右美托咪定调控PI3K/AKT/mTOR介导的自噬对糖尿病肾病足细胞炎症反应的影响

The effect of dexmedetomidine on PI3K/AKT/mTOR mediated autophagy on podocyte inflammation in diabetic nephropathy

目的探讨磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路介导的右美托咪定对高糖诱导的足细胞炎症反应的影响。方法体外培养人肾小球足细胞(HGPC),以5 mmol/L D-葡萄糖刺激细胞为对照,30 mmol/L D-葡萄糖刺激细胞建立糖尿病肾病模型,以25、50、100 nmol/L右美托咪定干预模型细胞,采用细胞计数试剂盒-8(CCK-8)测定细胞活力,筛选最佳药物浓度。然后将HGPC分为正常糖组(D-g组)、高糖组(H-g组)、右美托咪定组(Dex组)、激活剂组(A组)、右美托咪定+激活剂组(Dex+A组)及右美托咪定+抑制剂组(Dex+Y组)。干预24 h,测定细胞迁移数、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β及超氧化物歧化酶(SOD)、丙二醛(MDA)、自噬效应蛋白(Beclin1)、微管相关蛋白1轻链3-Ⅱ(LC3Ⅱ)及PI3K/AKT/mTOR通路蛋白水平。结果50 nmol/L右美托咪定为干预细胞活力最佳浓度。与D-g组比较,H-g组细胞活力及Beclin1、LC3Ⅱ、SOD、p-PI3K、p-AKT、p-mTOR水平下降,细胞迁移数及TNF-α、IL-1β、MDA水平升高,差异均有统计学意义(P<0.05);与H-g组比较,Dex组和A组的细胞活力及Beclin1、LC3Ⅱ、SOD、p-PI3K、p-AKT、p-mTOR水平升高,细胞迁移数及TNF-α、IL-1β、MDA水平下降,差异均有统计学意义(P<0.05);与Dex组比较,Dex+A组中TNF-α、IL-1β、MDA水平降低,SOD水平升高,细胞活力升高,细胞迁移数减少,差异均有统计学意义(P<0.05);与Dex组比较,Dex+Y组细胞中TNF-α、IL-1β及MDA水平升高,SOD水平降低,细胞活力降低,细胞迁移数增加,差异均有统计学意义(P<0.05)。结论右美托咪定能够通过激活PI3K/AKT/mTOR通路促进高糖环境下的足细胞自噬,并抑制细胞迁移、炎症及氧化应激损伤。

Objective To explore the effect of dexmedetomidine on the hyperglycemia-induced podocyte inflammatory response mediated by the phosphatidylinositol-3 kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway.Methods Human glomerular podocyte(HGPC)was cultured in vitro,and a diabetic nephrotic model was established with 5 mmol/L D-glucose-stimulated cells as control and 30 mmol/L D-glucose-stimulated cells.The model cells were interfered with with dexmedetomidine at 25,50 and 100 nmol/L,and cell viability was determined by cell counting kit 8(CCK-8).Screening for optimal drug concentration.The cells were then divided into the normal glucose group(D-g group),the high glucose group(H-g group),the dexmedetomidine group(Dex group),the activator group(A group),the dexmedetomidine+activator group(Dex+A group)and the dexmedetomidine+inhibitor group(Dex+Y group).After 24 h of intervention,cell migration number,tumor necrosis factor-α(TNF-α),interleukin-1β,superoxide dismutase(SOD),malondialdehyde(MDA),autophagy effect protein(Beclin1),microtubule-associated protein 1 light chain 3-Ⅱ(LC3Ⅱ)and PI3K/AKT/mTOR pathway protein levels were measured.Results Fifty nmol/L dexmedetomide was the optimal concentration to interfere with cell viability.The cell viability,Beclin1,LC3Ⅱ,SOD,p-PI3K,p-AKT and p-mTOR levels in the H-g group decreased compared with those in the D-g group(P<0.05),while the cell migration number,levels of TNF-α,IL-1β and MDA increased(P<0.05).Compared with those in the H-g group,the cell viability,Beclin1,LC3Ⅱ,SOD,P-PI3K,P-AKT and p-mTOR levels in the Dex group and the A group increased,while the cell migration number,TNF-α,IL-1β and MDA levels decreased,with statistical significance(P<0.05).Compared with those in the Dex group,the levels of TNF-α,IL-1β and MDA in the Dex+A group cells reduced,SOD levels increased,cell viability increased,and cell migration reduced,with statistical significance(P<0.05).Compared with those in the Dex group,the Dex+Y group showed increase in TNF-α,IL-1β and MDA levels,a decrease in SOD levels,a decrease in cell viability,and an increase in cell migration,the differences were statistically significant(P<0.05).Conclusion Dexmedetomidine can promote podocyte autophagy in hyperglycemic environment by activating PI3K/AKT/mTOR pathway,and inhibit cell migration,inflammation and oxidative stress damage.