| Objective: In recent years,intestinal flora is considered to play an important role in the pathophysiology of obesity,type 2 diabetes and other metabolic diseases,and fecal microflora transplantation can increase intestinal microbial diversity and improve insulin sensitivity in patients with metabolic syndrome.Based on the low incidence of Kazakh,the purpose of this study is to explore the reason and mechanism of Kazakh normal glucose tolerance human intestinal microorganism transplantation to improve the disorder of glucose and lipid metabolism in type 2 diabetic db/db mice.Methods:1)normal glucose tolerance db/m mice and type 2 diabetic db/db mice were used as research objects.Kazakh normal glucose tolerance human fecal microorganism group was transplanted into db/db mice,and the phenotypic changes of blood glucose and blood lipids in mice were observed.Fecal samples of mice from normal diet group(db/m+PBS),model group(db/db+PBS)and Kazakh normal glucose tolerance human fecal microorganism transplantation intervetion group(db/db+KNGT)were collected at 0,6 and 10 weeks after Kazakh normal glucose tolerance human fecal bacterial fluid transplantation.Fasting blood glucose,random blood glucose and blood lipids were detected in each group,and blood samples were collected and killed.The V3-V4 variable region sequencing technique of 16 S r RNA gene was used to sequence the human fecal samples of Kazak normal glucose tolerance and the fecal samples of db/db mice treated with Kazakh normal glucose tolerance human fecal bacteria solution for 6 weeks.The target bacteria were found through the comparison of relative abundance between groups.The level of target bacteria in fecal samples of mice treated with Kazakh normal glucose tolerance human faecal bacteria solution for0,6 and 10 weeks was detected by real-time fluorescence quantitative PCR(FQ-PCR).Pearson(Pearson)correlation analysis was used to analyze the correlation between intestinal microbial target bacteria and the changes of blood glucose and blood lipids related to insulin resistance.2)the p H value of fecal solution of mice treated with Kazakh normal glucose tolerance human fecal bacteria solution for 0 weeks,6 weeks and 10 weeks was detected,and the levels of acetic acid,propionic acid and butyric acid in feces and serum were detected by gas chromatography(GC)at different time points after intervention of Kazakh normal glucose tolerance human fecal bacteria solution for 0 weeks,6 weeks and 10 weeks.Pearson(Pearson)correlation analysis was used to analyze the relationship between acetic acid,propionic acid,butyric acid and intestinal target bacteria and the correlation between blood glucose and blood lipid levels associated with insulin resistance.3)Real-time fluorescence quantitative PCR and Western blot were used to detect the expression of related proteins in colon and hypothalamus of Kazakh mice treated with normal glucose tolerance human fecal bacteria solution for 0,6 and 10 weeks.Results: 1)compared with db/db+PBS group,the glucose and lipid metabolism of db/db+KNGT group was significantly improved after intervention with Kazakh normal glucose tolerance human fecal bacteria solution for 0,6 and 10 weeks.The results of variable region sequencing of Illumina 16 S r RNA gene showed that there was no significant difference in the relative abundance of bacteria among the three groups at all levels of genera and species of family family,but it could be seen that after the intervention of Kazakh normal glucose tolerance human fecal liquid,compared with db/db+PBS mice,the diversity and richness of intestinal flora in db/db+KNGT group increased,and the relative abundance of,Sutterella,Dorea,Coprococcus,Clostridium,Bacteroides_uniformis and Ruminococcus_gnavus increased at genus level and species level.It is closer to the normal group of db/m+PBS.The results of real-time fluorescence quantitative PCR showed that the levels of Clostridium,Bacteroides,Sutterella,Bacteroides_uniformis,Ruminococcus_gnavus and Faecalibacterium_prausnitzii in Kazakh normal glucose tolerance human feces showed an upward trend compared with db/db+PBS group,closer to db/m+PBS group,while Mucispirillum_schaedleri bacteria showed the opposite trend after 6 and 10 weeks of intervention.Correlation analysis showed that the logarithm of initial template of Bacteroides,Faecalibacterium_prausnitzii,Ruminococcus_gnavus and Sutterella was negatively correlated with the level of glycolipid,while the logarithm of initial template of Mucispirillum_schaedleri was positively correlated with the level of glycolipid.2)after 6 and 10 weeks of intervention with human fecal bacteria solution of Kazakh normal glucose tolerance,compared with db/db+PBS group,the p H value of fecal samples in db/db+KNGT group decreased significantly,while the contents of acetic acid and butyric acid increased significantly.In serum samples,the content of acetic acid increased significantly,and the content of butyric acid also showed an upward trend.Correlation analysis showed that in fecal samples,the content of acetic acid was positively correlated with the logarithm of initial template of Clostridium,negatively correlated with the logarithm of initial template of Mucispirillum_schaedleri,the content of propionic acid was positively correlated with the logarithm of initial template of Bacteroides,and the content of butyric acid was positively correlated with the logarithm of initial template of Clostridium and Bacteroides,respectively.In the blood sample,the content of acetic acid was positively correlated with the logarithm of the initial template of Clostridium,the logarithm of the initial template of Clostridium was positively correlated with the content of acetic acid and butyric acid,and the content of propionic acid was positively correlated with the logarithm of the initial template of Bacteroides.3)compared with db/db+PBS group,the m RNA expression of GPR43 and GLP-1 and the protein expression of GLP-1 in colon tissue of mice in db/db+KNGT group increased significantly after 6 and 10 weeks of intervention with normal glucose tolerance of Kazakh mice,and the m RNA and protein expression of tight junction proteins ZO-1 and Occludin also increased,but there was no statistical difference.The protein expression of ZO-1 increased significantly,and the difference was statistically significant.The protein levels of GLP-1,ZO-1 and Occludin were negatively correlated with the levels of glucose and lipids.The expression of preopioid melanocyte corticosteroid(POMC)in mouse hypothalamus increased after the intervention of Kazakh normal glucose tolerance human fecal bacteria for 10 weeks,but there was no statistical difference.Conclusion: 1)fecal bacteria from Kazakh normal glucose tolerance donor can improve the disorder of glucose and lipid metabolism and increase the diversity and richness of intestinal flora in type 2 diabetic db/db mice after 6 and 10 weeks of fecal bacteria transplantation.There were differences in specific flora among groups and there was a correlation between differential bacteria and glycolipid phenotype.2)Transplantation of Kazakh normal glucose tolerance human fecal bacteria solution effectively decreased the p H value of mouse feces,which may be increased by increasing the amount of short-chain fatty acids,such as acetic acid and butyric acid,which are metabolites of different intestinal flora of mice,thus improving the disorder of glucose and lipid metabolism in db/db mice.3)By verifying the pathway of improving blood lipid level related to short-chain fatty acids,it is concluded that part of the mechanism of Kazakh normal glucose tolerance human fecal liquid transplantation to improve the disorder of glucose and lipid metabolism is through activating GPR43,to increase the expression of GLP-1,tight junction protein ZO-1 and Occludin,improve insulin resistance and protect intestinal mucosal barrier. |
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