摘要
目的探讨下瘀血汤调控核苷酸结合寡聚化结构域样受体含pyrin结构域蛋白6(NLRP6)抑制高脂饮食(HFD)诱导的小鼠非酒精性脂肪性肝病(NAFLD)的作用机制。方法15只雄性C57BL/6小鼠随机分为低脂饮食(LFD)组、HFD组和下瘀血汤-HFD(XYXD)组,每组各5只。测量肝功能指标ALT和AST、血脂代谢指标TG、TC水平;肝组织经过HE染色、油红O染色,观察小鼠组织形态、脂滴沉积;实时荧光定量PCR检测肝组织中炎症因子TNF-α、IL-1β、IL-6及NLRP6表达水平;Western Blot检测NLRP6、NF-κB和NF-κB p65蛋白水平;免疫组化检测NLRP6和CD68表达。棕榈酸(PA)、脂多糖(LPS)和下瘀血汤含药血清处理鼠Raw264.7细胞,检测炎症情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果与LFD组比较,HFD组血清ALT、AST和TC、TG水平显著升高(P值均<0.05)。肝组织病理学显示,HFD组肝脂肪变性明显,NAS评分显著升高(P<0.05);实时荧光定量PCR结果显示,IL-1β、IL-18等炎症相关因子显著升高,NLRP6表达显著下调(P值均<0.05)。免疫组化显示NLRP6表达与巨噬细胞标志物CD68重合。Western Blot显示,NLRP6表达下调后,磷酸化的NF-κB p65(p-NF-κB p65)显著上调(P<0.05)。与HFD组相比,下瘀血汤可有效改善HFD小鼠的肝脏炎症,上调NLRP6的表达,下调p-NF-κB p65(P<0.05)。PA处理Raw264.7细胞后下调NLRP6,促进炎症进展(P<0.05);下瘀血汤处理可上调NLRP6,抑制炎症和NF-κB(P<0.05)。结论下瘀血汤可显著改善HFD诱导NAFLD小鼠模型的肝脂肪变性和炎症,调控NLRP6/NF-κB减轻巨噬细胞活化可能是其作用机制之一。
Objective To investigate the mechanism of action of Xiayuxue decoction in inhibiting nonalcoholic fatty liver disease(NAFLD)induced by high-fat diet in mice by regulating nucleotide binding oligomerization domain like receptor containing pyrin domain protein 6(NLRP6).Methods A total of 15 male C57BL/6 mice were randomly divided into low-fat diet(LFD)group,high-fat diet(HFD)group,and Xiayuxue decoction-HFD group(XYXD group),with 5 mice in each group.Liver function parameters(alanine aminotransferase[ALT]and aspartate aminotransferase[AST])and blood lipid metabolic indicators(triglycerides[TG]and total cholesterol[TC])were measured;HE staining and oil red O staining were performed for liver tissue to observe histomorpholoty and lipid droplet deposition;quantitative real-time PCR was used to measure the expression levels of inflammatory factors(tumor necrosis factor-α[TNF-α],interleukin-1β[IL-1β],interleukin-18[IL-18],and NLRP6)in liver tissue;Western blot was used to measure the protein expression levels of NLRP6,nuclear factor-kappa B(NF-κB),and NF-κB p65;immunohistochemistry was used to measure the expression of NLRP6 and CD68.Mouse Raw264.7 cells were treated with palmitic acid(PA),lipopolysaccharide,and serum containing Xiayuxue decoction to observe inflammation.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results Compared with the LFD group,the HFD group had significant increases in the serum levels of ALT,AST,TC,and TG(all P<0.05).Liver histopathological examination showed that the HFD group had marked hepatic steatosis and a signficant increase in NAS score(P<0.05),and quantitative real-time PCR showed significant increases in the inflammatory factors such as IL1βand IL-18 and a significant reduction in the expression of NLRP6(all P<0.05).Immunohistochemistry showed that the expression of NLRP6 showed a similar trend as that of the macrophage marker CD68.Western blot showed that after the downregulation of NLRP6 expression,there was a significant increase in phosphorylated NF-κB p65(P<0.05).Compared with the HFD group,Xiayuxue decoction effectively improved liver inflammation,upregulated the expression of NLRP6,and downregulated phosphorylated NF-κB p65 in HFD mice(all P<0.05).After Raw264.7 cells were treated with PA,NLRP6 was downregulated to promote the progression of inflammation(P<0.05),and treatment with Xiayuxue decoction could upregulate NLRP6 and inhibit inflammation NF-κB(P<0.05).Conclusion Xiayuxue decoction can effectively improve hepatic steatosis and liver inflammation in a mouse model of NAFLD,possibly by regulating NLRP6/NF-κB to alleviate macrophage activation.
作者
侯林圻
王知意
赵鑫
张洁
马文婷
刘旭凌
张玮
陶乐
刘成
吴柳
HOU Linqi;WANG Zhiyi;ZHAO Xin;ZHANG Jie;MA Wenting;LIU Xuling;ZHANG Wei;TAO Le;LIU Cheng;WU Liu(Laboratory of Experimental Center,Putuo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062,China;Department of Infectious Diseases,Putuo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062,China)
出处
《临床肝胆病杂志》
CAS
北大核心
2024年第4期712-719,共8页
Journal of Clinical Hepatology
基金
国家自然科学基金面上项目(82004106,81873136)
普陀医院人才类项目(2022-RCJC-04,2022-RCJC-02)
上海市自然基金(20ZR1450300)
上海市普陀区自主创新项目(ptkwws202223,ptkwws202112)
上海市第六人民医院医疗集团科研基金(21-ly-02)
成都中医药大学杏林学者(YYZX2020117)
上海市中医药高层次人才引领计划[(2021-2023)-0403]。
