汉黄芩苷调控NLRP3/SOCS3-TLR4-NF-κB炎症通路改善肝细胞胰岛素抵抗的研究

Study on regulation of NLRP3/SOCS3-TLR4-NF-κB inflammatory pathway by wogonoside to improve hepatic insulin resistance

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.