阿魏酸哌嗪预处理对脓毒症大鼠急性肾损伤的保护作用及机制

Protective effect and mechanism of piperazine ferulate pretreatment on acute kidney injury of rats with sepsis

目的探讨阿魏酸哌嗪(PF)对脓毒症大鼠急性肾损伤的保护作用及机制。方法将24只雄性Sprague Dawley大鼠随机分为假手术组、模型组、低剂量PF组和高剂量PF组,每组6只。低剂量PF组、高剂量PF组大鼠每天分别给予50、100 mg·kg^(-1)PF混悬液灌胃,连续7 d;假手术组、模型组大鼠每天给予9 g·L^(-1)氯化钠溶液(0.1 mL·kg^(-1))灌胃,连续7 d。假手术组大鼠行盲肠探查后关腹,不做盲肠结扎穿孔手术;模型组、低剂量PF组和高剂量PF组大鼠均行盲肠结扎穿孔手术制备急性肾损伤模型。造模后24 h,取各组大鼠颈动脉血并分离血清,应用生物化学法检测大鼠血清肌酐(Scr)、血尿素氮(BUN)水平,酶联免疫吸附试验法检测大鼠血清中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、白细胞介素-6(IL-6)、降钙素原(PCT)水平。断头处死各组大鼠,取肾脏,应用苏木精-伊红(HE)染色观察大鼠肾组织病理学变化,Western blot法检测肾组织中核因子κB(NF-κB)、Toll样受体4(TLR4)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)蛋白的相对表达量。结果模型组和低剂量PF组大鼠Scr、BUN及血清NGAL、IL-6、PCT水平显著高于假手术组(P<0.05);高剂量PF组大鼠Scr、血清PCT水平显著高于假手术组(P<0.05);高剂量PF组与假手术组大鼠BUN及血清NGAL和IL-6水平比较差异无统计学意义(P>0.05);低剂量PF组和高剂量PF组大鼠Scr、BUN及血清NGAL、IL-6、PCT水平显著低于模型组(P<0.05);高剂量PF组大鼠Scr、BUN及血清NGAL、IL-6、PCT水平显著低于低剂量PF组(P<0.05)。HE染色显示,假手术组大鼠肾组织无明显异常改变;模型组大鼠肾小管上皮细胞明显肿胀变性,细胞质内有较多红染物,可见肾小管充血、变性坏死,管腔有大量炎症细胞浸润,系膜毛细血管间细胞增殖;低剂量PF组大鼠部分肾小管上皮细胞呈现轻、中度肿胀,部分肾小管上皮细胞变性坏死,未见大量炎症细胞浸润;高剂量PF组大鼠肾小管上皮细胞轻度肿胀,未见明显变性坏死及炎症细胞浸润。模型组和低剂量PF组大鼠肾组织中TLR4、NF-κB、NLRP3蛋白水平显著高于假手术组(P<0.05);高剂量PF组大鼠肾组织中TLR4蛋白水平显著高于假手术组(P<0.05),高剂量PF组与假手术组大鼠肾组织中NF-κB、NLRP3蛋白水平比较差异无统计学意义(P>0.05);低剂量PF组和高剂量PF组大鼠肾组织中TLR4、NLRP3蛋白水平显著低于模型组(P<0.05);高剂量PF组大鼠肾组织中NF-κB水平显著低于模型组(P<0.05),低剂量PF组与模型组大鼠肾组织中NF-κB水平比较差异无统计学意义(P>0.05);高剂量PF组大鼠肾组织中TLR4、NLRP3蛋白水平显著低于低剂量PF组(P<0.05),高剂量PF组与低剂量PF组大鼠肾组织中NF-κB水平比较差异无统计学意义(P>0.05)。结论PF干预可有效降低脓毒症急性肾损伤大鼠Scr、BUN及血清NGAL、IL-6和PCT水平,对脓毒症急性肾损伤大鼠肾脏发挥一定保护作用;其作用机制可能与抑制TLR4、NF-κB、NLRP3的表达,进而调控TLR4/NF-κB/NLRP3信号通路有关。

Objective To investigate the protective effect and mechanism of piperazine ferulate(PF)on acute kidney injury of rats with sepsis.Methods Twenty-four male Sprague Dawley rats were randomly divided into sham operation group,model group,low-dose PF group and high-dose PF group,with 6 rats in each group.The rats in the low-dose PF group and high-dose PF group were given 50 and 100 mg·kg^(-1) PF suspension by gavage every day for 7 days,respectively.The rats in the sham operation group and model group were given 9 g·L^(-1) sodium chloride solution(0.1 mL·kg^(-1))by gavage every day for 7 days.The rats in the sham operation group underwent cecal exploration and closed their stomachs without undergoing cecal ligation and perforation surgery.The rats in the model group,low-dose PF group and high-dose PF group underwent cecal ligation and perforation surgery to prepare the acute kidney injury models.At 24 hours after modeling,carotid artery blood of rats in each group was collected and the serum was separated.The levels of serum creatinine(Scr)and blood urea nitrogen(BUN)were measured by biochemical methods,and the levels of neutrophil gelatinase related lipid transport protein(NGAL),interleukin-6(IL-6),and procalcitonin(PCT)were measured by enzyme-linked immunosorbent assay.The rats in each group were executed by decapitation,then the kidneys were taken.The pathological changes of the kidney tissues were observed by hematoxylin-eosin(HE)staining.The relative expression levels of nuclear factor-κB(NF-κB),Toll-like receptor 4(TLR4)and NOD-like receptor thermal protein domain associated protein 3(NLRP3)in renal tissues were detected by Western blot method.Results The levels of Scr,BUN and serum NGAL,IL-6 and PCT of rats in the model group and low-dose PF group were significantly higher than those in the sham operation group(P<0.05).The levels of Scr and serum PCT of rats in the high-dose PF group were significantly higher than those in the sham operation group(P<0.05);there was no statistically significant difference in the levels of BUN and serum NGAL and IL-6 of rats between the high-dose PF group and the sham operation group(P>0.05).The levels of Scr,BUN and serum NGAL,IL-6 and PCT of rats in the low-dose PF group and high-dose PF group were significantly lower than those in the model group(P<0.05).The levels of Scr,BUN and serum NGAL,IL-6 and PCT of rats in the high-dose PF group were significantly lower than those in the low-dose PF group(P<0.05).HE staining showed that there were no significant abnormal changes in the renal tissues of rats in the sham operation group.The renal tubular epithelial cells of rats in the model group showed significant swelling and degeneration,with a large amount of red staining in the cytoplasm;the renal tubular had congestion,degeneration,and necrosis,with a large number of inflammatory cells infiltrating the lumen,and cell proliferation in the mesangial-capillaries space.In the low dose PF group,some renal tubular epithelial cells of rats showed mild to moderate swelling,some renal tubular epithelial cells showed degenera-tion and necrosis,and no significant infiltration of inflammatory cells was observed.The renal tubular epithelial cells of rats in the high-dose PF group showed mild swelling,without significant degeneration,necrosis and inflammatory cell infiltration.The levels of TLR4,NF-κB and NLRP3 protein in renal tissues of rats in the model group and low-dose PF group were significantly higher than those in the sham operation group(P<0.05).The level of TLR4 protein in renal tissues of rats in the high-dose PF group was significantly higher than that in the sham operation group(P<0.05);there was no statistically significant difference in the levels of NF-κB and NLRP3 protein in the renal tissues of rats between the high-dose PF group and sham operation group(P>0.05).The levels of TLR4 and NLRP3 proteins in the renal tissues of rats in the low-dose PF group and high-dose PF group were significantly lower than those in the model group(P<0.05);the level of NF-κB in renal tissues of rats in the high-dose PF group was significantly lower than that in the model group(P<0.05);there was no statistically significant difference in the level of NF-κB in renal tissues of rats between the low-dose PF group and model group(P>0.05).The levels of TLR4 and NLRP3 proteins in tissues of rats in the high-dose PF group were significantly lower than those in the low-dose PF group(P<0.05);there was no statistically significant difference in the level of NF-κB in renal tissues of rats between the high-dose PF group and low-dose PF group(P>0.05).Conclusion PF intervention can effectively reduce the levels of Scr,BUN and serum NGAL,IL-6 and PCT of rats with septic acute kidney injury,and play a certain protective role on the kidneys of rats with septic acute kidney injury;its mechanism may be related to inhibiting the expression levels of TLR4,NF-κB and NLRP3,thereby regulating TLR4/NF-κB/NLRP3 signaling pathway.