| Background:TGF-βis a secreted polypeptide molecules which belongs to the TGF super-family.For human being,TGF-β has three subtypes like TGF-β1,2 and 3.Among which,TGF-β belongs to a kind of multi-functional cell growth factor which accounts for the most portion.In the pathogenesis procession of diabetic nephropathy,glomerular membrane cell phenotype trans-differentiation is the most prominent manifestations at the early stage.We can observe the proliferation of mesangial cells and the synthesis of extracellular matrix and abnormal accumulation,and the following glomerular sclerosis is the main pathological features of DN.However,glomerular membrane cell phenotype trans-differentiation mainly by the activation of TGF-β1.As one of the important modification mechanisms of epigenetic,DNA methylation plays a crucial role in regulating the expression of relevant genes about renal fibrosis and DN pathogenesis.Previous studies suggest that the key gene promoter methylation status and transcriptional activity,which led to DN in patients with renal fibrosis.But the relationship between the key factor TGF-β1 gene promoter region abnormal methylation of DNA and diabetic nephropathy has not been reported.The research group in order to investigate the relationship between the regulatory region of TGF-β1 gene methylation and serum TGF-β1,we had detected DNA methylation level of TGF-β1 regulatory region,and observed the level of TGF-β1protein,inflammatory cytokines and fibrosis factors,clinical biochemical indexes,the pathological changes of glomerular pathology in patients with diabetic nephropathy.At the end,we had analysised the changes of DNA methylation level in the regulatory region,serum TGF-β1 and other related indicators.Methods: According to the WHO 1999 guideline for diabetes mellitus diagnosis and classification standard,101 patients who were hospitalized in June 2013 to May 2015 and diagnosed as diabetes mellitus were elected(eGFR≥30ml/min/ 1.73 m2),simultaneous elimination of chronic nephritis,urinary tract infection,heart failure,primary hypertension and other diseases.According to urinary albumin / creatinine(UACR)ratio patients were divided into diabetic group(DM group,UACR < 30μg/mg,n=42),diabetic nephropathy group(DN group,30μg/mg<UACR<300μg/mg,n = 49),losartan in treating diabetic nephropathy group(LOS group,30μg/mg<UACR<300μg/mg,n= 20).All DN patients underwent renal biopsy and were confirmed as diabetic nephropathy.Referencing the DN pathological diagnosis and typing standard of pathological changes proposed by Tervaet in 2010,we classified pathological changes of DN group and LOS group.30 volunteers in our hospital over the same period healthy were selected as control group(Con),diabetes and other systemic diseases were excluded.This clinical study had obtained the approval of the ethics committee of Xinqiao Hospital of the Third Military Medical University,all subjects had signed the informed consent.Parameter detection:DNA was extracted from all the subjects’peripheral blood and modified by sodium bisulfite.DNA methylation status of TGF-β1 gene regulatory region was screened by methylation specific PCR and the DNA methylation level was detected by bisulfite sequencing PCR.Serum creatinine(Scr),blood urea nitrogen(BUN),fasting blood glucose(FBS),postprandial blood glucose(PBS),glycosylated hemoglobin(Hb A1c),serum levels of TGF-β1,C-reactive protein(CRP),leucocyte interleukin 6(IL-6),leukocyte interleukin 8(IL-8),tumor necrosis factor-a(TNF-a),urinary albumin/creatinine(UACR),urine monocyte chemoattractant protein-1/creatinine(UMCR)and urinary type IV collagen(C0L-IV)had been tested,using formula 186.3×(Scr/88.4)-1.154×年龄-0.203[×0.742(if female)]to calculate eGFR.Results:1.DNA methylation status of TGF-β1 gene regulatory region was screened by methylation specific PCR.The methylation rate of Con group was 73.3%,DM group decreased to 42.8%,DN group decreased to 12.2% furtherly,there were significant differences between all groups.The methylation rate of LOS group was 20%,compared to DN group,the difference was significant.2.Using BSP to detect the change of DNA methylation level in TGF-β1 gene regulation region,we had found that the methylation level of Con group was 66.7±9.1%,DM group was 35.6±6.0%,DN group was 12.5±8.1%,and the differences between all groups were significant.LOS group was 18.7±6.2%,and the methylation level was significantly higher than that of DN group.3.ELISA was used to test serum TGF-β1,Con group was 296.38±74.37 ng/L,DM group was 1367.22±126.13 ng/L,DN group was 2885.73±411.36 ng/L,there were significant differences between all groups.Serum TGF-β1 of LOS group was 1523.82±173.71 ng/L,compared to DN group,the difference was significant(P < 0.01).4.Detection of serum inflammatory cytokines showed that C reactive protein(CRP),Con group was 6.53±0.25 mg/L,DM group was 11.15±0.83 mg/L,DN group was 19.03±2.57 mg/L,LOS group was 12.84±1.62 mg/L;Interleukin-6(IL-6),Con group was 2.69±0.15 ng/L,DM group was 5.02±0.19 ng/L,DN group was 7.47±1.09 ng/L,LOS group was 5.41±0.51 ng/L;Interleukin-8(IL-8),Con group was 54.84±2.36 ng/L,DM group was 147.68±5.70 ng/L,DN group was 289.09±30.79 ng/L,LOS group was 160.13±14.61 ng/L;Tumor necrosis factor?(TNF?),Con group was 7.57±0.75 ng/L,DM group was 15.83±0.63 ng/L,DN group was 45.57±7.68 ng/L,LOS group was 22.64±7.61 ng/L.These indicators had significant differences between the three groups,LOS group was significantly lower than that in DN group.5.Urine analysis showed that Urinary type IV collagen(Col-IV),Con group was 39.59±12.56 pg/ng,DM group was 151.40±20.62 pg/ng,DN group was 374.16±81.09 pg/ng,LOS group was 170.63±13.43 pg/ng;Urinary monocyte chemotaxis cell protein-1/ creatinine ratio(UMCR),Con group was 7.73±0.45 /L g,DM group was 37.14±9.03 /L g,DN group was 93.59±27.68 /L g,LOS group was 50.76±10.46 /L.These indicators had significant differences between the three groups,LOS group was significantly lower than that in DN group.6.Pearson correlation analysis showed that the level of serum TGF-β1 positively correlated with FBS(r=0.942),PBS(r=0.682),UACR(r=0.718),BUN(r=0.479),Scr(r=0.756),CRP(r=0.731),IL-6(r=0.653),IL-8(r=0.675),TNF-a(r=0.729),UMCR(r=0.794)and Col-IV(r =0.746)(all P<0.01),while negatively correlated with eGFR(r=-0.805)and gene methylation(r=-0.925),all P<0.01.Further study was conducted by multiple stepwise regression analysis,with UACR、 BUN、Scr、FBS、PBS、CRP、IL-6、IL-8、TNF-a、UMCR、col IV,e GFR and gene methylation as independent variables.The results also showed serum TGF-β1 levels was related with e GFR(β=-0.690,P<0.01)and methylation level(β=-0.302,P<0.01),indicating they were possible factors for serum TGF-β1 level.7.Serum TGF-β1 was negatively correlated with DNA methylation level(r=-0.925,P<0.01),but positively correlated with severity of renal pathological scores(rs=0.847,P<0.01).Meanwhile the methylation level related to the pathological grade(χ2=23.667,P=0.04).Conclusions:1.With extension of the course of diabetes,the methylation level of TGF-β1 gene regulation region gradually decreased,and DN group was the lowest.Meanwhile,serum TGF-β1 was gradually increased,and DN group was the highest,which suggested that high glucose could induce demethylation in TGF-β1 regulation region and promote expression of TGF-β1gene,2.Angiotensin II-1 receptor inhibitors(Losartan)treatment could reduce serum TGF-β1,which may relate to the inhibition of demethylation in TGF-β1 gene regulation region. |
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