| Background and aims: Myocardial fibrosis is featured by excessive proliferation of cardiac fibroblast and collagen deposition.There is close relationship between myocardial fibrosis and various cardiovascular disease,such as hypertension,chronic heart failure,dilated cardiomyopathy and viral myocarditis.It is a potential risk factor for sudden death.At present the precise mechanism remains unclear.CD38 as an important NAD+ dependent ADPr cyclase and hydrolase in mammalian cells,its gain or loss of function could regulate multiple biological process through NAD+ or cADPr dependent signaling pathways,such as aging,cardiovascular injury,insulin release,energy metabolism and inflammatory reaction.Our previous study showed that,CD38 gene deficiency significantly alleviate Ang II induce cardiac hypertrophy and fibrosis,however,the effect of CD38 on myocardial fibrosis and its mechanism are not completely clear.Therefore,in this study,a CD38 gene over-expression NIH3T3 stable cell line was prepared and the in vitro fibrosis cell model induced by Ang II or TGF? was settled,so as to explain the role and mechanism of CD38 mediated myocardial interstitial fibrosis,and provide experimental basis for clinical prevention of myocardial remodeling after myocardial infarction.Methods 1 Preparation of CD38 over-expression NIH3T3 stable cell line.1)G418 with 7 different concentration gradients(0,100,200,400,600,800,1000ug/m L)were used to culture NIH3T3 cells for 14 days,and the optimum antibiotic screening concentration was determined by observing the cell state.2)The pc DNA3.1-CD38 plasmid was transfected into NIH3T3 cells,and G418 with the optimal concentration was used to screen cells for two weeks after 48 hours transfection,then the m RNA and protein levels of CD38 were detected in selected single clones.2 Construction and analysis of cell fibrosis model in vitro.1)Different concentration gradients of Ang II(0,0.1,1,2mmol/L)or TGF?(0,0.25,5,10ng/m L)were used to determine the suitable concentration for the treatment of NIH3T3 cell line,and the m RNA level of COL1 and COL3 was tested by q-PCR analysis,and protein expression of ?-SMA was determined by western blot.2)The m RNA level of COL1 and COL3 and the protein level of ?-SMA was detected in CD38 overexpressed cells after 48 hours' treatment of 1mmol /L Ang II or 10ng/m L TGF?.3)CCK8 analysis was used to detect TGF? induced proliferation between CD38 overexpression cells and control cells.4)Wound healing was used to detect the effect of CD38 in cell migration after TGF? treatment.3 Mechanism analysis of CD38 mediated fibrosis induced by TGF?.1)Western blot analysis was used to exam TGF? induced phosphorylation of SMAD3 in CD38 overexpressed NIH3T3 cells and primary rat cardiac fibroblasts.2)Exogenous cADPr was used in CD38 overexpression and control NIH3T3 cells,and Fluro-3am was used to detect intracellular calcium with a multi-mode micro-plate reader after TGF? stimulation.3)The protein level of ?-SMA was examined after exogenous cADPr supplemented and CD38 overexpressed NIH3T3 cells after TGF? stimulation.4)q-PCR analysis was used to detect the m RNA expression of fibrosis related genes FZ,ICAM1,PINK,SMA,SMEMB,TGF?,inflammation related gene TLR4 and oxidative stress related genes NOX1 and NOX2 in CD38 knockout and wild-type mouse embryonic fibroblasts.Results 1 After two weeks G418 treatment with an optimum antibiotic screening concentration of 400ug/m L,one single clone stable expressed CD38 was selected.2 CD38 overexpression promoted TGF? induced cell fibrosis of NIH3T3 1)Ang II and TGF? stimulation significantly increased the transcription of fibrosis marker genes COL1 and COL3 and the protein translation of ?-SMA with a dose dependent manner,and 1mmol /L Ang II or 10ng/m L TGF? was selected and as the best concentration for fibrosis stimulation.2)CD38 overexpression increased the m RNA level COL1 and COL3 and the protein level of ?-SMA after 48 hours' treatment with Ang II or TGF?.3)CD38 promoted TGF? induced cell proliferation,but no effect was observed in cell migration.3 CD38 gene overexpression increased TGF? induced phosphorylation of SMAD3 and intracellular calcium overload.1)CD38 gene overexpression increased TGF? induced phosphorylation of SMAD3 in CD38 overexpression NIH3T3 cells and primary rat cardiac fibroblasts.2)Exogenous cADPr and overexpression of CD38 promoted TGF? induced intracellular calcium overload and the protein expression of ?-SMA.3)CD38 gene knockdown inhibited the m RNA levels of fibrosis related genes FZ,ICAM1,PINK,SMA,SMEMB,TGF?,inflammation related gene TLR4 and oxidative stress related genes NOX1 and NOX2.Conclusion 1 CD38 promote the activation and proliferation of cardiac fibroblasts induced by TGF?.2 CD38 promote TGF? induced cardiac fibrosis through activating TGF?-SMAD3 signaling pathway and increase cADPr mediated calcium overloading... |
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