目的 探究温经汤调控内质网应激（endoplasmic reticulum stress，ERS）介导的活化转录因子6（activating transcription factor 6，ATF6）/转录因子C/EBP同源蛋白（C/EBP homologous protein，CHOP）信号通路改善卵巢储备功能下降（decreased ovarian reserve，DOR）的作用机制。方法 通过ig雷公藤多苷建立DOR大鼠模型，分为对照组、模型组、芬吗通组（采用第1～2天ig雌二醇片0.2 mg/kg，第3～5天ig雌二醇地屈孕酮片1.2 mg/kg的序贯疗法）及温经汤低、中、高剂量（4.85、9.70、19.40 g/kg）组，各组给药4周后计算卵巢及子宫指数；ELISA法检测血清抗缪勒管激素（anti-mullerian hormone，AMH）、卵泡刺激素（follicle stimulating hormone，FSH）、雌二醇（estradiol，E2）、促黄体生成素（luteinizing hormone，LH）水平；苏木素-伊红（Hematoxylin eosin，HE）染色观察卵巢病理结构，并对各级卵泡进行计数；qRT-PCR检测各组大鼠卵巢组织ERS标志物葡萄糖调节蛋白78（glucose-regulated protein 78，GRP78）、ATF6、CHOP mRNA表达；Western blotting检测GRP78、ATF6、CHOP、半胱氨酸天冬氨酸特异性蛋白酶-12（cysteine aspartic acid specific protease-12，Caspase-12）蛋白表达水平。采用CCK-8法筛选雷公藤多苷的造模浓度；将人类卵巢颗粒KGN细胞分为对照组、雷公藤多苷组、雷公藤多苷＋5%空白血清组、雷公藤多苷＋5%温经汤血清组、ERS激动剂毒胡萝卜素（thapsigargin，TG）组，CCK-8法检测温经汤对雷公藤多苷处理的KGN细胞活力的影响；Fluo-4 AM钙离子（Ca²⁺）荧光探针检测各组细胞中Ca²⁺浓度；Western blotting检测各组细胞GRP78、ATF6、CHOP、Caspase-12蛋白表达水平。结果 与对照组比较，模型组大鼠卵巢及子宫指数显著下降（P＜0.05、0.01），血清AMH、E2水平显著降低（P＜0.01），FSH、LH水平显著升高（P＜0.01），卵巢组织颗粒细胞数量及层数较少、排列稀疏、卵巢皮质空洞，发育期卵泡数量显著减少（P＜0.01），闭锁卵泡数量显著增加（P＜0.01）；卵巢组织GRP78、ATF6、CHOP mRNA及蛋白表达、Caspase-12蛋白表达水平显著升高（P＜0.05、0.01）；与模型组比较，芬吗通组及温经汤中、高剂量组大鼠卵巢及子宫指数显著升高（P＜0.05、0.01），各给药组大鼠血清AMH水平均显著升高（P＜0.01），FSH水平均降低（P＜0.01），芬吗通组及温经汤中、高剂量组大鼠血清LH水平显著降低（P＜0.01），E2水平显著升高（P＜0.05、0.01），各给药组卵巢组织发育期卵泡数量增多，闭锁卵泡数量减少（P＜0.05、0.01），芬吗通组及温经汤中、高剂量组大鼠卵巢组织GRP78、ATF6、CHOP mRNA及蛋白表达、Caspase-12蛋白表达水平均显著降低（P＜0.05、0.01）。体外实验表明40、80、120、200、500 μg/mL的雷公藤多苷能够显著抑制KGN细胞增殖（P＜0.05）；雷公藤多苷＋5%空白血清组细胞存活率、Ca²⁺含量，GRP78、ATF6、CHOP、Caspase-12蛋白表达水平无明显变化（P＞0.05）；雷公藤多苷＋5%温经汤血清组细胞存活率升高（P＜0.01），Ca²⁺显著降低（P＜0.01），GRP78、ATF6、CHOP、Caspase-12蛋白表达水平显著降低（P＜0.05、0.01）。结论 温经汤可显著提高大鼠卵巢储备功能，对DOR具有较好的治疗作用，其作用机制可能与调控ATF6/CHOP通路、抑制ERS有关。

Objective To explore the mechanism of Wenjing Decoction (温经汤) regulating the endoplasmic reticulum stress (ERS) mediated activating transcription factor 6/C/EBP homologous protein (ATF6/CHOP) pathway in the treatment of decreased ovarian reserve (DOR). Methods The DOR rat model was established by multi-glycosides of Tripterygium glycosides, then the rats were divided into control group, model group, femoston (FMT) group (Sequential therapy involving the administration of estradiol valerate suspension at a dosage of 0.2 mg/kg on days 1 to 2, followed by estradiol and dydrogesterone tablets at a dosage of 1.2 mg/kg on days 3 to 5), low-, medium- and high- dose groups (4.85, 9.70, 19.40 g/kg) of Wenjing Decoction. The ovarian and uterine indexes were calculated after four weeks of treatment, the levels of serum anti mullerian hormone (AMH), follicle stimulating hormone (FSH), estradiol (E2), and luteinizing hormone (LH) were detected by ELISA method, hematoxylin eosin (HE) staining was used to observe the pathological structure of ovary, and the follicles at all levels were counted, the mRNA expressions of ERS markers glucose regulatory protein 78 (GRP78), ATF6, CHOP in ovarian tissues of rats in each group were detected by qRT-PCR method, the protein expressions of GRP78, ATF6, CHOP and cysteine aspartic acid specific protease-12 (Caspase-12) were detected by Western blotting. The modeling concentration of GTW was screened using the CCK-8 method, and Human ovarian granulosa cells KGN cells were divided into control group, GTW group, GTW+5% blank serum group (GTW+KBXQ), GTW+ 5% Wenjing Decoction containing serum group (GTW+WJTXQ), and ERS agonists thapsigargin (TG) group. The effect of Wenjing Decoction on the viability of KGN cells treated with GTW was detected by CCK8 assay, then Ca²⁺ concentration in each group was detected by Fluo-4 AM calcium fluorescent probe, the protein expression levels of GRP78, ATF6, CHOP and Caspase-12 in cells of each group were analyzed by western blotting. Results Compared with control group, the ovarian and uterine indexes in the model group were significantly reduced (P < 0.05, 0.01), serum AMH and E2 levels were decreased (P < 0.01), FSH and LH levels were increased (P < 0.01), the number and layers of granulosa cells in the ovarian tissue of the model group were less, the arrangement was sparse, and the ovarian parenchyma was empty,and the number of developing follicles in the ovarian tissue of the model group decreased significantly (P < 0.01), and the number of atretic follicles were increased (P < 0.01), and the mRNA and protein expressions of GRP78, ATF6, and CHOP, and the protein expressions of Caspase-12 in ovarian tissue were significantly increased (P < 0.05, 0.01). Compared with the model group, the ovarian and uterine indexes in FMT group, WJTZ group, and WJTG group were increased obviously (P < 0.05, 0.01), the serum AMH level of rats in each administration group were significantly increased (P < 0.01), and the FSH level were significantly decreased (P < 0.01), and the serum LH level of rats in the FMT group, WJTZ group and WJTG group were significantly decreased (P < 0.01), and the E2 level of rats in the FMT group, WJTZ group and WJTG group were significantly increased (P < 0.05, 0.01).The number of developing follicles at all levels in the ovarian tissue of each administration group were increased, and the number of atretic follicles were decreased (P < 0.05, 0.01). The mRNA and protein expressions of GRP78, ATF6, and CHOP, and the protein expression of Caspase-12 in the FMT group, WJTZ group and WJTG group were significantly reduced (P < 0.05, 0.01). In vitro experiments showed that GTW of 40, 80, 120, 200 and 500 μg/mL could significantly inhibit KGN cells proliferation (P < 0.05). Compared with the GTW group, there was no significant change in cell viability, Ca²⁺ concentration, GRP78, ATF6, CHOP, and Caspase-12 protein expression levels in the GTW+KBXQ group (P > 0.05), while the cell viability was significantly increased (P < 0.01) , and the Ca²⁺ concentration and protein expression levels of GRP78, ATF6, CHOP, and Caspase-12 were significantly decreased in the GTW+WJTXQ group (P < 0.05, 0.01). Conclusion Wenjing Decoction could significantly improve the ovarian reserve function of rats and has a good therapeutic effect on DOR, the mechanism might be related to regulating the ATF6/CHOP pathway and inhibiting ERS.

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国家自然科学基金项目(82004400);国家自然科学基金项目(82174426);河北省自然科学基金项目(H2020423069);河北省自然科学基金项目(H2021423020)