阿托伐他汀调控PERK/eIF2α/CHOP通路减轻造影剂诱导的大鼠肾小管上皮细胞凋亡

doi:10.3877/cma.j.issn.2095-3216.2019.03.002

目的

研究造影剂肾病大鼠肾脏中葡萄糖调节蛋白78(GRP78)、内质网调节激酶(PERK)、真核起始因子2α(eIF2α)及C/EBP同源蛋白质(CHOP)的表达情况，探讨内质网应激在造影剂肾病发病中的作用及阿托伐他汀的干预作用。

方法

60只大鼠随机分为4组：对照组、模型组和高、低剂量阿托伐他汀组(80 mg，40 mg)，每组15只。分别于注射造影剂后24、48、72 h留取血清；检测各组大鼠的血清尿素氮(BUN)、血清肌酐(Scr)；TUNEL法及Western印迹法测casepase-3的表达检测肾小管上皮细胞凋亡；免疫组化和Western印迹法检测各组大鼠肾组织GRP78、p-eIF2α、p-PERK及CHOP的表达。

结果

与对照组相比，模型组大鼠BUN、Scr显著升高，细胞凋亡严重，GRP78、p-eIF2α、p-PERK及CHOP的表达均显著升高(P< 0.05);与模型组相比，高、低剂量阿托伐他汀组，BUN、Scr显著下降，凋亡指数降低，GRP78、p-eIF2α、p-PERK及CHOP的表达显著下调，但仍高于对照组，差异均达到统计学意义(P<0.05)；高、低剂量阿托伐他汀组之间上述各指标差异均不显著。

结论

PERK/eIF2α/CHOP通路介导的内质网应激可能参与大鼠造影剂肾病的发生发展；阿托伐他汀在造影剂诱导的肾脏损伤中发挥保护作用，这可能与其调节PERK/eIF2α/CHOP通路，从而减轻内质网应激有关。

关键词: 造影剂, 内质网应激, 葡萄糖调节蛋白78, 阿托伐他汀

Objective

To investigate the expression of glucose regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), and C/EBP homologous protein (CHOP), in order to evaluate the effect of endoplasmic reticulum stress in rats with contrast induced-acute kidney injury (CI-AKI), and observe the possible protective effect of atorvastatin in CI-AKI rats.

Methods

Sixty Wistar rats were randomly divided into control group, CI-AKI model group, high-dose atorvastatin group (80 mg), and low-dose atorvastatin group (40 mg) with 15 rats each. At 24 h, 48 h, and 72 h after the rat model was established, BUN and Scr were detected. TUNEL and Western blotting were used to detect the apoptosis of renal tubular epithelial cells and caspase-3 expression, respectively. Immunohistochemistry and Western blotting were used to detect the expression of GRP78, p-eIF2α, p-PERK, and CHOP in the rat renal tissues.

Results

Compared with the control group, the model group had higher levels of BUN and Scr, more apoptotic cells, and more expression of GRP78, p-eIF2α, p-PERK, and CHOP (P<0.05). Both the high- and low-dose atorvastatin groups had lower levels of BUN and Scr, fewer apoptotic cells, and less expression of GRP78, p-eIF2α, p-PERK, and CHOP than the model group, which were yet higher than those of the control group (P<0.05). The differences of the levels of BUN, Scr, apoptosis of tubular cells, and expression of GRP78, p-eIF2α, p-PERK and CHOP proteins were not significant between the high-dose and low-dose atorvastatin groups.

Conclusion

Endoplasmic reticulum stress mediated by the PERK/eIF2α/CHOP pathway may be involved in the development of contrast- enhanced nephropathy in rats. Atorvastatin may play a protective role in contrast-induced renal injury by its regulation of the PERK/eIF2α/CHOP pathway leading to reduced endoplasmic reticulum stress.

Key words: Contrast medium, Endoplasmic reticulum stress, Glucose-regulated protein 78, Atorvastatin

图1 大鼠肾组织光镜(HE 400×)及电镜(5 000×)图片

表1 造模后不同时间点各组大鼠BUN和Scr水平(±s, n＝15)

时间	血肌酐(μmol/L)				血尿素氮(mmol/L)
时间	对照组	模型组	高剂量阿托伐他汀组	低剂量阿托伐他汀组	对照组	模型组	高剂量阿托伐他汀组	低剂量阿托伐他汀组
术前	34.6±2.9	35.5±3.2	33.9±2.1	34.8±2.4	7.4±4.5	7.2±3.6	7.2±3.5	7.6±4.5
术后24 h	36.9±1.9	67.8±5.9^a	47.9±6.0^ab	48.7±4.2^ab	7.6±3.7	35.4±3.9^a	17.8±4.8^ab	18.1±3.9^ab
术后48 h	36.8±3.2	78.8±6.4^a	57.8±5.7^ab	59.6±6.1^ab	7.3±5.5	35.6±5.1^a	18.5±5.0^ab	18.3±5.7^ab
术后72 h	35.6±2.8	76.2±3.8^a	55.3±3.9^ab	57.1±2.1^ab	7.8±3.2	36.1±4.6^a	17.8±5.3^ab	18.2±4.4^ab

表1 造模后不同时间点各组大鼠BUN和Scr水平(±s, n＝15)

时间	血肌酐(μmol/L)				血尿素氮(mmol/L)
时间	对照组	模型组	高剂量阿托伐他汀组	低剂量阿托伐他汀组	对照组	模型组	高剂量阿托伐他汀组	低剂量阿托伐他汀组
术前	34.6±2.9	35.5±3.2	33.9±2.1	34.8±2.4	7.4±4.5	7.2±3.6	7.2±3.5	7.6±4.5
术后24 h	36.9±1.9	67.8±5.9^a	47.9±6.0^ab	48.7±4.2^ab	7.6±3.7	35.4±3.9^a	17.8±4.8^ab	18.1±3.9^ab
术后48 h	36.8±3.2	78.8±6.4^a	57.8±5.7^ab	59.6±6.1^ab	7.3±5.5	35.6±5.1^a	18.5±5.0^ab	18.3±5.7^ab
术后72 h	35.6±2.8	76.2±3.8^a	55.3±3.9^ab	57.1±2.1^ab	7.8±3.2	36.1±4.6^a	17.8±5.3^ab	18.2±4.4^ab

图2 造模48 h时各组大鼠肾脏细胞凋亡情况(TUNEL 400×)

图3 各组大鼠Caspase－3活性蛋白质表达(Western印迹)

图4 各组大鼠肾组织GRP78、p－eIF2α、p－PERK及CHOP蛋白的表达(Western印迹)

图5 各组大鼠肾组织GRP78、p－eIF2α、p－PERK及CHOP的表达(免疫组化400×)

图6 各组大鼠肾组织细胞色素c和Fas的蛋白表达(Western印迹)

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Atorvastatin decreased the contrast-induced renal tubular epithelial cell apoptosis through regulation of the PERK/eIF2α/CHOP signaling pathway

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Atorvastatin decreased the contrast-induced renal tubular epithelial cell apoptosis through regulation of the PERK/eIF2α/CHOP signaling pathway