目的 探究溫經(jīng)湯調(diào)控內(nèi)質(zhì)網(wǎng)應(yīng)激（endoplasmic reticulum stress，ERS）介導(dǎo)的活化轉(zhuǎn)錄因子6（activating transcription factor 6，ATF6）/轉(zhuǎn)錄因子C/EBP同源蛋白（C/EBP homologous protein，CHOP）信號(hào)通路改善卵巢儲(chǔ)備功能下降（decreased ovarian reserve，DOR）的作用機(jī)制。方法 通過(guò)ig雷公藤多苷建立DOR大鼠模型，分為對(duì)照組、模型組、芬嗎通組（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的序貫療法）及溫經(jīng)湯低、中、高劑量（4.85、9.70、19.40 g/kg）組，各組給藥4周后計(jì)算卵巢及子宮指數(shù)；ELISA法檢測(cè)血清抗繆勒管激素（anti-mullerian hormone，AMH）、卵泡刺激素（follicle stimulating hormone，F(xiàn)SH）、雌二醇（estradiol，E2）、促黃體生成素（luteinizing hormone，LH）水平；蘇木素-伊紅（Hematoxylin eosin，HE）染色觀察卵巢病理結(jié)構(gòu)，并對(duì)各級(jí)卵泡進(jìn)行計(jì)數(shù)；qRT-PCR檢測(cè)各組大鼠卵巢組織ERS標(biāo)志物葡萄糖調(diào)節(jié)蛋白78（glucose-regulated protein 78，GRP78）、ATF6、CHOP mRNA表達(dá)；Western blotting檢測(cè)GRP78、ATF6、CHOP、半胱氨酸天冬氨酸特異性蛋白酶-12（cysteine aspartic acid specific protease-12，Caspase-12）蛋白表達(dá)水平。采用CCK-8法篩選雷公藤多苷的造模濃度；將人類(lèi)卵巢顆粒KGN細(xì)胞分為對(duì)照組、雷公藤多苷組、雷公藤多苷＋5%空白血清組、雷公藤多苷＋5%溫經(jīng)湯血清組、ERS激動(dòng)劑毒胡蘿卜素（thapsigargin，TG）組，CCK-8法檢測(cè)溫經(jīng)湯對(duì)雷公藤多苷處理的KGN細(xì)胞活力的影響；Fluo-4 AM鈣離子（Ca²⁺）熒光探針檢測(cè)各組細(xì)胞中Ca²⁺濃度；Western blotting檢測(cè)各組細(xì)胞GRP78、ATF6、CHOP、Caspase-12蛋白表達(dá)水平。結(jié)果 與對(duì)照組比較，模型組大鼠卵巢及子宮指數(shù)顯著下降（P＜0.05、0.01），血清AMH、E2水平顯著降低（P＜0.01），F(xiàn)SH、LH水平顯著升高（P＜0.01），卵巢組織顆粒細(xì)胞數(shù)量及層數(shù)較少、排列稀疏、卵巢皮質(zhì)空洞，發(fā)育期卵泡數(shù)量顯著減少（P＜0.01），閉鎖卵泡數(shù)量顯著增加（P＜0.01）；卵巢組織GRP78、ATF6、CHOP mRNA及蛋白表達(dá)、Caspase-12蛋白表達(dá)水平顯著升高（P＜0.05、0.01）；與模型組比較，芬嗎通組及溫經(jīng)湯中、高劑量組大鼠卵巢及子宮指數(shù)顯著升高（P＜0.05、0.01），各給藥組大鼠血清AMH水平均顯著升高（P＜0.01），F(xiàn)SH水平均降低（P＜0.01），芬嗎通組及溫經(jīng)湯中、高劑量組大鼠血清LH水平顯著降低（P＜0.01），E2水平顯著升高（P＜0.05、0.01），各給藥組卵巢組織發(fā)育期卵泡數(shù)量增多，閉鎖卵泡數(shù)量減少（P＜0.05、0.01），芬嗎通組及溫經(jīng)湯中、高劑量組大鼠卵巢組織GRP78、ATF6、CHOP mRNA及蛋白表達(dá)、Caspase-12蛋白表達(dá)水平均顯著降低（P＜0.05、0.01）。體外實(shí)驗(yàn)表明40、80、120、200、500 μg/mL的雷公藤多苷能夠顯著抑制KGN細(xì)胞增殖（P＜0.05）；雷公藤多苷＋5%空白血清組細(xì)胞存活率、Ca²⁺含量，GRP78、ATF6、CHOP、Caspase-12蛋白表達(dá)水平無(wú)明顯變化（P＞0.05）；雷公藤多苷＋5%溫經(jīng)湯血清組細(xì)胞存活率升高（P＜0.01），Ca²⁺顯著降低（P＜0.01），GRP78、ATF6、CHOP、Caspase-12蛋白表達(dá)水平顯著降低（P＜0.05、0.01）。結(jié)論 溫經(jīng)湯可顯著提高大鼠卵巢儲(chǔ)備功能，對(duì)DOR具有較好的治療作用，其作用機(jī)制可能與調(diào)控ATF6/CHOP通路、抑制ERS有關(guān)。

Objective To explore the mechanism of Wenjing Decoction (溫經(jīng)湯) 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.

R285.5

國(guó)家自然科學(xué)基金項(xiàng)目(82004400);國(guó)家自然科學(xué)基金項(xiàng)目(82174426);河北省自然科學(xué)基金項(xiàng)目(H2020423069);河北省自然科學(xué)基金項(xiàng)目(H2021423020)