姜黄素通过“微生物-肠-肝”轴抑制肝脏炎症和细胞焦亡发生改善代谢功能障碍相关脂肪性肝病的机制

Curcumin mitigates MASLD by modulating the microbiota-gut-liver axis to suppress hepatic inflammation and pyroptosis

摘要:

背景 代谢功能障碍相关脂肪性肝病(metabolic dysfunction‐associated steatotic liver disease, MASLD)发生与“微生物-肠-肝”轴受损有关，姜黄素(curcumin, CUR)具有抗炎、抗氧化及调节肠道微生态等多种药理活性，但其通过“微生物-肠-肝”轴改善MASLD的分子机制尚未完全阐明。目的 探究8周CUR干预通过“微生物-肠-肝”轴改善高脂膳食诱导MASLD小鼠的肝脏炎症和细胞焦亡作用机制。方法 40只C57BL/6雄性小鼠随机分为对照组(control, CON)、高脂喂养组(high-fat diet, HFD)、高脂喂养+药物干预组(CUR, 100 mg/kg灌胃)。采用生物试剂盒检测血脂及肝功能指标变化；采用苏木精-伊红染色观察肝脏和结肠组织细胞形态；采用ELISA检测脂多糖(lipopolysaccharide, LPS)水平；采用Western blotting检测紧密连接蛋白1 (zonula occludens-1, ZO-1)、肝脏中核因子κB (nuclear factor kappa B, NF-κB)炎症信号通路和含NACHT结构域、富亮氨酸重复序列及吡啶结构域的NOD样受体蛋白3 (NACHT domain-, leucine-rich repeat- and pyrin domain containing protein 3, NLRP3)细胞焦亡信号通路相关蛋白的变化。结果 相较于CON组，HFD组小鼠体重、肝指数、体重指数(body mass index, BMI)、Lee’s指数显著增加，血脂异常，肝脏脂质沉积并出现肝功能损伤(P<0.05)，CUR干预可显著改善小鼠肥胖状态、肝功能和肝脏脂质沉积(P<0.05)。门水平肠道菌群分析发现HFD组厚壁菌门/拟杆菌门(Firmicutes/Bacteroidota)比值极显著升高(P<0.01)，而CUR组极显著降低(P<0.01)；在属水平上，CUR逆转了HFD组红蝽菌科(Coriobacteriaceae_UCG-002)和/大肠杆菌-志贺菌属(Escherichia-Shigella)异常增殖(P<0.01)。相较于CON组，HFD组肠道通透性显著增加，ZO-1表达显著降低，肠道内容物和血清LPS显著增加，CUR干预显著逆转上述指标值(P<0.05)。相较于CON组，HFD组脂多糖结合蛋白(lipopolysaccharide-binding protein, LBP)、Toll样受体4 (Toll-like receptor 4, TLR4)、NF-κB、白介素1β (interleukin-1β, IL-1β)及NLRP3细胞焦亡信号通路相关蛋白表达水平显著增加(P<0.05)，CUR干预则显著抑制上述信号通路蛋白表达水平(P<0.05)。相关性分析结果显示，Coriobacteriaceae_UCG-002和Escherichia-Shigella与肥胖、炎症及细胞焦亡部分指标呈显著正相关(P<0.05)。结论 中等剂量(100 mg/kg) CUR干预显著改善高脂饮食诱导的MASLD小鼠血脂异常、肝脏脂质沉积及肝功能损伤、重塑肠道菌群组成维持肠道微生态环境、减少肠源性LPS的产生、抑制炎症和细胞焦亡发生，相关性分析发现回调菌群Coriobacteriaceae_UCG-002、Escherichia-Shigella与LBP/TLR4/NF-κB及NLRP3/Caspase-1/GSDMD通路相关蛋白呈显著相关性，提示CUR可能通过“微生物-肠-肝轴”抑制炎症及细胞焦亡发生，进而延缓MASLD发生发展。

Abstract:

Background The occurrence of metabolic dysfunction-associated steatotic liver disease (MASLD) is associated with impairment of the microbiota-gut-liver axis. Curcumin (CUR) exhibits multiple pharmacological activities such as anti-inflammatory, antioxidant, and gut microecology-regulating activities. However, the molecular mechanism by which CUR ameliorates MASLD through the microbiota-gut-liver axis has not been fully elucidated.Objective To explore the mechanism of 8-week CUR intervention in mitigating liver inflammation and pyroptosis in the mouse model of MASLD induced by a high-fat diet through the microbiota-gut-liver axis.Methods We randomized forty male C57BL/6 mice into three groups: control (CON), model (HFD, high-fat diet), and a high-fat diet plus CUR (100 mg/kg by gavage) intervention. Biochemical kits were used to measure the levels of blood lipids and liver function indices. Hematoxylin-eosin staining was performed to observe liver and colon histomorphology. Gut microbiota was analyzed via 16S gene rRNA sequencing. Lipopolysaccharide (LPS) levels were measured by ELISA. Western blotting was employed to determine the expression levels of zonula occludens-1 (ZO-1), proteins in nuclear factor kappa B (NF-κB) signaling pathway, and proteins in the NACHT domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3)-mediated pyroptosis signaling pathway in the liver.Results Compared with the CON group, the HFD group showed increases in body weight, liver index, body mass index (BMI), and Lee’s index, dyslipidemia, hepatic lipid accumulation, and liver function impairment (P<0.05). Compared with the HFD group, CUR intervention mitigated the obesity status, improved the liver function, and alleviated liver lipid deposition in mice (P<0.05). At the phylum level, the Firmicutes/Bacteroidota ratio was increased in the HFD group compared with that in the CON group (P<0.01) but was decreased in the CUR group (P<0.01). At the genus level, CUR reversed the abnormal proliferation of Coriobacteriaceae_UCG-002 and Escherichia-Shigella in the HFD group (P<0.01). Compared with the CON group, the HFD group showed increased intestinal permeability, decreased ZO-1 expression, and elevated LPS levels in the intestinal contents and serum, all of which were reversed by CUR intervention (P<0.05). Additionally, the HFD group exhibited upregulated expression levels of lipopolysaccharide-binding protein (LBP), Toll-like receptor 4 (TLR4), NF-κB, interleukin-1β (IL-1β), and NLRP3 pyroptosis signaling pathway-related proteins compared with the CON group (P<0.05), while CUR intervention inhibited the expression of proteins in these signaling pathways (P<0.05). Correlation analysis showed that Coriobacteriaceae_UCG-002 and Escherichia-Shigella were positively correlated with obesity, inflammation, and pyroptosis indices (P<0.05).Conclusion Moderate-dose curcumin (100 mg/kg) markedly ameliorated dyslipidaemia, hepatic steatosis, and liver function impairment in the mouse model of MASLD induced by a high-fat diet. It concurrently reshaped the gut microbiota to restore gut micro-ecological balance, reduced gut-derived LPS translocation, and inhibited inflammation and pyroptosis. Coriobacteriaceae_UCG-002 and Escherichia-Shigella, whose relative abundance was rebalanced by curcumin, showed significant positive correlations with the expression of proteins in the LBP/TLR4/NF-κB signaling pathway and NLRP3/Caspase-1/GSDMD-mediated pyroptosis in the liver. These findings suggest that curcumin may regulate the microbiota-gut-liver axis to suppress inflammation and pyroptosis pathways, thereby slowing down the progression of MASLD.