The Effect Of Yiqi-huoxue Decoction（YHD） On Endothelial Cell Inflammation Mediated By P2X₄ Receptor Based On Intracellular Ca²⁺ Levels

ObjectiveTo observe the intervention effect of Yiqi Huoxue Decoction（YHD）on cell inflammation induced by P2X₄ receptor and explore its mechanism.Methods1.Effect of YHD on cell inflammation mediated by P2X4 receptor（1）Human umbilical vein endothelial cells（EAs）were used t o establish inflammation models of endothelial cells under the stimulation of AT P and Bz ATP,respectively.The cells were divided into blank group,ATP group,Bz ATP group,YHD+ATP group,and YHD+Bz ATP group.（2）The m RNA levels of IL-6 and TNF-αwere detected using q RT-PCR.2.YHD exerts anti-inflammatory effect by affecting the concentration of Ca²⁺（1）PSB-12062,a P2X₄ inhibitor,was used to observe the mechanism of YHD.The basic cell groups were:control group,Bz ATP group,YHD+Bz ATP group,PSB-12062+Bz ATP group,YHD+PSB-12062+Bz ATP group.（2）Fura 2-AM,a calcium indicator,was used to detect intracellular Ca²⁺concentration.（3）The expressions of P2X₄,KLF2,IL-6 and TNF-αwere detected by Western Blotting.Results（1）Compared with the control group,the m RNA expressions of IL-6 and TNF-αin ATP group increased（P<0.01 or P<0.05）.Compared with ATP group,the m RNA expressions of IL-6 and TNF-αin YHD+ATP group were lower（P<0.01 or P<0.05）.Similarly,compared with the control group,th e m RNA levels of IL-6 and TNF-αin Bz ATP group increased（P<0.01 or P<0.05）;compared with Bz ATP group,the m RNA levels of IL-6 and TNF-αin YHD+Bz ATP group decreased（P<0.01 or P<0.05）.（2）Under the treatment of Bz ATP,the intracellular Ca²⁺concentration increased,and YHD and PSB-12062 both could reduce the intracellular Ca²⁺concentration（P<0.01）.（3）Compared with the control group,the expressions of P2X ₄ and KLF2 in Bz ATP group increased（P<0.05）;compared with Bz ATP group,the expressions of P2X₄ and KLF2 in YHD+Bz ATP group and PSB-12062+Bz ATP group decreased（P<0.05）.（4）Compared with the control group,the m RNA levels of IL-6 and TNF-αin Bz ATP group were significantly higher in the presence of Ca²⁺recording media（P<0.05）.Compared with Bz ATP group,the m RNA levels of IL-6 and TNF-αin YHD+Bz ATP group decreased（P<0.05）,and as well the expression of KLF2（P<0.05）.In the absence of Ca²⁺recording media,compared with Bz ATP group,the expression of KLF2 in YHD+Bz ATP group had no difference（P>0.05）.ConclusionYHD has the function of inhibiting the inflammation in endothelial cells mediated by P2X₄ receptor,which may be related to the decrease of intracellular Ca²⁺concentration,providing a new strategy for the treatment of cardiovascular diseases related to inflammation.