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目的评价水飞蓟素(Sil)对棕榈酸(PA)诱导HepG2细胞IR的作用并进一步揭示其机制。方法实验分为正常对照(NC)组,棕榈酸(PA)组,Sil干预低剂量(12.5μmol/L,L-Sil)组、中剂量(25μmol/L,M-Sil)组和高剂量(50μmol/L,H-Sil)组。PA诱导HepG2细胞IR,分别给予12.5,25,50μmol/L Sil作用6 h,测定葡萄糖消耗量,使用活性氧簇(ROS)试剂盒测定ROS活性水平。采用Western blot方法检测AKT以及还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的NOX2、p67phox、p47phox蛋白表达水平。结果PA组与NC组比较,葡萄糖耗氧量降低[(41.2±6.1)%vs(100±12)%,P=0.0087],磷酸化AKT(p-AKT)蛋白表达量降低[(0.458±0.185)vs(1.00±0.13),P=0.0184],同时PA组ROS水平[(1567±189 vs(14±34)U/ml,P=0.0094]以及NAPDH氧化酶NOX2、p67、p47亚基的表达水平升高(P<0.05)。Sil各组与PA组比较可增加葡萄糖消耗量[(52.8±8.4)%vs(62.2±6.5)%vs(82.3±9.8)%],差异有统计学意义(P=0.041、0.039、0.0076);H-Sil组磷酸化AKT(p-AKT)蛋白表达量提高(0.793±0.249),与PA组比较,差异有统计学意义(P=0.0395);Sil可降低ROS水平[(1085±252)vs(851±184)vvs(405±35)],与PA组比较,差异有统计学意义(P=0.047、0.023、0.0015),以及降低NAPDH氧化酶NOX2、p67、p47亚基的表达水平(P<0.05),且作用强度随Sil剂量的增加而增强。结论Sil可能通过降低NADPH氧化酶活性降低氧化应激,从而改善HepG2细胞的IR。
Objective To evaluate the effect of silymarin (Sil) on the IR of HepG2 cells induced by palmitic acid (PA) and to reveal its mechanism. Methods The experiment was divided into normal control (NC) group, palmitic acid (PA) group, Sil-treated low dose 12.5μmol / L L-Sil group, middle dose 25μmol / L M-Sil group and high dose 50 μmol / L, H-Sil) group. PA induced HepG2 cells IR, 12.5, 25 and 50μmol / L Sil were given for 6 h respectively, and the glucose consumption was measured. The activity of ROS was measured by reactive oxygen species (ROS) kit. Western blot was used to detect the expression of NOX2, p67phox and p47phox in AKT and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Results Compared with NC group, the oxygen consumption of glucose decreased in group PA compared with NC group [(41.2 ± 6.1)% vs (100 ± 12)%, P = 0.0087], and the phosphorylated AKT protein expression decreased [(0.458 ± 0.185 ) vs (1.00 ± 0.13), P = 0.0184]. The ROS levels in PA group [(1567 ± 189 vs (14 ± 34) U / ml, P = 0.0094] and the expression of NAPDH oxidase NOX2, p67 and p47 subunits (52.8 ± 8.4)% vs (62.2 ± 6.5)% vs (82.3 ± 9.8)%], the difference was statistically significant (P <0.05) .Sil groups compared with PA group increased glucose consumption [(52.8 ± 8.4)% vs P = 0.041,0.039,0.0076). Compared with PA group, the expression of phosphorylated AKT protein in H-Sil group was significantly increased (0.793 ± 0.249, P = 0.0395) (P = 0.047,0.023,0.0015), as well as the decrease of NAPDH oxidase NOX2, p67 (P <0.05). Compared with PA group, there was significant difference , P47 subunits (P <0.05), and the intensity increased with the increase of Sil dose.Conclusion Sil could reduce the oxidative stress by decreasing the activity of NADPH oxidase and thus improve the IR of HepG2 cells.