地塞米松减轻肾脏缺血再灌注损伤的作用和机制研究

发布时间：2018-03-19 04:16

本文选题：缺血再灌注损伤　切入点：炎症　出处：《华中科技大学》2014年博士论文　论文类型：学位论文

【摘要】：目的探讨地塞米松对小鼠肾脏缺血再灌注损伤的作用及其可能的信号通路。方法采用8周龄C57BL/6小鼠,随机分为3组：(1)假手术组,假手术前1小时腹腔注射生理盐水；(2)模型组,建立小鼠肾脏缺血再灌注模型,术前1小时腹腔注射生理盐水；(3)实验组,建立小鼠肾脏缺血再灌注模型,术前1小时腹腔注射4mg/kg地塞米松。术后24小时,收集各组小鼠的血清标本和肾脏标本,分别检测血清中肌酐、尿素氮水平,PAS染色观察肾脏病理改变,免疫组化观察肾脏中髓过氧化物酶(MPO)的表达,实时定量PCR测定肾脏中KIM-1、 TNF-α、IL-6和IFN-γ的转录水平,Western-blot检测糖皮质激素受体总量和磷酸化程度,P13K p85亚基总量及磷酸化程度,以及AKT总量与磷酸化程度。结果假手术组小鼠的血肌酐和血尿素氮水平分别为(17±5)μmol/L和(15±1.4)mmol/L;模型组分别为(147±13)μmol/L和(70±4)mmol/L:实验组分别为：(60±7.6)μmol/L和(37±3)mmol/L。假手术组小鼠肾功能正常,模型组小鼠肾功能下降明显,实验组肾功能较模型组有明显好转(P0.05)。实验组肾组织KIM-1mRNA水平明显低于模型组,肾脏损伤程度较轻(P0.05)。肾组织PAS染色示,模型组病变以外髓部位为主,表现为弥漫性肾小管上皮细胞空泡变性,肾小管上皮多灶状坏死脱落,并可见大量蛋白质管型形成；地塞米松组仅示肾小管上皮细胞空泡变性,肾小管上皮细胞局灶状坏死脱落,未见蛋白管型形成。免疫组化MPO染色,假手术组没有MPO阳性细胞,实验组MPO阳性细胞数明显少于模型组,实验组肾组织的MPO阳性细胞浸润较模型组减轻(P0.05)。模型组与假手术组相比,TNF-α、IL-6及IFN-γmRNA水平显著增高,实验组三种细胞因子mRNA水平也有所增加,但明显低于模型组(P0.05)。糖皮质激素受体,PI3K p85亚基和AKT的总量在三组间没有差异。磷酸化的糖皮质激素受体在模型组和实验组均有明显升高,但实验组升高程度更显著(P0.05)。模型组p85亚基和AKT磷酸化程度明显增高,实验组较之显著下降(P0.05)。结论地塞米松通过糖皮质激素受体减轻肾脏缺血再灌注中的炎症反应,缓解肾脏损伤。PI3K/AKT信号通路可能参与这一作用。
[Abstract]:Objective to investigate the effect of dexamethasone on renal ischemia reperfusion injury in mice and its possible signal pathway. Methods eight week-old C57BL / 6 mice were randomly divided into 3 groups: sham operation group (n = 3). The model group was treated by intraperitoneal injection of normal saline 1 hour before sham-operation. The model of renal ischemia-reperfusion was established. The experimental group was treated with intraperitoneal injection of normal saline for 1 hour before sham-operation. The model of renal ischemia-reperfusion was established in mice. 4 mg / kg dexamethasone was injected intraperitoneally 1 hour before operation. 24 hours after operation, serum samples and kidney samples of each group were collected and serum creatinine was detected. The expressions of myeloperoxidase (MPO) and myeloperoxidase (MPO) in kidney were observed by using urea nitrogen and pas staining. The transcription levels of KIM-1, TNF- 伪, IL-6 and IFN- 纬 in kidney were measured by real-time quantitative PCR. The total amount and phosphorylation of glucocorticoid receptor and phosphorylation of P13K p85 subunit and the total amount and phosphorylation of AKT were detected by Western-blot. Results the levels of serum creatinine and blood urea nitrogen in sham operation group were 17 卤5 渭 mol/L and 15 卤1.4 mmol / L, respectively, and those in model group were #number0# 卤13 渭 mol/L and 70 卤4 mmol / L respectively: the experimental group was 60 卤7.6 渭 mol/L and 37 卤3 mmol / L 路L ~ (-1) respectively. Compared with the model group, the renal function in the experimental group was significantly improved (P 0.05), the level of KIM-1mRNA in the experimental group was significantly lower than that in the model group, and the degree of renal injury was lighter than that in the model group. The renal tissue PAS staining showed that the lesion was mainly located outside the medulla in the model group. Diffuse tubular epithelial cell vacuolation, multiple focal necrosis and exfoliation of renal tubular epithelium, and formation of a large number of protein tubules were observed in the dexamethasone group, while in the dexamethasone group, the vacuolar degeneration of the tubular epithelial cells was observed only in the dexamethasone group. There were no MPO positive cells in the sham-operated group, and the number of MPO positive cells in the experimental group was significantly lower than that in the model group, and the number of MPO positive cells in the experimental group was significantly lower than that in the model group. The infiltration of MPO positive cells in the experimental group was less than that in the model group (P 0.05). The levels of TNF- 伪 IL-6 and IFN- 纬 mRNA in the model group were significantly higher than those in the sham operation group, and the mRNA levels of the three cytokines in the experimental group were also increased. The total amount of PI3K p85 subunit and AKT in the model group was significantly lower than that in the model group. The phosphorylated glucocorticoid receptor was significantly increased in both the model group and the experimental group. The phosphorylation of p85 subunit and AKT in the model group was significantly higher than that in the experimental group, but the level of P0.05 in the experimental group was significantly lower than that in the control group. Conclusion Dexamethasone can attenuate the inflammatory reaction in renal ischemia reperfusion by glucocorticoid receptor and attenuate renal injury. PI3K / AKT signaling pathway may be involved in this role.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

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