The Study Of Curcumin Gypsum Preparation Intervention On Blood-glucose Of Diabetic Mice Based On Gut Microbiota

Objective:Type 2 diabetic mice which were induced by the high fat diet were treated with"curcuma gypsum capsule" formula.During the study period,survival state and of diabetic mice were observed and body weight,fasting blood glucose,plasma insulin,insulin resistance index,endotoxin,triglyceride,total cholesterol,high density lipoprotein,low density lipoprotein and intestinal microflora would be tested.Firstly,to verify the effect of the "curcuma gypsum capsule" on blood glucose and the optimal dosage was obtained by comparison;Secondly,based on Illumina Hiseq 2500 PE250 sequencing platforms and technology,changes of the abundance and diversity of intestinal microbiota between diabetic mice and normal mice were researched,and the recovery of intestinal flora after intervention in Chinese medicine were examined.Through LEfSe analysis,significant difference gut microbiota would be detected after intervention.Thirdly,to explore the characteristic intestinal microbial species and structural changes related to the metabolic disorders of diabetes,and provide references for the use of intestinal flora targeted to treat type 2 diabetes.Methods:54 healthy and clean male C57BL/6J mice were randomly divided into normal feed control group(NCD),high-fat model group(HFD),low dose Chinese medicine group(HFD+L),medium dose Chinese medicine group(HFD+M),high dose Chinese medicine group(HFD+H),metformin group(HFD+Met),and 9 mice in each group.After one week of adaptive feeding,D12450Bi low fat formula feed was used in group NCD,and the other groups were fed with D12492i high fat formula for 12 weeks.After successful modeling,the mice in Chinese medicine groups were perfused by different doses of curcuma gypsum suspension,and the positive drug control group was intragastric administration of metformin suspension for eight weeks.In the process of intervention,the survival state,body weight and blood glucose were observed and measured.Near the end of the experiment,the feces of mice were collected and the OGTT was carried out.At the end of the experiment,blood was collected from the orbit and used to detect plasma insulin,LPS,triglyceride,total cholesterol,low density lipoprotein and high-density lipoprotein.The plasma insulin level was detected by Mouse insulin(INS)Elisa assay kit.The LPS level of plasma was measured by Mouse endotoxin(ET)Elisa test kit,and the blood lipid profile was detected by automatic biochemical analyzer.Illumina Hiseg 2500 PE250 sequencing platform was used to sequence the 16S rDNA V3-V4 region of all microbes in the fecal samples and to analyze the species abundance and diversity of gut microbiota.Results:1.The mice in the NCD group had smooth hair,sensitive activity and normal diet,whereas the mice in the HFD group showed obesity,greasy hair,easy shedding,increased urine volume and decreased activity during the modeling process.At the end of the drug intervention,the symptoms were reduced to a certain extent.The detection results of serum biochemical index of mice showed that FBG,LPS,TG,TC and LDL-C levels were higher in the serum of HFD group than NCD group(P<0.01).Drug treatment decreased FBG,LPS TC and LDL-C levels in serum of HFD group mice significantly(P<0.05).Among the drug treatment groups,there was a certain concentration-response relationship between the three dose groups of curcuma gypsum capsule,and the low-dose group was better than the medium and high dose groups.According to the results of OGTT and HOMA-IR' values compared with NCD group,AUC value and HOMA-IR in HFD mice increased significantly(P<0.001),and the results showed insulin resistance in HFD mice.AUC and HOMA-IR of Chinese medicine groups and metformin group mice significantly lower than the HFD group(P<0.001).The effects of three dose groups of Chinese medicine displayed certain concentration-response relationship.2.16S rDNA Amplicon sequencing results showed that,compared with the NCD group,mice intestinal flora abundance and diversity of HFD group decreased significantly,and the composition of intestinal flora was disturbed.At the phylum level,the relative abundance of Firmicutes,Candidatus Saccharibacteria,Proteobacteria increased,and that of Bacteroidetes,Actinobacteria,Tenericutes decreased.Bacteroidetes/Firmicutes ratio decreased.At the family and genus levels,the relative abundance of Bifidobacterium,Streptococcus,Faecalibacterium,Flavonifractor,Clostridium IV,Odoribacter,Blautia decreased,and the relative abundance of Lachnospiraceae,Veillonellaceae,Escherichia/Shigella,Parasutterella increased.After the intervention of curcuma gypsum preparation,the intestinal bacteria abundance and diversity decreased again,and the degree of reduction was positively correlated with the dosage.At the phylum level,the relative abundance of Firmicutes,Proteobacteria,Deferribacteres increased,and the relative abundance of Bacteroidetes,Actinobacteria decreased.Bacteroidetes/Firmicutes ratio decreased.At the family and genus level,the relative abundance of Streptococcus,Flavonifractor,Clostridium IV,Odoribacter,Blautia,Lachnospiracea_incertae_sedis increased,meanwhile,the relative abundance of Lachnospiraceae,Veillonellaceae,Escherichia/Shigella,and Alistipes declined.Conclusion:1.Curcuma gypsum capsule has significant beneficial effect on body weight,fasting blood-glucose,TC,LDL-C,LPS,HOMA-IR and AUC in type 2 diabetic mice,and the overall effectiveness of low dose Chinese medicine is better than that of middle and high dose.2.The gut microbiota of the HFD mice were significantly changed compared with that of the NCD group,and the gut microbiota abundance and diversity decreased dramatically.The increase of Firmicutes and decrease of Bacteroidetes leaded to B/F ratio decreased.Bacteria producing LPS increased,such as Escherichia/Shigella,Veillonellaceae.The abundance of bacteria producing SCFA decreased,such as Bifidobacterium,Faecalibacterium.The structure of intestinal flora related to obesity was changed,which involved the increase of Lachnospiraceae,Prevotellaceae,Veillonellaceae,and the decrease of Odoribacter and Clostridaceae.3.Curcuma gypsum capsule can effectively improve the disorder of intestinal microflora structure in type 2 diabetic mice.Mainly in:(1)Generally reducing the intestinal flora abundance and species diversity,especially the reduction of gram negative bacilli,for example:Escherichia/Shigella,Veillonellaceae,Alistipes.(2)Improving the bacteria producing SCFA,such as:Flavonifractor,Clostridium ?.(3)The proportion of related bacteria that participated in the production and consumption of succinic acid was improved,being both the decreased abundance of Prevotellaceae and Veillonellaceae and the increased abundance of Odoribacter and Clostridaceae.(4)Reducing the abundance of Lachnospiraceae and promoting weight loss.Based on the above analysis,the improvement of body weight and blood glucose after curcuma gypsum capsule treatment may be achieved by adjusting the structure of gut microbiota,rather than targeting a certain bacterium.4.The results of multivariate linear stepwise regression analysis of the effect of intestinal flora on fast blood glucose showed that Flavonifractor and Odoribacter were negatively correlated with blood glucose,it's statistically significant(P<0.05;P<0.01).