益肾健脾泻浊中药对慢性肾脏病妊娠大鼠肾脏NLRP3/caspase-1/IL-1β信号通路及细胞焦亡的影响

Effect of Yiqi Jianpi Xiezhuo Decoction on NLRP3/caspase-1/IL-1βSignaling Pathway and Pyroptosis in Pregnant Rats with Chronic Kidney Disease

目的观察益肾健脾泻浊中药对慢性肾脏病妊娠大鼠肾脏NLRP3/caspase-1/IL-1β信号通路及细胞焦亡的影响。方法将雌性Wistar大鼠随机分为6组,分别为对照组(Sham)、对照组+妊娠组(SP)、单侧输尿管结扎组(UUO)、UUO+妊娠组(UP)、UUO+妊娠+益肾健脾泻浊中药组(TCM)、UUO+妊娠+依普利酮组(EPL)。UUO各组采用结扎单侧输尿管的方法复制慢性肾病模型,治疗组分别给予依普利酮100 mg(⋅kg⋅天)^(-1)和益肾健脾泻浊方3.11 g(⋅kg⋅天)^(-1)治疗。8周后妊娠各组大鼠按动情周期与雄鼠合笼,妊娠第19天处死母鼠。检测各组肾功能,醛固酮含量,采用SABC法及Western blot法检测核因子κB(Nuclear factor-kappa B,NF-κB)、核苷酸结合寡聚结构域样受体蛋白3 NOD[nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3]、天冬氨酸特异性半胱氨酸蛋白1[Caspase-1]、白介素1β(Interleukine-1 beta,IL-1β)的表达,TUNEL法检测肾细胞DNA损伤情况。结果与UUO组比较,UP组血清肌酐(Scr)、血尿素氮(BUN)和24 h尿蛋白及醛固酮含量较Sham组显著升高(P<0.05),治疗后,两给药组Scr、BUN、24 h尿蛋白、醛固酮显著降低(P<0.05)。与Sham组比较,SP组大鼠肾组织NF-κB、NLRP3、Caspase-1、IL-1β表达明显升高(P<0.05),TUNEL阳性细胞少量增加(P<0.05)。NLRP3炎症小体主要表达于浸润巨噬细胞及肾小管上皮细胞;Caspase-1和IL-1β主要表达于肾小管上皮细胞胞浆;TUNEL阳性细胞主要见于远端小管上皮细胞。与UUO组比较,UP组大鼠肾组织NF-κB、NLRP3、Pro-caspase-1、Caspase1、Pro-IL-1β、IL-1β指标表达也明显升高(P<0.05),TUNEL阳性细胞明显增多(P<0.05)。与UP组比较,EPL、TCM组大鼠肾组织上述指标表达明显降低(P<0.05),TUNEL阳性细胞明显减少(P<0.05)。两个给药组间各项指标无明显差异。结论益肾健脾泻浊方及依普利酮可缓解慢性肾病妊娠大鼠肾脏炎症损伤,下调肾脏NLRP3炎症小体信号通路分子表达,从而抑制细胞焦亡,减轻肾脏炎症损伤。

Objective To observe the effect of Yiqi Jianpi Xiezhuo Decoction on NF-κB/NLRP3-caspase-1-IL-1βsignaling pathway and pyroptosis in pregnant rats with chronic kidney disease.Methods Female Wistar rats were randomly divided into 6 groups:control group(Sham),control+pregnancy group(SP),unilateral ureteral ligation group(UUO),UUO+pregnancy group(UP),UUO+pregnancy+Yiqi Jianpi Xiezhuo Decoction group(TCM),and UUO+pregnancy+eplidone group(EPL).The chronic kidney disease model was replicated in each UUO group by ligation of the unilateral ureter,and the treatment groups were treated with eplerenone 100 mg⋅(kg⋅d)^(-1) and the formula for kidney strengthening and spleen laxative 3.11 g⋅(kg⋅d)^(-1).After 8 weeks of gestation,the rats in each group were caged together with the male rats according to the estrous cycle,and the females were executed on the 19th day of gestation.The renal function,aldosterone content,nuclear factor-kappa B(NF-κB),nucleotide-binding oligomerization domain-like receptor protein 3 NOD[nucleotide-binding oligomerization domain-like receptor protein 3]were measured by SABC and Western blot.domain-like receptor protein 3(NLRP3),aspartate-specific cysteine protein 1(Caspase-1)and interleukine-1 beta(IL-1β),and the TUNEL method was used to detect DNA damage in renal cells.Results Compared with the UUO group,serum creatinine(Scr),blood urea nitrogen(BUN)and 24 h urine protein and aldosterone levels were significantly higher in the UP group compared with the Sham group(P<0.05),and after treatment,Scr,BUN,24 h urine protein and aldosterone were significantly lower in both dosing groups(P<0.05).The expression of NF-κB,NLRP3,Caspase-1 and IL-1βwas significantly higher in the renal tissues of the SP group compared with the Sham group(P<0.05),and the expression of TUNEL-positive cells was increased in small amounts(P<0.05).Caspase-1 and IL-1βwere mainly expressed in the cytoplasm of renal tubular epithelial cells;TUNEL-positive cells were mainly found in the distal tubular epithelial cells.Compared with the UUO group,the expression of NF-κB,NLRP3,Pro-caspase-1,Caspase-1,Pro-IL-1βand IL-1βindexes in the renal tissues of the UP group was also significantly higher(P<0.05),and the TUNEL-positive cells were significantly increased(P<0.05).Compared with the UP group,the expression of the above indicators in the kidney tissues of EPL and TCM rats was significantly lower(P<0.05),and the TUNELpositive cells were significantly reduced(P<0.05).There was no significant difference in the indexes between the two drug administration groups.Conclusion Yiqi Jianpi Xiezhuo Decoction and eplerenone can alleviate renal inflammatory injury in pregnant rats with chronic kidney disease,down-regulate renal NLRP3 inflammatory vesicle signaling pathway molecule expression,thus inhibit cell scorching and reduce renal inflammatory injury.