下瘀血汤对高脂饮食诱导的非酒精性脂肪性肝病小鼠模型的治疗作用及机制

Abstract

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。

Alternate abstract:

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 IL1β and 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.