基于GPER/PI3K/AKT 通路探究四物汤对成骨细胞的雌激素样效应及其分子机制

目的：利用SD大鼠颅骨原代培养成骨细胞研究四物汤的雌激素样效应及其分子机理。方法：40只未性成熟SD大鼠，体重50 g，按照随机分组原则分为5组：正常对照组、雌激素组和四物汤高、中、低剂量组。四物汤灌胃给药，持续4天后腹主动脉取血，制备含药血清。利用出生72 h内的乳鼠提取并培养原代成骨细胞（rats osteoblast，ROBs），并选取第2或3代的细胞进行实验研究。用四物汤含药血清作用ROBs细胞，并分别加入G蛋白偶联雌激素受体（GPER）的特异性激动剂G1和特异性拮抗剂G15作为工具药，通过MTT法检测各组含药血清对ROBs细胞增殖的影响；通过细胞免疫荧光法检测含药血清对ROBs PI3K、AKT、p-Akt表达的影响。结果：显微镜下形态学观察和ALP结果表明，原代ROBs提取和培养成功；四物汤含药血清可促进ROBs增殖（P<0.05或P<0.01），该促增殖作用在加入拮抗剂G15后明显减弱（P<0.01），加入激动剂G1后明显增强（P<0.05或P<0.01）；利用细胞免疫荧光技术检测发现四物汤含药血清可提高PI3K、p-Akt在ROBs细胞的荧光表达强度，且该效应在加入G1后增强（P<0.05或P<0.01），加入G15后减弱（P<0.05）。结论：四物汤可通过GPER通路发挥雌激素样效应，促进成骨细胞增殖，提高GPER介导下游信号分子PI3K、p-Akt的表达水平。

Study on the Estrogenic Effects and Molecular Mechanism of Siwu Decoction onOsteoblast via GPER/PI3K/AKT Pathway

To study the estrogenic effects and its molecular mechanism of Siwu Decoction on primary osteslastsof SD rats. Method: 40 immature SD rats, weighing 50 g, were randomly divided into 5 groups according to the principleof random distribution law: normal control group, estrogen group, high dose Siwu group, middle dose Siwu group and lowdose Siwu group. Gavage administration for 4 days. Then the rats were sacrificed. The blood was taken from theabdominal aorta and the pharmacological serum was prepared. The primary osteslast (rats osteoblast, ROBs) wereextracted from new born rats within 72 h and the secind or third generation cells were selected for experimental study.With GPER agonists G1 and GPER antagonists G15 as tools, the proliferation rate of ROBs influenced by Siwu Decoctionwas determined by MTT assay. The expression level of PI3K, AKT and p-Akt in ROBs cells affected by Siwu Decoctionwere detected by cell immunofluorescence test. Result: microscopic observation and ALP results showed that the primaryROBs were successfully extracted and cultured. Siwu Decoction could promote proliferation rate of ROBs (P<0.05 or P<0.01). Such effect could be inhibited significantly by G15(P<0.01) and enhanced significantly by G1(P<0.05 or P<0.01).The pharmacological serum of Siwu Decoction could improve the expression level of PI3K and p-Akt in ROBs cells (P<0.05 or P<0.01). Such effect could also be decreased significantly by G15(P<0.05 or P<0.01) and enhanced significantlyby G1(P<0.05). Conclusion: Siwu Decoction could play estrogenic effects through GPER pathway. It could improveROBs proliferation and promote the expression level of GPER mediated downstream signal molecules including PI3K andp-Akt.