtubule-associated protein1 light chain 3-II/I,LC3-II/LC3-I)、苄氯素1(recombinant beclin1,Beclin1)、PTEN、HIF-1α蛋白表达量均降低(P<0.01);与Bpv组相比,Bpv+SE组细胞自噬小体及溶酶体数量减少(P<0.01),自噬小体荧光强度减弱(P<0.01),p62、p-mTOR/mTOR蛋白表达量升高(P<0.01或P<0.05),LC3-II/LC3-I、Beclin1、PTEN、HIF-1α蛋白表达量降低(P<0.01)。提示,SE在0.1μmol/L浓度下能够达到抗神经元自噬损伤最佳效果,发挥抑制OGD-PC12细胞自噬损伤的作用;通过加入PTEN抑制,能够上调mTOR,继而下调HIF-1α,并且在SE与PTEN抑制剂联合用药后效果更为明显。本研究可为地方天然民族产物SE研发和其抗缺血性脑卒中(ischemic stroke,IS)缺血损伤的分子机制提供一定实验室依据,并对地方民族天然产物服务人类健康、促进地方经济发展累积经验。This study aims to investigate the mechanism of the regulation of oxygen-glucose deprivation(OGD)in PC12 cells by schisanlactone E(SE)via phosphatase and tensin homolog(PTEN)/mammalian target of rapamycinm(mTOR)/hypoxia-inducible factor 1α(HIF-1α)pathway.OGD-PC12 cells models were constructed in vitro,which were divided into control group(Con),OGD group,SE group,bisperoxovanadium(Bpv)group,and Bpv+SE group.Relevant indexes were detected by thiazolyl blue staining(MTT)method,flow cytometry,transmission electron microscopy,monodansylcadaverine(MDC),and western blot.The results indicated that,compared to the OGD group,the SE group exhibited a marked improvement in cell survival rate(P<0.01)and a corresponding decrease in apoptosis rate(P<0.01),both the SE and Bpv groups showed a reduction in the number of autophagosomes and lysosomes(P<0.01),as well as a diminished fluorescence intensity of autophagosomes(P<0.01),there was an elevation in the expression levels of sequestosome1(p62)and p-mammalian target of rapamycinm(p-mTOR)/mTOR proteins(P<0.01 or P<0.05),the microtubule-associated protein1 light chain 3-II/I(LC3-II/LC3-I),recombinant beclin1(Beclin1),PTEN,and HIF-1αprotein levels were significantly decreased(P<0.01).The Bpv+SE group showed a decrease in the number of autophagosomes and lysosomes compared to the Bpv group(P<0.01),the fluorescence intensity of autophagosomes also decreased(P<0.01),while the expression levels of p62 and p-mTOR/mTOR proteins increased(P<0.01 or P<0.05).Additionally,the expression levels of LC3-II/LC3-I,Beclin1,PTEN,and HIF-1αproteins decreased(P<0.01).At a concentration of 0.1μmol/L,SE can achieve the optimal effect in protecting neurons from autophagic injury and exert an inhibitory effect on OGD-PC12 cell autophagic damage.By adding PTEN inhibitor,mTOR can be upregulated,leading to downregulation of HIF-1α.The combined use of SE and PTEN inhibitor shows more significant effects.This study can provide a certain experimental basis for the research and development of local natura

关 键 词：血筒素 氧糖剥夺 细胞自噬 PTEN/mTOR/HIF-1α PTEN抑制剂 

分 类 号：R932[医药卫生—生药学]