Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK
Metformin inhibits testosterone-induced endoplasmic reticulum stress in ovarian granulosa cells via inactivation of p38 MAPK
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二甲双胍通过 p38 MAPK 失活抑制睾酮诱导的卵巢颗粒细胞内质网应激
DOI：
10.1093/humrep/deaa077
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发表时间：
2020-05-01
影响因子：
6.1
通讯作者：
Zhang, Songying
中科院分区：
文献类型：
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作者：
Jin, Jiamin;Ma, Yerong;Zhang, Songying
摘要研究问题：在体内和体外，二甲双胍是否能抑制雄激素诱导的小鼠颗粒细胞(GC)内质网(ER)过度应激？摘要：二甲双胍通过抑制卵巢GCs中p38MAPK的磷酸化，抑制睾酮诱导的内质网应激和未折叠蛋白反应(UPR)激活。已知的多囊卵巢综合征(PCOS)与高雄激素血症有关。过量的睾酮诱导人卵丘细胞ER应激和UPR激活，导致细胞凋亡。二甲双胍对内质网应激和UPR激活有潜在的抑制作用，这在人胰腺β细胞和肥胖小鼠中得到了证实。研究设计、大小、持续时间、卵丘细胞和卵泡液来自我们体外受精中心的25名PCOS患者和25名对照组。建立双氢睾酮(DHT)诱导的多囊卵巢综合征(PCOS)小鼠模型。用睾酮、二甲双胍、p38MAPK抑制剂或p38MAPK小干扰RNA培养原代小鼠GCs和卵丘-卵母细胞复合体(COCs)。对象/材料、地点、方法采用实时定量聚合酶链式反应(QPCR)和免疫印迹法检测PCOS患者卵丘细胞中UPR受体蛋白和UPR相关基因的表达水平。取PCOS组小鼠卵巢、卵母细胞、GCs和COCs，并与对照组比较。用定量聚合酶链式反应、免疫印迹和免疫荧光法检测内质网应激标志物和p38MAPK磷酸化的表达。采用原代培养的GCs和COCs进行体外分析，通过qPCR和Western印迹方法证实二甲双胍对内质网应激激活的影响。最后，通过定量聚合酶链式反应和COC扩张法检测内质网应激激活对GCs和COCs的影响及其与黄体生成素反应性的关系。PCOS患者卵丘细胞ER应激标志物GRP78、CHOP和XBP1s的表达及UPR感受器蛋白p-IRE1α、p-eIF2α和GRP78的表达水平均高于对照组。与对照组相比，DHT处理的PCOS小鼠的卵巢、GCs和COCs显示出ER应激标记基因和蛋白的水平增加。PCOS小鼠卵巢中的高雄激素也诱导了COCs和GCs中p38MAPK的磷酸化。二甲双胍抑制内质网应激激活与降低p-p38MAPK水平有关。在体外实验中，二甲双胍或p38MAPK抑制原代培养的GCs和COCs可减轻睾酮诱导的内质网应激。在黄体生成素作用下，COCs迅速膨胀，排卵相关基因Areg、Ereg、Ptgs2、Sult1e1、Ptx3和Tnfaip6在COCs和GCs中有较强的表达。这些效应可被内质网应激抑制剂二甲双胍或p38MAPK基因敲除所逆转。局限性，谨慎的理由本研究中的多囊卵巢综合征患者的数量很少。研究结果的更广泛意义这项研究为二甲双胍作为多囊卵巢综合征的治疗提供了进一步的证据。研究经费/竞争利益(S)本研究得到了中国国家重点研究发展计划(2018YFC1004800)、浙江省重点研究发展计划(2017C03022)、浙江省医学科技计划项目(2017KY085、2018KY457)、中国国家自然科学基金(31701260、81401264、81701514)和中华医学会临床医学研究专项基金(16020320648)的资助。作者报告说，在这项工作中没有利益冲突，也没有任何可披露的信息。试用注册号N/A
Abstract STUDY QUESTION Does metformin inhibit excessive androgen-induced endoplasmic reticulum (ER) stress in mouse granulosa cells (GCs) in vivo and in vitro? SUMMARY ANSWER Metformin inhibits testosterone-induced ER stress and unfolded protein response (UPR) activation by suppressing p38 MAPK phosphorylation in ovarian GCs. WHAT IS KNOWN ALREADY Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism. Excessive testosterone induces ER stress and UPR activation in human cumulus cells, leading to cell apoptosis. Metformin has potential inhibitory effects on ER stress and UPR activation, as demonstrated in human pancreatic beta cells and obese mice. STUDY DESIGN, SIZE, DURATION Cumulus cells and follicular fluid were collected from 25 women with PCOS and 25 controls at our IVF centre. A dihydrotestosterone (DHT)-induced PCOS mouse model was constructed and treated with or without metformin. Primary mouse GCs and cumulus-oocyte complexes (COCs) were cultured with testosterone, metformin, a p38 MAPK inhibitor, or p38 MAPK small interfering RNA. PARTICIPANTS/MATERIALS, SETTING, METHODS The levels of UPR sensor proteins and UPR-related genes were measured in cumulus cells from PCOS and control patients by real-time quantitative PCR (qPCR) and western blot. The ovaries, oocytes, GCs and COCs were collected from PCOS mice treated with metformin and controls. The expressions of ER stress markers and p38 MAPK phosphorylation were assessed by qPCR, western blot and immunofluorescence. A subsequent in vitro analysis with primary cultured GCs and COCs was used to confirm the influence of metformin on ER stress activation by qPCR and western blot. Finally, the effects of ER stress activation on GCs and COCs in relation to LH responsiveness were examined by qPCR and COC expansion. MAIN RESULTS AND THE ROLE OF CHANCE The expression of the ER stress markers GRP78, CHOP and XBP1s in the cumulus cells was higher in PCOS patients than in control patients, as were the levels of the UPR sensor proteins p-IRE1α, p-EIF2α and GRP78. Compared to those of control mice, the ovaries, GCs and COCs of DHT-treated PCOS mice showed increased levels of ER stress marker genes and proteins. Hyperandrogenism in PCOS mouse ovaries also induced p38 MAPK phosphorylation in COCs and GCs. Metformin inhibited ER stress activation was associated with decreased p-p38 MAPK levels. In vitro experiments, testosterone-induced ER stress was mitigated by metformin or p38 MAPK inhibition in primary cultured GCs and COCs. COCs expanded rapidly in the presence of testosterone during LH administration, and ovulation-related genes, namely, Areg, Ereg, Ptgs2, Sult1e1, Ptx3 and Tnfaip6, were strongly expressed in the COCs and GCs. These effects were reversed by treatment with metformin, an ER stress inhibitor or by knockdown of p38 MAPK. LIMITATIONS, REASONS FOR CAUTION The number of PCOS patients in this study was small. WIDER IMPLICATIONS OF THE FINDINGS This study provides further evidence for metformin as a PCOS treatment. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the National Key Research and Developmental Program of China (2018YFC1004800), the Key Research and Development Program of Zhejiang Province (2017C03022), the Zhejiang Province Medical Science and Technology Plan Project (2017KY085, 2018KY457), the National Natural Science Foundation of China (31701260, 81401264, 81701514), and the Special Funds for Clinical Medical Research of the Chinese Medical Association (16020320648). The authors report no conflict of interest in this work and have nothing to disclose. TRIAL REGISTRATION NUMBER N/A.