甜橙黄酮体外抑制氧化应激减轻肾小球微血管内皮细胞焦亡的机制研究

Study on the Mechanism of Sinensetin-mediated Reduction of Glomerular Microvascular Endothelial Cells Pyroptosis Through Inhibition of Oxidative Stress in Vitro

关键词:  糖尿病肾脏疾病  甜橙黄酮  氧化应激  焦亡

Key words:  diabetic kidney disease  sinensetin  oxidative stress  pyroptosis

摘要:

目的 探讨甜橙黄酮对糖尿病肾脏疾病的治疗作用及其作用机制。方法 采用硫氧还蛋白外源性抑制剂Px-12孵育人肾小球微血管内皮细胞(human renal glomerular microvascular endothelial cells,HRGEC)在体外建立细胞氧化损伤模型。分别应用甜橙黄酮、氧化应激抑制剂N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine,NAC)和细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK1/2)抑制剂PD98059进行预处理,而后与Px-12共同孵育细胞。设立对照组、Px-12组、甜橙黄酮组、甜橙黄酮+Px-12组、NAC组、NAC+Px-12组、PD98059组、PD98059+Px-12组。观察细胞形态,CCK-8测定细胞活性,并使用活性氧(reactive oxygen species,ROS)/O₂^-荧光探针测定各组HRGEC细胞氧化应激水平的差异。通过透射电镜观察各组细胞膜形态的变化,Western blotting检测膜穿孔蛋白(gasdermin D,GSDMD)、ERK1/2磷酸化和间隙连接蛋白43(Connexin 43,Cx43)蛋白表达。结果 Px-12可诱导HRGEC细胞膜完整性破坏,并可使GSDMD蛋白剪切暴露其NT端,出现细胞焦亡(P<0.01);Px-12可明显上调ROS/O₂^-的表达,激活氧化应激水平(P<0.01);甜橙黄酮可明显抑制Px-12诱导的ROS/O₂^-增加(P<0.05),同时能够减少Px-12诱导的GSDMD-NT的形成(P<0.01)和ERK的磷酸化(P<0.05);Px-12可诱导Cx43的表达明显增加(P<0.01),PD98059则可以明显抑制Cx43的表达(P<0.01),而甜橙黄酮与其作用相似(P<0.01)。结论 甜橙黄酮可减轻Px-12诱导的HRGEC细胞焦亡,可能与调控ERK-MAPK/Cx43信号通路相关。

Abstract:

OBJECTIVE To explore the therapeutic effect and mechanism of sinensetin on diabetic kidney disease. METHODS In vitro cell injury model was constructed by incubating human renal glomerular microvascular endothelial cells (HRGEC) using the reducing body Trx1 exogenous inhibitor of Px-12. Control group, Px-12 group, sinensetin group, sinensetin+Px-12 group, N-acetyl-L-cysteine(NAC) group, NAC+Px-12 group, PD98059 group and PD98059+Px-12 group were established. Cells were pretreated with sinensetin, the oxidative stress inhibitor NAC, and the extracellular regulatory protein kinase(ERK1/2) inhibitor PD98059, and then co-incubated with Px-12. Cell morphology was observed, and CCK-8 was used to determine cell viability. The among-group difference level of oxidative stress of HRGEC in each group was determined by using reactive oxygen species(ROS)/O₂^- fluorescence probe. The changes in membrane morphology in each group were observed by transmission electron microscopy, and the protein expression of gasdermin D(GSDMD), ERK1/2 phosphorylation, and gap junction protein 43(Connexin 43, Cx43) proteins were detected by Western blotting. RESULTS Px-12 could induce the breakdown of HRGEC membrane integrity and expose the NT terminal of GSDMD, leading to pyroptosis(P<0.01). Px-12 could significantly induce the upregulation of ROS/O₂^- and activate the oxidative stress level(P<0.01). On the contrary, sinensetin could markedly inhibit Px-12-induced increase of ROS/O₂^-(P<0.05) and reduce Px-12-induced formation of GSDMD-NT and ERK phosphorylation(P<0.01 or P<0.05). Px-12 could induce an enhancement of Cx43 expression(P<0.01), while the inhibitor of ERK signaling inhibitor could significantly inhibit Cx43 expression(P<0.01). Coincidently, sinensetin had the similar function (P<0.01). CONCLUSION Sinensetin can alleviate Px-12-induced HRGEC pyroptosis, which is probably related to the regulation of the ERK-MAPK-Cx43 signaling pathway.