Explore The Mechanism Of Yiqi Yangyin Huoxue Huatan Recipe On Macrophage Polarization On The Basis Of Inflammatory Response Theory Of Coronary Heart Disease

Objectives To observe the effect of Yiqi Yangyin Huoxue Huatan recipe on the expression of inflammatory factors,mRNA and protein of M1/M2 type macrophages,to evaluate the effects of Yiqi Yangyin Huoxue Huatan recipe on the polarization regulation of macrophages and to explore whether the mechanism is related to the p38MAPK/NF-κB signaling pathway on the basis of inflammatory response theory of coronary heart disease.Methods:LPS was used to induce RAW264.7 cells to construct an in vitro inflammatory model.RAW264.7 cells were divided into blank group,LPS inflammatory model group and TCM group with different concentrations.The morphological changes of each group were observed under electron microscope.MTT assay was used to determine the effect of different concentrations of Yiqi Yangyin Huoxue Huatan recipe on the proliferation of RAW264.7 cells.Griess method was used to detected the release of nitric oxide.FCM method was used to detect the expression of CD86 and CD206 on the surface markers of M1/M2 macrophages.Enzyme linked immunosorbent assay was used to detect the release of M1 pro-inflammatory factors TNF-α.IL-6、IL-1β、iNOS and M2 anti-inflammatory cytokines IL-10、TGF-β、Arg-1 in cell supernatant.Real-time PCR was used to detect the expression of the pro-inflammatory factor genes of M1-macrophages and the anti-inflammatory factor genes of M2-macrophages.Western blot was used to determine the effects of protein expression levels of TNF-α、IL-6、IL-1β、iNOS and the expression of key proteins in p38MAPK/NF-κB signaling pathways.Results:(1)Under electron microscope:compared with blank group,cell morphology was altered in LPS inflammatory model group;compared with LPS-induced group,cell morphology was improved obviously in Chinese medicine groups.(2)Results of MTT showed that Yangyin Huoxue Huatan recipe with the dose of 2.0 g·L-1 and below had no effect on the cell proliferation.(3)Results of Griess indicated,compared with blank group,the release of NO of LPS-induced group was increased(P<0.01);compared with LPS-induced group,Chinese medicine group could both reduce the release of NO(P<0.01).(4)Results of FMC showed that the expression of CD86 was up-regulated in LPS inflammatory model group and the expression of CD206 had no significant change.It is suggested that LPS mainly induces the transformation of macrophages into M1 phenotype.(5)Results of ELISA showed,compared with blank group,the release of M1/M2 inflammatory cytokines of LPS-induced group were both up-regulated(P<0.01);compared with LPS-induced group,the secretion of M1-macrophages inflammatory cytokines TNF-α、IL-6、IL-1β、iNOS were down-regulated in Chinese medicine group(P<0.01).But M2-macrophages inflammatory cytokines IL-10、TGF-β、Arg-1 weren’t change.(6)Results of RT-PCR indicated,compared with blank group,the expression of M1/M2 were both increased(P<0.01,P<0.05);compared with LPS-induced group,the expression levels of M1-macrophages were down-regulated in Chinese medicine group(P<0.01);anti-inflammatory factor genes of M2-macrophages had no significant change.(7)Results of Western blot showed,compared with blank group,protein expression levels of TNF-α、IL-6、IL-1β、iNOS were up-regulated(P<0.01);compared with LPS-induced group,the protein expression of TNF-α、IL-6.IL-1β、iNOS were down-regulated in Chinese medicine group(P<0.01);compared with blank group,the protein expression levels of P-p38、P65、IκB were up-regulated(P<0.01);compared with LPS-induced group,the protein expression of P-p38、P65%IκB were down-regulated in Chinese medicine group(P<0.01)Conclusion:(1)Yangyin Huoxue Huatan Recipe can play the role of immune regulation by inhibiting the polarization of macrophages to M1 subtype and could effectively reduce the expression of pro-inflammatory factors,like TNF-α、IL-6、IL-1β、iNOS,to play a good anti-inflammatory effect in vitro.(2)The mechanism of CHD treatment by Yangyin Huoxue Huatan Recipe may be realized by regulating the p38MAPK/NF-κB signaling pathway,inhibiting the phosphorylation reaction of p38MAPK,blocking the transfer of NF-κB from the cytoplasm to the intracellular nucleus and thereby inhibiting the transcription function of NF-κB.