核苷酸结合寡聚化结构域样受体蛋白3炎性小体在脓毒症急性肾损伤大鼠肾脏组织的表达及其影响

目的探讨核苷酸结合寡聚化结构域样受体蛋白（NLRP3）炎性小体在脓毒症急性肾损伤（AKI）大鼠中的作用及其机制。 方法采用随机数字表法将18只雄性Wistar大鼠分为脂多糖（LPS）组、LPS +抑制剂组及阴性对照组3组，每组6只。LPS组大鼠给予腹腔内注射LPS（3.5 mg/kg），LPS +抑制剂组大鼠腹腔内注射LPS（3.5 mg/kg）及半胱氨酸天冬氨酸蛋白酶1（Caspase-1）抑制剂Belnacasan（VX-765，100 mg/kg），而阴性对照组大鼠则给予腹腔内注射0.3 mL等渗NaCl溶液。比较3组大鼠建模前及建模后24 h血清肌酐水平变化，采用酶联免疫吸附测定（ELISA）法检测3组大鼠建模24 h后血清白细胞介素1β（IL-1β）、IL-18及肿瘤坏死因子α（TNF-α）表达水平，采用Western-blotting检测3组大鼠建模24 h后NLRP3及Caspase-1蛋白表达水平。 结果3组大鼠建模前后血清肌酐水平比较，差异有统计学意义（F = 146.910，P < 0.001）。进一步两两比较发现，建模后，LPS +抑制剂组及阴性对照组大鼠血清肌酐水平均较LPS组显著降低（1.57 ± 0.20）、（0.54 ± 0.39）、（2.31 ± 0.24）mg/L]，且阴性对照组大鼠血清肌酐水平较LPS +抑制剂组更低（P均< 0.05）；而LPS组（2.31 ± 0.24）mg/L vs.（0.53 ± 0.16）mg/L]及LPS +抑制剂组（1.57 ± 0.20）mg/L vs.（0.56 ± 0.08）mg/L]大鼠建模后血清肌酐水平均较建模前显著升高（P均< 0.05）。LPS组、LPS +抑制剂组及阴性对照组大鼠血清IL-1β （9.33 ± 1.16）、（1.93 ± 0.30）、（1.88 ± 0.30）ng/L]、IL-18 （23.8 ± 2.8）、（19.6 ± 1.5）（16.6 ± 1.2）ng/L]、TNF-α （42.3 ± 2.1）、（23.9 ± 2.5）、（23.5 ± 1.3）ng/L]及NLRP3蛋白（2.04 ± 0.25）、（2.04 ± 0.27）、（1.49 ± 0.28）]和Caspase-1蛋白（0.62 ± 0.07）、（0.51 ± 0.09）、（0.50 ± 0.09）]表达水平比较，差异均有统计学意义（F = 217.015、20.590、168.766、8.723、3.905，P均< 0.05）。进一步两两比较发现，LPS +抑制剂组及阴性对照组大鼠血清IL-1β、IL-18、TNF-α表达水平均较LPS组大鼠显著降低，且阴性对照组大鼠IL-18表达水平较LPS +抑制剂组更低（P均< 0.05）。阴性对照组大鼠NLRP3蛋白表达水平均较LPS组及LPS +抑制剂组显著降低，LPS +抑制剂组及阴性对照组大鼠Caspase-1蛋白表达水平均较LPS组显著降低（P均< 0.05）。 结论大鼠发生脓毒症时，通过激活炎性小体NLRP3，进而通过Caspase-1通路造成急性肾损伤。

Effect of nucleotide binding oligomerization domain-like receptor protein 3 inflammasome on kidney tissues in septic rats with acute kidney injury

Objective To investigate the role and mechanism of nucleotide binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome in septic rats with acute kidney injury(AKI).Methods A total of 18 male Wistar rats were randomly divided into a lipopolysaccharide(LPS)group,a LPS+inhibitor group,and a negative control group,6 in each group.Rats in the LPS group were given an intraperitoneal injection of LPS(3.5 mg/kg),and rats in the LPS+inhibitor group were given an intraperitoneal injection of LPS(3.5 mg/kg)and cysteinyl aspartate specific proteinase-1(Caspase-1)inhibitor Bernacasan(VX-765,100 mg/kg),while rats in the negative control group were given an intraperitoneal injection of isotonic NaCl solution(0.3 mL).Serum creatinine levels of rats in the 3 groups were compared before and after modeling for 24 h.The expression levels of serum interleukin-1beta(IL-1β),IL-18,and tumor necrosis factor-alpha(TNF-α)of rats in the 3 groups were measured by enzyme-linked immunosorbent assay(ELISA)after modeling for 24 h.In the meantime,the expression levels of NLRP3 and Caspase-1 proteins were detected by Western-blotting after modeling for 24 h.Results The serum creatinine levels of rats in the 3 groups were statistically significantly different before and after modeling(F=146.910,P<0.001).Further pairwise comparisons showed that after modeling,the serum creatinine levels in the LPS+inhibitor group and negative control group were both significantly lower than those in the LPS group(1.57±0.20),(0.54±0.39),(2.31±0.24)mg/L],and the serum creatinine level in the negative control group(0.54±0.39)mg/L vs.(1.57±0.20)mg/L]was lower than that in the LPS+inhibitor group,whereas the serum creatinine levels in the LPS group(2.31±0.24)mg/L vs.(0.53±0.16)mg/L]and LPS+inhibitor group(1.57±0.20)mg/L vs.(0.56±0.08)mg/L]after modeling were both significantly higher than those before modeling(all P<0.05).The expression levels of serum IL-1β(9.33±1.16),(1.93±0.30),(1.88±0.30)ng/L],IL-18(23.8±2.8),(19.6±1.5),(16.6±1.2)ng/L],TNF-α(42.3±2.1),(23.9±2.5),(23.5±1.3)ng/L],NLRP3 proteins(2.04±0.25),(2.04±0.27),(1.49±0.28)],and Caspase-1 proteins(0.62±0.07),(0.51±0.09),(0.50±0.09)]were statistically significantly different among the LPS group,LPS+inhibitor group and negative control group(F=217.015,20.590,168.766,8.723,3.905;all P<0.05).Further comparisons showed that the serum levels of IL-1β,IL-18,and TNF-αin the LPS+inhibitor group and negative control group were significantly lower than those in the LPS group,and the IL-18 level in the negative control was lower than that in the LPS+inhibitor group(all P<0.05).The levels of NLRP3 protein in the negative control group were significantly lower than those both in the LPS group and the LPS+inhibitor group(both P<0.05).In addition,the expression levels of Caspase-1 protein in the LPS+inhibitor group and negative control group were both significantly lower than those in the LPS group(both P<0.05).Conclusion In septic rats,the NLRP3 inflammasome is activated and then acute kidney damage occurs through the Caspase-1 pathway.