摘要
该实验研究汉黄芩苷对抗炎改善肝细胞胰岛素抵抗(IR)的降糖作用及相关抗炎分子机制。1×10-9mol·L-1胰岛素和3. 75×10-6mol·L-1地塞米松联合诱导48 h,建立稳定IR-HepG2细胞模型。采用葡萄糖氧化酶法在不同时间点(30,36,48,54 h)检测1,5,10,20,50μmol·L-1汉黄芩苷干预IR-HepG2细胞葡萄糖消耗量的变化,确定最佳起效时间点;蒽酮法检测细胞内糖原含量;CCK-8法检测各浓度汉黄芩苷对细胞活力的影响;Western blot法分别检测IR-HepG2细胞炎症小体隐热蛋白3(NLRP3)、细胞因子信号转导抑制蛋白3(SOCS3)、Toll样受体4(TLR4)、核转录因子-κB(NF-κB)、白细胞介素-1β(IL-1β)、白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)、瘦素(leptin)、瘦素受体(Ob-R)、磷酸化胰岛素受体底物2[p-IRS2 (Ser731)]、胰岛素受体底物2(IRS2)、磷酸化磷脂酰肌醇3-激酶p85[p-PI3K(Tyr607)]、磷脂酰肌醇3-激酶p85[PI3K(p85)]、磷酸化蛋白激酶B[p-Akt(Ser473)]、蛋白激酶B(Akt)和葡萄糖转运蛋白1/2/4(GLUT1/2/4)的蛋白含量。结果显示,最佳起效时间点为48 h。与IR模型组比较,20和50μmol·L-1汉黄芩苷明显上调IR-HepG2细胞葡萄糖消耗量(P<0. 001)。20和50μmol·L-1汉黄芩苷干预48 h增加IR-HepG2细胞糖原含量,且50μmol·L-1差异极其显著(P<0. 001)。各浓度汉黄芩苷对细胞活力均没有明显影响。汉黄芩苷明显抑制炎症通路中重要核转录因子NLRP3,SOCS3,TLR4和NF-κB,并减弱下游炎症效应因子IL-1β,IL-6和TNF-α的表达。除此之外,汉黄芩苷下调受SOCS3调控的leptin水平,激活胰岛素降糖通路中Ob-R,IRS2,p-PI3K/PI3K (p85),p-Akt/Akt和GLUT1/2/4蛋白表达。汉黄芩苷可促进Hep G2细胞葡萄糖摄取和增加糖原合成改善肝IR,其降糖机制可能与调控NLRP3/SOCS3-TLR4-NF-κB炎症通路降低肝IR细胞炎症因子表达,激活胰岛素降糖通路来加强葡萄糖摄取和利用相关。
This study was to investigate the hypoglycemic effect of wogonoside to improve hepatic insulin resistance(IR)and its relative anti-inflammatory mechanism.The stable IR-Hep G2 cell model was established by the combination of 1×10-9 mol·L-1 insulin and 3.75×10-6 mol·L-1 dexamethasone for 48 hours.The changes of glucose consumption in IR-Hep G2 cells with different concentrations of wogonoside(1,5,10,20,50μmol·L-1)at different time points(30,36,48,54 h)were detected by glucose oxidase assay to determine the optimal onset time.Glycogen content and cell viability were respectively detected by ketone method and CCK-8 method.Cryptothermal protein 3(NLRP3),suppressor of cytokine signaling 3(SOCS3),Toll-like receptor 4(TLR4),nuclear factor(NF-κB),interleukin(IL-1β),IL-6,tumor necrosis factor(TNF-α)involving in the inflammatory signaling pathway,as well as leptin,Ob-R,p-IRS2/IRS2,p-PI3 K/PI3 K(p85),p-Akt/Akt and glucose transporter(GLUT1/2/4)involving in the insulin signaling pathway were detected in IR-HepG2 cells by Western blot.Results showed that 20 and 50μmol·L-1 wogonoside significantly up-regulated the glucose consumption of IR-HepG2 cells(P<0.001)as compared with IR model group,and the optimal onset time was 48 h.Wogonoside had no obvious effect on the cell viability of Hep G2 cells.Further studies showed that 20,50μmol·L-1 wogonoside respectively increased the glycogen content of IR-HepG2 cells after 48 h treatment,especially in 50μmol·L-1 group(P<0.001).Compared with IR model group,wogonoside not only inhibited the protein expression of inflammatory nuclear transcriptional factors NLRP3,SOCS3,TLR4,NF-κB,but also decreased the expression of downstream inflammatory effect factors IL-1β,IL-6 and TNF-α.In addition,wogonoside elevated Ob-R,p-IRS2/IRS2,p-PI3 K/PI3 K(p85),p-Akt/Akt and GLUT1/2/4 protein expression,whereas it suppressed leptin expression that was regulated by SOCS3.Wogonoside could promote glucose uptake and increase glycogen content to enhance insulin sensitivity in IR-Hep G2 cells.The hypoglycemic effect may be related to the intervention of NLRP3/SOCS3-TLR4-NF-κB inflammatory pathway and decrease of inflammatory factor expression.
作者
朱水兰
吴青华
涂珺
ZHU Shui-lan;WU Qing-hua;TU Jun(Jiangxi Province Key Laboratory of Traditional Chinese Medicine Etiopathogenisis,Research Center for Differentiation and Development of Traditional Chinese Medicine Basic Theory,Jiangxi University of Traditional Chinese Medicine,Nanchang 330004,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2019年第20期4504-4510,共7页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81460621)
江西省自然科学基金项目(20143ACB20010,20171BAB205094)
江西省卫生厅中医药科技计划项目(2017Z017)
关键词
汉黄芩苷
胰岛素抵抗
炎症信号通路
胰岛素信号通路
wogonoside
insulin resistance(IR)
inflammatory signaling pathway
insulin signaling pathway