目的 探讨中介素(intermedin,IMD)通过抑制内质网应激(endoplasmic reticulum stress,ERS)途径的细胞凋亡,发挥对大鼠肾小管上皮细胞NRK-52E缺氧复氧(hypoxia/reoxygenation,H/R)损伤的保护机制。方法 NRK-52E细胞随机分为对照组;H/R组;空质粒(H/R+pIRES2)组;IMD(H/R+IMD)组。四甲基偶氮唑蓝(MTT)法检测细胞活力;Annexin V-FITC/PI双染细胞凋亡检测试剂盒检测细胞凋亡;Realtime PCR 法和Western-blot法分别检测ERS相关经典分子GRP78、CHOP、Caspase 12在mRNA及蛋白水平的表达;ER特异性荧光染料Dapoxyl用于观察ER形态变化。结果 MTT结果显示,H/R组NRK-52E细胞存活率较对照组明显下降,而IMD转染后细胞存活率提高;肾小管上皮细胞NRK-52E经H/R处理后出现凋亡,细胞凋亡率显著增加,同时H/R可导致ERS途径经典分子GRP78、CHOP、Caspase 12的mRNA和蛋白表达均显著上调;IMD转染后可显著降低NRK-52E细胞凋亡,细胞凋亡率降低,ERS途径经典分子GRP78、CHOP、Caspase 12的mRNA和蛋白表达均明显下降。内质网染色结果显示,H/R可以导致内质网结构受损,Dapoxyl染料聚集,荧光强度加强,内质网颗粒感增强,荧光颗粒分布不均、浓集,并有空泡;IMD转染后内质网结构受损减轻。结论 大鼠肾小管上皮细胞NRK-52E H/R损伤过程中可能存在ERS凋亡途径的活化,IMD可以通过抑制ERS相关的凋亡信号通路,从而减少肾小管上皮细胞凋亡,减轻肾脏IRI。
Abstract
OBJECTIVE To investigate intermedin protects against at rat renal tubular epithelial cells(NRK-52E) hypoxia-reoxygenation injury through suppresses endoplasmic reticulum stress(ERS)-related apoptosis. METHODS The NRK-52E cells were divided into 4 groups. One of them was control group; the other three model groups were exposed to H/R condition, following the intervention of single H/R, primitive vector and highly expressed was used IMD vector, respectively. The cell survival rate was detected by MTT; annexin V-FITC/PI double-stained cell apoptosis detection kit for detection of apoptosis; real-time PCR and Western-blot were used to detect expression of ERS molecules such as GRP78, CHOP and Caspase 12. ER specific resident fluorochrome dapoxyl was used to observe ER morphological changes. RESULTS MTT RESULTS showed that the survival rate of NRK-52E cells in H/R group was significantly decreased compared with the control group, and the cell survival rate was increased significantly in IMD group. The apoptosis rate of H/R increased significantly, and the mRNA and protein expressions of GRP78, CHOP and Caspase 12 in H/R group were all up-regulated significantly, IMD gene transfer markedly decreased the apoptosis index compared with that of H/R. The mRNA and protein expressions of GRP78, CHOP and Caspase 12 in IMD group were all down-regulated significantly. Dapoxyl staining demonstrated that the structure of the ER was severely destroyed in H/R, the fluorescence density showed nonuniform distribution, accompanied by notable vacuoles in the ER; Over-express IMD decreased the injury of the ER. CONCLUSION During rat renal tubular epithelial cells NRK-52E H/R injury may occur activation of ERS apoptotic pathway, IMD inhibites ERS-related apoptosis, reduces renal tubular epithelial cell apoptosis and reduces renal IRI.
关键词
中介素 /
缺氧复氧损伤 /
内质网应激 /
凋亡
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Key words
intermedin /
ischemia-reperfusion injury /
endoplasmic reticulum stress /
apoptosis
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中图分类号：
R965
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参考文献
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基金
国家自然科学基金项目资助(81500518,81500529);山西省青年科技应用基础研究项目资助(201601D021147);山西医科大学博士启动基金项目资助(03201302,03201403);山西医科大学科技创新基金资助(01201403)
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