| Objective:To investigate the inhibitory effects of oxymatrine?OMT?on aldosterone?ALD?induced neonatal rat cardiac fibroblasts proliferation and differentiationin vitro,and then explore the underlying mechanisms.Methods:The primary cardiac fibroblasts wereseperated and purified by trypsin digestion methods and differential adhension from 1-3 days of SD neonatal rat.The cellular morphology was observed by inverted microscope.Vimentin of CFs was identified by immunocytochemical methods.The relationship of CaN,PKC and p38MAPK were assayed with inhibitor of CsA,R0-31-8220 and SB203580 as reverse probe.The dose-effect and time-effect relationship of ALD was determined by MTT.Inhibitory effect of OMT on CFs proliferation was detected by the MTT assay.The dose-effect relationship of ALD was randomly divided into groups as following:control group?serum free DMEM?,model group(1×10-10mol/L,1×10-9mol/L,1×10-8 mol/L,1×10-7mol/L,ALD).The time-effect relationship of ALD were randomly divided into groups as following:control group?serum free DMEM?,model group(1×10-7 mol/L,ALD)with4 designing time points as following:12 h,24 h,36 h,48 h.Inhibitory effect of OMT on CFs proliferation were randomly divided into 6 groups as following:control group?Ctrl.serum free DMEM?,model group(1×10-7 mol/L,ALD),OMT low dose group(3.78×10-4 mol/L),OMT high dose group(7.56×10-4 mol/L),Spi(1×10-6 mol/L),Asp(1.11×10-6 mol/L).Inhibitory effect ofCaN,PKC,p38MAPK inhibitor on CFs proliferation were randomly divided into 5 groups as following:control group?serum free DMEM?,model group(1×10-7 mol/L,ALD),CaN I(1×10-7mol/L),PKC I(5×10-8mol/L),p38MAPK I?7?mol/L?.Gimsa staining wasused to assay the morphology.Picric acid Sirius red staining was analyzed the deposition of collagen fiber.The mRNA and protein expression were analyzedby qRT-PCR and Western blot for CaN,PKC,p38MAPK.Results:MTT results showed that ALD(1×10-7mol/L)stimulated the cardiac fibroblasts proliferation?P<0.01?and the optimal treatment duration effect time was 24 h?P<0.05?.The results confirmed that OMT could attenuate proliferation of cardiac fibroblasts induced by aldosterone?ALD??P<0.01?.The CaN,PKC,p38MAPK inhibitor could inhibit cardiac fibroblasts proliferation induced by aldosterone?ALD??P<0.05?.Gimsa staining results suggestedthe violet of nucleus and light pink of cytoplasm of CFs,and the cells were significantly increase compared to control group.Pretreatment with OMT,Spi,Asp,alleviated the pathological change.The results of picric acid Sirius red stainingindicated that the red collagen fiber increased significantly in ALD group,and the treatment drug could decrease the collagen fiber expression and the cell numbers compared to the single ALD groups.qRT-PCR results showed that ALD induced CaNmRNA increased in CFs?P<0.01?,OMT,CaN inhibitor and PKC inhibitor treatment attenuated CaNmRNA expression in CFs?P<0.05?.There is no obvious effect of OMT on ALD induced PKCmRNA and p38MAPKmRNA expression in CFs.Western blot results showed that ALD caused an increase in CaN,p-PKC and p-p38MAPK protein expression?P<0.01?,but it has no effect of PKC and p38MAPK.OMT treatment attenuated cardiac fibroblasts proliferation induced by ALD and decreased CaN,p-PKC and p-p38MAPK protein expression in CFs?P<0.05?,andCaN inhibitor and PKC inhibitor treatment decreased the elevation of p-PKC and p-p38MAPK in CFs?P<0.05?.The Western blot results suggested that onlyinhibitory effect of p38MAPK inhibitor on ALD induced p-p38MAPK decreased in CFs.Conclusion:ALD induced proliferation and differentiation of CFs which maybe involve in regualting CaN,phosphorylated PKC and phosphorylated p38MAPK.There is crosstalk between CaN and PKC,and p38MAPK depending on CaN and PKC activity.OMT ameliorated the collagen deposition;CaN,p-PKCand p-p38MAPK induced by ALD,which mechanism may be involved in inhibiting CaN and p-PKC.p38MAPK has no effection. |
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