肠道菌群对小鼠放射敏感组织稳态维持和再生修复的影响

Role of gut microbiota in homeostatic maintenance and promoting regeneration of radiosensitive tissues in mice following radiation exposure

目的探讨肠道菌群(gut microbiota, GM)对小鼠骨髓造血和肠上皮稳态维持及放射损伤修复的影响。方法 6~8周龄C57BL/6J小鼠分为2组:Abx组饮用抗生素(antibiotics, Abx)饮水清除GM;对照组给予正常饮水。测定实验后21 d小鼠体质量等基础状况改变。检测外周血和骨髓HE染色评估清除GM对骨髓造血稳态维持的影响;HE染色、BrdU免疫组化、qPCR、类器官培养等方法评估清除GM对肠道干细胞稳态的影响。8 Gy全身照射小鼠后3、7 d,取材小肠,通过形态学和组织染色评估清除GM对小鼠全身放射损伤后骨髓造血和肠道再生修复过程的影响。结果清除GM后21 d,Abx组小鼠体质量较对照组显著降低(P<0.01);外周血中白细胞、红细胞、血红蛋白和血小板水平出现明显波动,小鼠骨髓巨核细胞计数较对照组显著增加(P<0.01)。与对照组比较,清除GM后21 d,Abx组小肠隐窝缩短,BrdU阳性隐窝细胞减少,Lgr5、Olfm4、Axin2等肠干细胞相关基因mRNA水平显著下降,肠隐窝形成类器官能力降低(P<0.05)。8 Gy放射损伤后,Abx组小鼠存活时间较对照组显著缩短(P<0.01),同时骨髓和小肠再生速度减缓。放射损伤后7 d,Abx组小肠绒毛中杯状细胞数量显著增多,隐窝中内分泌肠上皮细胞显著减少。结论肠道菌群有助于维持小鼠骨髓造血和肠道干细胞稳态,并促进放射损伤后骨髓造血重建和肠道再生修复。

Objective To investigate the role of gut microbiota(GM) in maintaining hematopoietic function and intestinal epithelial homeostasis function and promoting regeneration of hematopoietic tissues and intestinal epithelium in mice radiation exposure. Methods C57 BL/6 J mice(6~8 weeks) were treated with antibiotics added in drinking water for 21 d to deplete the GM(Abx group), and the control mice were given normal drinking water. The changes in body weight and general condition of the mice were recorded. The effect of GM depletion on hematopoietic homeostasis was evaluated by peripheral blood analysis and HE staining of the bone marrow, and the changes in intestinal homeostasis were determined with HE staining, BrdU immunohistochemistry, RT-qPCR, and enteroid culture. The mice with or without GM depletion were subjected to X-ray exposure at the total body irradiation(TBI) dose of 8 Gy, and the regeneration of the radiosensitive tissues were assessed at 3 and 7 d after the exposure. Results GM depletion induced by 21-day treatment with antibiotics resulted in severe weight loss of the mice(P<0.01) and significant changes in peripheral white blood cells(WBC), red blood cells(RBC), hemoglobin and platelets, and markedly increased number of megakaryocytes in the bone marrow(P<0.01). GM depletion induced an obvious decrease in crypt depth and the number of BrdU^+ cryptal cells and significantly down-regulated the expression of intestinal stem cell-related genes including Lgr5, Olfm4, and Axin2. The mice with GM depletion showed significantly lowered enteroid formation capacity(P<0.05) and shortened survival(P<0.01) with obviously lowered rate of hematopoietic and intestinal tissue regeneration after radiation exposure. At 7 d after the radiation, the mice with GM depletion showed increased villous goblet cells and decreased cryptal enteroendocrine cells in the intestines. Conclusion Normally functioning GM are essential for maintaining hematopoietic and intestinal stem cell homeostasis and promoting regeneration of the radiosensitive tissues after radiation exposure.