| Objective:PERK exerts pro-apoptotic or anti-apoptotic effects through activating downstream e IF2αand Nrf-2 signaling pathways. Endoplasmic reticulum stress pathway of the PERK, which is involved in the pathogenesis of many diseases through the regulation of apoptosis. Recent studies have shown that diabetic kidney tissue is brought about endoplasmic reticulum stress and PERK/e IF2α pathway activation, however, the relationship with the two kinds of stress pathways and renal cell apoptosis are unclear. This study was based on the preliminary work of the GRP78/PERK/ e IF2αendoplasmic reticulum stress signaling pathway and the increase of apoptosis in the diabetic kidney, we use a technique called RNA interference down-regulated e IF2α and Nrf-2 gene expression by in vitro experiments. We try to clear:(1)whether cell apoptosis in diabetic kidney has relation to PERK/e IF2α signaling pathway activation;(2)High glucose and angiotensin induced renal tubular Ⅱepithelial cells apoptosis whether depends on PERK/e IF2α and(or) PERK/Nrf-2 signaling pathway activation;(3) e IF2α and Nrf-2, what is a key molecule in the induction of apoptosis. The significance of this project is screening new effective molecular intervention targets in the treatment of diabetic nephropathy. Methods:The renal tubular epithelial cells(NRK-52E) were cultured in vitro, then which were singly transfected lentiviral vector for e IF2αand Nrf-2 si RNA. The transfection efficiency was observed by fluorescence microscopy, and the value of MOI was determined. After transfection, NRK-52 E cells were stimulated with high glucose(30mmol/L) and different concentrations of angiotensin II(10-5、10-6、10-7、10-8、10-9mol/L). The observation time was 12 h, 24 h, and 48 h. Apoptosis was detected by flow cytometry, and the optimal concentration and time of action of angiotensin II were screened out. The experiments were divided into four groups: normal control group(glucose 5.5mmol/L), high glucose and angiotensin II stimulation group, high glucose and angiotensin II + empty body group, high glucose and angiotensin II +Si RNA group. The expression of e IF2α、p-e IF2α and Nrf-2 of the renal tubular epithelial cells were detected by immunohistochemistry and Western assay. The renal tubular epithelial cells apoptosis were detected by TUNEL. Results:1.Fluorescence microscopy showed a large number of green fluorescent expression, transfection efficiency at least 80% or more, MOI value was 10.2.The optimal action concentration of angiotensin II(10-7 mol/L)was screened by flow cytometry, and the optimal action time was 24 hours.3.The expression of e IF2α and Nrf-2 in NRK-52 E cells was confirmed by immunohistochemistry and Western blot. After adding high glucose, vascular angiotensin II induced cell apoptosis, the expression of p-e IF2α increased significantly(p<0.05). After e IF2α-Si RNA interference, the expression of e IF2α、p-e IF2α was obviously inhibited(p<0.05). After Nrf-2-Si RNA interference, the expression of Nrf-2 was significantly inhibited(p<0.05).4.The apoptosis of NRK52 E cells was increased after the addition of high glucose and angiotensin II to TUNEL. After e IF2-Si RNA interference, apoptosis was significantly lower than that of normal control group and high glucose, Ang II group(p<0.05). After Nrf-2-Si RNA interference, apoptosis was significantly higher than that of the normal control group and the high glucose group, the Ang II group(p<0.05). Conclusion:1.Under normal circumstances, the expression of Nrf-2 and e IF2α is in NRK52 E cells,PERK/e IF2 signaling pathway plays a pro-apoptotic role in the ERS response. PERK/Nrf-2 signaling pathway plays an anti-apoptotic role in the ERS response.2.High glucose and angiotensin II induced renal tubular epithelial cells apoptosis may depend on the PERK/e IF2α and(or) PERK/Nrf-2 signaling pathway activation. |
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