艾司洛尔对脓毒症急性肾损伤大鼠的保护作用

Protective effect of Esmolol for sepsis-induced acute kidney injury in rats

目的基于细胞周期阻滞探讨艾司洛尔对脓毒症急性肾损伤(SAKI)大鼠保护作用的可能机制。方法 48只雄性SD大鼠,按随机数字表法分为假手术组(Sham组)、模型组(CLP组)、试验组(ES组),每组16只又随机平均分为6 h组和24 h组。Sham组采用盲肠探查法,CLP组与ES组采用盲肠结扎穿刺术法建立脓毒症模型。造模成功后Sham组与CLP组分别经颈内静脉置管泵入生理盐水(速度为1 mL/h),ES组泵入艾司洛尔[给药量为15 mg/(kg·h),速度为1 ml/h]。泵药6 h后分别于术后6、24 h相应时间点处死各组大鼠,留取血清及组织标本。大鼠血液离心后采用ELISA法检测血清尿素氮(BUN)、肌酐(Cr)含量;摘取两侧肾脏,其中左侧肾脏行p53免疫组化染色,观察细胞周期阻滞情况;右侧肾脏组织置于-80℃超低温保存箱保存,待组织匀浆后一部分行Western blot分析,检测Toll样受体4(TLR-4)、核因子κB(NF-κB)、毛细血管扩张性共济失调症突变蛋白(ATM)的表达水平,另一部分肾脏组织行ELISA检测基质金属蛋白酶组织抑制剂-2(TIMP-2)、胰岛素样生长因子结合蛋白-7(IGFBP-7)水平。结果 (1)TLR-4与NF-κB水平:ES 6 h组和CLP 6 h组较Sham 6 h组升高(P<0.05),CLP 24 h组较Sham 24 h组升高(P<0.05),ES 24 h组TLR-4水平较CLP 24 h组下降(P<0.05)。(2)大鼠血清Cr、BUN水平:ES 6 h组和CLP 6 h组较Sham 6 h组明显升高(P<0.05),ES 24 h组较CLP 24 h组降低(P<0.05)。(3)在术后6 h和24 h中,与Sham组相比,CLP组与ES组大鼠肾脏组织IGFBP-7、TIMP-2、ATM、p53水平均明显升高(P<0.05),同时ES组上述因子水平较CLP组降低(P<0.05)。结论艾司洛尔可能通过影响TLR-4/NF-κB/ATM/p53通路关键蛋白,减轻炎症反应和肾小管上皮细胞周期阻滞,促进SAKI大鼠肾功能恢复,进而对SAKI大鼠起到保护作用。

Objective To investigate the underlying mechanism of Esmolol to attenuate sepsisinduced acute kidney injury(SAKI).Methods Forty-eight male SD rats were randomly and equally divided into a sham operation group(Sham group),a sepsis group(CLP group),and an Esmolol group(ES group).In each group,16 rats were further randomly divided into either a 6-h group or a 24-h group.Rats in the Sham group received only cecal exploration,and the rats in the CLP and ES groups received the cecal ligation and puncture.When the model was established,internal jugular vein catheterization was performed,and normal saline(1 ml/h)was infused through the internal jugular vein in the Sham and CLP groups,while the ES group was infused with Esmolol[15 mg/(kg·h),1 ml/h].After infusing the drug for 6 hours,the rats in each group were sacrificed at 6 h or 24 h after the operation.Blood and kidney tissue samples were collected for further examinations.Serum was isolated by centrifugation to detect the contents of BUN and Cr by ELISA.For kidney tissue samples,left kidney sample was used for p53 immunohistochemical detection,while right kidney sample from the same rat was used for Western blot analysis of the protein expression levels of TLR-4,NF-κB,and ATM,and for ELISA to detect the TIMP-2 and IGFBP-7 level.Results(1)TLR-4 and NF-κB in the ES-6 h group and CLP-6 h group were significantly higher than those of the Sham-6 h group(P<0.05).And these two factors in the CLP-24 h group were also significantly higher than those of the Sham-24 h group(P<0.05).TLR-4 level in the ES-24 h group was significantly lower than that of the CLP-24 h group(P<0.05).(2)Serum Cr and BUN in the ES-6 h group and the CLP-6 h group were also significantly higher than those of the Sham group(P<0.05),but the ES-24 h group showed a decreasing trend compared with the CLP-24 h group(P<0.05).(3)The levels of IGFBP-7,TIMP-2,ATM,and p53 in the CLP group and ES group were significantly higher than those in the Sham group at each time(P<0.05),and they were significantly lower in the ES group than in the CLP group(P<0.05)at each time point.Conclusion Esmolol reduces inflammation and renal tubular epithelial cell cycle block,promotes the recovery of renal function in SAKI rats,and thus protects SAKI rats possibly by affecting the expression of key proteins of the TLR-4/NF-κB/ATM/p53 pathway.