赖永森.谷氨酰胺抑制NLRP3/Caspase-1通路减轻坏死性小肠结肠炎大鼠肠损伤[J].内科急危重症杂志,2022,28(6):508-512
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| DOI：10.11768/nkjwzzzz20220617 |
| 中文关键词: 谷氨酰胺 坏死性小肠结肠炎 新生大鼠 炎症反应 NLRP3/Caspase-1通路 |
| 英文关键词: |
| 基金项目:深圳市龙华区医疗卫生机构区级科研项目（No：2020097） |
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| 中文摘要: |
| 摘要 目的：探究谷氨酰胺（Gln）对坏死性小肠结肠炎（NEC）新生大鼠肠损伤的保护作用，及其与核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）/半胱氨酸天冬氨酸蛋白水解酶1（Caspase-1）通路之间的关系。方法：将新生SD大鼠随机分成对照组、模型组、谷氨酰胺（Gln）组和Gln+NLRP3激活剂（DDC）组，每组12只。除对照组外，其他3组均采用缺氧和冷刺激建立NEC模型。在造模的同时，Gln组大鼠给予Gln 0.3g/kg灌胃，DDC组大鼠给予Gln 0.3 g/kg和二乙基二硫代氨基甲酸酯（DDC）30 mg/kg灌胃，给药1次/d，连续3 d，末次灌胃空腹24 h后，采用酶联免疫吸附法（ELISA）检测4组大鼠血清中白介素-18（IL-18）、白介素-1β（IL-1β）、肿瘤坏死因子α（TNF-α）水平；心脏取血后打开腹腔，取回盲部近端肠组织1~2 cm，苏木素-伊红（HE）染色检测大鼠肠组织病理学变化并进行损伤评分；TUNEL试剂盒检测大鼠肠细胞凋亡指数；蛋白印迹（WB）法检测肠组织NLRP3、Caspase-1蛋白表达水平。结果：模型组、Gln组和DDC组大鼠在造模24 h后，相继出现不同程度的腹胀、腹泻、活动减少等现象；与模型组比较，Gln组大鼠临床症状出现较晚且程度减轻，活动度较好。与对照组比较，模型组大鼠肠组织可见大量炎性细胞浸润、结构坏死等病变，肠组织损伤评分、细胞凋亡指数、IL-18、IL-1β、TNF-α含量及NLRP3、Caspase-1蛋白相对表达量显著升高（P均＜0.05）。与模型组比较，Gln组肠组织病变减轻，肠组织损伤评分、细胞凋亡指数、IL-18、IL-1β、TNF-α含量及NLRP3、Caspase-1蛋白相对表达量显著降低（P均＜0.05）。与Gln组比较，DDC组大鼠肠组织病变加重，肠组织损伤评分、细胞凋亡指数、IL-18、IL-1β、TNF-α含量及NLRP3、Caspase-1蛋白相对表达量显著升高（P均＜0.05）。结论：Gln可减轻NEC大鼠的炎症水平，保护肠组织结构，其机制可能与抑制NLRP3/Caspase-1信号通路有关。 |
| 英文摘要: |
| Abstract Objective: To explore the protective effect of glutamine (Gln) on intestinal injury in neonatal rats with necrotizing enterocolitis (NEC) and its relationship with the nucleotide binding oligomeric domain-like receptor protein 3(NLRP3)/cysteine aspartic proteolytic enzyme 1(Caspase-1) pathway. Methods: Neonatal SD rats were randomly divided into control group, model group, Gln group and glutamine + NLRP3 activator (diethyldithiocarbamate, DDC) group, with 12 rats in each group. Except the control group, the NEC model was established by hypoxia and cold stimulation in other groups. At the same time of modeling, rats in Gln group were given Gln 0.3 g/kg by gavage, rats in DDC group were given Gln 0.3 g/kg and DDC 30 mg/kg by gavage. The levels of inflammatory cytokines [interleukin-18 (IL-18), IL-1β and tumor necrosis factor-α (TNF-α)] in serum were detected by enzyme-linked immunosorbent assay (ELISA). After taking blood from the heart, the abdominal cavity was opened, and the intestinal tissue at the proximal end of the cecum for 1-2 cm was retrieved. The hematoxylin eosin (HE) staining was used to detect the pathological changes of intestinal tissue and the injury score was evaluated. TUNEL kit was used to detect the apoptosis index of intestinal cells. The expression levels of NLRP3 and Caspase-1 were detected by Western blotting. Results: At 24 h after modeling, the rats in model group, Gln group and DDC group successively showed abdominal distension, diarrhea and decreased activity in different degrees. Compared with the model group, the clinical symptoms of Gln group appeared later and the degree was alleviated, the degree of activity was better. Compared with the control group, a large number of inflammatory cell infiltration and structural necrosis were found in the intestinal tissue of the model group, the intestinal injury score, apoptosis index, contents of IL-18, IL-1β, TNF-α and the relative protein expression of NLRP3 and Caspase-1 were significantly higher (all P< 0.05). Compared with model group, the pathological changes of intestinal tissue in Gln group were alleviated, the intestinal injury score, apoptosis index, contents of IL-18, IL-1β, TNF-α and the relative protein expression of NLRP3 and Caspase-1 were significantly lower (all P< 0.05). Compared with Gln group, the intestinal lesions in DDC group were more severe, the intestinal injury score, apoptosis index, contents of IL-18, IL-1β, TNF-α and the relative protein expression of NLRP3 and Caspase-1 were significantly higher (all P< 0.05). Conclusions: Gln can reduce the inflammatory level and protect the intestinal structure of NEC rats. The mechanism may be related to the inhibition of NLRP3/Caspase-1 signaling pathway. |
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