摘要
目的:探讨麻芍平喘汤(MSPC)通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对脂多糖(LPS)诱导的人支气管气道上皮样细胞(16HBE)自噬的影响。方法:选用人支气管上皮细胞16HBE作为研究对象,采用细胞增殖与活性检测(CCK-8)法检测LPS诱导的16HBE活性影响和麻芍平喘含药血清对16HBE细胞活性影响;以CCK-8法筛选出的适宜条件的LPS诱导16HBE细胞,并测定肿瘤坏死因子-α(TNF-α)含量鉴定模型成立,制备麻芍平喘汤含药血清作用于LPS诱导的16HBE,将细胞分为正常组、LPS组、LPS+MSPC组、LY294002+LPS组、LY294002+LPS+MSPC组。透射电镜观察细胞的自噬囊泡和超微结构的变化;蛋白免疫印迹法(Western blot)检测细胞PI3K、磷酸化PI3K(p-PI3K)、Akt、p-Akt、mTOR、p-mTOR及微管相关蛋白1轻链3B(LC3B)的蛋白表达水平;酶联免疫吸附测定法(ELISA)检测5组炎症因子白细胞介素(IL)-5、IL-6、TNF-α、IL-10的表达水平。结果:LPS对16HBE细胞的抑制率呈剂量依赖性;与正常组比较,模型组作用24 h能显著升高促炎因子TNF-α表达(P<0.05);麻芍平喘汤含药血清对16HBE的促进作用呈浓度依赖性;与正常组比较,模型组自噬体形成增多,麻芍平喘汤可以一定程度抑制自噬,改善细胞状态;Western blot结果显示,与正常组比较,LPS组p-PI3K、p-Akt、p-mTOR蛋白的表达明显降低(P<0.05),LC3B蛋白的表达显著升高(P<0.01);与LPS组比较,含药血清组p-PI3K、p-Akt、p-mTOR蛋白表达水平明显升高(P<0.05),LC3B表达水平明显降低(P<0.05),与LPS+LY294002组比较,LY294002+LPS+MSCP组p-PI3K、p-Akt、p-mTOR蛋白表达水平明显升高(P<0.05),LC3B明显降低(P<0.05)。ELISA结果显示,与正常组比较,LPS组细胞炎症IL-5、IL-6、TNF-α、IL-10水平显著升高(P<0.01),用药处理后,与LPS组比较,TNF-α、IL-6、IL-8含量均显著下降(P<0.01),IL-10升高(P<0.01)。结论:麻芍平喘汤可能通过激活PI3K/Akt/mTOR信号�
Objective:To investigate the effect of Mashao Pingchuan decoction(MSPC)on lipopolysaccharides(LPS)-induced autophagy in human bronchial airway epithelial cells(16HBE)via the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signaling pathway.Method:16HBE cells were selected for the study,and cell counting kit-8(CCK-8)was used to detect the activity of of LPS-induced 16HBE cells and the effect of MSPC-containing serum on the cells.Suitable LPS-induced 16HBE cells were screened by the CCK-8 method,and the content of tumor necrosis factor-α(TNF-α)was measured to identify the established model.And MSPC-containing serum was prepared.The cells were divided into normal group,LPS group,LPS+MSPC group,LY294002+LPS group and LY294002+LPS+MSPC group.Transmission electron microscopy was performed to observe the changes in autophagic vesicles and ultrastructure of the cells.Western blot was performed to detect the protein expressions of PI3K,phosphorylated PI3K(p-PI3K),Akt,phosphorylated Akt(p-Akt),mTOR,phosphorylated mTOR(p-mTOR)and microtubule-associated protein 1 light chain 3B(LC3B),and enzyme-linked immunosorbent assay(ELISA)was used to detect the expressions of inflammatory factors interleukin-5(IL-5),IL-6,TNF-αand IL-10 in the five groups.Result:LPS inhibited the 16HBE cells in a dose-dependent manner.Compared with the normal group,the LPS group(150 mg·L-1of LPS)increased the expression of pro-inflammatory factor TNF-αafter 24 h of treatment(P<0.05)and facilitated the autophagosome formation,and MSPC-containing serum exerted a concentration-dependent promotion effect on the 16HBE cells,inhibited the autophagy to a certain degree and enhanced the cell status.Western blot revealed that the protein expressions of p-PI3K,p-Akt and p-mTOR in the model group were lower(P<0.05)and the protein expression of LC3B was higher(P<0.01)than those in the normal group.Compared with the conditions in the LPS group,the protein expressions of p-PI3K,p-Akt and p-mTOR in the LPS+MSPC group we
作者
任燕群
王小乐
刘桐
张璐
王心恒
吴迪
丁焕章
李泽庚
REN Yanqun;WANG Xiaole;LIU Tong;ZHANG Lu;WANG Xinheng;WU Di;DING Huanzhang;LI Zegeng(Anhui University of Chinese Medicine,Hefei 230031,China;Key Laboratory of Xin'an Medicine,Ministry of Education,Hefei 230031,China;Key Laboratory of Chinese Medicine for Prevention and Treatment of Major Diseases in Pulmonary System,Key Laboratoryof Anhui Provincial Education Department,Hefei 230031,China;Institute of Chinese Medicine for Prevention and Treatment of Respiratory Diseases,Anhui Academy of Chinese Medicine,Hefei 230031,China;The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei 230031,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2023年第3期88-95,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金区域创新重点项目(U20A20398)
安徽省自然科学基金项目(2108085QH369)
安徽省教育厅重点项目(KJ2019A0469)。
