摘要
目的:观察补肾助孕方(补肾法)、逍遥丸(疏肝法)含药血清对人子宫内膜微血管内皮细胞(HEMECs)PI3K/Akt通路的影响,探讨两法改善子宫内膜容受性的可能作用机制。方法:常规传代人子宫内膜微血管内皮细胞,2%正常人低血清培养24h后,分别给予补肾助孕方、逍遥丸人含药血清预孵育24h、以VEGF165细胞因子(40 ng/mL)刺激、PI3K/Akt通路抑制剂LY294002孵育,设为正常组、VEGF组、补肾组、疏肝组、补肾+VEGF组、疏肝+VEGF组、抑制剂+VEGF组、补肾+抑制剂+VEGF组、疏肝+抑制剂+VEGF组。Western Blot法检测血管内皮生长因子(VEGF)、血管内皮生长因子受体2(VEGFR-2)、内皮型一氧化氮合酶(eNOS)、蛋白激酶-B(Akt)、磷酸化蛋白激酶-B(p-Akt)蛋白表达,实时荧光定量PCR法检测VEGF、VEGFR-2、eNOSmRNA表达,硝酸还原酶法检测培养液中一氧化氮(NO)含量。结果:补肾法、疏肝法上调HEMECs中VEGF、VEGFR-2、eNOS、Akt、p-Akt的蛋白表达,同时疏肝法上调eNOS蛋白表达优于补肾法(P<0.05)。加入PI3K/Akt通路抑制剂后,疏肝组、补肾组的p-Akt、eNOS蛋白表达均降低(P<0.05)。结论:疏肝法、补肾法能够增加VEGF的表达,激活PI3K/Akt信号通路,上调靶基因eNOS表达,从而促进血管通透性增加,达到改善子宫内膜容受性、提高胚胎着床率的作用。且提示疏肝法在提高血管通透性方面优于补肾法。
Objective: To observe the effects of serum containing Bushen Zhuyun Formula and Xiaoyao Pills on PI3K/Akt pathway of human endometrial microvascular endothelial cells, and to explore the possible mechanism of improving endometrial receptivity. Methods: Routinely passaged human endometrial microvascular endothelial cells were cultured with 2% normal human low serum for 24 h, then pre-incubated with Bushen Zhuyun Formula and Xiaoyao Pills human drug-containing serum for 24 h, and stimulated with VEGF165 cytokine(40 ng/mL), and incubated with the PI3K/Akt pathway inhibitor LY294002. The experiment was divided into normal group, VEGF group, Bushen group, Shugan group, Bushen+VEGF group,Shugan+VEGF group, inhibitor+VEGF group, Bushen+inhibitor+VEGF group, Shugan+inhibitor+VEGF group. The protein expressions of vascular endothelial growth factor(VEGF), vascular endothelial growth factor receptor 2(VEGFR-2), endothelial nitric oxide synthase(eNOS), protein kinase-B(Akt) and phosphorylated protein kinase-B by Western Blot(p-Akt) were detected by Western Blot. The mRNA expressions of VEGF, VEGFR-2 and eNOS were detected by real-time PCR. The content of nitric oxide(NO) was detected by nitrate reductase. Results: Bushen method and Shugan method upregulated the expression of VEGF,VEGFR-2, eNOS, Akt and p-Akt in HEMECs. And the protein expression of eNOS upregulated by Shugan method was better than that of Bushen method(P<0.05). After adding PI3K/Akt pathway inhibitor, the protein expression of p-Akt and eNOS in Shugan group and Bushen group were decreased(P<0.05). Conclusion: Shugan and Bushen method can increase the expression of VEGF and activate PI3K/Akt signal pathway, up-regulate the expression of target gene eNOS, so as to increase vascular permeability, improve endometrial receptivity and embryo implantation rate. Shugan method is better than Bushen method in improving vascular permeability.
作者
耿丹丹
李丽
魏学聪
范丽洁
贺明
杜惠兰
GENG Dan-dan;LI Li;WEI Xue-cong;FAN Li-jie;HE Ming;DU Hui-lan(Hebei University of Chinese Medicine,Shijiazhuang 050091,China;The Fourth Hospital of Shijiazhuang,Shijiazhuang 05001l,China;Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Hebei Reproductive Hospital,Shijiazhuang 050000,China)
出处
《中华中医药杂志》
CAS
CSCD
北大核心
2022年第9期5344-5350,共7页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金项目(No.81473719)。
作者简介
通信作者:杜惠兰,河北省石家庄市桥西区新石南路326号河北中医学院橘泉校区,邮编:050091,电话:0311-89926008,E-mail:duhuilan@163.com。
