巴氏灭活嗜黏蛋白阿克曼氏菌对ox-LDL诱导的HAEC细胞损伤的保护作用及机制初探

目的:本研究以巴氏灭活的嗜黏蛋白阿克曼氏菌（pasteurized Akkermansia muciniphila，PAKK）为研究对象，探究其对氧化低密度脂蛋白（oxidized low density lipoprotein，ox-LDL）诱导的人主动脉内皮细胞（human aortic endothelial cells，HAEC）损伤的保护作用。方法:首先利用MTT法评价PAKK对细胞活性的影响并确定合适的剂量。建立ox-LDL诱导的细胞损伤模型，并通过测定细胞乳酸脱氢酶、活性氧等评价PAKK对细胞损伤的保护作用，最后通过测定抗氧化酶活力以及Nrf2抗氧化通路相关基因及蛋白表达等，探讨PAKK改善HAEC细胞氧化损伤的可能机制。结果:菌数为105和106 CFU/mL的PAKK不影响HAEC细胞的活性；能显著降低ox-LDL诱导HAEC细胞的乳酸脱氢酶释放率；显著降低细胞内的活性氧水平，提高超氧化物歧化酶、过氧化氢酶的活力，以及细胞的总抗氧化能力；显著上调细胞内转录因子核因子-E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）、血红素氧合酶-1（hemeoxygenase-1, HO-1）、谷胱甘肽巯基转移酶（glutathione S-transferase，GST）以及NADPH醌氧化还原酶1 （NADPH quinine oxidoreductase-1，NQO1）的mRNA表达量；显著上调Nrf2、HO-1、NQO1蛋白的表达。结论:本研究表明巴氏灭活的嗜黏蛋白阿克曼氏菌可缓解ox-LDL对HAEC细胞造成的损伤，且其保护作用可能是通过调节Nrf2信号通路从而增强抗氧化相关基因表达。本研究为开发基于嗜黏蛋白阿克曼氏菌的可用于预防动脉粥样硬化的益生菌相关产品奠定理论基础。

Protective Effects of Pasteurized Akkermansia muciniphila on Ox-LDL-Induced Cell Injury in Human Aortic Endothelial Cells and Underlying Mechanisms

Objective: In this study, pasteurized Akkermansia muciniphila (PAKK) was examined for its protective effect on oxidized low-density lipoprotein (ox-LDL)-induced injury in human aortic endothelial cells (HAEC). Methods: MTT method was used to evaluate the effect of PAKK on HAEC activity and determine the appropriate dose. A model of ox-LDL-induced cell injury was established, and the protective effect of PAKK on HAEC cell injury was evaluated by measuring lactate dehydrogenase and reactive oxygen species. By measuring the activities of antioxidant enzymes and the expression of genes and proteins related to the Nrf2 antioxidant pathway, the possible mechanisms by which PAKK ameliorated HAEC oxidative damage was discussed. Results: PAKK at the doses of 105 and 106 CFU/mL did not affect the viability of HAEC, but significantly reduced the release rate of lactate dehydrogenase induced by ox-LDL. The reactive oxygen species level was decreased, while the activity of superoxide dismutase, catalase and the total antioxidant capacity of cells were significantly increased. The mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), glutathione S-transferase (GST) and NADPH quinone oxidoreductase-1 (NQO1) were significantly up-regulated. Nrf2, HO-1 and NQO1 protein expression were also significantly enhanced. Conclusion: These findings indicated that PAKK could alleviate the injury of HAEC caused by ox-LDL, and its protective effect was partly mediated through enhanced expression of antioxidant related enzymes by regulating Nrf2 signaling pathway. This study would provide theoretical basis for development of Akkermansia muciniphila-based probiotic or postbiotic for prevention of atherosclerosis.