Mild hypothermia alleviates renal ischemia-reperfusion injury by up-regulating the expression of cold-shock protein RBM3
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摘要:
目的 探讨亚低温对肾脏缺血-再灌注损伤(IRI)的作用，以及RNA结合基序蛋白3(RBM3)及其下游效应分子在这一过程中的表达情况。 方法 健康SD雄性大鼠18只，随机分为正常对照(NC)组、IRI组和亚低温预处理(MHP)组，每组6只。通过检测血清肌酐水平评价肾功能，苏木素-伊红(HE)染色检测肾脏组织损伤情况，蛋白质印迹法检测肾脏组织的RBM3、Yes相关蛋白1(YAP1)、核因子E2相关因子2(Nrf2)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白相对表达量，免疫组织化学染色进一步检测RBM3及YAP1蛋白的表达情况，采用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记(TUNEL)法检测肾脏组织细胞凋亡情况，通过测定丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性检测肾脏组织氧化应激水平。 结果 与NC组相比较，IRI组和MHP组血清肌酐水平、肾脏组织病理损伤评分均升高，RBM3、YAP1和Nrf2蛋白表达水平均升高，Bcl-2/Bax比值均降低，细胞凋亡率均升高，MDA含量均升高，SOD活性均下降，差异均有统计学意义(均为P < 0.05)；与IRI组相比较，MHP组血清肌酐水平、肾脏组织病理损伤评分均降低，RBM3、YAP1和Nrf2蛋白表达水平均升高，Bcl-2/Bax比值升高，细胞凋亡率降低，MDA含量降低，SOD活性升高，差异均有统计学意义(均为P < 0.05)。 结论 亚低温对肾脏IRI具有保护作用，可减轻IRI所致细胞凋亡和氧化应激损伤，其机制可能为亚低温上调RBM3及其下游效应分子YAP1和Nrf2的表达水平。 -
关键词:
- 亚低温 /
- RNA结合基序蛋白3(RBM3) /
- Yes相关蛋白1(YAP1) /
- 核因子E2相关因子2(Nrf2) /
- 肾移植 /
- 缺血-再灌注损伤 /
- 氧化应激 /
- 丙二醛(MDA) /
- 超氧化物歧化酶(SOD)
Abstract:Objective To evaluate the effect of mild hypothermia on the renal ischemia-reperfusion injury (IRI), and the expression profile of RNA-binding motif protein 3(RBM3) and its downstream effector molecules during this process. Methods Eighteen healthy SD male rats were randomly divided into the normal control (NC) group, IRI group and mild hypothermia pretreat (MHP) group, with 6 rats in each group. Serum creatinine level was measured to evaluate the renal function. Hematoxylin-eosin (HE) staining was performed to assess the renal tissue injury. Western blot was used to determine the relative expression levels of RBM3, Yes-associated protein 1(YAP1), nuclear factor E2-related factor 2(Nrf2), B cell-lymphoma-2(Bcl-2) and Bcl-2-associated X protein (Bax) in the kidney tissues. Immunohistochemical staining was employed to further detect the expression levels of RBM3 and YAP1 proteins. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was adopted to detect the cell apoptosis of kidney tissues. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were evaluated to determine the oxidative stress level of kidney tissues. Results Compared with the NC group, the serum creatinine level, the pathological injury score of kidney tissues and the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably lower, the apoptosis rate was remarkably elevated, the MDA content was significantly increased and the SOD activity was dramatically reduced in the IRI and MHP groups (all P < 0.05). Compared with the IRI group, the serum creatinine level and the pathological injury score of kidney tissues were significantly decreased, the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably higher, the apoptosis rate was significantly decreased, the MDA content was significantly decreased and the SOD activity was considerably elevated in the MHP group (all P < 0.05). Conclusions Mild hypothermia may exert protective effect upon renal IRI and it could alleviate cell apoptosis and oxidative stress injury induced by IRI, probably by up-regulating the expression level of RBM3 and its downstream effector molecules of YAP1 and Nrf2. -
图 1 各组大鼠肾脏组织病理学表现
注: A图为各组大鼠肾皮质和肾髓质组织病理学图片(HE，× 200)；B图为各组大鼠肾脏组织病理损伤评分，与NC组比较，aP<0.05，与IRI组比较，bP<0.05。
Figure 1. Histopathological findings of kidney tissues in rats among each group
图 2 各组大鼠肾脏组织蛋白表达水平比较
注：A图示蛋白质印迹法检测各组蛋白表达水平；B图为蛋白质印迹法检测的定量分析结果，与NC组比较，aP<0.05，与IRI组比较，bP<0.05。
Figure 2. Comparison of protein expression levels in kidney tissues of rats among each group
图 3 各组大鼠肾脏组织RBM3和YAP1阳性细胞分布情况(免疫组织化学，× 200)
注：棕黄色代表阳性细胞。
Figure 3. Distribution of RBM3 and YAP1 positive cells in kidney tissues of rats among each group
图 4 各组大鼠肾脏组织细胞凋亡率比较
注：A图为各组大鼠肾脏组织细胞TUNEL染色图片，绿色荧光代表凋亡细胞(TUNEL，× 200)；B图为各组大鼠肾脏组织细胞凋亡率比较，与NC组比较，aP<0.05，与IRI组比较，bP<0.05。
Figure 4. Comparison of the apoptosis rate in kidney tissues of rats among each group
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