| Objective:Diabetes mellitus(DM)threatening human health seriously is the third of non-infectious chronic disease following the cancer and cardiovascular disease.Today,382 million people suffer from DM worldwide and the number of diabetic patients is up to 592 million by 2035.In China,there are about 100 million people suffering from DM,which is equivalently around a quarter of the global diabetic patients.As all know,nearly one-third of patients with DM develop diabetic nephropathy(DN),which is a leading cause of end-stage renal disease.DN is the main cause of death in diabetic patients with no therapeutic methods to cure.So far,the molecular mechanisms of DN are still not fully elucidated.Hence,it is urgent and important to research the pathogenesis of DN.The pathological changes of DN include thickening of the glomerular basement membrane,accumulation of mesangial extracellular matrix and eventually leading to glomerulosclerosis.In mesangial extracellular matrix,type IV collagen is the major component.It has six genetically different α(Ⅳ)chains:α1(Ⅳ)to α6(Ⅳ).In DN,the transforming growth factor-β1(TGF-β1)signaling is a key regulator for the development and progression of fibrosis.TGF-β1 could regulate the expression of type Ⅳ collagen by activating the transcription factor Smad3.Our previous study also had demonstrated that TGF-β1/Smad3 signaling pathway was activated to induce the over expressions of type Ⅳ collagen at the early stage of mesangial cell exposed to high glucose.In the 1960s,C-peptide was first described by Steiner et al.when they study the biosynthesis of insulin.C-peptide is predominantly synthesized byβ-cells in the vertebrate pancreas.C-peptide serves to link the A-and B-chains of the insulin molecule.The molecular weight of human C-peptide is 3020Da and its isoelectric point is 3.0.With a half-life of about 30 minutes,human C-peptide composed of 31 amino acid residues is mainly cleared by the kidney.The normal serum concentration of human C-peptide is around 0.4nmol/L.In recent years,overwhelming research evidence suggests that C-peptide can exert a variety of biological effects.C-peptide could bring therapeutic effect on diabetic complications,especially on DN.In STZ-diabetic rats,C-peptide could ameliorate renal function by reducing the expansion of glomerular basement membrane and mesangium.In type 1 diabetic patients,the renal function could be improved by pancreatic islet transplantation or combination of insulin and C-peptide.Thus,we assume that as an endogenous component,C-peptide has unique therapeutic effect and unparalleled advantages on DN compared to exogenous drugs.It means that it’s necessary and urgent to clarify the therapeutic mechanism of C-peptide for DN.DN was caused by the accumulation of type IV collagen directly,while C-peptide could reverse renal morphology of DN.Therefore,we hypothesize that the beneficial effect of C-peptide on DN is closely related to the metabolism of type IV collagen.Although it had been confirmed that C-peptide could prevent the expansion of mesangium through inhibiting the accumulation of type IV collagen in the STZ-diabetic rats,the molecular mechanism remains unclear.The.aim of this study was to investigate whether C-peptide regulates TGF-β1/Smad3-induced the expression of type Ⅳcollagen to reverse fibrosis in DN.In present study,the rat glomerular mesangial cell line(HBZY-1)was used.To determine the effects of high,glucose on the expressions of type Ⅳcollagen and TGF-β1,cells were exposed to various concentrations of D-glucose(5.6,20,25,30,35,or 40mmol/L)and different times(3h,6h,12h).To research the effects of C-peptide on the expressions of type Ⅳ collagen and TGF-β1,cells were stimulated with high D-glucose(35mmol/L)in the absence or presence of C-peptide at various concentrations for.3h.real-time-PCR was performed to assay the mRNA expressions of al(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1.Meanwhile,the protein level of TGF-β1 in culture media was measured in HBZY-1 cells using enzyme linked immunosorbent assay(ELISA)detection kits.In addition,the cell immunofluorescent staining application was carried out to detect the nuclear translocation of Smad3.At the end of this study,to confirm the bindings of Smad3 to the promoter regions of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ)or α5(Ⅳ)genes,a chromatin immunoprecipitation.(ChIP)assay was performed.All in all,this study aimed to explore the major molecular mechanism of C-peptide reversing fibrosis in DN,which would provide experimental evidence for C-peptide treatment of DN in clinic.Moreover,diabetes was induced in healthy male Sprague-Dawley(SD)rats by intraperitoneal injection of streptozotocin(STZ)in this study.In this experiment,some diabetic rats were treated with human C-peptide.Body weight and blood glucose in all animals were measured before or after the experiment.The level of 24-hour urinary albumin in all animals was also calculated in the experiment.Morphological changes in renal tissues were observed by optical and transmission electron microscopy.In a word,this experiment aimed to further explore that C-peptide could exert the beneficial effects on STZ-induced rats to reverse DN.Methods:1 Cell culture:The rat mesangial cell line HBZY-1 was purchased from China Center for Type Culture Collection(CCTCC).HBZY-1 cells were maintained at 37℃ in a humidified incubator with 5%CO2 and propagated in Dulbecco’s modified Eagle’s medium(DMEM)containing 100mg/L D-glucose and 10%fetal bovine serum(FBS).Cells were inoculated in 150ml flasks and passaged with 0.25%trypsin.Prior to C-peptide or scrambled C-peptide treatment,some cells were exposed to 35mmol/L high D-glucose DMEM for 24h.Subsequently,these cells were cultured with 35mmol/L high D-glucose DMEM plus 0.5nmol/L C-peptide or 0.5nmol/L scrambled C-peptide,respectively.2 Experimental design and cell grouping2.1 To determine the effect of high D-glucose at various concentrations on HBZY-1 cells,the mRNA expressions of α1(Ⅳ),a2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1.At the same time,the protein level of TGF-β1 in culture media was measured.HBZY-1 cells were exposed to different concentrations of D-glucose for 24-hour.According to this,the experiment was divided into six groups:Con(control)group(with medium containing 5.6mmol/L D-glucose),20mM group(with medium containing 20mmol/L D-glucose),25mM group(with medium containing 25mmol/L D-glucose),30mM group(with medium containing 30mmol/L D-glucose),35mM group(with medium containing 35mmol/L D-glucose)and 40mM group(with medium containing 40mmol/L D-glucose).2.2 To investigate the effect of 35mmol/L high D-glucose on HBZY-1 cells,the mRNA expressions of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1 were assayed.Simultaneously,the protein level of TGF-β1 in culture media was detected.HBZY-1 cells were incubated in 35mmol/L high D-glucose DMEM with different times(3h,6h,or 12h).Cells were divided into four groups:Con(control)group(cells were incubated in 35mmol/L high D-glucose DMEM for Oh),3h group(cells were incubated in 35mmol/L high D-glucose DMEM for 3-hour),6h group(cells were incubated in 35mmol/L high D-glucose DMEM for 6-hour)and 12h group(cells were incubated in 35mmol/L high D-glucose DMEM for 12-hour).2.3 To explore the effects of C-peptide on HBZY-1 cells,the mRNA expressions of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1.Meanwhile,the protein level of TGF-β1 in culture media was assayed.According to different concentrations of C-peptide,the experiment was divided into five groups:Con(control)group(cells were incubated in 5.6mmol/L D-glucose DMEM),OC(osmotic control)group(cells were incubated in 35mmol/L L-glucose DMEM for 3-hour),HG group(cells were incubated in 35mmol/L D-glucose DMEM for 3-hour),CP1 group(cells were incubated in 35mmol/L D-glucose DMEM plus 0.lmmol/L C-peptide for 3-hour),CP5 group(cells were incubated in 35mmol/L D-glucose DMEM plus 0.5nmol/L C-peptide for 3-hour)and CP9 group(cells were incubated in 35mmol/L D-glucose DMEM plus 0.9nmol/L C-peptide for 3-hour).2.4 To observe the nuclear translocation of Smad3 of HBZY-1 cellsCells were divided into seven groups:Con(control)group(cells were incubated in 5.6mmol/L D-glucose DMEM),HG1 group(cells were incubated in 35mmol/L D-glucose DMEM for 1-hour),HG2 group(cells were incubated in 35mmol/L D-glucose DMEM for 2-hour),HG3 group(cells were incubated in 35mmol/L D-glucose DMEM for 3-hour),CP1 group(cells were incubated in 35mmol/L D-glucose DMEM plus 0.5nmol/L C-peptide for 1-hour),CP2 group(cells were incubated in 35mmol/L D-glucose DMEM plus 0.5nmol/L C-peptide for 2-hour)and CP3 group(cells were incubated in 35mmol/L D-glucose DMEM plus 0.5nmol/L C-peptide for 3-hour).2.5 To evaluate the binding of Smad3 to the promoter regions of al(IV),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ)or α5(Ⅳ)genes in HBZY-1 cells.Five groups was divided:Con(control)group(cells were incubated in 5.6mmol/L D-glucose DMEM),OC(osmotic pressure group as a control)group(cells were incubated in 35mmol/L L-glucose DMEM for 3-hour),HG group(cells were incubated in 35mmol/L D-glucose DMEM for 3-hour),CP group(cells were incubated in 35mmol/L D-glucose DMEM plus 0.5nmol/L C-peptide for 3-hour)and ScCP group(cells were incubated in 35mmol/L D-glucose DMEM plus 0.5nmol/L scrambled C-peptide for 3-hour).3 The changes of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1 genes transcriptional level were detected in HBZY-1 cells.Total RNA was isolated using a one-step Trizol reagent in HBZY-1 cells.real-time PCR analysis for the expression of mRNA for α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1 genes with β-actin as a control reference was carried out.4 The cell immunofluorescent staining application was performed to detect the nuclear translocation of Smad3 in HBZY-1 cells.5 The ChIP assay was performed to confirm the binding of Smad3 to the promoter regions of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ)or α5(Ⅳ)genes.6 Experimental design and animal grouping:Healthy male SD rats(n=80)were used.Diabetes was induced in healthy male SD rats by intraperitoneal injection of STZ(n=72).The other non-diabetic animals served as a control group(n=8).Diabetes was confirmed by measuring blood glucose levels at three days after the STZ-injection.Animals with blood glucose higher than 16.7mmol/L were classified as diabetic.Then the diabetic rats(n=68)were divided randomly into four groups:untreated diabetic group(DM group,n=17),diabetic prevention group(C-peptide administration at once after the STZ-injection;DP group,n=17),and two diabetic treatment groups,receiving either C-peptide(CP group,n=17)or scrambled C-peptide(ScCP group,n=17).All animals housed under the standard environment(20-25 ℃).Rats were treated twice daily(12h-interval)with subcutaneous injections of human C-peptide at a dosage of 130nmol/kg for six weeks in DP group.After the diabetic rats housed six weeks,they were treated twice daily(12h-interval)with subcutaneous injections of human C-peptide or scrambled C-peptide at a dosage of 130nmol/kg for six weeks in CP group or ScCP group,respectively.At the end of the experiment,all the rats were sacrificed by femoral artery bleeding.6.1 Blood glucose and 24-hour urinary were detectedNon-fasting blood was obtained from the tail vein in the morning.At week 12 as well as the third day before or after injection STZ,blood glucose was measured using the glucometer.For urinary measurements,24-hour urinary was collected to detect 24-hour urinary albumin.6.2 To observe renal morphologyThe renal cortexes were observed under optical and transmission electron microscopy.Results:1 real-time-PCR was performed to assay the mRNA expressions of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1in HBZY-1 cells stimulated with different concentrations of D-glucose for 24-hour.1.1 The α1(Ⅳ)mRNA relative expression level in the 20mM(2.25 ± 0.12,P<0.05),25mM(2.69 ± 0.19,P<0.01),30mM(2.51 ± 0.10,P<0.01),35mM(4.02 ± 0.52,P<0.01)and 40mM(3.20 ± 0.36,P<0.01)groups was significantly higher than those in the Con group.1.2 The α2(Ⅳ)mRNA relative expression level in the 20mM(2.98 ± 0.22,P<0.01),25mM(2.57 ± 0.38,P<0.05),30mM(2.57 ± 0.30,P<0.05),35mM(4.28 ±0.54,P<0.01)and 40mM(2.40 ± 0.32,P<0.05)groups was significantly higher than those in the Con group.1.3 The α3(Ⅳ)mRNA relative expression level in the 20mM(2.42 ± 0.10,P<0.05),25mM(2.54 ± 0.29,P<0.01),30mM(2.58 ± 0.27,P<0.01),35mM(4.28 ± 0.54,P<0.01)and 40mM(2.59 ± 0.40,P<0.01)groups was significantly higher than those in the Con group.1.4 The α4(Ⅳ)mRNA relative expression level in the 20mM(3.33 ± 0.16,P<0.01),25mM(3.39 ± 0.25,P<0.01),30mM(3.32 ± 0.44,P<0.01),35mM(4.68 ± 0.44,P<0.01)and 40mM(3.50 ± 0.22,P<0.01)groups was significantly higher than those in the Con group.1.5 The α5(Ⅳ)mRNA relative expression level in the 20mM(2.13 ± 0.15,P<0.01),25mM(2.31 ± 0.09,P<0.01),30mM(2.17 ± 0.05,P<0.01),35mM(3.39 ± 0.05,P<0.01)and 40mM(2.192 ± 0.09,P<0.01)groups was significantly higher than those in the Con group.1.6 The TGF-β1 mRNA relative expression level in the 20mM(3.10 ± 0.29,P<0.01),25mM(3.14 ± 0.14,P<0.01),30mM(2.78 ± 0.32,P<0.01),35mM(3.76 ± 0.27,P<0.01)and 40mM(2.84 ± 0.14,P<0.01)groups was significantly higher than those in the Con group.2 To investigate the effects of 35mmol/L high D-glucose on HBZY-1 cells at different time points,the mRNA expressions of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1 were assayed by realtime-PCR.2.1 The levels of α1(Ⅳ)mRNA relative expression in the 3h(4.63 ± 0.61,P<0.01),6h(2.48 ± 0.26,P<0.05),and 12h(2.86 ± 0.17,P<0.01)groups were significantly higher than those in the Con group.2.2 The level of α2(Ⅳ)mRNA relative expression in the 3h(2.97 ± 0.05,P<0.05),6h(2.43 ± 0.13,P<0.01),and 12h(2.66 ± 0.12,P<0.01)groups was significantly higher than those in the Con group.2.3 The level of a3(IV)mRNA relative expression in the 3h(4.00 ± 0.56,P<0.01),6h(3.05 ± 0.49,P<0.01),and 12h(3.07 ± 0.50,P<0.01)groups was significantly higher than those in the Con group.2.4 The level of α4(Ⅳ)mRNA relative expression in the 3h(2.94 ± 0.82,P<0.01),6h(2.13 ± 0.07,P<0.01),and 12h(2.44 ± 0.07,P<0.01)groups was significantly higher than those in the Con group.2.5 The level of α5(Ⅳ)mRNA relative expression in the 3h(3.41 ± 0.16,P<0.01),6h(2.40 ± 0.11,P<0.01),and 12h(2.42 ± 0.06,P<0.01)groups was significantly higher than those in the Con group.2.6 The level of TGF-β1 mRNA relative expression in the 3h(4.00 ± 0.56,P<0.01),6h(3.05 ± 0.49,P<0.05),and 12h(3.07 ± 0.50,P ＜ 0.05)groups was significantly higher than those in the Con group.3 To explore the effect of C-peptide on HBZY-1 cells,the levels of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ),α5(Ⅳ)and TGF-β1 mRNA relative expression were assayed via realtime-PCR.3.1 The relative expression of al(IV)mRNA was no overt significance in the OC(1.16 ± 0.11,P>0.05)and CP5(1.18 ± 0.16,P>0.05)groups compared with the Con group;Whereas that was significantly increased in the HG(3.59±0.57,P<0.01),CPI(3.59 ± 0.57,P<0.01)and CP9(3.82 ± 0.55,P<0.01)groups in contrast with Con group.3.2 The relative expression of α2(Ⅳ)mRNA was no overt significance in the OC(0.96 ± 0.10,P>0.05)and CP5(0.92 ± 0.12,P>0.05)groups compared with the Con group;Whereas that was significantly increased in the HG(3.12± 0.48,P<0.01),CPI(2.53 ± 0.28,P<0.01)and CP9(2.25 ± 0.28,P<0.05)groups in contrast with Con group.3.3 The relative expression of α3(Ⅳ)mRNA was no overt significance in the OC(0.85 ± 0.07,P>0.05)and CP5(1.04 ± 0.13,P>0.05)groups compared with the Con group;Whereas that was significantly increased in the HG(2.67± 0.45,P<0.05),CP1(2.46 ± 0.52,P<0.05)and CP9(2.42 ± 0.40,P<0.05)groups in contrast with Con group.3.4 The relative expression of a4(IV)mRNA was no overt significance in the OC(1.05 ± 0.06,P>0.05)and CP5(1.12 ± 0.10,P>0.05)groups compared with the Con group,whereas that was significantly increased in the HG(2.18± 0.18,P<0.01),CP1(2.22 ± 0.15,P<0.01)and CP9(2.26 ± 0.15,P<0.01)groups in contrast with Con group.3.5 The relative expression of a5(IV)mRNA was no overt significance in the OC(1.10 ± 0.12,P>0.05)and CP5(1.08 ± 0.07,P>0.05)groups compared with the Con group,whereas that was significantly increased in the HG(2.30± 0.07,P<0.01),CPI(1.71 ± 0.05,P<0.01)and CP9(2.04 ± 0.11,P<0.01)groups in contrast with Con group.3.6 The relative expression of TGF-β1 mRNA was no overt significance in the OC(1.04 ± 0.07,P>0.05)and CP5(1.06 ± 0.14,P>0.05)groups compared with the Con group,whereas that was significantly increased in the HG(2.90± 0.09,P<0.01),CP1(2.04 ± 0.13,P<0.01)and CP9(2.21 ± 0.21,P<0.01)groups in contrast with Con group.4 ELISA assay was performed to assess the protein level of TGF-β1 in culture media in HBZY-1 cells stimulated with various media in the presence of D-glucose at various concentrations.The protein level of TGF-β1 in culture media was no overt significance in the 20mM(485.55 ± 12.49 pg/ml)(P>0.05)group compared to the Con(506.60 ± 37.44 pg/ml)group,while that was significantly lower in 25mM(364.12 ± 18.12 pg/ml)(P<0.01),30mM(385.25 ± 20.42 pg/ml)(P<0.01),35mM(307.29 ± 6.66 pg/ml)(P<0.01)and 40mM(352.22 ± 14.98 pg/ml)(P<0.01)groups than the Con group.5 ELISA assay was carried out to evaluate the protein level of TGF-β1 in culture media in HBZY-1 cells exposed to 35mmol/L high D-glucose DMEM at different time.points.The protein level of TGF-β1 in culture media was significantly lower in 3h(322.77 ± 22.52 pg/ml)(P<0.01)and 6h(447.69 ±47.68 pg/ml)(P<0.01)groups than that in the Con(798.972 ± 37.367 pg/ml)group,while it was no significance in the 12h(738.40 ± 64.53 pg/ml)(P>0.05)group in contrast with Con group.6 To test the effect of C-peptide on HBZY-1 cells,the protein level of TGF-β1 in culture media in HBZY-1 cells was determined by ELISA assay.The protein level of TGF-β1 in culture media was no significantly in the OC(1228.90 ± 57.79 pg/ml)(P>0.05)and CP5(1197.19 ± 13.75 pg/ml)(P>0.05)groups compared with the Con(1228.90 ± 57.79 pg/ml)group,while it was significance in the HG(1076.29 ± 38.320 pg/ml)(P<0.05),CP1(1365.66 ±45.84 pg/ml)(P>0.01)and CP9(1459.49 ± 45.52 pg/ml)(P<0.05)groups in contrast with Con group.7 The cell immunofluorescent staining application was utilized to detect the nuclear translocation of Smad3 in HBZY-1 cells.In the cell immunofluorescent staining application,the Smad3 protein was no expressed in HBZY-1 nuclei in the Con group.However,the expression of Smad3 protein increased significantly in HBZY-1 nuclei in the H1,H2 and H3 groups,especially in the H3 group.Nevertheless,the Smad3 protein was low expressed in HBZY-1 nuclei in the CPI,CP2 and CP3 groups,especially in the CP3 group.8 ChIP assay for Smad3 binding to the promoter regions of α1(Ⅳ),α2(Ⅳ),α3(Ⅳ),α4(Ⅳ)or α5(Ⅳ)genes in HBZY-1 cells.8.1 Smad3 binding to the promoter regions of α1(Ⅳ)and α2(Ⅳ),it was no overt significance in the OC(1.08 ± 0.11,P>0.05)and CP(1.18 ± 0.11,P>0.05)groups compared to the Con group;Whereas it showed much greater stability of smad3 binding to the promoter regions of al(IV)and a2(IV)in the HG(2.58 ± 0.21,P<0.01)and ScCP(2.04 ± 0.12,P<0.01)groups in contract with the Con group.8.2 Smad3 binding to the promoter regions of α3(Ⅳ)and α4(Ⅳ),it was no overt significance in the OC(1.16 ± 0.06,P>0.05)and CP(1.41 ± 0.26,P>0.05)groups compared to the Con group;Whereas it showed much greater stability of Smad3 binding to the promoter regions of α3(Ⅳ)and α4(Ⅳ)in the HG(109.33 ± 2.85,P<0.01)and ScCP(66.09 ± 6.51,P<0.01)groups in contract with the Con group.8.3 Smad3 binding to the promoter regions of α5(Ⅳ),it was no overt significance in the OC(1.23 ± 0.17,P>0.05)and CP(1.59 ± 0.15,P>0.05)groups compared to the Con group;Whereas it showed much greater stability of Smad3 binding to the promoter regions of α5(Ⅳ)in the HG(13.08 ± 0.83,P<0.05)and ScCP(10.26 ± 0.90,P<0.05)groups in contract with the Con group.9 The symptoms of diabetic rats and the effects of C-peptide on blood glucose and body weight in DM rats.The symptoms of diabetic rats include loss of body weight,dull,coarse hair,lethargy,polyuria,polydipsia,polyphagia and growth retardation and so on.With the experiment,the symptoms of diabetic rats were more and more apparent in DM and ScCP groups,while the symptoms of diabetic rats had significantly improved in DP and CP groups.There were no significant differences with body weight and blood glucose in all experimental rats.Throughout the experiment,there was no overt significance at the average blood glucose level among DP(29.96 ± 0.52 mmol/L)(P>0.05),CP(28.9 ± 0.74 mmol/L)(P>0.05),ScCP(29.74 ± 0.66 mmol/L)(P>0.05)groups and the DM(29.23 ± 0.66 mmol/L)group,but it was higher in all of the four groups than the Con(6.4 ± 0.25 mmol/L)(P<0.01)group.At the end of C-peptide intervention treatment,there was no overt significance at the average blood weight among CP(271.82 ± 8.76 g)(P>0.05),ScCP(248.35 ± 7.19 g)(P>0.05)and DM(249.00 ± 7.13 g)groups,yet it was decreased significantly in all of the four groups compared to the Con(515.50 ± 14.42 g)(P<0.01)group.10 Collect 24-hour urinary to detect 24-hour urinary albuminThroughout the experiment,the average of 24-hour urinary albumin was lower in DP(55.21 ± 4.06 mg)and CP(70.2 ± 9.46 mg)groups than DM(327.93 ± 32.58 mg)(P<0.05)group,while there was no overt significance at the average 24-hour urinary albumin level between ScCP group(293.79±49.40 mg)and DM group(P>0.05).All in all,it was higher in all of the four groups than the Con(15.42 ± 4.06 mg)(P<0.01)group.11 The observation of the morphological changes in rat renal tissueObservation of glomeruli under optical microscopy(x400):The Con group showed normal structure of glomeruli;the DM and ScCP groups showed the hardening of the glomeruli and glomerular-capsule adhesion,besides,glomerular necrosis was observed in ScCP group;the shrinking of glomerular-capsule adhesion was observed in DP and CP groups with other renal lesions improved in contrast with the DM group,and they were better in DP group than those in CP group.Observation of glomeruli under transmission electron microscopy(×20K):The Con group showed normal structure of glomeruli.The basement membrane showed local irregular severe thickening,with local fusion of podocytic processes severe,vascular endothelial cell proliferation severe and endothelial fenestrae shrinking in DM and ScCP groups;the basement membrane and vascular endothelium showed normal nearly with local fusion of podocytic processes mildly in DP and CP groups with other renal lesions improved in contrast with the DM group,and they were better in DP group than those in CP group.Conclusion:1 The molecule mechanisms of C-peptide reversing fibrosis by regulating the expression of type IVcollagen in DN are as follows:(1)C-peptide prevented the over-expressions of type IV collagen a chains and TGF-β1 mRNA obviously in mesangial cells induced by high glucose(35mmol/L).It was 0.5nmol/L C-peptide that had the best inhibitory effect,yet the function of C-peptide was not enhanced with increasing concentration of C-peptide.(2)C-peptide suppressed the expression of type IV collagen by inhibiting Smad3 binding to the promoter regions of type IV collagen a-chain genes.2 C-peptide could improve the glomeruli morphology in DM rats. |
PDF Full Text Request