枯草杆菌二联活菌肠溶胶囊调节2型糖尿病合并昼夜节律紊乱小鼠糖脂代谢的作用机制

doi:10.12122/j.issn.1673-4254.2025.07.15

摘要/Abstract

摘要：

目的分析枯草杆菌二联活菌肠溶胶囊（LCBE）对2型糖尿病合并昼夜节律紊乱糖脂代谢的影响以及作用机制。方法将KM小鼠随机分为3组，正常组8只，给予正常饲料喂养，高脂组8只，给予高脂饲料喂养，节律干预组16只，给予节律干预和高脂喂养，8 周后给予链脲佐菌素100 mg/kg腹腔注射，将节律干预糖尿病造模成功的小鼠随机分为2组，灌胃干预的2型糖尿病合并昼夜节律紊乱组（n=8）和2型糖尿病合并昼夜节律紊乱对照组（n=8），给予灌胃干预组225 mg/kg的LCBE灌胃干预，其余3组给予等体积的生理盐水，持续8 周。使用鱼跃血糖仪测小鼠血糖，ELISA法检测Bmal 1、PER 2、胰岛素、C肽和血脂水平，HE染色观察小鼠结肠变化，油红O染色观察对肝脏脂代谢的影响，液相色谱-质谱法检测粪便短链脂肪酸的水平，qPCR和Western blotting法检测小鼠GPR43和GLP-1基因及蛋白表达情况。结果与CRD-DM组比较，LCBE-CRD-DM组体质量、周期节律调节因子2（PER2）、胰岛素、C肽、T-CHO、TC和LDL-C水平明显下降（P<0.001），Bmal 1和HDL-C水平明显升高（P<0.001）；灌胃干预后，LCBE-CRD-DM组短链脂肪酸、GPR43和GLP-1基因和蛋白表达水平明显高于CRD-DM组（P<0.001）。结论 LCBE能够降低节律紊乱2型糖尿病的体质量，改善小鼠的血脂水平，LCBE对节律紊乱2型糖尿病小鼠糖脂代谢的影响可能与SCFAs/GPR43/GLP-1通路有关。

关键词: 2型糖尿病, 昼夜节律, 枯草杆菌二联活菌肠溶胶囊, 短链脂肪酸, 糖脂代谢

Abstract:

Objective To investigate the effects of live combined Bacillus subtilis and Enterococcus faecium (LCBE) on glucose and lipid metabolism in mice with type 2 diabetes mellitus (T2DM) and circadian rhythm disorder (CRD) and explore the possible mechanisms. Methods KM mice were randomized into normal diet (ND) group (n=8), high-fat diet (HFD) group (n=8), and rhythm-intervention with HFD group (n=16). After 8 weeks of feeding, the mice were given an intraperitoneal injection of streptozotocin (100 mg/kg) to induce T2DM. The mice in CRD-T2DM group were further randomized into two equal groups for treatment with LCBE (225 mg/kg) or saline by gavage; the mice in ND and HFD groups also received saline gavage for 8 weeks. Blood glucose level of the mice was measured using a glucometer, and serum levels of Bmal1, PER2, insulin, C-peptide and lipids were determined with ELISA. Colon morphology and hepatic lipid metabolism of the mice were examined using HE staining and Oil Red O staining, respectively, and fecal short-chain fatty acids (SCFAs) was detected using LC-MS; GPR43 and GLP-1 expression levels were analyzed using RT-qPCR and Western blotting. Results Compared with those in CRD-T2DM group, the LCBE-treated mice exhibited significant body weight loss, lowered levels of PER2, insulin, C-peptide, total cholesterol (TC) and LDL-C, and increased levels of Bmal1 and HDL-C levels. LCBE treatment significantly increased SCFAs, upregulated GPR43 and GLP-1 expressions at both the mRNA and protein levels, and improved hepatic steatosis and colon histology. Conclusion LCBE ameliorates lipid metabolism disorder in CRD-T2DM mice by reducing body weight and improving lipid profiles and circadian regulators possibly via the SCFAs/GPR43/GLP-1 pathway.

Key words: type 2 diabetes mellitus, circadian rhythm, live combined Bacillus subtilis and Enterococcus faecium, short-chain fatty acids, glucolipid metabolism

韩瑞敏, 赵曼可, 袁俊芳, 史振红, 王珍, 王德峰. 枯草杆菌二联活菌肠溶胶囊调节2型糖尿病合并昼夜节律紊乱小鼠糖脂代谢的作用机制[J]. 南方医科大学学报, 2025, 45(7): 1490-1497.

Ruimin HAN, Manke ZHAO, Junfang YUAN, Zhenhong SHI, Zhen WANG, Defeng WANG. Live combined Bacillus subtilis and Enterococcus faecium improves glucose and lipid metabolism in type 2 diabetic mice with circadian rhythm disruption via the SCFAs/GPR43/GLP-1 pathway[J]. Journal of Southern Medical University, 2025, 45(7): 1490-1497.

图/表 12

表1 GPR43/GLP-1通路基因的引物序列

Tab. 1 Primer sequences for genes in the GPR43/GLP-1 pathway

Primer name	Primer sequence (5' to3')
Primer name	Forward primer	Reverse primer
GPR43	CTTCTTTCTTGGCAATTACTGGC	CCGAAATGGTCAGGTTTAGCAA
GCG	AAGAGGAACCGGAACAACATTG	GCCCTCCAAGTAAGAACTCACA
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA

图1 小鼠节律因子水平

Fig. 1 Levels of rhythmic factors in different groups. A: Level of Bmal 1 in the mice. B: Level of Per 2 in the mice. *P<0.05 vs NC.

图2 小鼠体质量变化趋势图

Fig. 2 Trends of body mass changes of the mice. ***P<0.001 vs DM group.

图3 小鼠糖耐量曲线

Fig.3 Glucose tolerance curves in different groups. **P<0.01, ***P<0.001 vs CRD-DM group.

图4 各组小鼠节律因子水平

Fig. 4 Levels of rhythmic factors in the mice in each group. A: Level of Bmal 1 in the mice. B: Level of Per 2 in the mice. **P<0.001 vs NC group; ##P<0.001.

图5 各组小鼠胰岛素和C肽水平

Fig. 5 Insulin (A) and C-peptide (B) levels of the mice in each group. **P<0.001 vs NC group; ##P<0.001.

图6 各组小鼠血脂水平比较

Fig.6 Comparison of lipid levels among the groups. A: Total cholesterol levels of the mice in each group. B: Triglyceride levels of the mice in each group. C: Low-density lipoprotein cholesterol levels of the mice in each group. D: High-density lipoprotein cholesterol levels of the mice in each group. **P<0.001 vs NC group; ##P<0.001.

图7 各组结肠HE染色结果比较

Fig. 7 HE staining of the colon tissue in each group.

图8 各组肝脏油红O染色结果比较

Fig. 8 Oil red O staining of the liver tissue in each group.

图9 各组短链脂肪酸结果比较

Fig. 9 Fecal levels of short-chain fatty acid in each group. A: Acetic acid level. B: Propanoic acid level. C: Butyric acid level. D: Pentanoic acid level. *P<0.05, **P<0.001 vs NC group; #P<0.05, ##P<0.001.

图10 各组结肠GPR43和GLP-1基因表达结果

Fig.10 Colonic GPR43 (A) and GLP-1 (B) mRNA expression in each group. **P<0.001 vs NC group; ##P<0.001.

图11 各组小鼠结肠GPR43和GLP-1蛋白水平结果

Fig. 11 GPR43 and GLP-1 protein levels in the colon of the mice in each group detected by Western blotting. *P<0.05, **P<0.001 vs NC group; ##P<0.001.

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