| Atherosclerosis is a chronic inflammatory response.Macrophages participate in the innate immune response and specific immune response in the body,and play an important role in promoting AS.Polarization of macrophages is the transformation of their own phenotypes made by macrophages according to the different microenvironments.M1 polarization has the role of promoting the inflammation.The JAK1-STAT1-SOCS1 pathway is an important signaling pathway leading to M1-type polarization.mi R-155 is considered to be closely related to the inflammatory response,and one of its downstream targets is SOCS1.This research group has long been devoted to the research of paeonol anti-AS,and achieved certain results.This experiment mainly explored the effect and mechanism of paeonol on M1 type polarization of macrophages.OBJECTIVETo investigate the effect of paeonol on M1-type polarization of macrophages,and to explore whether this effect is caused by the regulation of mi R-155 and the effect on the downstream JAK1-STAT1 pathway.Put forward new ideas for the study of paeonol anti-atherosclerosis mechanism.METHODSIn the experiment,li-popolysaceharides?LPS?and interferon-??IFN-??were used to stimulate RAW264.7 cells for 24 hours to establish a macrophage M1polarization model.Paeonol gives cells pre-protection 24 hours before stimulation.to detect the damage effect of LPS and IFN-?co-stimulation on cells by CCK-8 method.For the expression of M1 Surface Markers F4/80 and CD86 was detected by Flow Cytometry.ELISA method to detect the secretion of interleukin-6?IL-6?and Tumor necrosis factor-??TNF-??in cell supernatant?Inhibition and overexpression of mi RNA-155 by kit transfection.RT-PCR detection of mi R-155 expression in each group of cells.Western blot was used to detect protein expression on the JAK1-STAT1-SOCS1 pathway.RESULTS1.Co-stimulation of IFN-?and LPS to establish M1 polarization model of macrophages1.1 Effects of IFN-?and LPS on the survival rate of macrophagesWhen the IFN-?concentration is 20 ng·m L-1 and the LPS concentration range is 50-250 ng·m L-1,the survival rate of RAW264.7 cells is not less than 80%,which is not significant compared with the blank group reduce?P>0.05?It shows that the stimulant has no obvious damage to the cells in this concentration range,and further experimental research can be performed on this basis.1.2 Effects of IFN-?and LPS on inflammatory response of macrophagesThe concentration of IFN-?was 20 ng·m L-1,and LPS was in the range of100-250 ng·m L-1.The inflammatory factors IL-1?and IL-6 secreted by macrophages were significantly enhanced compared with the blank group?P<0.05?.1.3 Effects of IFN-?and LPS on macrophage surface markers CD86 and F4/80When IFN-?was 20 ng·m L-1 and LPS concentrations were 100 and 150 ng·m L-1,the fluorescence intensity of CD86 and F4/80 increased significantly?P<0.05?.From the perspective of the availability of the reagent and the effect of the effect,when the concentration of IFN-?was 20 ng·m L-1 and the concentration of LPS was100 ng·m L-1,PBS was used as the dissoving agent to co-stimulate the cells for 24hours.The M1 polarization model was successfully established.2.Inhibitory effect of paeonol on M1-type polarization of macrophages2.1 Effect of paeonol on inflammatory response of macrophagesPaeonol pre-protected M1 polarized model of RAW264.7 cells significantly reduced the levels of inflammatory factors IL-6 and TNF-?,indicating that paeonol can effectively inhibit the inflammatory response caused by macrophage stimulation.2.2 Effects of paeonol on the expression of M86-type marker factors CD86 and F4/80on the surface of macrophagesFurthermore,the effects of paeonol on the expression of CD86 and F4/80 on the surface of macrophages were detected by flow cytometry.Paeonol could significantly inhibit the M1 type polarization of macrophages caused by the co-stimulation of IFN-?and LPS.3.Paeonol inhibits mi R-155 expression in macrophagesThe level of mi R-155 was detected by RT-PCR to verify the expression of mi R-155 in polarized RAW264.7 cells and the effect of paeonol on it.The experimental results showed that compared with the blank group,the macrophages in the model group after M1 polarization were highly expressed mi R-155,and the mi R-155 levels in the cells protected by paeonol were significantly decreased compared with the model group.Paeonol can reduce the high expression of mi R-155on the surface of M1 macrophages caused by IFN-?and LPS.4.Inhibition of paeonol on JAK1-STAT1 pathwayBecause macrophages have IFN-?receptors on the surface,JAK1-STAT1pathway will be activated after binding to the receptor.Western blot was used to detect the protein expression on JAK1-STAT1 pathway.The experimental results showed that compared with the blank group,the protein phosphorylation degree of JAK1 and STAT1 was significantly enhanced,while the expression of SOCS1 was significantly inhibited.Compared with the model group,the degree of phosphorylation of JAK1 and STAT1 protein was significantly reduced and the degree of inhibition of SOCS1 protein expression was reduced in macrophages treated with paeonol.Compared with the model group,paeonol significantly inhibited the JAK1-STAT1 pathway.5.Paeonol inhibits the JAK1/STAT1 pathway by down-regulating the expression of mi R-155,thereby down-regulating the inflammatory response.In order to investigate whether paeonol's target of inhibiting M1 polarization of macrophages is mi R-155 or a protein on the JAK1-STAT1 pathway,RAW264.7 cells were transfected with mi R-155-inhibitor and mi R-155-mimic.Furthermore,the expression of mi R-155 and the expression of IL-6,IL-1?and TNF-?in the supernatant of each group of cells were detected.Compared with the blank group,mi R-155 expression in the mi R-155-inhibitor group was significantly reduced,and mi R-155 expression in the mi R-155-mimic group was significantly increased?P<0.01?.Compared with the paeonol group and paeonol+stimulant group,the expression of mi R-155 decreased significantly.When paeonol was applied to cells simultaneously with stimulants and mi R-155-inhibitor,there was no significant difference compared with paeonol group+stimulant group,indicating that the effect of paeonol on mi R-155 expression was blocked?P>0.05?.The above results indicate that paeonol has a direct regulating effect on mi R-155.The expression of IL-6,IL-1?and TNF-?was detected by ELISA.The experimental results showed that the secretion of three inflammatory factors in the cell supernatant of the mi R-155-inhibitor group was significantly reduced compared with the model group,The mi R-155-mimic group is significantly higher than the model group.Paeonol combined with mi R-155-inhibitor and stimulant macrophages had no significant difference with inflammatory factors secreted by inhibitor-transfected macrophages.mi R-155 inhibitor inhibited the expression of mi R-155,and at the same time inhibited effect of phenol on inflammatory response.The results indicate that the inhibitory effect of paeonol on the inflammatory response is likely to be achieved by regulating mi R-155.CONCLUSION1.Paeonol can inhibit macrophage polarization caused by IFN-?and LPS,while inhibiting the inflammatory response of macrophages.2.Paeonol may down-regulate the expression of mi R-155 in macrophages,promote the expression of SOCS1,inhibit downstream JAK1-STAT1 pathway conduction,and then inhibit macrophage M1 type polarization,and reduce the occurrence of macrophage inflammation. |
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