Effects Of Carbohydrate Diet With Different Contents On Gut Microbiota And Aortic Calcification In Diabetic Mice

ObjectiveVascular calcification widely existed in diabetic patients and is one of the main causes of cardiovascular and cerebrovascular accidents in diabetic patients.So far,there is no effective treatment for vascular calcification in clinical practice.Therefore,it is urgent to develop some new methods to prevent or treat vascular calcification.The aim of this study is to investigate the effects of carbohydrate-rich diet on gut microbiota and aortic calcification in diabetic Apo E^-/-mice.Methods8-week-old male Apo E^-/-mice were injected intraperitoneal with a low dose of streptozotocin[40 mg/（kg·d）]for five consecutive days to establish the diabetes model.After two weeks,mice with blood glucose level≥300 mg/dl were included in the experiment.They were randomly divided into four groups:ketogenic diet group（ketogenic diet,KD group,n=6）,low carbohydrate diet group（ow carbohydrate diet,LCD group,n=6）,medium carbohydrate diet group（medium carbohydrate diet,MCD group,n=6）,and high carbohydrate diet group（high carbohydrate diet,HCD group,n=6）and had been fed for 6 months with blood glucose,blood ketone and body weight monitored monthly.After six months fed,total cholesterol（TC）,triglyceride（TG）,high-density lipoprotein cholesterol（HDL-C）and low-density lipoprotein cholesterol（LDL-C）in the mice serum were detected by biochemical analyzer.The tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）and interleukin-10（IL-10）were detected by ELISA kit.The morphology of vascular plaque was observed by total oil red staining,hematoxylin-eosin staining and Masson staining.The vascular calcification was evaluated by total alizarin red staining,Von Kossa and local alizarin red staining,and calcium content determination.HE and PAS staining were used to observe intestinal morphology and the number of goblet cells.The expressions of occludin,ZO-1 and claudin-1 proteins in ileum were detected by Western-blot and immunohistochemical staining.Feces of mice were collected and subjected to Illumina Mi Seq sequencing.The changes of intestinal flora at various levels were analyzed by bioinformatics,and the metabolites of intestinal flora were quantitatively detected by ELISA.Results（1）The fasting blood glucose（8.77±4.45 mmol/L）and the area under the curve of intraperitoneal glucose injection（37.89±1.15 h·mmol/L）in the KD group were lower than those in the other three groups within 6 months,and the blood ketone level（2.06±0.63 mmol/L）was significantly increased.（2）Compared with the three rest groups,KD mice intestinal villi weren’t broken,and not seen obviously ulcer without inflammatory cell infiltration.The scores of intestinal mucosal injury in KD group（2.2±0.75）were significantly lower than the MCD group（4.20±0.40）and HCD group（4.20±0.74）.Moreover,the number of goblet cells in the intestine of diabetic mice was significantly increased in KD group.（3）Occludin,Claudin-1,ZO-1 protein expression level of KD group were higher than the rest of the three groups.At the same time,in the intestinal immunofluorescence of the KD group,ZO-1 and Occludin proteins were found to be distributed on the surface of the intestinal mucosal epithelium in wavy lines,closely continuous with smooth edges and strong fluorescence intensity.（4）Gut microbiota sequencing results showed that the KD group reduced the intestinal flora alpha diversity,and beta diversity analysis showing four groups existed differences between groups.At the phylum level,KD increased the relative abundance of Firmicutes and decreased the relative abundance of Bacteroidetes.At the genus level,the relative abundance of Allobaculum was significantly increased in the KD group.（5）Compared with MCD group and HCD group,the aortic artery atheromatous plaque area and calcium salt deposition of mice in the LCD and KD group were significantly reduced.ConclusionKetogenic diet can delay aortic calcification and improve intestinal barrier function in diabetic Apo E^-/-mice.