| Objective:To evaluate the clinical efficacy as well as the safety of Jiedu Tongluo Tiaogan Formula(JTTF)intervention in type 2 diabetes mellitus(T2DM)combined with non-alcoholic fatty liver disease(NAFLD)with heat stagnation syndrome in the liver and stomach;to uncover the metabolic profile and potential biomarkers of the T2DM combined with NAFLD population based on non-targeted metabolomics;Using non-targeted metabolomics to explore the core differential metabolites and possible mechanisms of action of JTTF in patients with T2DM combined with NAFLD with heat stagnation syndrome in the liver and stomach.Methods:The first part of the clinical study used a parallel,randomized controlled clinical trial to include 96 patients with T2DM combined with NAFLD with hepatic and gastric depression and heat,randomly divided into Chinese medicine group and control group,the control group was given conventional western medical treatment,the Chinese medicine group added JTTF to the control group.to observe the glycated hemoglobin(HbA1c),fasting blood glucose(FPG),postprandial blood glucose(2hPG),insulin resistance index(HOMA-IR),total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),body mass index(BMI),visceral fat area(VAT),subcutaneous fat area(SAT),severity of fatty liver,Chinese medicine evidence score,and liver fibrosis score(FIB-4).Clinical efficacy and safety were observed and evaluated after 12 weeks of intervention,and fasting blood and urine specimens were collected from patients before and after the intervention.In the second part of the metabolomics study,serum and urine samples were collected from healthy individuals(Healthy),type 2 diabetes mellitus alone(T2DM),nonalcoholic fatty liver disease alone(NAFLD)and T2DM combined with NAFLD(DM-NAFLD),in 4 groups of 30 patients each.An observational study was conducted to detect and analyze metabolites in serum and urine samples using LC-MS/MS after metabolite extraction,sample quality control,data pre-processing,multivariate statistical analysis,data screening,ROC curve analysis,and differential metabolite enrichment analysis.In the third part,based on the previous clinical trial study,fasting serum and urine specimens were collected from healthy subjects,before and after the JTTF intervention,and before and after the control group intervention,and the analysis methods were the same as in the second part,and the metabolites in serum and urine samples were detected and analyzed using LC-MS/MS.Results:In the first part of the clinical study,compared with the control group,the TCM evidence score,HbA1c,FPG,2hPG,TG,and VAT were significantly decreased in the Chinese medicine group after treatment(p<0.01),the severity of fatty liver and HOMA-IR were significantly reduced,which was statistically significant(p<0.05).The TCM efficacy efficiency and total clinical efficiency of the TCM group were significantly better than those of the control group(p<0.05);when comparing the difference between the two groups before and after treatment,the TCM group was significantly better than the control group in terms of the TCM symptom score,TG,VAT,and BMI(p<0.01),and the 2hPG,LDL-C,and SAT improved better than those of the control group(p<0.05).Compared with the pre-treatment period,the severity of fatty liver,TCM symptom score,HbA1c,FPG,2hPG,HOMA-IR,TC,TG,LDL-C,VAT,SAT,and BMI were significantly lower and HDL-C was significantly higher in the Chinese medicine group after treatment,all of which were significantly different(p<0.01);the TCM symptom score,HbA1c,2hPG,VAT,and BMI were significantly lower in the control group after treatment,VAT,BMI decreased significantly(p<0.01),fatty liver severity,FPG,TC,SAT decreased and HDL-C increased,which were statistically significant(p<0.05),HOMA-IR,TG,LDL-C were not statistically significant(p>0.05).The observation index FIB-4 was not statistically significant before and after treatment in both groups(p>0.05).No abnormalities were observed for safety indicators.The results of the second part of the metabolomic study in patients with T2DM combined with NAFLD showed that the differential metabolites in the DM-NAFLD group were mainly concentrated in the categories of alcohols,amino acids,carbohydrates and fatty acids.the NAFLD group was mainly concentrated in the categories of alcohols and amino acids.the T2DM group was mainly concentrated in the categories of carbohydrates and amino acid metabolites.Finally,55 potential biomarkers were screened for the T2DM combined with NAFLD population,including 39 serum metabolites and 16 urine metabolites.These included 2-ketobutyric acid,L-erythrulose,6-hydroxymelatonin,17a-hydroxypregnenolone,dimethylglycine,2,3-dinitro-8-isomeric prostaglandin F1α,vanillylmandelic acid,acetoacetic acid,and phosphoserine in serum,and piperic acid,aminoadipic acid,phosphoserine,3-hydroxybenzoic acid,phenylpyruvic acid,benzamide,β-glycerophosphate in urine.The results of ROC curve analysis were all greater than 0.7 and were accurate.The metabolites 2-ketobutyric acid,L-erythrulose,dimethylglycine,2,3-dinitro-8-isoprostane F1α,vanillylmandelic acid(VM-A),acetoacetate,3-indoleacrylate and β-glycerophosphate were compared between groups in the combined disease,healthy population,T2DM population and NAFLD populations with significant levels of difference between groups.Serum differential metabolites were mainly enriched in the pathways of glycine,serine and threonine metabolism,cysteine and methionine metabolism,linoleic acid metabolism,PPAR signaling pathway and tyrosine metabolism.Metabolic differences occur mainly in metabolic pathways such as amino acid metabolism,lipid metabolism and carbohydrate metabolism;organismal systems such as digestive,nervous and endocrine systems;and human diseases are concentrated in endocrine and metabolic diseases.Urinary differential metabolites were mainly annotated and enriched in the pathways of lysine degradation,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,prolactin signaling pathway,and tryptophan metabolism.Metabolic differences mainly occurred in metabolic pathways such as amino acid metabolism,lipid metabolism and carbohydrate metabolism;organismal systems such as digestive system,nervous system,and endocrine system;and human diseases were concentrated in endocrine and metabolic diseases.In the third part,77 core targets were screened in serum,including cyclic AMP,O-acetyl serine,glycyl leucine,ornithine,L-homocysteine,etc.,and 16 in urine samples,including 3,4-dihydroxymandelic acid,diaminoheptanedioic acid,methylmalonic acid,succinic semialdehyde,and 5-hydroxyindoleacetic acid.These included Luteolin and Rhein,the main blood-entry components of the formula,as well as the potential biomarkers of the disease like dimethylglycine,6-hydroxy melatonin,andα-dimercaptoacetic acid.The core targets were mainly enriched in the PPAR signaling pathway,arginine biosynthesis,Hedgehog signaling pathway,tyrosine metabolism,metabolism of alanine,aspartate and glutamate,NAFLD,insulin signaling pathways and other pathways,among which PPAR signaling pathway had the smallest p-value and the strongest enrichment.Conclusion:1.JTTF combined with basic therapy can alleviate the severity of fatty liver and the manifestation of T2DM combined with NAFLD in patients with hepatic and gastric depression and heat,regulate the disorder of glucolipid metabolism,reduce IR level and fat content,with good overall clinical efficacy and good safety.2.The metabolic characteristics of different populations are different,the metabolic characteristics of T2DM population are highlighted in the changes of carbohydrate class,NAFLD patients are specific to the metabolic abnormalities of alcohols,there are significant metabolic differences between T2DM combined with NAFLD population and healthy population,T2DM population and NAFLD population,mostly focused on the metabolic disorders of amino acids,carbohydrates and fatty acids,a collection of The metabolic abnormalities occurring in single diseases were more diverse and increased in severity,with abnormal metabolism among amino acid metabolites occurring in all three diseases.Multiple potential biomarkers of this combined disease were identified in serum and urine samples.The core differential metabolites and the main blood components of the formula in which JTTF acts were found to be enriched in the PPAR signaling pathway,arginine biosynthesis,Hedgehog signaling pathway,tyrosine metabolism and other pathways,probably acting mainly on the PPARα/γ dual signaling pathway to exert efficacy,providing a basis for further validation.Meanwhile,JTTF can act on potential biomarkers and key signaling pathways of T2DM combined with NAFLD.4.JTTF was found to significantly improve the metabolic environment in T2DM combined with NAFLD patients,mainly by regulating the disorders of amino acid,lipid and carbohydrate metabolism in T2DM combined with NAFLD patients,thus achieving the effects of improving glucolipid metabolism,reducing fat content,regulating IR levels,protecting liver function and inhibiting fibrosis.These changes may be the specific manifestation of the efficacy of this formula to regulate qi,detoxify the liver and eliminate turbidity,and dredge the meridians and channels. |
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