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目的研究西罗莫司对肿瘤坏死因子α(TNF-α)诱导的HepG2细胞脂质积聚的影响。方法将棕榈酸(PA)负荷的HepG2细胞分为3组:对照组[0.2%牛血清白蛋白(BSA)+0.16 mmol·mL~(-1)PA],模型组(0.2%BSA+0.16 mmol·mL~(-1)PA+25 ng·mL~(-1)TNF-α),实验组(0.2%BSA+0.16mmol·mL~(-1)PA+25 ng·mL~(-1)TNF-α+10 ng·mL~(-1)西罗莫司)。各组处理24h后,以油红O染色观察HepG2细胞脂质蓄积情况;以蛋白质印迹法检测哺乳动物雷帕霉素靶蛋白(mTOR)及其下游核糖体蛋白P70S6激酶(P70S6K)总蛋白和磷酸化蛋白水平;以实时荧光定量聚合酶链式反应检测固醇调节原件结合蛋白1(SREBP1)、乙酰辅酶A羧化酶(ACC)、脂肪酸合成酶(FAS)mRNA的表达。结果在炎症状态下,西罗莫司可以明显抑制TNF-α诱导的HepG2细胞脂质积聚。同时,西罗莫司降低了TNF-α诱导的磷酸化mTOR(p-mTOR)及磷酸化P70S6K(p-P70S6K)蛋白表达:对照组、模型组及实验组的p-mTOR蛋白相对水平分别为1.00±0.20,3.11±0.60,2.38±0.50;这3组的p-P70S6K蛋白相对水平分别为1.00±0.30,3.67±0.60,1.62±0.50,模型组与对照组相比或者实验组与模型组相比,差异均有统计学意义(均P<0.05)。西罗莫司还下调了SREBP1、ACC、FAS的mRNA表达:对照组、模型组及实验组的FAS mRNA相对水平分别是1.04±0.32,2.85±0.90,1.68±0.38;这3组的ACC mRNA相对水平分别是1.04±0.30,3.23±1.33,2.07±0.52;这3组的SREBP1的mRNA相对水平分别是1.01±0.16,3.85±1.30,2.82±0.57,模型组与对照组相比或者实验组与模型组相比,差异均有统计学意义(均P<0.05)。结论西罗莫司通过抑制mTOR信号通路,减轻TNF-α诱导的HepG2细胞脂质积聚。
Objective To study the effect of sirolimus on lipid accumulation in HepG2 cells induced by tumor necrosis factor α (TNF-α). Methods HepG2 cells loaded with palmitic acid (PA) were divided into three groups: control group (0.2% BSA + 0.16 mmol · mL -1 PA), 0.2% BSA + 0.16 mmol · PA + 25 ng · mL -1 · mL-1 PA + 25 ng · mL -1 in the experimental group (0.2% BSA + 0.16 mmol · mL -1 PA + TNF-α + 10 ng · mL -1 sirolimus). After 24 h of treatment, the lipid accumulation of HepG2 cells was observed by oil red O staining. The total protein of mammalian target protein of rapamycin (mTOR) and its downstream P70S6 kinase (P70S6K) The levels of SREBP1, ACC and FAS mRNA were detected by real-time fluorescence quantitative polymerase chain reaction. Results In the inflammatory state, sirolimus significantly inhibited the TNF-α-induced HepG2 cell lipid accumulation. Simultaneously, sirolimus reduced the expression of phosphorylated mTOR (p-mTOR) and phosphorylated P70S6K (p-P70S6K) induced by TNF-α: The relative levels of p-mTOR protein in control group, model group and experimental group were 1.00 ± 0.20,3.11 ± 0.60,2.38 ± 0.50. The relative levels of p-P70S6K protein in these three groups were 1.00 ± 0.30, 3.67 ± 0.60 and 1.62 ± 0.50, respectively. Compared with the control group or the model group The differences were statistically significant (all P <0.05). Sirolimus also down-regulated the mRNA expression of SREBP1, ACC and FAS: The relative levels of FAS mRNA in control group, model group and experimental group were 1.04 ± 0.32, 2.85 ± 0.90 and 1.68 ± 0.38, respectively; The levels of SREBP1 mRNA in these three groups were 1.01 ± 0.16, 3.85 ± 1.30 and 2.82 ± 0.57, respectively. Compared with the control group or the experimental group and the model Compared with the control group, the differences were statistically significant (all P <0.05). Conclusion Sirolimus can reduce the lipid accumulation of HepG2 cells induced by TNF-α by inhibiting the mTOR signaling pathway.