目的 探讨丁酸钠（sodium butyrate,NaB）对葡聚糖硫酸钠（dextran sulfate sodium salt,DSS）诱导的炎症性肠病模型小鼠的保护作用及其机制。方法 将38只7 w 龄的 C57BL/6J 小鼠随机分为对照组（control）、模型组（model）、NaB组[500 mg/（kg·d ）ig]和阳性药物 5-氨基水杨酸（5-aminosalicylic acid,5-ASA）组[150 mg/（kg·d）ig ]。造模开始前3d开始给NaB组小鼠灌胃NaB并直至实验结束,造模期间对照组饮用灭菌水,其余3组均饮用2.5% DSS,6 d后换为灭菌水,第7日处死小鼠并测量结肠长度和称重,比较体重、疾病活动指数（disease activity index,DAI）、结肠长度,HE染色观察结肠病理改变,AB-PAS染色观察结肠杯状细胞数量改变,测定谷胱甘肽（glutathione,GSH）含量、亚铁离子（Fe2+）含量、丙二醛（malondialdehyde,MDA）含量以及超氧化物歧化酶（superoxide dismutase,SOD）活性,WB法检测谷胱甘肽过氧化物酶4（glutathione peroxidase 4, GPX4）、胱氨酸/谷氨酸反向转运体（solute carrier family 7 member 11, SLC7A11）表达以及细胞外调节蛋白激酶（extracellular regulated protein kinases,ERK）和信号换能器和转录激活因子 3（signal transducer and activator of transcription 3,STAT3）的磷酸化水平。结果 与对照组相比,模型组小鼠体重下降（P<0.001）,DAI升高（P<0.001）、结肠长度缩短（P<0.001）,HE染色显示结肠细胞浸润、隐窝变浅、绒毛结构消失,AB-PAS染色显示杯状细胞明显减少,上述结果提示DSS成功诱导小鼠IBD模型;ERK和STAT3磷酸化程度降低（P<0.05, P<0.05）,GPX4和SLC7A11表达降低（P<0.05,P<0.05）,表明ERK、STAT3磷酸化被抑制以及铁死亡的发生;与模型组比,NaB组小鼠DAI下降（P<0.01）、结肠长度增加（P<0.001）,HE染色显示结肠结构恢复,AB-PAS染色显示杯状细胞数量增加,GSH含量和SOD活力增加（P<0.01,P<0.01）,MDA和Fe2+含量减少（P<0.01,P<0.01）,GPX4及SLC7A11蛋白表达增加（P<0.01,P<0.05）,ERK 和 STAT3 磷酸化程度明显增强（P<0.05,P<0.05）。结论 丁酸钠可能通过 ERK 和 STAT 3 磷酸化及抑制铁死亡发挥其防治炎症性肠病作用。
Abstract
Objective To investigate the protective effects and mechanism of sodium butyrate (NaB) on dextran sulfate sodium salt (DSS) induced inflammatory bowel disease in mice. Methods Thirty-eight 7-week-old C57BL/6J mice were randomly divided into control group, model group, NaB (500 mg/(kg·d) ig) group and 5-aminosalicylic acid (5-ASA) group (150mg/(kg·d )ig). NaB administration commenced 3 days before DSS induction and continued throughout the experimental period. During molding with DSS, the control group received sterile water, while the other three groups were given 2.5% DSS for 6 days and followed by sterile water until sacrifice. On day 7, mice were euthanized for colon length measurement and tissue weight analysis. The body weight, disease activity index (DAI) and colon length among different groups were compared. Colon pathology was evaluated via HE staining, and the number of goblet cells in the colon was observed after AB-PAS staining. To assess the degrees of ferroptosis, GSH, Fe2+, MDA levels and SOD activity were measured. The WB method was used to detect the expression of GPX4 and SLC7A11 and the phosphorylation levels of ERK and STAT3. IHC was used to detect the expression level of GPX4. Results Compared to the control group, the model group exhibited significant weight loss (P<0.001), elevated DAI (P<0.001), and shortened colon length (P<0.001). HE staining showed colon cell infiltration, shallow crypt, disappeared villi structure. The AB-PAS staining showed that the number of goblet cells was decreased. Meanwhile, both decreased expression of GPX4 and SLC7A11(P<0.05, P<0.05) and suppressed phosphorylation of ERK and STAT3 (P<0.05, P<0.05) indicated that ERK and STAT3 phosphorylation were inhibited and ferroptosis occured. Compared to the model group, the NaB group displayed reduced DAI (P<0.01), increased colon length (P<0.001). HE staining showed that colon structure was recoved AB-PAS staining indicated increased goblet cells. GSH level and SOD activity were increased (P<0.01, P<0.01), while MDA and Fe2+ levels decreased significantly (P<0.01, P<0.01). GPX4 and SLC7A11 protein expressions were increased (P<0.01, P<0.05), and the phosphorylation of ERK and STAT3 was significantly enhanced (P<0.05, P<0.05). Conclusion Sodium butyrate may play a protective role in inflammatory bowel disease through ERK and STAT3 phosphorylation and ferroptosis inhibition.
关键词
丁酸钠 /
炎症性肠病 /
铁死亡 /
细胞外调节蛋白激酶 /
信号换能器和转录激活因子 3
Key words
sodium butyrate /
ferroptosis /
inflammatory bowel disease /
extracellular regulated protein kinases (ERK) /
ignal transducer and activator of transcription 3 (STAT3)
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基金
国家自然科学基金（No.81773429）; 广东省自然科学基金（No.2022A1515011631）; 广东省自然科学基金（No.2024A1515012175）
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