本研究旨在探讨汉黄芩苷改善肝脏胰岛素抵抗(IR)的降血糖作用及其相关抗炎机制。1×10-9mol·L-1胰岛素与3.75×10-6mol·L-1地塞米松48小时。用葡萄糖氧化酶法检测不同浓度的Wogonoside(1,5,10,20,50μmol·L-1)在不同时间点(30,36,48,54h)对IR-HepG2细胞葡萄糖消耗的变化,以确定最佳起效时间。用酮法和CCK-8法分别检测糖原含量和细胞活力。潜热蛋白3(NLRP3),细胞因子信号抑制因子3(SOCS3),Toll样受体4(TLR4),核因子(NF-κB),白细胞介素(IL-1β),IL-6、肿瘤坏死因子(TNF-α)参与炎症信号通路,以及瘦素,Ob-R,p-IRS2/IRS2,p-PI3K/PI3K(p85),Westernblot检测IR-HepG2细胞中参与胰岛素信号通路的p-Akt/Akt和葡萄糖转运蛋白(GLUT1/2/4)。结果:20和50μmol·L-1黄芩苷显著上调IR-HepG2细胞的葡萄糖消耗(P<0。001)与IR模型组相比,最佳起效时间为48h。Wogonoside对HepG2细胞的细胞活力无明显影响。进一步的研究表明,20,50μmol·L-1黄芩苷处理48h后分别增加IR-HepG2细胞的糖原含量,特别是50μmol·L-1组(P<0。001).与IR模型组相比,Wogonoside不仅抑制炎性核转录因子NLRP3、SOCS3、TLR4、NF-κB的蛋白表达,同时也降低了下游炎症效应因子IL-1β的表达,IL-6和TNF-α。沃戈诺赛德升高的Ob-R,p-IRS2/IRS2,p-PI3K/PI3K(p85),p-Akt/Akt和GLUT1/2/4蛋白表达,而它抑制了SOCS3调节的瘦素表达。Wogonoside可以促进IR-HepG2细胞的葡萄糖摄取,增加糖原含量,从而增强胰岛素敏感性。其降糖作用可能与干预NLRP3/SOCS3-TLR4-NF-κB炎症通路、降低炎症因子表达有关。
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( 1,5,10,20,50 μmol·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 50 μmol·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 20,50 μmol·L-1 wogonoside respectively increased the glycogen content of IR-HepG2 cells after 48 h treatment,especially in 50 μmol·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.