丁苯酞通过下调NF-κB信号通路抑制细胞焦亡减轻大鼠肾缺血-再灌注损伤

Dl-3-N-butylphthalide alleviates renal ischemia-reperfusion injury by down-regulating NF-κB signaling pathway and inhibiting cell pyroptosis in rat models

    摘要
  • 摘要:
      目的  探讨丁苯酞对大鼠肾缺血-再灌注损伤（IRI）的作用机制。
      方法  将40只SD大鼠随机分为假手术组（Sham组）、模型组（IRI组）、NF-κB抑制剂吡咯烷二硫代氨基甲酸酯（PDTC）组、丁苯酞低剂量组（NBP-L组）及丁苯酞高剂量组（NBP-H组），每组8只。检测各组大鼠血清肌酐（Scr）、血清胱抑素C（Cys-C）、血尿素氮（BUN）和血清白细胞介素（IL）-1β、IL-18水平，苏木素-伊红（HE）染色观察各组肾组织病理损伤情况，采用蛋白质印迹法和免疫组织化学法检测肾组织中炎症因子、核因子（NF）-κB信号通路及细胞焦亡相关蛋白表达水平。
      结果  与Sham组比较，IRI组肾组织损伤较为严重，Scr、Cys-C、BUN和血清IL-1β、IL-18水平均升高，蛋白质印迹法结果显示NOD样受体蛋白（NLRP3）、Gasdermin D（GSDMD）、半胱氨酸天冬氨酸蛋白酶（Caspase）-1、IL-18、IL-1β、NF-κB p65、p-NF-κB p65蛋白相对表达量均增加，免疫组织化学染色结果显示NF-κB p65、p-NF-κB p65、IL-1β、IL-18和NLRP3蛋白表达均增多。与IRI组比较，PDTC组、NBP-L组和NBP-H组肾组织的损伤程度均减轻，Scr、Cys-C、BUN和血清IL-18、IL-1β水平均下降，蛋白质印迹法结果显示NLRP3、GSDMD、Caspase-1、IL-1β、IL-18、NF-κB p65、p-NF-κB p65蛋白表达均减少，免疫组织化学染色结果显示NF-κB p65、p-NF-κB p65、IL-1β、IL-18和NLRP3蛋白表达均下降。与NBP-L组比较，NBP-H组肾组织的损伤程度减轻，Scr、Cys-C、BUN和血清IL-18、IL-1β水平均下降，蛋白质印迹法结果显示NLRP3、GSDMD、Caspase-1、IL-1β、IL-18、NF-κB p65、p-NF-κB p65蛋白表达均减少，免疫组织化学染色结果显示NF-κB p65、p-NF-κB p65、IL-1β、IL-18和NLRP3蛋白表达均下降。
      结论  丁苯酞可下调NF-κB/NLRP3信号通路的活性，降低肾IRI后焦亡相关蛋白的表达水平及炎症因子水平，进而抑制细胞焦亡，减轻肾IRI。

     

    Abstract:
      Objective  To elucidate the mechanism of dl-3-N-butylphthalide (NBP) on renal ischemia-reperfusion injury (IRI) in rat models.
      Methods  Forty SD rats were randomly divided into the sham operation group (Sham group), model group (IRI group), NF-κB inhibitor pyrrolidine dithiocarbamate group (PDTC group), low-dose NBP group (NBP-L group) and high-dose NBP group (NBP-H group), with 8 rats in each group. Serum creatinine (Scr), serum cystatin C(Cys-C), blood urea nitrogen (BUN) and serum interleukin (IL)-1β and IL-18 levels were detected in all groups. Pathological injury of renal tissues in each group was observed by Hematoxylin-eosin (HE) staining. The expression levels of inflammatory factors and nuclear factor (NF)-κB signaling pathway and cell pyroptosis-related proteins in renal tissues were measured by Western blot and immunohistochemical staining.
      Results  Compared with the Sham group, renal tissue injury was more severe, and the levels of Scr, Cys-C, BUN and serum IL-1β and IL-18 were all up-regulated in the IRI group. Western blot showed that the relative expression levels of NOD-like receptor protein (NLRP3), Gasdermin D(GSDMD), cysteinyl aspartate specific proteinase (Caspase)-1, IL-18, IL-1β, NF-κB p65 and p-NF-κB p65 proteins were all up-regulated, and immunohistochemical staining revealed that the expression levels of NF-κB p65 and p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were all up-regulated in the IRI group. Compared with the IRI group, renal tissue injury was alleviated, and the levels of Scr, Cys-C, BUN and serum IL-18 and IL-1β were down-regulated in the PDTC, NBP-L and NBP-H groups. Western blot showed that the expression levels of NLRP3, GSDMD, Caspase-1, IL-1β, IL-18, NF-κB p65 and p-NF-κB p65 proteins were down-regulated, and immunohistochemical staining indicated that the expression levels of NF-κB p65, p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were down-regulated in the PDTC, NBP-L and NBP-H groups, respectively. Compared with the NBP-L group, renal tissue injury was mitigated, and the levels of Scr, Cys-C, BUN, serum IL-18 and IL-1β were all down-regulated in the NBP-H group. Western blot showed the expression levels of NLRP3, GSDMD, Caspase-1, IL-1β, IL-18, NF-κB p65 and p-NF-κB p65 proteins were down-regulated in the NBP-H group. Immunohistochemical staining indicated that the expression levels of NF-κB p65, p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were down-regulated in the NBP-H group.
      Conclusions  NBP may down-regulate the activity of NF-κB/NLRP3 signaling pathway and reduce the expression levels of cell pyroptosis-related proteins and inflammatory factors after renal IRI, thereby suppressing cell pyroptosis and alleviating renal IRI.

     

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