The Effect And Mechanism Of Pinocembrin On The Synthesis Of Extracellular Matrix In AngⅡ Induced Rat Mesangial Cells

Objective:Diabetic nephropathy is one of the most common microvascular complications of diabetes and has now risen to the leading cause of end-stage renal disease.High blood glucose,as the initial factor in DN,activates renal RAAS,inflammation response,oxidative stress,etc,resulting in diffuse thickening of the glomerular basement membrane,proliferation of mesangial matrix,eventually leading to glomerulosclerosis,tubular atrophy and tubulointerstitial fibrosis.At present,the clinical control of blood glucose,blood lipids,blood pressure,proteinuria and other treatment methods have failed to completely prevent the progress of DN,therefore searching for new methods for the treatment of DN has been a hot topic in the field of kidney disease research.Pb is a natural flavonoid compound.Recent studies have found that Pb can inhibit the AngⅡ/AT1R axis-mediated rat aortic ring contraction.In addition,studies have suggested that Pb may improve diabetic brain disease,cerebral ischemia-reperfusion injury,and acute lung injury by inhibiting NF-kappaB-mediated inflammatory responses.However,whether Pb can be used for the treatment of DN has not been reported yet.We found that Pb could effectively improve proteinuria and renal mesangial proliferation in rat DN in the previous study.However the mechanism of action of Pb is not yet clear.This study used rat mesangial cells as the research object to study the effect of Pb on the synthesis of extracellular matrix in AngⅡ induced rat mesangial cells and its action mechanism,to provid a new method for the prevention and treatment of DN.Methods:1.Detecting the effect of Pb on synthesis of extracellular matrix in AngⅡ-induced rat mesangial cells:The cultured cells were divided into the following five groups:normal control group(NC),10-6 mol/L AngⅡ group(Ng),10-6 mol/L AngⅡ+DMSO group(Ng-D),10-6 mol/L AngⅡ+30 umol/L Pb group(Ng-Pb)(the result of preliminary experiment showed that the optimum concentration of pinocembrin was 30 umol/L)and 10’6 mol/L AngII+10 umol/L valsartan group(Ng-Val);immunofluorescence was used to detect the level of intracellular Col Ⅳ,FN,TGF-β1,Smad3,p-Smad3,NF-κb,and p-NF-κb,ELISA was used to detect the level of TNF-α and IL-6 in the supernatant.The PCR was used to detect the intracellular mRNA level of Col Ⅳ,FN,TGF-β1,TNF-α and IL-6.2.The analysis and processing of data was performed by SPSS 17.0 statistical software.Results:1.Pb inhibited the synthesis of Col Ⅳ and FN in AngⅡ induced rat mesangial cells:at the protein level,the result of immunofluorescence showed that the green fluorescence of Ng group was obviously higher than that of NC group,while the green fluorescence of Ng-Pb group was distinctly lower than that of Ng group;the result of western blot showed that the expression levels of Col Ⅳ and FN in the Ng group were significantly higher than those in the NC group(P<0.05),while the expression of Col IV and FN in Ng-Pb group was significantly lower than that in Ng group(P<0.05).At the transcriptional level,the result of qPCR showed that the mRNA levels of Col Ⅳ and FN in the Ng group were significantly higher than those in the NC group(P<0.05),while the mRNA levels of the Col Ⅳand FN in the Ng-Pb group were significantly lower than those in the Ng group(P<0.05).2.Pb suppressed the activation of TGF-β1/Smad3/NF-κb signaling pathway in AngⅡ induced rat mesangial cells:(1)At the protein level,the result of western blot showed that the expression of TGF-β1 in Ng group was significantly higher than that in NC group(P<0.05),while the expression level of TGF-β1 in Ng-Pb group was significantly lower than that in Ng group(P<0.05).At the transcription level,the result of qPCR showed that the mRNA level of TGF-β1 in Ng group was significantly higher than that in NC group(P<0.05),yet the mRNA level of TGF-β1 in Ng-Pb group was significantly lower than that in Ng group(P<0.05).(2)The result of western blot showed that the level of p-Smad3/Smad3 increased significantly in Ng group when compared with NC group(P<0.05),while the level of p-Smad3/Smad3 decreased significantly in Ng-Pb group when compared with Ng group(P<0.05).(3)The result of western blot showed that the level of p-NF-κb/NF-κb increased significantly in Ng group compared with NC group(P<0.05),but the level of p-NF-κb/NF-κb decreased significantly in Ng-Pb group when compared with Ng group(P<0.05).3.Pb restrained the synthesis and secretion of IL-6 and TNF-α in AngⅡ induced rat mesangial cells:at the protein level,the result of ELISA showed that the levels of IL-6 and TNF-α increased significantly in Ng group when compared with NC group(P<0.05),but the levels of IL-6 and TNF-α in the Ng-Pb group were significantly lower than those in the Ng group(P<0.05).At the transcriptional level,The result of qPCR showed that mRNA levels of IL-6 and TNF-α in the Ng group were significantly higher than those in the NC group(P<0.05),but mRNA levels of IL-6 and TNF-α in Ng-Pb group were significantly lower than those in Ng group(P<0.05).Conclusion:1.Pb can inhibit the synthesis of extracellular matrix in AngⅡ-induced rat-mesangial cells.2.Pb can inhibit the synthesis of extracellular matrix in AngⅡ-induced rat mesangial cells through suppressing TGF-β1/Smad3/NF-κb signaling pathway.