胃饥饿素对HK-2细胞缺氧/复氧损伤后的保护作用及机制

Protective Effect and Mechanism of Ghrelin on Hypoxia/Reoxygenation Injury in HK-2 Cells

目的探讨胃饥饿素(ghrelin)对HK-2细胞缺氧/复氧的保护作用及机制。方法以HK-2细胞为研究对象,建立缺氧/复氧模型,模拟肾缺血再灌注损伤过程。胃饥饿素预处理后,采用CCK-8试剂盒检测不同质量浓度胃饥饿素(0,12.5,25,50,100,200,400,800,1600 ng/m L)对缺氧/复氧及正常HK-2细胞活力的影响。将细胞分为正常对照组(N组),缺氧/复氧损伤组(H/R组),胃饥饿素(400 ng/m L)预处理组+缺氧/复氧损伤组(ghrelin+H/R组)。造模成功后,采用原位末端转移酶标记(TUNEL)试剂盒检测细胞凋亡,蛋白质印迹法(WB)检测各组细胞外调节蛋白激酶(ERK)及磷酸化细胞外调节蛋白激酶(p-ERK)、P38促分裂素原活化蛋白激酶(P38)及磷酸化P38促分裂素原活化蛋白激酶(p-P38)的表达。结果缺氧4 h,复氧12 h能有效诱导HK-2细胞缺氧/复氧损伤模型;与胃饥饿素质量浓度为0 ng/m L时相比,质量浓度为12.5,25,50 ng/m L时对缺氧/复氧损伤的HK-2细胞无影响;胃饥饿素质量浓度为100,200,400,800 ng/m L预处理能有效提高细胞活力(P<0.01),质量浓度为1600 ng/m L时HK-2细胞的活力开始降低;胃饥饿素质量浓度大于800 ng/m L对正常HK-2细胞活力有影响(P<0.05);与N组相比,H/R组细胞凋亡明显,ERK,p-ERK,P38及p-P38表达无差异;与H/R组相比,ghrelin+H/R组细胞凋亡水平下降,ERK,p-ERK,P38表达无差异,p-P38表达增高。结论胃饥饿素预处理能对HK-2细胞缺氧/复氧损伤起到保护作用,可能是通过激活P38信号通路发挥其功能。

Objective To explore the protective effect and mechanism of ghrelin on hypoxia/reoxygenation of HK-2 cells.Methods A hypoxia/reoxygenation model was established with HK-2 cells as the research object to simulate the renal ischemia-reperfusion injury process.After pretreatment of ghrelin,CCK-8 kits were used to detect the effects of different concentrations of ghrelin on hypoxia/reoxygenation and normal HK-2 cell viability.Respectively,ghrelin concentration of 0,12.5,25,50,100,200,400,800 and 1600 ng/m L.HK-2 cells were divided into three group:normal control group(N),hypoxia/reoxygenation injury group(H/R),ghrelin(400 ng/m L)pretreatment group+hypoxia/reoxygenation injury group(ghrelin+H/R).Apoptosis was detected by TdT-mediated dUTP nick end labeling(TUNEL)kit,and the expressions of extracellular regulated protein kinase(ERK),phosphorylated extracellular regulated protein kinase(p-ERK),P38 kinogen-activated protein kinase(P38)and phosphorylated P38 kinogen-activated protein kinase(p-P38)were detected by Western Blot(WB).Results The H/R injury model in HK-2 cells was effectively induced by 4 h of hypoxia and 12 h of reoxygenation.Compared with Ghrelin concentration of 0,concentration of 12.5,25 and 50 ng/m L had no significantly effect on HK-2 cells injured by H/R injury.Pretreatment with ghrelin concentration of 100,200,400 and 800 ng/m L could significantlyincreasedcell viability(P<0.01).However,the viability of HK-2 cells was reduced when the ghrelin concentration was 1600 ng/m L.Ghrelin significantly affected the viability of normal HK-2 cells when the concentration was greater than 800 ng/m L(P<0.05).Compared with the N group,the H/R group had obvious apoptosis,but there was no difference in the expression of ERK,p-ERK,P38 and p-P38 protein.Compared with the H/R group,the apoptosis level of ghrelin+H/R group was decreased,and there was no difference in the expression of ERK,p-ERK,P38 protein,but the expression of p-P38 protein was significantly increased.Conclusion Ghrelin pretreatment may protect hypoxia/reoxygenation injury of HK-2 cells by activating the P38 signaling pathway.