肠道菌群改变影响CG-IUGR大鼠糖代谢的初步研究

doi:10.11958/20230026

摘要/Abstract

摘要：

目的对生长追赶的宫内发育迟缓（CG-IUGR）大鼠进行正常大鼠肠道菌群干预，分析其肠道菌群改变与糖代谢功能降低的关系。方法将SD大鼠分为Control组、CG-IUGR组和CG-IUGR+肠菌组，每组8只。采用低热量饮食法复制CG-IUGR大鼠模型，CG-IUGR+肠菌组大鼠在其3周龄时移植Control组大鼠的肠菌，每周移植1次，共6次。测量3组大鼠出生后至8周龄的体质量和身长，计算体质指数（BMI）；检测3组大鼠糖代谢相关指标：空腹血糖（FBG）及空腹胰岛素（FINS）水平，葡萄糖耐量试验和胰岛素耐量试验，以及葡萄糖负荷后15 min的血清胰岛素（INS）水平；采用实时荧光定量聚合酶链式反应（qPCR）和Western blot法检测3组大鼠骨骼肌糖原合成酶（GYS）1和肝脏GYS2的mRNA和蛋白表达水平；检测骨骼肌和肝脏GYS酶活性。采用16S rDNA测序法检测3组大鼠肠道菌群的多样性和结构组成。结果与Control组相比，CG-IUGR组大鼠在生长发育过程中身长、体质量和BMI均明显增加，血清FINS水平下降，血清FBG无明显变化，血糖水平在糖负荷和INS负荷后明显上调，且血清INS水平在糖负荷后的15 min显著上调；骨骼肌GYS1和肝脏GYS2的mRNA和蛋白表达水平均降低，其GYS酶活性亦下降（P<0.05）；CG-IUGR+肠菌组大鼠的上述变化均较CG-IUGR组改善（P<0.05）。CG-IUGR组大鼠肠道菌群ACE、Chao1指数低于Control组（P<0.05）；与CG-IUGR组相比较，CG-IUGR+肠菌组大鼠肠道双歧杆菌相对丰度增加（P<0.05）。结论 IUGR可通过下调骨骼肌和肝脏GYS的表达及活性降低CG-IUGR大鼠糖代谢功能，CG-IUGR大鼠糖代谢功能异常可能与肠道菌群改变有关。

关键词: 胃肠道微生物群, 糖尿病, 糖原合酶, 葡萄糖代谢障碍, 宫内发育迟缓

Abstract:

Objective To analyze the relationship between intestinal flora and glucose metabolism changes in CG-IUGR rats by interventing CG-IUGR rats with the intestinal flora of normal rats. Methods SD rats were divided into three groups: the control group, the CG-IUGR group and the CG-IUGR+intestinal bacteria group (n=8 in each group). The CG-IUGR rat model was established by low-calorie diet. The intestinal flora of rats in the control group were transplanted into CG-IUGR rats in the CG-IUGR+intestinal bacteria group at 3-week-old (once a week, 6 times in total). From birth to 8 weeks, the body weight and body length were measured in three groups of rats, and body mass index (BMI) was calculated. The relevant indexes of glucose metabolism were detected in three groups, including fasting blood glucose (FBG), serum fasting insulin (FINS) and serum insulin (INS) at 15 min after glucose loading and tolerance test of glucose and insulin. The mRNA and protein expression levels of skeletal muscle glycogen synthase (GYS)1 and liver GYS2 were detected by real-time fluorescence quantitative polymerase chain reaction (qPCR) and Western blot assay respectively. The activities of GYS in skeletal muscle and liver were detected. 16S rDNA sequencing was used to detect the diversity and composition of intestinal flora in rats of three groups. Results Compared with the control group, the body length, body weight and BMI of rats in the CG-IUGR group were all increased significantly during the process of growth and development. Its serum FINS level decreased, and serum FBG had no significant change. The blood glucose levels of rats in the CG-IUGR group were significantly up-regulated after glucose and INS loading. The serum INS level of rats in the CG-IUGR group was significantly up-regulated at 15 min after glucose loading. The mRNA and protein expression levels of skeletal muscle GYS1 and liver GYS2 in rats of the CG-IUGR group were decreased, and the activity of GYS was also decreased (P<0.05). These above changes of rats in the CG-IUGR+intestinal bacteria group were all improved (P<0.05). Compared with the control group, ACE and Chao1 indices of intestinal bacteria in rats of the CG-IUGR group were lower (P<0.05). Compared with the CG-IUGR group, the relative abundance of intestinal bifidobacterium in rats of the CG-IUGR+intestinal bacteria group was increased (P<0.05). Conclusion IUGR can attenuate the glucose metabolism fuction of CG-IUGR rats by down-regulating the expression and activity of GYS in skeletal muscle and liver tissue. The abnormal glucose metabolism in CG-IUGR rats is related to changes of intestinal flora.

Key words: gastrointestinal microbiome, diabetes mellitus, glycogen synthase, glucose metabolism disorders, fetal growth retardation

中图分类号:

R363

图/表 12

表1 引物序列

Tab.1 Primer sequences

基因名称	引物序列（5′→3′）	产物大小/bp
GYS1	上游：CCTTGACTGTATTGGCTGTG	219
GYS1	下游：ATTTCTTTATCGCTGGACG	219
GYS2	上游：TAAAGCCATCCGTCGTTG	101
GYS2	下游：GGGCAGATATTACCAGCAT	101
β-actin	上游：CCTAGACTTCGAGCAAGAGA	140
β-actin	下游：GGAAGGAAGGCTGGAAGA	140

图1 3组大鼠0—8周龄的身长变化 n=8；＊P<0.05，＊＊P<0.01；a与Control组比较，b与CG-IUGR组比较，P<0.05；F组间=26.305＊＊，F时间=2 079.122＊＊，F交互=2.997＊＊；F0周龄=3.691＊，F1周龄=0.430，F2周龄=2.176，F3周龄=23.910＊＊，F4周龄=5.475＊，F5周龄=27.807＊＊，F6周龄=3.250，F7周龄=8.579＊＊，F8周龄=4.133＊。

Fig.1 Changes of body length from birth to 8 weeks in three groups of rats

图2 3组大鼠0—8周龄的体质量变化 n=8；＊P<0.05，＊＊P<0.01；a与Control组比较，b与CG-IUGR组比较，P<0.05；F组间=76.452＊＊，F时间=1 553.272＊＊，F交互=12.319＊＊； F0周龄=16.812＊＊，F1周龄=4.763＊，F2周龄=34.385＊＊，F3周龄=109.168＊＊，F4周龄=58.587＊＊，F5周龄=72.251＊＊，F6周龄=23.472＊＊，F7周龄=49.411＊＊，F8周龄=56.071＊＊。

Fig.2 Changes of body weight from birth to 8 weeks in three groups of rats

图3 3组大鼠0—8周龄的BMI变化 n=8；＊P<0.05，＊＊P<0.01；a与Control组比较，b与CG-IUGR组比较，P<0.05；F组间=46.259＊＊，F时间=1 019.633＊＊，F交互=3.958＊＊；F0周龄=0.009，F1周龄=9.016＊＊，F2周龄=25.455＊＊，F3周龄=109.996＊＊，F4周龄=17.314＊＊，F5周龄=27.020＊＊，F6周龄=11.272＊＊，F7周龄=19.062＊＊，F8周龄=31.524＊。

Fig.3 Changes of body mass index from birth to 8 weeks in three groups rats

表2 各组大鼠FBG及FINS水平比较

Tab.2 Comparison of FBG and FINS levels between three groups（n=6，$\bar{x}±s$）

组别	FBG/（mmol/L）	FINS/（mIU/L）
Control组	5.22±0.52	55.45±5.76
CG-IUGR组	5.62±0.44	45.54±2.81^a
CG-IUGR+肠菌组	5.35±0.29	55.61±4.50^b
F	1.367	9.755^＊＊

图4 3组大鼠GTT血糖的变化 n=6；＊P<0.05，＊＊P<0.01；a与Control组比较，b与CG-IUGR组比较，P<0.05；F组间=36.832＊＊，F时间=149.680＊＊，F交互=2.394＊；F0 min=1.936，F15 min=8.584＊＊，F30 min=20.806＊＊，F60 min=33.528＊＊，F90 min=21.889＊＊，F120 min=14.944＊＊。

Fig.4 Changes of GTT blood glucose in 3 groups of rats

图5 3组大鼠ITT血糖的变化 n=6；＊＊P<0.01；a与Control组比较，b与CG-IUGR组比较，P<0.05；F组间=22.601＊＊，F时间=122.433＊＊，F交互=1.397；F0 min=0.875，F15 min=20.678＊＊，F30 min=24.965＊＊，F60 min=17.808＊＊，F90 min=17.796＊＊，F120 min=14.800＊＊。

Fig.5 Changes of ITT blood glucose in 3 groups of rats

表3 3组大鼠骨骼肌GYS1和肝脏GYS2 mRNA表达水平比较

Tab.3 Comparison of mRNA expression levels of GYS1 in skeletal muscle and GYS2 in liver between three groups（n=6，$\bar{x}±s$）

组别	GYS1	GYS2
Control组	1.00±0.00	1.00±0.00
CG-IUGR组	0.70±0.19^a	0.56±0.17^a
CG-IUGR+肠菌组	0.97±0.25^b	0.88±0.20^b
F	4.822^＊	13.126^＊＊

图6 3组大鼠骨骼肌GYS1和肝脏GYS2的蛋白表达水平 A：Control组；B：CG-IUGR组；C：CG-IUGR+肠菌组。

Fig.6 Protein expression of GYS1 in skeletal muscle and GYS2 in liver of rats in each group

表4 3组大鼠骨骼肌GYS1和肝脏GYS2蛋白表达水平比较

Tab.4 Comparison of skeletal muscle GYS1 and liver GYS2 protein expression between three groups （n=6，$\bar{x}±s$）

组别	GYS1	GYS2
Control组	1.04±0.15	0.83±0.13
CG-IUGR组	0.66±0.06^a	0.53±0.08^a
CG-IUGR+肠菌组	0.92±0.12^b	0.68±0.08^b
F	16.105^＊＊	13.605^＊＊

表5 3组大鼠骨骼肌和肝脏GYS酶活性比较

Tab.5 Comparison of skeletal muscle and liver GYS enzyme activity between three groups

组别	骨骼肌	肝脏
Control组	30.49±4.67	24.29±3.54
CG-IUGR组	15.98±2.22^a	11.37±4.05^a
CG-IUGR+肠菌组	22.47±2.23^ab	17.92±2.82^ab
F	30.005^＊＊	20.337^＊＊

表6 各组大鼠肠道菌群的多样性分析

Tab.6 Diversity analysis of intestinal flora in each group （n=5，$\bar{x}±s$）

组别	OTU	ACE指数	Chao1指数	Shannon指数	Simpson指数
Control组	834.60±119.19	1 295.55±184.59	1 177.70±138.77	3.85±0.43	0.07±0.02
CG-IUGR组	740.40±64.72	1 052.62±116.26^a	1 003.46±104.74^a	3.80±0.34	0.08±0.02
CG-IUGR+肠菌组	844.40±71.11	1 324.59±189.60^b	1 182.60±88.66^b	3.86±0.30	0.08±0.05
F	2.109	4.005^＊	4.101^＊	0.046	0.176

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