Salvianolic Acid B Ameliorated Angiotensin Ⅱ Induced Rat Cardiac Fibroblast-myofibroblast Transformation (FMT) Via Inhibiting NF-κB Activation In Vitro

Objective:To investigate the inhibitory effects of Salvianolic acid B（Sal B）on transformation of cardiac fibroblast-myofibroblast of neonatal Sprague Dawley rat cardiac fibroblasts（CFs）induced by AngiotensinⅡ（AngⅡ）and then explore the underlying mechanisms in vitro.Methods:The primary CFs were were seperated and purified by trypsin digestion methods and differential adhension from1-3-day-old neonatal rats.CFs were passaged after confluence in cultured for 3-4days.2rd or 3th passage CFs were used in the experiments.Cellular morphology was observed by inverted microscope.The cultured cell was identified with anti-vimentin by immunocytochemistry.The relationship of Sal B on FMT induced by Ang Ⅱ and the activation of NF-κB were assayed with inhibitor of PDTC as reverse probe.The dose-effect and time-effect relationship of Ang Ⅱ was determined by MTT.Inhibitory effect of Sal B on CFs proliferation was detected by the MTT assay.Inhibitory effect of Sal B on CFs proliferation were randomly divided into 5 groups as following:control group（Ctrl.serum free DMEM）,model group(1×10^-6 mol/L,Ang Ⅱ),Sal B low dose group（12.5 umol/L+1×10^-66 mol/L Ang Ⅱ）,Sal B medium dose group（25umol/L+1×10^-66 mol/L Ang Ⅱ）,Sal B high dose group（50 umol/L+1×10^-66 mol/L Ang Ⅱ）.Wound scratch assay was used to investigate CFs migration rate.The expression of phosphorylated-IκBα（p-IκBα）,IκBα,phosphorylated-p65（p-p65）,p65,nuclear p65,cytoplasmic p65,alpha-smooth muscle actin（α-SMA）,collagen I（Coll I）,fibronectin（FN）and Connective tissue growth factor（CTGF）were detected by Western blot.The mRNA of NF-κB were analyzed by RT-PCR.The concentration of hydroxyproline was detected by commercial kit.the expression ofα-SMA protein was observed by immunofluorescence staining.Results:MTT results showed that Ang Ⅱ（1×10^-66 mol/L）stimulated the cardiac fibroblasts proliferation（P<0.01）and the optimal treatment duration effect time was 24 h（P<0.01）.The results confirmed that Sal B could attenuate proliferation of cardiac fibroblasts induced by Angiotensin Ⅱ（Ang Ⅱ）（P<0.05）.PDTC could also inhibit the proliferation of CFs induced by Ang Ⅱ.Wound scratch assay revealed Ang Ⅱ evidently increased migration rate of CFs compared with control group（P<0.01）,Sal B significantly attenuates Ang Ⅱ-induced CFs migration rate compared with Ang Ⅱ group（P<0.01）.Western blot result revealed that the expression ofα-SMA,Coll I,FN,CTGF,p-IκBαand p-p65 in Ang Ⅱ group significantly increased,Sal B suppressed Ang Ⅱ-induced Coll I,FN,CTGF,p-IκBαand p-p65 up-regulation.The expression of Coll I protein decreased after incubating with Sal B,PDTC,Sal B and PDTC compared with Ang Ⅱ group（P<0.05）.IκBαand p65 expression had no significant in each group.In addition,Sal B inhibited Ang Ⅱ-induced NF-κB p65 from the cytoplasm to the nucleus,RT-PCR results showed that Ang Ⅱ induced NF-κB mRNA increased in CFs（P<0.05）,Sal B inhibited Ang Ⅱ-induced NF-κB mRNA increased（P<0.05）.The concentration of hydroxyproline increased in Ang Ⅱ group（P<0.01）compared with the control group,decreased after incubating with Sal B,PDTC,or Sal B and PDTC compared with Ang Ⅱ group（P<0.05）.Immunofluorescence staining observed that fluorescence staining signals forα-SMA expression（green）,nuclei were stained with DAPI（blue）,it exhibited bright fluorescence staining signals forα-SMA expression（green）compared with the control group,while the fluorescence staining signals was markedly inhibited by coincubation with Sal B,PDTC,or Sal B and PDTC.Conclusion:Sal B could alleviate Ang Ⅱ-induced FMT and its mechanism may be involved in inhibiting NF-κB Activation.