摘要
目的:在前期研究的基础上,探究瓜蒌薤白半夏汤正丁醇部位对缺血缺氧的骨髓间充质干细胞(BMSCs)凋亡的保护作用及机制。方法:90%乙醇回流提取+正丁醇萃取法制备瓜蒌薤白半夏汤正丁醇部位,传统黄酒加水煎煮法制备该方总提取物。分离培养大鼠BMSCs,将细胞分为正常对照组、模型对照组、瓜蒌薤白半夏汤150μg/mL组和瓜蒌薤白半夏汤正丁醇部位50μg/mL组。造模组以无血清的培养液、缺氧气体(5%CO2+94%N2+1%O2)处理24 h诱导细胞凋亡。以相应药物处理细胞24 h。通过倒置相差显微镜观察细胞形态,MTT法检测细胞存活率,TUNEL法检测细胞凋亡率,免疫荧光、Western Blot检测BMSCs中磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(AKT)蛋白的表达位点及磷酸化水平,分析磷酸化/非磷酸化蛋白的比值。结果:与正常对照组相比,模型对照组BMSCs存活率显著降低、凋亡率显著升高,PI3K、AKT蛋白的表达及磷酸化水平显著降低(P<0.01);与模型对照组相比瓜蒌薤白半夏汤正丁醇部位50μg/mL、瓜蒌薤白半夏汤150μg/mL均能显著提高细胞存活率,降低细胞凋亡率;同时显著上调细胞内PI3K、AKT蛋白表达及磷酸化水平;且瓜蒌薤白半夏汤正丁醇部位的上述作用均优于瓜蒌薤白半夏汤总提取物(P<0.05或P<0.01)。结论:瓜蒌薤白半夏汤正丁醇部位可能通过激活PI3K/AKT信号通路,逆转缺血缺氧诱导的BMSCs凋亡,提高缺血缺氧状态下干细胞的生存率。这可能是该方祛痰散结功效的部分生物学机制。
Objective:To explore the protective effect and mechanism of n-butanol fraction from Gualou Xiebai Banxia Decoction against ischemia and hypoxia-induced apoptosis of bone marrow mesenchymal stem cells(BMSCs) on the basis of previous research. Methods: Gualou Xiebai Banxia Decoction was reflux-extracted with 90% ethanol and then with n-butyl alcohol to obtain the N-butanol fraction. Besides, Gualou Xiebai Banxia Decoction was also treated with the traditional yellow rice wine and then decocted with water for yielding the total extract. BMSCs were isolated from rats, cultured, and then divided into the normal control group, model control group, 150 μg/mL Gualou Xiebai Banxia Decoction group, and 50 μg/mL n-butanol fraction from Gualou Xiebai Banxia Decoction group. The cells were cultured for 24 h with serum-free culture medium and hypoxic gas(5%CO2+94%N2+1%O2) for inducing apoptosis, and then treated with the corresponding drugs for 24 h. Cell morphology was observed under an inverted phase contrast microscope. The cell survival rate was detected by MTT method and cell apoptosis rate by TUNEL method. The expression sites and phosphorylation levels of phosphatidylinositol 3-kinase(PI3 K) and protein kinase B(AKT) in BMSCs were assayed by Western Blotting, followed by the analysis of phosphorylated/non-phosphorylated protein ratio. Results: Compared with the normal control group, the model control group displayed significantly reduced survival rate of BMSCs, increased apoptosis rate, and down-regulated PI3 K and AKT protein expression and phosphorylation levels(P<0.01). Compared with the model control group, both the 150 μg/mL Gualou Xiebai Banxia Decoction group and 50 μg/mL n-butanol fraction from Gualou Xiebai Banxia Decoction group significantly increased the cell survival rate, decreased the apoptosis rate, and up-regulated the protein expression and phosphorylation levels of PI3 K and AKT. Moreover, the effects of n-butanol fraction from Gualou Xiebai Banxia Decoction were superior to those of the total ext
作者
姜晶晶
傅旎旎
桑晓宇
郑书臣
赵启韬
Jiang Jingjing;Fu Nini;Sang Xiaoyu;Zheng Shuchen;Zhao Qitao(Shandong University of Traditional Chinese Medicine,College of Pharmacy,Jinan 250355;Shandong University of Traditional Chinese Medicine,College of Traditional Chinese Medicine,Jinan 250355)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2021年第4期2-7,共6页
Pharmacology and Clinics of Chinese Materia Medica
基金
国家自然科学基金项目(编号:81573852)
山东省重点研发计划公益类项目(编号:2018GSF119009)
山东省自然科学基金(编号:ZR2020MH342)。
关键词
瓜蒌薤白半夏汤
骨髓间充质干细胞
细胞凋亡
磷脂酰肌醇3-激酶
蛋白激酶B
Gualou Xiebai Banxia Decoction
bone marrow mesenchymal stem cells(BMSCs)
apoptosis
phosphatidylinositol 3-kinase(PI3K)
protein kinase B(AKT)
