清热祛浊胶囊对非酒精性脂肪肝炎小鼠NF-κB/NLRP3信号通路的调控机制研究

Study on the regulatory mechanism of Qingre Quzhuo Capsule on NF-κB/NLRP3 signaling pathway in mice with non-alcoholic steatohepatitis

[目的]研究清热祛浊胶囊(QRQZ)对非酒精性脂肪肝炎(NASH)模型小鼠的治疗效果及对核转录因子-κB(NF-κB)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)信号通路的影响。[方法]通过蛋氨酸和胆碱缺乏(MCD)饮食诱导建立NASH小鼠模型,并灌胃不同剂量的QRQZ。通过检测各组小鼠体质量、肝指数、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、三酰甘油(TG)、总胆固醇(TC),肝苏木素-伊红(HE)染色及油红O染色评估QRQZ对NASH模型小鼠的治疗作用;通过检测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)水平评估QRQZ对NASH模型小鼠氧化应激水平;通过酶联免疫吸附测定(ELISA)检测白细胞介素-1β(IL-1β)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平评估QRQZ对NASH模型小鼠炎症的影响;通过检测磷酸化核因子κB抑制蛋白(p-IκB)、磷酸化核转录因子κB P65(p-NF-κB P65)、NLRP3、凋亡相关斑点样蛋白(ASC)、活化的半胱氨酸天冬氨酸蛋白酶1(cleaved Caspase-1)、mature IL-1β的蛋白水平以及NLRP3、ASC、Caspase-1、IL-1β的mRNA表达来评估QRQZ对NASH小鼠NF-κB/NLRP3信号通路的影响。[结果] QRQZ可改善MCD引起的体质量减轻,降低肝指数,降低血清ALT、AST活性及TC、TG水平,同时改善肝组织病理学变化,提示QRQZ对NASH模型小鼠具有治疗作用;此外,QRQZ提升肝组织中SOD、GSH-Px活性,降低了MDA水平,降低了IL-6、IL-1β、iuTNF-α炎症因子水平,提示了QRQZ的抗氧化及抗炎作用;进一步研究发现QRQZ降低了IκB与P65的磷酸化水平,减少NLRP3、ASC、Caspase-1、IL-1β蛋白及基因表达。提示QRQZ干预抑制了NASH小鼠NF-κB/NLRP3信号通路活化。[结论] QRQZ对NASH模型小鼠具有治疗作用,并且可以提升抗氧化能力改善炎症,其作用机制可能与抑制NF-κB/NLRP3信号通路活化有关。

[Objective]To study the therapeutic effect of Qingre Quzhuo Capsule(QRQZ)on non-alcoholic steatohepatitis(NASH)and impact on the nuclear factor kappa-B(NF-κB)/nucleotide binding oligomerization domain like receptor protein 3(NLRP3)signaling pathway.[Methods]NASH mice were induced by methionine and choline deficiency(MCD)diet and given different doses of QRQZ.Evaluated the therapeutic effect of QRQZ on NASH model mice by detecting body weight,liver index,alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG)and triglyceride(TC).Hematoxylin-Eosin(HE)and oil red O staining.The levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)were detected to evaluate the oxidative stress of QRQZ in NASH model mice.Through ELISA,the levels of interleukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)were used to evaluate the effect of QRQZ on the inflammation of NASH mice.By detecting phospho-inhibitor kappa B(P-IκB),phospho-nuclear factor kappa-B(p-NF-κB P65),NLRP3,apoptosine-associated speck-like protein(ASC),cleaved cysteinyl aspartate specific proteinase-1(cleaved Caspase-1),mature The protein level of IL-1βand mRNA expression of NLRP3,ASC,Caspase-1 and IL-1βto evaluate the effect of QRQZ on NF-κB/NLRP3 signaling pathway in NASH mice.[Results]QRQZ can improve the weight loss caused by MCD,decrease liver index,decrease serum ALT,AST activities,TC,TG levels,and improve liver histopathological changes,suggesting that QRQZ has therapeutic effect on NASH model mice.In addition,QRQZ increased the activity of SOD and GSH-Px in liver tissue,decreased the level of MDA,and decreased the levels of IL-6,IL-1β,TNF-αinflammatory factors,suggesting the antioxidant and anti-inflammatory effects of QRQZ.Further studies showed that QRQZ reduced the phosphorylation levels of IκB and P65,and decreased the expression of NLRP3,ASC,Caspase-1,IL-1βprotein and gene.These results suggest that QRQZ inhibits the activation of NF-κB/NLRP3 signaling pathway in NASH mice.[Conclusion]QRQZ has therapeutic effect on NASH model mice,and can enhance antioxidant capacity and improve inflammation,the mechanism of which may be related to inhibiting the activation of NF-κB/NLRP3 signaling pathway.