背景:非酒精性脂肪性肝炎(NASH)是非酒精性脂肪性肝病(NAFLD)进展的关键阶段,以明显的炎症和纤维化为特征。由于其发病率高、后果严重,NASH正在成为一个全球性的健康问题。内毒素易位对NASH的影响正受到关注。作为一种有效改善肝脏炎症的传统中草药,樟脑(衢州产地,FAQ)广泛应用于NASH的临床治疗。然而,FAQ对reg3g及相关内毒素易位的干预机制尚不清楚。
目的:研究回肠再生家族成员3γ(reg3g)缺乏和随后的内毒素易位对NASH进展的影响机制;阐明FAQ治疗NASH的疗效和机制。
方法:临床血清,回肠组织,和动态NASH模型相关分析共同证实reg3g是与NASH相关的关键基因。Reg3g-/-小鼠用于评估reg3g对肝损伤的影响,炎症,和纤维化,以及所涉及的潜在机制。体外研究阐明了FAQ对reg3g的调节作用,肠屏障功能,和肠道通透性。随后,在NASH小鼠模型中研究了FAQ的功效.病理检查结合蛋白质印迹(WB),免疫组织化学(IHC),和多重免疫组织化学(mIHC)分析用于评估FAQ对粘膜修复和屏障功能的影响。跨上皮电阻(TEER),异硫氰酸荧光素-葡聚糖4(FD-4)实验,结合酶联免疫吸附试验(ELISA)和显色LAL内毒素测定来确定肠通透性和内毒素易位。WB和mIHC的结果反映了肝脏中内毒素募集和M1巨噬细胞极化的水平。体重等参数,转氨酶,和胆固醇用于评估FAQ的代谢作用。
结果:reg3g表达降低与NASH的进展有关。reg3g回肠缺乏导致肠屏障和通透性受损,导致内毒素通过“肠-肝”轴募集到肝脏,导致M1巨噬细胞的极化,炎症因子的释放,过度炎症,和激活肝星状细胞(HSC),导致纤维化。FAQ显著上调小鼠回肠reg3g的表达和肠屏障相关蛋白紧密连接蛋白1(ZO-1)和闭塞蛋白(OLCN)的表达(p<0.05),从而提高肠屏障功能和通透性。肠通透性降低导致进入血流并在肝脏中积累的内毒素减少(p<0.05)。CD68的表达表明M1巨噬细胞的极化减少。肌动蛋白α2、平滑肌肌动蛋白(α-SMA)和细胞外基质(ECM)相关蛋白的表达水平也降低,表明改善肝纤维化。
结论:FAQ通过上调reg3g的表达改善NASH。reg3g的上调有助于肠屏障和通透性的修复,减少内毒素的募集和随后M1巨噬细胞的极化,过度炎症,和纤维化。
BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a critical stage in the progression of non-alcoholic fatty liver disease (NAFLD), characterized by obvious inflammation and fibrosis. Because of its high incidence rate and serious consequences, NASH is becoming a global health problem. The influence of endotoxin translocation on NASH is receiving attention. As a traditional Chinese herb that effectively improves hepatic inflammation, Fructus Aurantii (Quzhou origin, FAQ) is widely used in the clinical treatment of NASH. However, the intervention mechanism of FAQ on reg3g and related endotoxin translocation remains unclear.
OBJECTIVE: To study the mechanism of the impact by which ileal regenerating family member 3 gamma (reg3g) deficiency and subsequent endotoxin translocation impact the progression of NASH; To elucidate the efficacy and mechanism of FAQ in the treatment of NASH.
METHODS: Clinical serum, ileal tissue, and dynamic NASH model-related analyses collectively confirmed that reg3g is a pivotal gene associated with NASH. Reg3g-/- mice were used to assess the impact of reg3g on liver injury, inflammation, and fibrosis, as well as the underlying mechanism involved. In vitro studies elucidated the regulatory effects of FAQ on reg3g, intestinal barrier function, and intestinal permeability. Subsequently, the efficacy of FAQ was investigated in NASH mouse models. Pathological examinations combined with Western blotting (WB), immunohistochemistry (IHC), and multiplex immunohistochemical (mIHC) analyses were used to evaluate the effects of FAQ on mucosal repair and barrier function. Transepithelial electrical resistance (TEER), fluorescein isothiocyanate-dextran 4 (FD-4) experiments, coupled with enzyme linked immunosorbent assay (ELISA) and chromogenic LAL endotoxin assay were used to confirm intestinal permeability and endotoxin translocation. The results of WB and mIHC reflected the levels of endotoxin recruitment and M1 macrophage polarization in the liver. Parameters such as body weight, transaminases, and cholesterol were utilized to assess the metabolic effects of FAQ.
RESULTS: Decreased expression of reg3g was associated with the progression of NASH. Ileal deficiency in reg3g resulted in damage to the intestinal barrier and permeability, leading to the recruitment of endotoxins via the \'gut-liver\' axis to the liver, causing the polarization of M1 macrophages, release of inflammatory factors, excessive inflammation, and activation of hepatic stellate cells (HSCs), leading to fibrosis. FAQ significantly upregulated ileal reg3g expression and the expression of intestinal barrier-related proteins tight junction protein 1 (ZO-1) and occludin (OLCN) in mice (p < 0.05), thereby improving intestinal barrier function and permeability. Reduced intestinal permeability led to decreases in endotoxins entering the bloodstream and accumulating in the liver (p < 0.05). The expression of CD68 suggested reduced polarization of M1 macrophages. Expression levels of actin alpha 2, smooth muscle actin (α-SMA) and extracellular matrix (ECM)-related proteins also decreased, indicating improved liver fibrosis.
CONCLUSIONS: FAQ ameliorates NASH by upregulating the expression of reg3g. The upregulation of reg3g contributes to the repair of the intestinal barrier and permeability, reducing the recruitment of endotoxins and subsequent polarization of M1 macrophages, excessive inflammation, and fibrosis.