Song Ya-Jing, Li Dan-Dan, Lyu Jing-Fang, Jiang Min, DU Hui-Lan
Institute of Integrated Chinese and Western Medicine, Hebei University of Chinese Medicine Shijiazhuang 050091, China Hebei Collaborative Innovation Center of Integrated Traditional and Western Medicine on Reproductive Disease Shijiazhuang 050091, China Hebei Key Laboratory of Liver and Kidney Diseases of Integrated Traditional Chinese and Western Medicine Shijiazhuang 050091, China.
Zhongguo Zhong Yao Za Zhi. 2024 Apr;49(8):2169-2177. doi: 10.19540/j.cnki.cjcmm.20231213.403.
This study aims to explore the mechanism of Shoutai Pills in treating threatened abortion. According to the random number table method, ICR female mice were randomized into a normal group, a model group, a dydrogesterone group, and a Shoutai Pills group, with 15 mice in each group. Mice were administrated with normal saline(normal and model groups) or the suspension of Shoutai Pills or dydrogesterone by gavage at 9:00 am every day. At 16:00 every day, mice in the normal group were administrated with an equal volume of distilled water, while those in the model, Shoutai Pills, and dydrogesterone groups were administrated with hydrocortisone solution by gavage for 4 consecutive days. ICR female and male mice were caged in a ratio of 2∶1 during the pre-estrous or estrous period. From the first day of pregnancy, drug administration was continued for 5 consecutive days. On day 6, mice were administrated with mifepristone by gavage to establish the model of kidney deficiency-induced abortion. On day 6 of pregnancy, 10 female ICR mice were randomly selected from each group, and the uterus was collected for observation of the pathological changes of trophoblasts at the maternal-fetal interface by hematoxylin-eosin(HE) staining. The protein levels of key enzymes of glycolysis, hexokinase 2(HK2), enolase 1(ENO1), pyruvate kinase M2(PKM2), and lactate dehydrogenase A(LDHA), were determined by Western blot and immunofluorescence. The expression of apoptosis-related proteins including B cell lymphoma-2(Bcl-2), Bcl-2-associated protein X(Bax), and cysteinyl aspartate-specific proteinase-3(caspase-3) was determined by Western blot and real-time PCR. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling was employed to examine apoptosis. The embryo loss rate of the remaining five female mice was calculated by trypan blue staining method on day 14 of pregnancy. On day 14 of pregnancy, the embryo loss rate of the normal group was 5.00%, which was lower than that(27.78%) in the model group(P<0.05). Dydrogesterone and Shoutai Pills groups showed reduced embryo loss rates(10.26% and 7.50%, respectively) compared with the model group. On day 6 of pregnancy, compared with the normal group, the model group showed down-regulated expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and up-regulated expression of Bax and caspase-3(P<0.05). Compared with the model group, dydrogesterone and Shoutai Pills up-regulated the expression of HK2, ENO1, PKM2, LDHA, and Bcl-2 and down-regulated the expression of Bax and caspase-3(P<0.05). Compared with that in the normal group, the apoptosis rate in the model group increased(P<0.05). Compared with the model group, dydrogesterone and Shoutai Pills reduced the apoptosis rate(P<0.05). In conclusion, Shoutai Pills can reduce the embryo loss rate and protect embryos by promoting aerobic glycolysis at the maternal-fetal interface and inhibiting the apoptosis of trophoblasts in mice.
本研究旨在探讨寿胎丸治疗先兆流产的机制。采用随机数字表法，将ICR雌性小鼠随机分为正常组、模型组、地屈孕酮组和寿胎丸组，每组15只。每天上午9:00，正常组和模型组小鼠灌胃生理盐水，寿胎丸组和地屈孕酮组小鼠分别灌胃寿胎丸混悬液或地屈孕酮。每天16:00，正常组小鼠灌胃等体积蒸馏水，模型组、寿胎丸组和地屈孕酮组小鼠连续4天灌胃氢化可的松溶液。在动情前期或动情期，将ICR雌性和雄性小鼠按2∶1比例合笼。从妊娠第1天开始，连续给药5天。第6天，小鼠灌胃米非司酮以建立肾虚型流产模型。妊娠第6天，每组随机选取10只雌性ICR小鼠，取子宫，采用苏木精-伊红（HE）染色观察母胎界面滋养层细胞的病理变化。采用蛋白质免疫印迹法和免疫荧光法检测糖酵解关键酶己糖激酶2（HK2）、烯醇化酶1（ENO1）、丙酮酸激酶M2（PKM2）和乳酸脱氢酶A（LDHA）的蛋白水平。采用蛋白质免疫印迹法和实时荧光定量PCR法检测凋亡相关蛋白B细胞淋巴瘤- 2（Bcl-2）、Bcl-2相关蛋白X（Bax）和半胱天冬酶-3（caspase-3）的表达。采用末端脱氧核苷酸转移酶介导的缺口末端标记法检测细胞凋亡。妊娠第14天，采用台盼蓝染色法计算其余5只雌性小鼠的胚胎丢失率。妊娠第14天，正常组胚胎丢失率为5.00%，低于模型组（27.78%）（P<0.05）。与模型组相比，地屈孕酮组和寿胎丸组胚胎丢失率降低（分别为10.26%和 7.50%）。妊娠第6天，与正常组相比，模型组HK2、ENO1、PKM2、LDHA和Bcl-2表达下调，Bax和caspase-3表达上调（P<0.05）。与模型组相比，地屈孕酮和寿胎丸上调HK2、ENO1、PKM2、LDHA和Bcl-2的表达，下调Bax和caspase-3的表达（P<0.05）。与正常组相比，模型组细胞凋亡率升高（P<0.05）。与模型组相比，地屈孕酮和寿胎丸降低了细胞凋亡率（P<0.05）。综上所述，寿胎丸可降低小鼠胚胎丢失率，通过促进母胎界面有氧糖酵解和抑制滋养层细胞凋亡来保护胚胎。
