摘要
目的探讨麦冬皂苷D(OPD)对脂多糖(LPS)诱导的胃癌细胞分泌细胞因子的影响及可能机制。方法体外培养人胃癌HGC-27细胞,用不同剂量(2.5、10、40μmol/L)的OPD作用于LPS诱导的HGC-27细胞24 h,酶联免疫吸附法检测细胞培养上清液中TGF-β、VEGF和IL-6表达,RT-qPCR检测细胞中miR-448表达,蛋白质印迹法检测细胞中TLR4蛋白表达。转染miR-448模拟物/抑制剂、TLR4小干扰RNA至HGC-27细胞,上述相同方法观察miR-448和TLR4对LPS诱导的HGC-27细胞培养上清液中TGF-β、VEGF和IL-6表达的影响。双荧光素酶报告基因实验验证miR-448和TLR4靶向调控关系。结果LPS促进HGC-27细胞分泌TGF-β、VEGF和IL-6,而OPD呈剂量依赖性降低了LPS诱导的HGC-27细胞分泌TGF-β、VEGF和IL-6。OPD促进LPS诱导的HGC-27细胞中miR-448的表达,而抑制TLR4表达。过表达miR-448或敲减TLR4均可抑制LPS诱导的HGC-27细胞分泌TGF-β、VEGF和IL-6。miR-448靶向负调控TLR4。敲减miR-448降低OPD对LPS诱导的HGC-27细胞TGF-β、VEGF和IL-6分泌的抑制作用。结论OPD可能通过上调细胞中miR-448表达进而抑制TLR4表达,最终抑制LPS诱导的胃癌HGC-27细胞分泌TGF-β、VEGF和IL-6。
This study was performed to explore the effect of ophiopogonin D(OPD)on the expression of immunosuppressive cytokines in gastric cancer cells induced by lipopolysaccharide(LPS)and its possible mechanism.Human gastric cancer cell line HGC-27 cells were cultured in vitro,then induced by LPS and treated by different doses of OPD(2.5,10,40μmol/L)for 24 h.Enzyme-linked immunosorbent assay was used to detect the expression of TGF-β,VEGF and IL-6 in the cell culture supernatant;RT-qPCR was used to detect the expression of miR-448 in cells;Western blot was used to detect the expression of TLR4 protein in cells.miR-448 mimics/inhibitors or TLR4 small interfering RNA was transfected into HGC-27 cells,and the same method described above were used to observe the effects of miR-448 or TLR4 on the expression of TGF-β,VEGF and IL-6 in the culture supernatant of LPS-induced HGC-27 cells.Furthermore,dual luciferase reporter gene experiment was performed to verify the targeted regulatory relationship between miR-448 and TLR4.Data showed that LPS promoted the secretion of TGF-β,VEGF and IL-6 in HGC-27 cells,while OPD dose-dependently reduced the secretion of TGF-β,VEGF and IL-6 in LPS-induced HGC-27 cells.OPD promoted the expression of miR-448 in LPS-induced HGC-27 cells,but inhibited the expression of TLR4.Overexpression of miR-448 or knockdown of TLR4 inhibited the secretion of TGF-β,VEGF and IL-6 in LPS-induced HGC-27 cells.miR-448 negatively regulated the expression of TLR4,and knockdown of miR-448 reduced the inhibitory effect of OPD on the secretion of TGF-β,VEGF and IL-6 in LPS-induced HGC-27 cells.Taken together,OPD may inhibit the secretion of TGF-β,VEGF and IL-6 in LPS-induced gastric cancer HGC-27 cells by up-regulating the expression of miR-448 and inhibiting the expression of TLR4.
作者
康振朝
李晓
朱晓梅
王伟
盛志伟
孙瑞
KANG Zhenchao;LI Xiao;ZHU Xiaomei;WANG Wei;SHENG Zhiwei;SUN Rui(Department of Radiotherapy,Wuwei Liangzhou Hospital,Wuwei 733000,China;Department of Neurology,Wuwei Liangzhou Hospital,Wuwei 733000,China;Department of Urology,Wuwei Liangzhou Hospital,Wuwei 733000,China;Department of Oncology,First People's Hospital of Lanzhou City,Lanzhou 730050,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2022年第5期416-423,共8页
Immunological Journal
作者简介
通信作者:孙瑞,E-mail:sunrui0931@163.com。
