目的 探究甘草酚（glycyrol, GC）是否通过调节肠道菌群及其代谢产物激活自噬减轻结肠损伤。方法 雄性Balb/C小鼠（6 w龄）适应性喂养1 w后,随机分为对照组(N)：灌胃生理盐水14 d+腹腔注射生理盐水3 d;结肠损伤模型组(5-FU)：灌胃生理盐水14 d+腹腔注射5-氟尿嘧啶（5-fluorouracil,5-FU,100 mg/kg) 3 d;甘草酚干预组(GC)：灌胃GC (20 mg/kg) 14d+腹腔注射生理盐水3 d;GC+5-FU组：灌胃GC（20 mg/kg）14 d+腹腔注射5-FU (100 mg/kg) 3d;抗生素组(AB) ：灌胃抗生素（200 μl）14d+腹腔注射生理盐水3 d;GC+5-FU+AB组：灌胃抗生素（200 μl）14 d+灌胃GC (20 mg/kg) 14d+腹腔注射5-FU（100 mg/kg）3d,每组8只。采用HE染色、Western blot分析等,确定GC对小鼠结肠损伤的保护作用;通过16S rDNA高通量测序、代谢组学等分析GC对小鼠肠道菌群结构、肠道菌群代谢产物、结肠自噬水平的影响;利用相关性分析,阐明GC是否通过调节肠道菌群及其代谢产物激活自噬保护结肠损伤的作用机制。结果 与5-FU组相比,GC可显著增加小鼠的体重、摄食量,提高结肠超氧化物歧化酶 (superoxide Dismutase,SOD)、过氧化氢酶（catalase,CAT）活力,显著降低结肠白细胞介素-1β (interleukin 1 beta, IL-1β)、白细胞介素-6 (interleukin 6, IL-6) 和肿瘤坏死因子-α (tumor necrosis factor alpha, TNF-α)的表达水平,并明显减轻小鼠结肠组织病理学损伤;GC还可以改善小鼠肠道菌群结构,上调厚壁菌门和弯曲菌门的丰度、下调变形菌门和拟杆菌门的丰度,恢复拟杆菌科、毛螺旋菌科和普雷沃氏菌科等的异常变化,并显著下调胆钙化醇等6种肠道菌群代谢产物的水平,明显上调TNK等8种肠道菌群代谢产物的水平;相关性分析结果显示,多种肠道菌群代谢产物与小鼠结肠自噬水平有相关性。结论 GC激活自噬减轻结肠损伤与其调节肠道菌群及其代谢产物有关。
Abstract
Objective To explore whether glycyrol (GC) can protect against colon damage by regulating gut microbiota and metabolite to activate autophagy. Methods Male Balb/C mice (6 weeks old) were adaptively fed for 1 week and randomly divided into control group (N): gavage of physiological saline for 14 days+intraperitoneal injection of physiological saline for 3 days; Colonic injury model group (5-FU): gavage of physiological saline for 14 days+intraperitoneal injection of 5-fluorouracil (5-FU, 100 mg/kg) for 3 days; GC intervention group (GC): gavage of GC (20 mg/kg) for 14 days+intraperitoneal injection of physiological saline for 3 days; GC+5-FU group: gavage of GC (20 mg/kg) for 14 days+intraperitoneal injection of 5-FU (100 mg/kg) for 3 days; Antibiotic group (AB): gavage of antibiotics (200 μl) for 14 days+intraperitoneal injection of physiological saline for 3 days; GC+5-FU+AB group: Administer antibiotics (200 μl) by gavage for 14 days, GC (20 mg/kg) by gavage for 14 days, and intraperitoneal injection of 5-FU (100 mg/kg) for 3 days, with 8 animals in each group. HE staining and Western blot analysis were used to determine the protective effect of GC on colon injury in mice. High-throughput 16S rDNA sequencing and metabolomics were conducted to investigate the effects of GC on gut microbiota structure, gut metabolites, and colon autophagy levels in mice colon damaged mice; Using correlation analysis to elucidate the mechanism by which GC protects against colon injury by regulating gut microbiota and its metabolites to activate autophagy. Results Compared with the 5-FU group, GC intervention can significantly improve the body weight, food intake, and colon superoxide dismutase (SOD) and catalase (CAT) activity. Significantly reduces colon interleukin 1 beta (IL-1β), Interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) level in mice, and significantly reduce damage to colon tissue. GC can also improve the structure of gut microbiota, upregulate the abundance of Firmicutes and Campylobacter, downregulate the abundance of Proteobacteria and Bacteroidetes, restore abnormal changes in Bacteroidetes, Spirochetes, and Prevotellaceae. GC significantly downregulated the levels of six gut microbiota metabolites, including cholecalciferol, and significantly upregulated the levels of eight gut microbiota metabolites, including TNK. The correlation analysis results show that there is a correlation between various gut microbiota metabolites and the level of autophagy in the mouse colon. Conclusion GC activates autophagy to protect colon injury, which is related to regulating gut microbiota and its metabolites.
关键词
甘草酚 /
结肠 /
自噬 /
肠道菌群 /
代谢产物
Key words
colon /
autophagy /
gut microbiota /
gut microbiota derived metabolites /
glycyrol
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基金
国家自然科学基金青年科学基金项目（No.82304147）; 山西省应用基础研究计划青年项目（No.202103021223215）
