| Background:Cardiac fibrosis is a crucial pathological process that results in cardiac failure in the development of multiple cardiovascular diseases.It is characterized by excessive deposition of extracellular matrix with collagen as the main component,resulting in ventricular remodeling.In recent years,more and more studies have found that inflammatory response plays a key role in triggering adverse ventricular remodeling.Seeking drugs to inhibit the cascade waterfall effect caused by inflammatory cytokines may be an effective means to interfere with the development of cardiac fibrosis.Puerarin has anti-inflammatory and anti-oxidation effects,and it is reported that puerarin could inhibit cardiac fibrosis.While the exact mechanism of puerarin on cardiac fibrosis has not been clearly enough.Objective:To observe the effect of puerarin on cardiac fibrosis and inflammation during its formation,and research the changes of TLR4-NF-?B proinflammatory pathways focusing on PARP-1 and HMGB1 after cardiac fibrosis has been treaded with puerarin,then explore the mechanism of puerarin in inhibiting cardiac fibrosis.Methods:1.To analyse the relationship between cardiac fibrosis and inflammatory cytokines HMGB1,PARP-1,TNF-?,IL-6.A.The separated primary cardiac fibroblasts(CFs)were induced by lipopolysaccharide(LPS),and related tests were carried out.B.?-SMA was detected by immunofluorescence to assess the activation of CFs.C.The levels of TNF-?,IL-6,HMGB1,PARP-1,?-SMA,collagen-1 and collagen-3 were examined by ELISA,RT-qPCR and western blot.2.To observe the influence of puerarin on cardiac fibrosis and the levels of HMGB1,PARP-1,TNF-?,IL-6.A.Cardiac fibrosis rats,model were established using transverse aortic constriction(TAC)and treated by puerarin.The degree of fibrosis was evaluated by masson staining.In addition,the levels of hydroxyproline was examined by ELISA,and the levels of ?-SMA,collagen-1 and collagen-3 were examined by western blot and immunohistochemistry assays.Besides,western blot was used to examine the expression of HMGB1 and PARP-1.B.The separated rat primary CFs were induced by LPS and treated with puerarin.The levels of TNF-?,IL-6,HMGB1,PARP-1,?-SMA,collagen-1 and collagen-3 were examined by ELISA,immunofluorescence,RT-qPCR and western blot.3.Exploring the mechanism of puerarin inhibiting cardiac fibrosis.A.In vivo,the protein levels of TLR4,p65,p-p65,I?B? and p-I?B? in rats'heart tissue were detected by western blot.B.In vitro,primary CFs transfected with PARP-1 overexpression plasmids and/or HMGB1 siRNA were pretreated with puerarin and/or LPS.Afterwards,the level of HMGB1 was detected by ELISA,RT-qPCR and western blot.The expression of TLR4,p65,p-p65,I?B? and p-I?B? were detected by western blot.The levels of?-SMA,collagen-1,collagen-3 were examined by immunofluorescence,RT-qPCR and western blot.Results:1.The expression of PARP-1,HMGB1,TNF-?,IL-6 and fibrosis-related proteins(?-SMA,collagen-1,collagen-3)increased significantly in rat CFs induced by LPS.2.Puerarin effectively inhibited the production of ?-SMA,collagen-1 and collagen-3 in rat CFs induced by LPS.Further more,puerarin significantly alleviated cardiac fibrosis in TAC rats.3.Puerarin inhibited the expression of PARP-1,HMGB1 and inflammatory cytokines(TNF-?,IL-6),which were induced by LPS in primary CFs.In addition,we proved that puerarin also restrained the expression of PARP-1and HMGB1 in TAC rats.4.Overexpression of PARP-1 could upregulate the level of HMGB1 and reverse the protective effect of puerarin in rat CFs induced by LPS.While silencing HMGB1 inhibited the expression of fibrosis-related proteins(?-SMA,collagen-1,collagen-3)notably.5.After puerarin intervention,the TLR4-NF-?B signaling pathway was obviously restrained.This effect could be attenuated by silencing HMGB1.Conclusion:Puerarin has a significant protective effect on cardiac fibrosis in rats.In terms of mechanism,this effect is related to the inhibition of HMGB1-mediated TLR4-NF-?B signaling pathway which follows after the down-regulation of PARP-1. |
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