| 摘要: |
| 目的：探索茯苓酸调节腺苷酸活化蛋白激酶（AMPK）/糖原合成酶激酶3β（GSK-3β）/核因子E2相关因子2（Nrf2）信号通路对子宫内膜异位症（EM）大鼠基质细胞铁死亡的影响。方法：将51只SD大鼠于腹腔内异位移植子宫内膜组织构建EM大鼠模型，并将其随机分为模型组、茯苓酸低剂量（3.5 mg/kg）组、茯苓酸高剂量（7.0 mg/kg）组、茯苓酸高剂量（7.0 mg/kg）+盐酸多索啡（0.2 mg/kg）组，每组各12只，另取12只SD大鼠只开腹不移植，设为假手术组，药物分组处理后测定各组大鼠异位子宫内膜体积、异位子宫内膜组织铁死亡指标［谷胱甘肽（GSH）、丙二醛（MDA）水平、铁含量］、血清及异位子宫内膜组织炎症因子［肿瘤坏死因子α（TNF-α）、白细胞介素-6（IL-6）］水平、异位子宫内膜组织铁死亡相关蛋白［长链脂酰辅酶A合成酶4（ACSL4）、环加氧酶2（PTGS2）］与AMPK/GSK-3β/Nrf2通路相关蛋白表达。原代培养EM大鼠子宫内膜基质细胞，并将其随机分为对照组、茯苓酸低浓度（40 mg/L）组、茯苓酸高浓度（80 mg/L）组、茯苓酸高浓度（80 mg/L）+盐酸多索啡（10 μmol/L）组，以药物分组处理后，以CCK-8法、流式细胞术分别测定各组细胞增殖及凋亡；以试剂盒检测各组细胞铁死亡指标GSH及MDA水平、铁含量；以免疫印迹实验检测各组细胞铁死亡相关蛋白及AMPK/GSK-3β/Nrf2通路相关蛋白表达。结果：与假手术组相比，模型组大鼠异位子宫内膜体积、MDA水平及铁含量、血清及异位子宫内膜组织TNF-α及IL-6水平、ACSL4及PTGS2蛋白表达升高（P<0.05），GSH水平、p-AMPK/AMPK、p-GSK-3β/GSK-3β及Nrf2蛋白表达降低（P<0.05）；与模型组相比，茯苓酸低、高剂量组大鼠异位子宫内膜体积、MDA水平及铁含量、血清及异位子宫内膜组织TNF-α及IL-6水平、ACSL4及PTGS2蛋白表达降低（P<0.05），GSH水平、p-AMPK/AMPK、p-GSK-3β/GSK-3β及Nrf2蛋白表达升高（P<0.05），且高剂量茯苓酸作用更强；盐酸多索啡可减弱高剂量茯苓酸对EM模型大鼠的作用。与对照组相比，茯苓酸低、高浓度组大鼠子宫内膜基质细胞活力、MDA水平及铁含量、ACSL4及PTGS2蛋白表达降低（P<0.05），凋亡率、GSH水平、p-AMPK/AMPK、p-GSK-3β/GSK-3β及Nrf2蛋白表达升高（P<0.05），且高浓度茯苓酸作用更强；盐酸多索啡可减弱高剂量茯苓酸对EM大鼠内膜基质细胞的作用。结论：茯苓酸可减轻EM大鼠炎症及铁死亡，抑制其子宫内膜基质细胞增殖及异位内膜生长，其作用机制可能是通过激活AMPK/GSK-3β/Nrf2信号通路来实现的。 |
| 关键词: 茯苓酸 AMPK/GSK-3β/Nrf2 子宫内膜异位症 子宫内膜基质细胞 铁死亡 |
| DOI： |
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| Effect of pachymic acid on ferroptosis of endometrial stromal cells in rats with endometriosis by regulating the adenosine monophosphate-activated protein kinase/glycogen synthase kinase-3β/nuclear factor erythroid 2-related factor 2 signaling pathway |
| LI Lin,MA Benling,HE Bing |
| (The Second Affiliated Hospital of Hunan University of Chinese Medicine，Changsha 410005，Hunan，China) |
| Abstract: |
| Objective：To investigate the effect of pachymic acid on ferroptosis of endometrial stromal cells in rats with endometriosis (EM) by regulating the adenosine monophosphate-activated protein kinase (AMPK)/glycogen synthase kinase-3β (GSK-3β)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway.Methods：A total of 51 Sprague-Dawley rats were given ectopic transplantation of endometrial tissue in the abdominal cavity to establish a rat model of EM，and then they were randomly divided into model group，low-dose pachymic acid (3.5 mg/kg) group，high-dose pachymic acid (7.0 mg/kg) group，high-dose pachymic acid (7.0 mg/kg)+dorsomorphin dihydrochloride (0.2 mg/kg) group，with 12 rats in each group；another 12 Sprague-Dawley rats were given laparotomy without transplantation and were established as sham-operation group.Related indicators were measured after grouping and treatment，including the volume of ectopic endometrium，ferroptosis markers in ectopic endometrium (glutathione ［GSH］，malondialdehyde ［MDA］，and iron content)，inflammatory factors in serum and ectopic endometrium (tumor necrosis factor-α ［TNF-α］ and interleukin-6 ［IL-6］)，ferroptosis-related proteins in ectopic endometrium (acyl-CoA synthetase long-chain family member 4 ［ACSL4］ and prostaglandin endoperoxide synthase 2 ［PTGS2］)，and the expression of proteins associated with the AMPK/GSK-3β/Nrf2 pathway.Primary cultured endometrial stromal cells of EM rats were randomly divided into control group，low-concentration pachymic acid (40 mg/L) group，high-concentration pachymic acid (80 mg/L) group，high-concentration pachymic acid (80 mg/L)+dorsomorphin dihydrochloride (10 μmol/L) group.After grouping and treatment，CCK-8 assay and flow cytometry were used to measure cell proliferation and apoptosis；kits were used to measure the levels of the ferroptosis markers GSH and MDA and the content of iron；Western blotting was used to measure the expression of ferroptosis-related proteins and proteins associated with the AMPK/GSK-3β/Nrf2 pathway.Results：Compared with the sham-operation group，the model group had significant increases in the volume of ectopic endometrium，the level of MDA，the content of iron，and the levels of TNF-α and IL-6 and the protein expression levels of ACSL4 and PTGS2 in serum and ectopic endometrium (P<0.05)，as well as significant reductions in the level of GSH and the protein expression levels of p-AMPK/AMPK，p-GSK-3β/GSK-3β，and Nrf2 (P<0.05).Compared with the model group，the low- and high-dose pachymic acid groups had significant reductions in the volume of ectopic endometrium，the level of MDA，the content of iron，and the levels of TNF-α and IL-6 and the protein expression levels of ACSL4 and PTGS2 in serum and ectopic endometrium (P<0.05)，as well as significant increases in the level of GSH and the protein expression levels of p-AMPK/AMPK，p-GSK-3β/GSK-3β，and Nrf2 (P<0.05)，and high-dose pachymic acid tended to have a stronger effect；dorsomorphin dihydrochloride could alleviate the effect of high-dose pachymic acid on EM model rats.Compared with the control group，the low- and high-concentration groups had significant reductions in the viability of endometrial stromal cells，the level of MDA，the content of iron，and the protein expression levels of ACSL4 and PTGS2 (P<0.05) and significant increases in apoptosis rate，the level of GSH，and the protein expression levels of p-AMPK/AMPK，p-GSK-3β/GSK-3β，and Nrf2 (P<0.05)，and high-dose pachymic acid tended to have a stronger effect；dorsomorphin dihydrochloride could alleviate the effect of high-dose pachymic acid on the endometrial stromal cells of EM rats.Conclusion：Pachymic acid can alleviate inflammation and ferroptosis in EM rats and inhibit the proliferation of endometrial stromal cells and the growth of ectopic endometrium，possibly by activating the AMPK/GSK-3β/Nrf2 signaling pathway. |
| Key words: pachymic acid adenosine monophosphate-activated protein kinase/glycogen synthase kinase-3β/nuclear factor erythroid 2-related factor 2 endometriosis endometrial stromal cells ferroptosis |
