双歧杆菌三联活菌对肥胖小鼠慢性低度炎症的影响

Effects of bifid triple viable on chronic low-grade inflammation in obese mice induced by high fat diet

目的 观察双歧杆菌三联活菌对高脂饲料诱导的肥胖小鼠慢性低度炎症和胰岛素抵抗的影响,并探讨可能的作用机制。方法 6周龄雄性C57BL/6J小鼠18只,适应性喂养2周后,按体重随机分为:对照组6只,予以常规饲料饲养,另12只60%高脂饲料饲养。饲养8周,确认高脂饲养小鼠体重和空腹血糖值均显著高于对照组小鼠后(P<0.001),再根据体重和血糖随机分为高脂组和双歧杆菌三联活菌处理组,每组6只。高脂组继续给予高脂饲料进一步诱导肥胖,双歧杆菌三联活菌组在继续高脂饲料饲养同时给予24 mg/kg双歧杆菌三联活菌混悬液灌胃,对照组和高脂组给予等体积蒸馏水,每日1次。给药4周后,尾静脉取血,全自动血糖仪检测空腹血糖。透射电镜观察回肠黏膜上皮细胞间紧密连接情况,鲎试剂检测血清内毒素水平;免疫组化法对肝脏和附睾白色脂肪组织中巨噬细胞标志物F4/80进行染色;酶联免疫法检测血清胰岛素水平,肝脏和附睾白色脂肪组织中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interlukin-6,IL-6)含量,附睾白色脂肪组织中单核细胞趋化蛋白-1(monocyte chemotactic protein-1,MCP-1)含量;计算胰岛素抵抗稳态模型(homeostasis model assessment of insulin resistance, HOMA-IR)指数。结果与对照组相比,高脂组小鼠回肠黏膜上皮细胞间紧密连接结构疏松;血清内毒素,肝脏TNF-α、IL-6,附睾白色脂肪组织TNF-α、IL-6、MCP-1含量均显著升高(P<0.05);肝脏和附睾白色脂肪组织中巨噬细胞数量增多;空腹血糖、空腹胰岛素以及HOMA-IR(P<0.01)均显著升高。与高脂组相比,双歧杆菌三联活菌干预组小鼠回肠黏膜上皮细胞间紧密连接结构排列紧密,没有明显受损;血清内毒素,肝脏TNF-α、IL-6,附睾白色脂肪组织中TNF-α、IL-6、MCP-1含量均显著降低(P<0.05);肝脏和附睾白色脂肪组织中浸润的巨噬细胞数量明显减少;空腹血糖没有差异,空腹胰岛素和HOMA-IR均显著下降(P<0.05)。结论 双歧杆菌三联活菌可能通过保护肠道黏膜屏障,减轻代谢性内毒素血症,从而改善高脂饲料诱导的肥胖小鼠的肝脏和脂肪组织慢性低度炎症,进而缓解胰岛素抵抗,部分恢复了胰岛素的敏感性。

OBJECTIVE To explore the effects and possible mechanism of bifid triple viable(BTV) on chronic low-grade inflammation and insulin resistance in obese mice induced by high fat diet(DIO). METHODS A total of 18 male C57 BL/6 J mice aged 6 weeks were randomly divided into two groups according to their body weight after adaptive feeding for 2 weeks. Six mice in one group were fed with normal rodent diet as control group. Twelve mice in the other group were fed with high fat diet(60% kcal% fat)(HF). After 8 weeks, it was confirmed that body weight and fasting blood glucose of the mice fed with high fat diet were significantly higher than those of the control group(P<0.001), and then they were randomly divided into HF group and HF+BTV group according to the body weight and fasting blood glucose. Six mice in the HF group continued to be fed with high fat diet. Six mice in the HF+BTV group were fed with high fat diet and administered bifid triple viable suspension by gavage(24 mg/kg body weight), once a day. Mice in the control group and HF group were given equal volume of water as control treatment. After 4 weeks of treatment, blood was collected from tail vein, and the fasting blood glucose was measured by automatic glucose meter. Then the animals were killed and collected blood, distal ileum, liver and epididymal white adipose tissue. The tight junctions between the epithelial cells of ileum mucosa were observed by transmission electron microscopy. Serum endotoxin(also called lipopolysaccharide, LPS) levels were determined by limulus amebocyte lysate assay. Immunohistochemical staining was used to observe the macrophage marker F4/80 in liver and epididymal white adipose tissue. Serum fasting insulin levels, tumor necrosis factor-α(TNF-α), interlukin-6(IL-6) and monocyte chemotactic protein-1(MCP-1) in liver and/or epididymal white adipose tissue were detected by ELISA. The index of homeostasis model assessment of insulin resistance(HOMA-IR) was calculated by formula.RESULTS Compared with the mice fed with normal rodents diet, the tight junctions between the epithelial cells of ileum mucosa were loose, the levels of serum endotoxin, live TNF-α and IL-6, epididymal white adipose tissue TNF-α, IL-6, MCP-1 were significantly elevated(P<0.05), the number of macrophages increased in liver and epididymal white adipose tissue, fasting glucose, fasting insulin levels and HOMA-IR were significantly up-regulated(P<0.01) in the HF group. In comparison with the HF group, the structures of tight junctions between the epithelial cells of ileum mucosa were normalized, the levels of serum endotoxin, liver TNF-α, IL-6 and epididymal white adipose tissue TNF-α, IL-6, MCP-1 were obviously decreased(P<0.05), the number of macrophages reduced in liver and epididymal white adipocytes, fasting insulin levels and HOMA-IR were significantly down-regulated(P<0.05), but had no effect on fasting blood glucose levels in HF+BTV group.CONCLUSION Bifid triple viable may protect intestinal mucosa barrier, alleviate metabolic endotoxemia, thus improve chronic low-grade inflammation in liver and adipose tissue, and partially restore insulin sensitivity in DIO mice by regulating gut microbiota.