| Objective To compare the changes of clinical indexes and to identify the expression of serum differential proteins by label free quantitative proteomics method after taking dapagliflozin in patients with type 2 diabetes.To verify the differential proteins by ELISA and to compare serum differential proteins and clinically relevant indicators among the post-treatment group,the control group post-treatment of saxagliptin,metformin and the control group of diet control and exercise whose HbA1 c meets the standard(HbA1c<7.0%).To find new differential proteins involved in mechanisms such as glucose and lipid metabolism in diagnosis,treatment,and progression of type 2 diabetes。Methods Twenty patients with type 2 diabetes mellitus whose HbA1 c ranged from7.0% to 8.5% in the Department of Endocrinology,Affiliated Hospital of Inner Mongolia Medical University from January 1,2017 to December 1,2018,were selected as the experimental group.At the same time,20 patients with type 2 diabetes mellitus who were treated with saxagliptin or metformin and 20 patients with type 2diabetes mellitus whose blood sugar was controlled by diet control and exercise alone were selected as the experimental group.The experimental group was treated with oral dapagliflozin(10 mg/tablet,1 tablet/day)for 3 months and HbA1 c < 7.0%was up to the standard.The post-treatment of metformin,and metformin was taken regularly in the past 3 months;the diet control and exercise group did not take any hypoglycemic agents in the past 3 months and only controlled blood sugar through diet and exercise.Blood samples were collected from the treatment and post-treatment groups of dapagliflozin and saxagliptin group,metformin group and diet control and exercise group.The clinical indicators of blood sample determination included fasting blood glucose(FBG),glycosylated hemoglobin(HbA1c),liver function,Renal function,triglyceride(TG),cholesterol(TC),high-density lipoprotein(HDL-C),low-density lipoprotein(LDL-C),fasting insulin(FINS),fasting C-peptide(FCP),retinal Alcohol-binding protein 4(RBP4),homocysteine(HCY),C-reactive protein(CRP),free fatty acid(NEFA).According to the principle of heterogeneityhomogeneity,serum samples from 5 patients before and after treatment of dapagliflozin were selected for label-free quantitative proteomics to screen differential proteins,which would be clinically meaningful and type 2 diabetes closely related Plasma alpha-L-fucosidase(AFU),αIIb integrin and Podocalyxin(PCX)were tested by ELISA in serum samples of the experimental group and the control group,and it was compared to the differential protein expression between the experimental group and the control group.The situation was compared before and after treatment and inter-group analysis combined with the relevant clinical collected data.At the same time,the quantitative differential proteins were retrieved from the database for bioinformatics analysis,including annotation of proteins from biological processes,cell composition and molecular functions,functional analysis of differential proteins and sub-cellular structure analysis of differential proteins.Results(1)Analysis of clinical data: compare with before treatment,BMI,waist circumference and waist-to-hip ratio decreased after treatment of dapagliflozni(P<0.05);blood glucose-related indicators FBG,HbA1 c,FCP,FINS,HOMA2-IR decreased(P<0.05),HOMA2-S%,HOMA2-β increased(P<0.05);lipid metabolism related indicators TC,non-HDL-C decreased(P<0.05),ApoA1 increased(P<0.05);other related indicators Retinol binding protein(RBP),blood homocysteine(HCY),free fatty acid(NEFA)decreased(P<0.05),and urine sugar and urine ketone body increased(P<0.05).Compared saxagliptin after treatment with dapagliflozin,waist-to-hip ratio,HCY,RBP4,FINS decreased(p<0.05),HOMA2-β increased(p<0.05);compared with metformin group,HCY,RBP4,and FINS decreased(p<0.05),HOMA2-S%,and HOMA2-β increased(p<0.05).Compared with the simply diet control and exercise group,HCY,RBP4,and FINS decreased(p<0.05),ApoA1.HOMA2-S% was elevated(p<0.05).(2)Serum differential protein expression before and after dapagliflozin treatment: 19 differential protein such as AFU,alpha II B integrin and PCX were screened from 5patients with homogeneous heterogeneous serum samples by label free quantitative proteomics.Among them,18 differential protein such as AFU and alpha II B integrin were down-regulated and one differential protein PCX was up-regulated after the treatment of dapagliflozin.(3)ELISA results:the expression of AFU and alpha II B integrin was down-regulated(P < 0.05)and PCX was up-regulated(P < 0.05)in 20 patients of experimental group compared with before treatment.PCX protein was up-regulated(P < 0.05),AFU and PCX protein were up-regulated(P < 0.05)and down-regulated(P < 0.05)in the after treatment of dapagliflozin.group compared with the after treatment of shagliptin group,and compared with the metformin-treated group,AFU and PCX protein were up-regulated(P < 0.05).(4)bioinformatics analysis results: protein annotation results,differential proteins in the classification of biological processes,cell processes accounted for the most proportion;in cell composition classification,differential proteins are expressed in organelles;in molecular function classification,binding molecules have the largest proportion of functional categories.In subcellular localization,differential proteins are mainly located in cytoplasm and nuclei.In functional enrichment,homocytic adhesion pathway,lymphocyte activation pathway and muscle cell skeleton were found to be significantly enriched.Conclusion(1)Dapagliflozin has a significant effect on reducing blood sugar,and it also has a good effect on the weight loss,lipid metabolism and islet function of patients.(2)Using label-free quantitative proteomics technology,we found that there were differences in serum protein expression levels in type 2 diabetes mellitus patients after treatment with dapagliflozin.The down-regulated differential proteins were 18 proteins such as AFU,alpha II beta integrin,and the up-regulated differential proteins were PCX proteins.ELISA confirmed that the differential proteins of AFU,alpha II B integrin and PCX did exist in all samples of the study groups.The basic information and the metabolic pathways involved can be understood through the annotation and functional enrichment of differential expressed proteins.Whether these pathways are the therapeutic mechanisms of dapagliflozin remains to be verified.(3)The changes of AFU,alpha II beta integrin and PCX may be related to oxidative stress,insulin resistance and lipid metabolism,among which the expression of PCX is up-regulated after treatment with dapagliflozin and metformin.It is speculated that dapagliflozin can exert renal protective effect by regulating the expression of PCXprotein,and its specific mechanism needs further study.These hypotheses can be used to reduce blood sugar of dapagliflozin,and the study of mechanism provides more ideas and guidance for clinical drug use. |
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