【摘要】 目的 探讨狄氏副拟杆菌（Parabacteroides distasonis）长链脂酰辅酶A合成酶基因PdfadD的功能，及其对高脂饮食诱导的胰岛素抵抗小鼠糖脂代谢紊乱的改善作用与机制。方法 以P. distasonis ATCC 8503基因组为模板克隆PdfadD基因，构建pBAD24M-PdfadD与pET-21a(+)-PdfadD重组质粒，并分别转化至大肠埃希菌JW1794与BL21(DE3)中，所得工程菌分别用于异源互补验证PdfadD基因功能及后续动物实验。将50只C57BL/6J雄性小鼠随机分为正常饮食组、高脂饮食组、高脂饮食+狄氏副拟杆菌组、高脂饮食+工程菌组及高脂饮食+空载体对照组，每组10只。各细菌组干预时间与高脂饮食同步，小鼠自由采食，持续12周。检测小鼠体重变化，使用ELISA试剂盒测定胰岛素敏感性，通过全自动生化分析仪检测血脂水平，苏木精-伊红染色后高倍镜下观察肝、脂肪和胰腺组织病理变化，并结合16S rRNA基因高通量测序与粪便非靶向代谢组学分析其相关作用机制。结果 异源互补实验证实PdfadD基因可恢复JW1794菌株的长链脂肪酸利用能力。工程菌能显著抑制高脂饮食诱导小鼠体重增长（t=4.588,P<0.001），改善胰岛素抵抗状态（t=6.357,P<0.001），降低血浆低密度脂蛋白胆固醇水平（Z=2.179,P=0.029），并减轻肝脏空泡样变、脂肪细胞肥大及胰岛结构损伤。此外，工程菌可调节小鼠肠道菌群结构，提高拟杆菌门相对丰度（Z=0.220,P=0.575），降低厚壁菌门/拟杆菌门比值（Z=0.472,P=0.637），并促进鼠李乳杆菌增殖（LDA=5.382,P=0.003），且该菌的丰度与胆汁酸合成和组胺代谢的关键代谢物水平显著相关（r=0.542,P=0.002）。结论 PdfadD基因在狄氏副拟杆菌中编码长链脂酰辅酶A合成酶，表达该基因的工程菌能显著改善高脂饮食小鼠的胰岛素抵抗，其作用可能与调节肠道菌群，促进鼠李乳杆菌增殖及关联胆汁酸代谢通路有关。更多还原

【Abstract】 Objective To explore the function of long-chain acyl-CoA synthetase gene PdfadD of Parabacteroides distasonis, and its improving effect and mechanism on the disorder of glucose and lipid metabolism in insulin-resistant mice induced by high-fat diet. Methods The PdfadD gene was cloned using the genome of P. distasonis ATCC 8503 as a template. The recombinant plasmids pBAD24M-PdfadD and pET-21a(+)-PdfadD were constructed, and transformed into Escherichia coli JW1794 and BL21(DE3) respectively. The obtained engineered bacteria were used for heterologous complementation to verify the function of the PdfadD gene and subsequent animal experiments. A total of 50 male C57 BL/6J mice were randomly divided into a normal diet group, a high-fat diet group, a high-fat diet + P. distasonis group,a high-fat diet + engineered bacteria group, and a high-fat diet + empty vector control group, 10 in each group. The intervention time of each bacterial group in the mice was synchronized with the high-fat diet. The mice were allowed to freely feed and the experiment lasted for 12 weeks. The weight changes of mice were detected, insulin sensitivity were determined by using ELISA kit, and blood lipid levels were detected through automatic biochemical analyzer. After hematoxylin-eosin staining, the pathological changes under high-power microscopy of liver, fat, and pancreatic tissues were observed. The relevant mechanisms of action were analyzed by combining 16S rRNA gene high-throughput sequencing and fecal non-targeted metabolomics. Results Heterologous complementation experiments confirmed that PdfadD restored the long-chain fatty acid utilization ability of strain JW1794. The engineered bacteria significantly inhibited the body weight gain of mice induced by high-fat diet(t=4.588, P<0.001), improved the insulin resistance state(t=6.357, P<0.001), reduced the level of plasma low-density lipoprotein cholesterol(Z=2.179, P=0.029), and alleviated liver vacuolization, adipocyte hypertrophy and pancreatic islet structural damage. The engineered bacteria regulated the structure of intestinal flora of mice, increased the relative abundance of Bacteroidetes(Z=0.220, P=0.575), and reduced the Firmicutes/Bacteroidetes ratio(Z=0.472, P=0.637). Moreover, it promoted the proliferation of Lactobacillus murinus(LDA=5.382, P=0.003), the abundance of which was significantly correlated with the levels of key metabolites in bile acid synthesis and histamine metabolism(r=0.542, P=0.002). Conclusion The PdfadD encodes long-chain acyl-CoA synthetase in Parabacteroides distasonis. The engineered bacteria expressing this gene can significantly improve insulin resistance in mice with high-fat diet; the effect may be related to regulating the intestinal flora, promoting the proliferation of Lactobacillus murinus, and associating bile acid metabolism pathways.更多还原