虾青素缓解新生大鼠坏死性小肠结肠炎发展的机制

Mechanism of Astaxanthin in alleviates necrotizing enterocolitis development in neonatal rats

摘要

目的：探讨虾青素上调FGF21/PGC-1α通路改善新生大鼠坏死性小肠结肠炎（NEC）模型肠道黏膜线粒体功能和坏死性凋亡的机制。方法：妊娠第14天对大鼠进行剖宫产手术，取出早产SD大鼠，雌雄不限。早产大鼠分为3组：对照组、NEC模型组和虾青素组。通过qPCR检测各组大鼠FGF21/PGC-1α表达。通过HE染色分析大鼠小肠组织病理学。ELISA检测大鼠血清中白细胞介素-1β（IL-1β）、白细胞介素6（IL-6）、肿瘤坏死因子-α（TNF-α）含量及大鼠肠道组织中丙二醛（MDA）、超氧化物歧化酶（SOD）和谷胱甘肽（GSH）含量。通过TUNEL试剂盒检测细胞凋亡率。采用Western blot法检测炎症及凋亡相关蛋白半胱天冬酶-3（Caspase 3）、TNF-α、诱导型一氧化氮合酶（iNOS）、核因子-κB（NF-κB）、B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X（BAX）、Toll样受体4（TLR4）的表达。采用荧光素酶法和qPCR技术检测肠道组织三磷酸腺苷（ATP）和线粒体DNA（mtDNA）水平。通过JC-1双色荧光染色法检测线粒体膜电位（MMP）。通过qPCR检测线粒体相关基因核呼吸因子1（NRF1）和线粒体转录因子A（TFAM）的表达。通过吸光度变化分析NEC线粒体复合酶活性。结果：NEC模型组FGF21和PGC-1α mRNA表达较对照组降低（P<0.05），虾青素组较NEC模型组升高（P<0.05）。NEC模型组FGF21和PGC-1α蛋白表达较对照组降低，虾青素组较NEC模型组升高（P<0.05）。NEC模型组HE评分较对照组升高（P<0.05），虾青素组较NEC模型组降低（P<0.05），虾青素减轻NEC病理改变。NEC模型组IL-6、IL-1β和TNF-α水平较对照组升高，虾青素组较NEC模型组降低（P<0.05）。NEC模型组SOD和GSH水平较对照组降低，虾青素组较NEC模型组升高（P<0.05）；NEC模型组MDA水平较对照组升高，虾青素组较NEC模型组降低（P<0.05），虾青素抑制NEC氧化应激升高。NEC模型组肠道组织细胞凋亡率较对照组升高，虾青素组细胞凋亡率较NEC模型组降低（P<0.05）。NEC模型组肠道组织Caspase 3表达较对照组升高，虾青素组较NEC模型组降低（P<0.05）。NEC模型组TNF-α、iNOS、NF-κB、BAX、TLR4表达高于对照组，虾青素组低于NEC模型（P<0.05）；NEC模型组Bcl-2表达较对照组降低，虾青素组较NEC模型组升高（P<0.05）。NEC模型组ATP、mtDNA、MMP、NRF1和TFAM mRNA表达较对照组降低（P<0.05），虾青素组较NEC模型组升高（P<0.05）。NEC模型组复合物Ⅰ、复合物Ⅲ和复合物Ⅳ活性较对照组降低，虾青素组较NEC模型组升高（P<0.05）。结论：虾青素通过上调FGF21和PGC-1α通路，改善NEC模型肠道黏膜细胞线粒体功能，并减少细胞的坏死性凋亡。

Abstract

Objective: To explore the mechanism by which Astaxanthin upregulates the FGF21/PGC-1α pathway and improves intestinal mucosal mitochondrial function and necrotizing apoptosis in a neonatal rat model of necrotizing enterocolitis. Methods: On the 14th day of pregnancy, a cesarean section was performed on the rats to remove the premature SD rats, regardless of gender. Premature rats were divided into three groups: the control group, the necrotizing enterocolitis (NEC) model group, and the Astaxanthin group (NEC+ Astaxanthin group). The expression of NEC FGF21/PGC-1α was detected by qPCR. The histopathology of the small intestine in rats was analyzed by HE staining. ELISA was used to detect the contents of interleukin-1 β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor -α (TNF-α) in rat serum, as well as the contents of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) in rat intestinal tissue. The apoptosis rate of cells was detected by the TUNEL kit. The inflammation and apoptosis-related protein Caspase 3, TNF-α, inducible nitric oxide synthase (iNOS), nuclear factor -κB (NF-κB), B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X (BAX), and Toll-like receptor 4 (TLR4) were detected by Western blot. The levels of adenosine triphosphate (ATP) and mitochondrial DNA (mtDNA) in intestinal tissues were detected by luciferase method and qPCR technology. Mitochondrial membrane potential (MMP) was detected by JC-1 dual-color fluorescence staining. The expression of mitochondrial-related genes NRF1 and TFAM was detected by qPCR. Biochemical analysis of mitochondrial complex enzyme activity in NEC. Results: The expressions of FGF21 and PGC-1α mRNA in the NEC model group were lower than those in the control group (P<0.05), and the expressions of FGF21 and PGC-1α mRNA in the astaxanthin group were higher than those in the NEC model group (P<0.05). The expressions of FGF21 and PGC-1α in the NEC model group were lower than those in the control group, while the expressions of FGF21 and PGC-1α in the astaxanthin group were higher than those in the NEC model group (P<0.05). The HE score of the NEC model group was higher than that of the control group, and the HE score of the Astaxanthin group was lower than that of the NEC model group. Astaxanthin alleviated the pathological changes of NEC (P<0.05). The levels of IL-6, IL-1β and TNF-α in the NEC model group were higher than those in the control group, while the levels of IL-6, IL-1β and TNF-α in the Astaxanthin group were lower than those in the NEC model group (P<0.05). The levels of SOD and GSH in the NEC model group were lower than those in the control group, while the levels of SOD and GSH in the Astaxanthin group were higher than those in the NEC model group. The level of MDA in the NEC model group was higher than that in the control group, and the level of MDA in the Astaxanthin group was lower than that in the NEC model group. The inhibition of oxidative stress in NEC by Astaxanthin increased. The apoptosis rate of intestinal tissue cells in the NEC model group was higher than that in the control group, while the apoptosis rate of Astaxanthin group was lower than that in the NEC model group (P<0.05). The expression of Caspase 3 in intestinal tissue of the NEC model group was higher than that of the control group, while the expression of Caspase 3 in the Astaxanthin group was lower than that in the NEC model group (P<0.05). The expressions of TNF-α, iNOS, NF-κB, BAX and TLR4 in the NEC model group were higher than those in the control group. The expressions of TNF-α, iNOS, NF-κB, BAX and TLR4 in the Astaxanthin group were lower than those in the NEC model group. The expression of Bcl-2 in the NEC model group was lower than that in the control group. The expression of Bcl-2 in the Astaxanthin group was higher than that in the NEC model group (P<0.05). The levels of ATP and mtDNA in the NEC model group were lower than those in the control group, while those in the Astaxanthin group were higher than those in the NEC model group (P<0.05). The MMP in the NEC model group was lower than that in the control group, and the MMP in the Astaxanthin group was higher than that in the NEC model group (P<0.05). The expressions of NRF1 and TFAM mRNA in the NEC model group were lower than those in the control group, while the expressions of NRF1 and TFAM mRNA in the Astaxanthin group were higher than those in the NEC model group (P<0.05). The activities of complex I, complex III and complex IV in the NEC model group were lower than those in the control group, while those in the Astaxanthin group were higher than those in the NEC model group (P<0.05). Conclusion: Astaxanthin improves mitochondrial function of intestinal mucosal cells in the NEC model by up-regulating the FGF21 and PGC-1α pathways, and reduces necrotic apoptosis of cells.

关键词

虾青素 / FGF21/PGC-1α / 坏死性小肠结肠炎 / 线粒体功能 / 核呼吸因子1 / 线粒体转录因子A / 大鼠

Key words

Astaxanthin / FGF21/PGC-1α / Necrotizing enterocolitis / Mitochondrial function / Nuclear respiratory factor 1 / Mitochondrial transcription factor A / Rats

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基金资助

山西省卫生健康委员会重点攻关专项(2020XM29)