二甲双胍对2型糖尿病大鼠结直肠癌发生率及其肠道菌群的影响

Effects of Metformin on Incidence of Colorectal Cancer and Gut Microbiota in Type 2Diabetic Rats

目的 探究二甲双胍(MET)对2型糖尿病大鼠结直肠癌发生率及肠道菌群的影响和相关机制。方法 采用高脂高糖饮食联合腹腔低剂量注射链脲佐菌素(STZ)诱导2型糖尿病大鼠模型,在此基础上腹腔注射二甲基肼(DMH)诱导大鼠结直肠癌的发生。通过MET灌胃和控制摄入饮食量的方法模拟临床2型糖尿病患者高血糖的治疗方案。将大鼠分为正常组(NC组),对照组(Control组)和实验组(MET组)。在DMH注射32周末,解剖所有大鼠,记录结直肠肿瘤的发生率、个数,苏木精-伊红(HE)染色观察结直肠组织病理变化,实时定量聚合酶链式反应(qRT-PCR)检测大鼠结直肠组织与肿瘤组织中IL-17的表达,收集大鼠粪便进行16S rRNA测序。结果 (1)成功诱导2型糖尿病大鼠模型并控制实验组大鼠的高血糖。(2)成功诱导2型糖尿病大鼠结直肠癌的发生。(3)实验组和对照组的肿瘤发生率和平均肿瘤数有显著差异。(4)HE染色结果显示二甲双胍干预可缓解2型糖尿病大鼠结直肠正常组织和癌前病变组织中炎性细胞浸润。(5)qRT-PCR检测结果显示肿瘤组织中IL-17的表达显著高于正常组织;对照组正常肠组织中IL-17表达显著高于正常组;而实验组与对照组肿瘤组织中IL-17的表达无统计学差异。(6)二甲双胍干预可降低由DMH诱导的2型糖尿病结直肠癌大鼠肠道菌群的Alpha多样性;NMDS显示实验组与正常组肠道菌群群落组成差异最大,实验组与对照组群落组成相似。组间物种差异结果显示,在门水平上实验组与对照组无明显差异;属水平上,实验组的Ligilactobacillus、Ruminococcus、[Eubacterium]_siraeum_group、Prevotellaceae_NK3B31_group、Alistipes、[Eubacterium]_fissicatena_group、UCG-005丰度均低于对照组(均P<0.05)。结论 二甲双胍干预可降低由DMH诱导的2型糖尿病大鼠结直肠癌的发生率,并影响其肠道菌群多样性和菌群构成。同时二甲双胍降低由DMH诱导的2型糖尿病结直肠癌大鼠正常肠组织中的IL-17表达,但无法抑制其肿瘤组织中IL-17的表达。

Objective To investigate the effect of metformin(MET)on the incidence of colorectal cancer and gut microbiota in type 2 diabetic rats and possible mechanism.Methods Type 2 diabetic rat model was established by high-fat and high-sugar diet combined with a low-dose intraperitoneal injection of streptozotocin(STZ).Intraperitoneal injection of dimethylhydrazine(DMH)was used to induce colorectal cancer in rats.The treatment of hyperglycemia in patients with type 2 diabetes was simulated by MET intragastric administration and dietary intake control.The rats were divided into the normal group, the control group(Control group),and the experimental group(MET group).After 32 weeks of DMH injection, all rats were dissected to record the incidence and number of colorectal tumors.HE staining was used to observe the pathological changes of colorectal tissues, and qRT-PCR was used to detect the expression of IL-17 in colorectal tissues and tumor tissues of rats, and rat feces was collected for 16 S rRNA sequencing.Results(1) Type 2 diabetes rat model was successfully established and hyperglycemia was well controlled in experimental rats.(2) Colorectal cancer model was successfully established in type 2 diabetic rats.(3) There were differences in incidence and average number of tumors between the experimental group and the control group.(4) HE staining results showed that metformin intervention could alleviate inflammatory cell infiltration in normal colorectal tissues and precancerous tissues of type 2 diabetic rats.(5) qRT-PCR results showed that the expression of IL-17 in tumor tissues of both the control group and the experimental group were significantly higher than that in normal tissues.The expression of IL-17 in normal intestinal tissue in the control group was significantly higher than that in the normal group, while there was no significant difference in the expression of IL-17 in normal intestinal tissue between the normal group and the experimental group.(6) Metformin can reduce the alpha diversity of gut microbiota in DMH-induced colorectal cancer rats with type 2 diabetes;NMDS showed that the difference in microbiota between the experimental group and the normal group was the most significant, and the microbiota compositions of the experimental group and the control group were similar.There was no significant difference between the control group and the experimental group at the phylum level.At the genus level, Ligilactobacillus,Ruminococcus,[Eubacterium]_siraeum_group,Prevotellaceae_NK3B31_group,Alistipes,[Eubacterium]_Fissicatena_groupandUCG-005 in the experimental group were all lower than those in the control group(P<0.05).Conclusion Metformin can down regulate the expression of IL-17 in normal intestine tissues of DMH-induced type 2 diabetic colorectal cancer rats, but it cannot inhibit the expression of IL-17 in tumor tissues.Meanwhile, metformin can reduce the incidence of colorectal cancer in DMH-induced type 2 diabetic rats, and affect the diversity and composition of gut microbiota.