| Part 1.Characteristics of intestinal flora and serum metabolites in patients with diabetic cardiomyopathyObjective:By including patients meeting the diagnostic criteria for diabetic cardiomyopathy and healthy subjects,fecal and serum samples were collected for metagenomics and metabolomics detection and analysis,respectively,to screen the characteristic intestinal flora and serum metabolites of diabetic cardiomyopathy patients,and preliminary explore the correlation between diabetic cardiomyopathy and intestinal flora and serum metabolites.Methods:1.Patients who met the diagnostic criteria for diabetic cardiomyopathy were included as the case group,and healthy volunteers matched for age,sex,and body mass index were included as the control group,and demographic and clinical data were collected from both study groups.2.Fecal and serum samples were collected.Metagenomics sequencing was performed on fecal samples and untargeted metabolomics was performed on serum samples to screen for characteristic intestinal flora and serum metabolites of patients with diabetic cardiomyopathy.3.LEfSe analysis of metagenomic data was performed to screen candidate biomarkers for intestinal flora that might distinguish diabetic cardiomyopathy patients from healthy volunteers.Results:1.In this study,a total of 27 diabetic cardiomyopathy patients were included in the case group and 13 healthy volunteers were included in the control group.There was no statistical difference between the two study groups in terms of age,gender,body mass index,smoking history,and liver and kidney function(p>0.05).However,fasting glucose levels,total cholesterol levels,and low-density lipoprotein levels were higher in patients with diabetic cardiomyopathy(p<0.05).In addition,with regard to echocardiographic indexes,the LA and LVEDD were larger,the IVST was thicker,and the LVEF was lower in patients with diabetic cardiomyopathy(p<0.05).2.Metagenomics analysis revealed characteristic alterations in the structure of the intestinal flora of patients with diabetic cardiomyopathy compared to healthy controls.Further functional annotation of the metagenomics data in the Kyoto Encyclopedia of Genes and Genomes(KEGG)database revealed that the functions of the characteristic intestinal flora of patients with diabetic cardiomyopathy were mainly related to the biosynthesis of phenylalanine,tyrosine and tryptophan,arginine and proline metabolism,lysine biosynthesis,terpenoid skeleton biosynthesis,bacterial population sensing,polyketide unit biosynthesis,peptidoglycan biosynthesis,pantothenic acid and coenzyme A biosynthesis and ABC transporter protein.3.To further screen for intestinal flora biomarkers that can identify patients with diabetic cardiomyopathy,we performed LEfSe analysis of metagenomics data,which showed that:s_Megamonas_rupellensis,o_Burkholderiales,c_Betaproteobacteria,f_Sutterellaceae,s_Bifidobacterium_longum,s_Burkhol deriales_bacterium and g_Prevotella are core bacteria in the intestinal flora of diabetic cardiomyopathy patients and can be used as candidate biomarkers to distinguish diabetic cardiomyopathy patients from healthy volunteers.4.The results of non-targeted metabolomics analysis showed that a total of 204 differential metabolites were identified in diabetic cardiomyopathy patients compared to healthy controls.97 differential metabolites were identified in positive ion mode,of which 89 were up-regulated and 8 were down-regulated;107 differential metabolites were identified in negative ion mode,of which 97 were up-regulated and 10 were down-regulated.Further KEGG functional analysis of the differential metabolites showed that the functions of the differential metabolites in diabetic cardiomyopathy patients were significantly enriched in the modules of ketone body synthesis and degradation,lipopolysaccharide biosynthesis,pantothenic acid and coenzyme A biosynthesis,bile secretion,methyl butyrate metabolism,ornithine,lysine and nicotinic acid alkaloid biosynthesis,AMPK signaling pathway,cAMP signaling pathway,etc.5.Combined metagenomic and metabolomic analyses showed an association between alterations in serum metabolites and alterations in characteristic bacterial strains in the intestinal flora of patients with diabetic cardiomyopathy,and that chenodeoxycholic acid 3-sulfate and 1,5-anhydro-d-glucitol are serum metabolites closely associated with diabetic cardiomyopathy.Conclusions:There may be characteristic alterations in intestinal flora and serum metabolites in patients with diabetic cardiomyopathy,and there may be an interaction between the two.The characteristic altered intestinal flora may serve as biomarkers for identifying diabetic cardiomyopathy.Part 2.Fecal microbiota transplantation may induced diabetic cardiomyopathy related manifestations in pseudo-germ-free ratsObjective:The intestinal flora of diabetic cardiomyopathy patients and healthy volunteers were transplanted into pseudo-germ-free rats to observe whether pseudo-germ-free rats in diabetic cardiomyopathy group would develop diabetic cardiomyopathy related manifestations,so as to further verify the correlation between intestinal flora and diabetic cardiomyopathy.Methods:1.Two patients with diabetic cardiomyopathy and two healthy volunteers were selected as "donors" from the clinical study subjects,and their fecal samples were collected and divided into centrifuge tubes and frozen in the refrigerator at-80℃.2.Pseudo-germ-free rats model,called "recipients",were constructed by feeding antibiotic solution.The recipients were matched by baseline blood glucose level,body weight,echocardiography and other indicators,and divided into diabetic cardiomyopathy group and healthy control group,with 6 recipients in each group.3.The fecal samples of the donor were prepared as fecal fluid,and the intestinal flora were transplanted into the intestine of the recipients by gavage at a ratio of 1:3(i.e.,each donor fecal fluid was gavaged to 3 recipients),and gavage was administered every other day for 20 weeks,during which the blood glucose,body weight and other indicators were regularly monitored.To avoid interference,recipients receiving the same donor fecal bacteria transplantation were kept in the same rat cage.4.After 20 weeks of fecal microbiota transplantation,fasting blood glucose levels were measured again in both groups,and intraperitoneal glucose and insulin tolerance tests were performed.Using the single blind method,the rats were examined by echocardiography and the relevant diameters were measured by a professional technician(No information about grouping).5.The rats were executed,and the heart,kidney,pancreas and colon tissues were collected for histopathological observation.Serum samples were collected for ELISA to detect the expression level of inflammatory indexes.Results:1.Compared with the control group,20 weeks after fecal microbiota transplantation,the pseudo-germ-free rats in diabetic cardiomyopathy group had a higher fasting blood glucose level(p<0.05),and a larger area under the intraperitoneal glucose tolerance test time-glucose curve(p<0.05),suggesting reduced insulin sensitivity.2.Compared with the control group,after 20 weeks of fecal microbiota transplantation,AWTd、AWTs、PWTs were thicker in the diabetic cardiomyopathy group(p<0.05).3.ELISA results showed that serum concentrations of LPS,IL-1β,IL-6 and TNF-α in the diabetic cardiomyopathy group were higher than those in the control group(p>0.05).4.HE staining of the heart tissue indicated that the pseudo-germ-free rats in the diabetic cardiomyopathy group had mild epicardial edema,loose connective tissue arrangement,vascular dilatation,a small amount of lymphocyte infiltration,and irregular arrangement of surrounding myocardial cells.Masson staining indicated that the percentage of pseudo-germ-free rats with fibrosis positive area in diabetic cardiomyopathy group was larger(p<0.05).HE staining of the pancreatic tissue indicated that there were different degrees of edema and inflammatory cell infiltration in the pancreas of pseudo-germ-free rats in diabetic cardiomyopathy group.There were no significant differences in the histomorphology of kidney and colon between the two groups.Conclusions:Colonization of intestinal flora from diabetic cardiomyopathy patients into pseudo-germ-free rats may induce increased fasting glucose level,decreased insulin sensitivity,left ventricular wall thickening,myocardial fibrosis and other diabetic cardiomyopathy-related manifestations,further verifying the correlation between intestinal flora and diabetic cardiomyopathy. |
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