参芪复方对糖尿病葡萄糖激酶大鼠肠道菌群及代谢影响的实验研究

Effect of Shenqi Compound(参芪复方)on Intestinal Flora and Metabolism in Diabetic GK Rats

目的 探讨参芪复方对糖尿病葡萄糖激酶(glucokinase, GK)大鼠血糖的影响及其潜在的效应机制。方法 40只5月龄糖尿病GK大鼠适应性喂养1周后将两次随机血糖≥11.1 mmol/L的大鼠随机分为模型组、参芪复方组、西格列汀组,每组各8只,予以高脂饲料喂养;8只5月龄Wistar大鼠设为空白组,予以普通饲料喂养。分组完成后模型组及空白组予以生理盐水5 mL/(kg·d)灌胃,参芪复方组予以参芪复方浸膏1.44 g/(kg·d)灌胃,西格列汀组给予西格列汀片悬液16 mg/(kg·d)灌胃,连续干预8周。在整个实验过程中观察各组大鼠的一般状况,并于灌胃前、灌胃4周、灌胃8周检测各组大鼠的随机血糖,在干预结束后观察结肠组织病理切片,无菌采集肠道粪便进行肠道菌群宏基因组学技术测序。结果 与空白组比较,灌胃前、灌胃4、8周、模型组、参芪复方组、西格列汀组的随机血糖水平显著升高(P<0.05);灌胃4周,参芪复方组、西格列汀组较模型组随机血糖无统计学意义(P>0.05);灌胃8周,参芪复方组、西格列汀组较模型组随机血糖显著降低(P<0.05);结肠组织病理显示模型组大鼠腺体结构破坏,杯状细胞减少,肠绒毛坏死脱落,有大量的炎性细胞浸润,部分甚至可见溃疡;参芪复方组仅在黏膜固有层及隐窝可见炎性细胞浸润,伴有黏膜下水肿;西格列汀组大鼠肠黏膜上皮完整,腺体结构排列规则,仅有少量的炎性细胞浸润。结肠组织损伤指数(TDI)评分结果显示,与空白组比较,模型组TDI更高(P<0.05);而参芪复方组、西格列汀组较模型组TDI有所降低(P<0.05);宏基因组学测序显示糖尿病GK大鼠较空白Wistar大鼠的肠道菌群更为丰富,但主要优势菌群组成基本一致;与模型组比较,参芪复方能显著增加门水平B/F比值,增加Fusobacteria丰度(P<0.01)、科水平上Marinifilaceae丰度(P<0.05)及属水平上Bacteroides、Prevotella丰度;同时KEGG分析发现差异肠道菌群的基因功能主要与物质代谢中的碳水化合物代谢相关,参芪复方能通过影响脂质代谢通路、次级代谢产物的合成、异生物素的降解和代谢这三条通路功能基因的表达来发挥治疗T2DM的作用。结论 益气健脾活血的参芪复方具有改善糖代谢紊乱的作用,通过调节肠道菌群,抑制免疫炎症,恢复内环境稳态可能是其发挥抗糖尿病潜在途径。

Objective To investigate the effect of Shenqi Compound(参芪复方)on blood glucose in diabetic Goto-Kakizaki(GK)rats and its potential mechanism.Methods Forty 5-month-old diabetic GK rats were randomly divided into model group,Shenqi Compound group and sitagliptin group after adaptive feeding for one week.Eight rats in each group were fed with high-fat diet.Eight 5-month-old Wistar rats were set as blank group and fed with normal diet.Then the model group and blank group were given normal saline[5 mL/(kg·d)]by gavage.The Shenqi compound group was given Shenqi Compound extract[1.44 g/(kg:d)]by gavage,and the sitagliptin group was given sitagliptin tablet suspension[16 mg/(kg·d)]by gavage for 8 weeks.During the whole experiment,the general condition of rats in each group was observed,and the random blood glucose of rats in each group was detected before gavage,4 and 8 weeks of gavage.After the intervention,the pathological sections of colon tissue were observed,and the intestinal feces were aseptically collected for intestinal flora metagenomics technology testing.Results Compared with those of the blank group,the random blood glucose levels of the model group,Shenqi Compound group and sitagliptin group were significantly higher before gavage,4 and 8 weeks after gavage(P<0.05).After 4 weeks of gavage,compared with that of the model group,there was no significant diference in the random blood glucose of the Shenqi Compound group and the sitagliptin group(P>0.05).After 8 weeks of gavage,compared with that of the model group,the random blood glucose of Shenqi Compound group and the sitagliptin group was significantly lower(P<0.05).Colonic histopathology showed that in the model group,the gland structure was destroyed,goblet cells were reduced,intestinal villi were necrotic and exfoliated,a large number of inflammatory cells were infiltrated,and some ulcers were even seen.In the Shenqi Compound group,only inflammatory cell infiltration and submucosal edema were found in the lamina propria and crypt of mucosa;In the sitagliptin group,the intestinal mucosa epithelium was intact,the glandular structure was arranged regularly,and only a small amount of inflammatory cells were infiltrated.Compared with that of the blank group,the tissues damage index(TDI)of the model group was higher(P<0.05).The TDI values of the Shenqi Compound group and the sitagliptin group were lower than that of the model group(P<0.05).Metagenomic sequencing showed that the intestinal flora of diabetic GK rats was more abundant than that of blank Wistar rats,but the composition of the main dominant flora was basically the same.Compared with the model group,Shenqi Compound could significantly increase the abundance of fusobacteria at portal level(P<0.01)and mariniflaceae at branch level(P<0.05).At the same time,KEGG analysis found that the gene function of intestinal flora was mainly related to carbohydrate metabolism in material metabolism.Shenqi Compound can play a role in the treatment of diabetes by affecting the expressions of functional genes in lipid metabolism pathway,secondary metabolite synthesis,degradation of heterobiotin and metabolism.Conclusion The effect of Shenqi Compound on improving glucose metabolism disorders may be achieved by regulating the intestinal flora,suppressing immune inflammation and restoring the homeostasis.