miR-100过表达通过增强AMPK-mTOR-p70S6K信号通路介导的自噬发挥抗脓毒症作用的研究

miR-100 inhibition of cell model sepsis by the activation of AMPK-mTOR-p70S6K signaling-induced autophagy

目的探讨miR-100过表达通过增强AMPK-mTOR-p70S6K信号通路介导的自噬,进而发挥抗脓毒症的作用机制研究。方法利用脂多糖(LPS)刺激大鼠心肌细胞H9C2构建脓毒症体外细胞模型。将大鼠心肌细胞H9C2分为对照组、LPS组、LPS+miR-100激动剂组和LPS+miR-100拮抗剂组。细胞通过转染miR-100的激动剂或抑制剂使miR-100在细胞内过表达或敲低,并用qRTPCR检测细胞内miR-100的表达水平的改变;ELISA检测不同细胞处理组上清中炎症因子IL-1β、IL-6和TNF-α的释放;细胞免疫荧光检测LC3蛋白在细胞中的表达;Western blot检测自噬相关蛋白(p62、LC3和beclin-1)和AMPK-mTOR-p70S6K通路相关蛋白(AMPK、p-AMPK、mTOR、pm TOR、p70S6K和p-p70S6K)的表达。结果与对照组比较,在LPS处理心肌细胞H9C2的脓毒症细胞模型中,血清炎症因子分泌显著增加[TNF-α:(145.05±0.15)pg/mL比(50.35±0.05)pg/mL;IL-6:(35.47±0.08)pg/mL比(12.15±0.11)pg/mL;IL-1β:(141.38±0.11)pg/mL比(72.46±0.09)pg/mL,P<0.05];与LPS组比较,LPS+miR-100激动剂组上调miR-100表达能够诱导增强细胞自噬,并且减弱炎症反应[TNF-α:(71.22±0.06)pg/mL比(145.05±0.15)pg/mL;IL-6:(12.36±0.09)pg/mL比(35.47±0.08)pg/mL;IL-1β:(78.48±0.13)pg/mL比(141.38±0.11)pg/mL,P<0.05];相反,LPS+miR-100拮抗剂组下调miR-100表达减弱了自噬,并且增强了炎症反应[TNF-α:(241.33±0.19)pg/mL比(145.05±0.15)pg/mL;IL-6:(49.78±0.12)pg/mL比(35.47±0.08)pg/mL;IL-1β:(205.69±0.08)pg/mL比(141.38±0.11)pg/mL,P<0.05];进一步研究发现,miR-100能够引起自噬相关信号通路AMPKmTOR-p70S6K中相关蛋白表达变化。结论 miR-100过表达可能通过上调AMPK-mTOR-p70S6K信号通路介导的自噬水平,发挥抗炎作用,进而在脓毒症中发挥保护作用。

Objective To explore whether miR-100 inhibiting sepsis was through the activation of the AMPKmTOR-p70 S6 K signaling-induced autophagy. Methods The rat cardiomyocytes H9 C2 were grown and stimulated with lipopolysaccharide(LPS) to establish a cell sepsis model in vitro and divided into the control, LPS, LPS+miR-100 agonist, and LPS+miR-100 inhibitor groups. The overexpression or knockdown of miR-100 was achieved by transfecting cells with miR-100 agonists or inhibitors. Level of miR-100 was detected using qRT-PCR and levels of IL-1β, IL-6, and TNF-α in the supernatant were detected using ELISA. Level of the autophagy-related LC3 protein was assessed using immunofluorescence and other autophagy-related proteins(p62, LC3-Ⅰ, LC3-Ⅱ, and beclin-1) and the AMPK-mTOR-p70 S6 K pathway-related proteins(AMPK, p-AMPK, mTOR, p-mTOR, p70 S6 K, and p-p70 S6 K)were detected using Western blot. Results Compared with the control cells, LPS treated H9 C2 cells showed a significantly increase in the secretion of pro-inflammatory factors(TNF-α, 145.05±0.15 vs. 50.35±0.05 pg/mL;IL-6, 35.47±0.08 vs. 12.15±0.11 pg/mL;IL-1β, 141.38±0.11 vs. 72.46±0.09 pg/mL;all P<0.05). Compared with the LPS-treated cells, miR-100 expression was able to induce H9 C2 cell autophagy, but decrease levels of the inflammatory response proteins(TNF-α, 71.22 ±0.06 vs. 145.05 ±0.15 pg/mL;IL-6, 12.36 ±0.09 vs. 35.47 ±0.08 pg/mL;IL-1β, 78.48±0.13 vs. 141.38±0.11 pg/mL;all P<0.05) in LPS+miR-100 agonist-treated cells. In contrast, knockdown of miR-100 expression was able to reduce H9 C2 cell autophagy, but increase levels of the inflammatory response proteins(TNF-α: 241.33 ±0.19 vs. 145.05 ±0.15 pg/mL;IL-6, 49.78 ±0.12 vs. 35.47 ±0.08 pg/mL;IL-1β,205.69±0.08 vs. 141.38±0.11 pg/mL;all P<0.05)in LPS+miR-100 inhibitor-treated cells. Moreover, miR-100 expression was able to activate the AMPK-mTOR-p70 S6 K signaling and in turn to promote H9 C2 cell autophagy. Conclusion The data form the current study revealed that miR-100 possesses an anti-inflammatory activity by activation of the AMPK-mTOR-p70 S6 K signaling and autophagy in H9 C2 cells;thus, plays a protective role in sepsis.