双氢青蒿素抑制TLR4/NF-κB信号通路对高糖刺激的足细胞功能障碍的改善作用  

Dihydroartemisinin Improves High Glucose-Stimulated Podiatocyte Dysfunction by Inhibiting TLR4/NF-κB Signaling Pathway

作　　者：张云[1] 陈小青[2] 李月婷[1] 施书涵[1] 林长达 ZHANG Yun;CHEN Xiaoqing;LI Yueting;SHI Shuhan;LIN Changda(Department of Nephrology,the Second Affiliated Hospital of Fujian Medical University,Quanzhou 362000 Fujian,China;Department of Rheumatology and Immunology,the Second Affiliated Hospital of Fujian Medical University,Ouanzhou 362000 Fujian,China)

机构地区：[1]福建医科大学附属第二医院肾脏内科,福建泉州362000 [2]福建医科大学附属第二医院风湿免疫科,福建泉州362000

出　　处：《中药新药与临床药理》2023年第8期1075-1082,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：福建省自然科学基金项目(2021J01250);福建省自然科学基金项目(2020J01231)。

摘　　要：目的探究双氢青蒿素通过调控Toll样受体4(TLR4)/核因子-κB(NF-κB)信号通路对D-葡萄糖诱导的人肾小球足细胞(HGPC)炎症、增殖、迁移和侵袭的影响。方法体外培养HGPC细胞,将HGPC细胞分为对照组(5 mmol·L^(-1)D-葡萄糖)、高糖组(30 mmol·L^(-1)D-葡萄糖)和不同浓度双氢青蒿素组(30 mmol·L^(-1)D-葡萄糖+5、10、20、40μmol·L^(-1)双氢青蒿素),筛选出合适的双氢青蒿素作用浓度;随后将细胞分为对照组、高糖组、双氢青蒿素组(30 mmol·L^(-1)D-葡萄糖+20μmol·L^(-1)双氢青蒿素)、抑制剂组(30 mmol·L^(-1)D-葡萄糖+5μmol·L^(-1)NF-κB通路抑制剂BAY 11-7082)、双氢青蒿素+抑制剂组(30 mmol·L^(-1)D-葡萄糖+20μmol·L^(-1)双氢青蒿素+5μmol·L^(-1)BAY 11-7082)和双氢青蒿素+激活剂组(30 mmol·L^(-1)D-葡萄糖+20μmol·L^(-1)双氢青蒿素+1μmol·L^(-1)NF-κB通路激活剂Prostratin),干预24 h。细胞计数试剂盒细胞8(CCK-8)法检测细胞活力;酶联免疫吸附试验(ELISA)法检测炎症因子白细胞介素(IL)-1β和IL-8的表达水平;5-乙炔基-2’脱氧尿嘧啶核苷(EdU)法检测细胞的增殖能力;Transwell小室法检测细胞迁移和侵袭能力;蛋白免疫印迹(Western Blot)法检测细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶9(MMP-9)和TLR4/NF-κB通路相关蛋白表达水平。结果CCK-8法检测结果显示20μmol·L^(-1)双氢青蒿素对D-葡萄糖HGPC细胞活力恢复效果最好,因此选择20μmol·L^(-1)双氢青蒿素用于后续实验。与对照组比较,高糖组细胞增殖率和CyclinD1蛋白相对表达量明显降低(P<0.05),炎症因子(IL-1β和IL-8)、迁移数、侵袭数、MMP-9、TLR4和磷酸化(p)-NF-κB p65蛋白相对表达量明显升高(P<0.05);双氢青蒿素组、抑制剂组、双氢青蒿素+抑制剂组及双氢青蒿素+激活剂组中双氢青蒿素和BAY11-7082明显抑制了D-葡萄糖对HGPC细胞的上述作用(P<0.05);与双氢青蒿素组比较,双氢青蒿素+抑制剂组中BAY11-7082增强了双�Objective To investigate the effects of dihydroartemisinin on D-glucose-induced inflammation,proliferation,migration and invasion of human glomerular podocytes(HGPC)by regulating Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway.Methods HGPC cells were cultured in vitro and divided into control group(5 mmol·L^(-1)D-glucose),high glucose group(30 mmol·L^(-1)D-glucose)and dihydroartemisinin groups(30 mmol·L^(-1)D-glucose+5,10,20,40μmol·L^(-1)dihydroartemisinin),the appropriate concentration of dihydroartemisinin was screened.The cells were then divided into control group,high glucose group,dihydroartemisinin group(30 mmol·L^(-1)D-glucose+20μmol·L^(-1)dihydroartemisinin),inhibitor group(30 mmol·L^(-1)D-glucose+5μmol·L^(-1)NF-κB inhibitor BAY11-7082),dihydroartemisinin+inhibitor group(30 mmol·L^(-1)D-glucose+20μmol·L^(-1)dihydroartemisinin+5μmol·L^(-1)BAY11-7082),and dihydroartemisinin+activator group(30 mmol·L^(-1)D-glucose+20μmol·L^(-1)dihydroartemisinin+1μmol·L^(-1)NF-κB activator Prostratin)and intervened for 24 hours.Cell counting kit-8(CCK-8)was used to detect cell viability.The expression levels of interleukin-1βand interleukin-8 were detected by enzymelinked immunosorbent assay(ELISA).The proliferation ability of cells was detected by 5-ethynyl-2'-deoxyuridine(EdU)method.Cell migration and invasion ability were detected by Transwell cell assay.The expression levels of CyclinD1,matrix metalloproteinase-9(MMP-9)and TLR4/NF-κB pathway related proteins were detected by Western Blot(WB).Results CCK-8 assay showed that 20μmol·L^(-1)dihydroartemisinin,which had the best effect in restoring viability of D-glucose HGPC cells,was selected for the follow-up experiment.Compared with the control group,cell proliferation rate and the relative expression of CyclinD1 protein in the hyperglycemic group were significantly decreased(P<0.05),while migration number,invasion number,the relative expression of inflammatory factors(IL-1β and IL-8),MMP-9,TLR4 and phosphorylated(p)-NF-κ

关 键 词：糖尿病肾病 人肾小球足细胞 D-葡萄糖 双氢青蒿素 Toll样受体4/核因子-κB信号通路(TLR4/NF-κB) 炎症 增殖 迁移 侵袭 

分 类 号：R285.5[医药卫生—中药学]