AngⅡ Induces Macrophage M1 Polarization By Up-regulating Cx43-activated NF-κB Signaling Pathway

Objective:In this study,angiotensin Ⅱ(Ang Ⅱ)was used to interfere with monocyte macrophages in RAW264.7 mice to simulate chronic inflammation,and Cx43 specific blocker Gap26/Gap19 and NF-kappa B(p65)signaling pathway inhibitor BAY117028 were given to explore the role of connexin 43(Cx43)and NF-kappa B signaling pathway in Ang Ⅱ-induced M1 polarization of RAW264.7 mice monocyte macrophages.Methods :RAW264.7 mouse mononuclear macrophages were divided into control group,Ang Ⅱ(10-6mol/L incubation for 12h)treatment group,Gap26 pretreatment group(10-5 mol/L Gap26 pretreatment for 30 min and then added 10-6 Mol/L Ang Ⅱ was incubated for 12 h),Gap19 pretreatment group(10-5 mol/L Gap19 pretreatment for 30 min,10-6 mol/L Ang Ⅱ was added for12 h).Flow cytometry was used to detect the expression frequency of M1/M2 marker CD86/CD206 in macrophages.Immunofluorescence was used to observe the localization and expression of Cx43 and macrophage M1 markers i NOS and CD86,and q-PCR was used to detect macrophages.m RNA expression of cell M1 markers i NOS,TNF-α,IL-1β,IL-6 and CD86;detection of macrophage Cx43,M1 marker i NOS,CD86 and p65 phosphorylated protein by Western Blot Expression of cytokines(TNF-α,IL-1β,IL-6)in macrophage supernatants by ELISA.Results :(1)To investigate the effect of Ang Ⅱ on macrophage activity at different concentrations(10-9,10-8,10-7,10-6,10-5 mol/L).CCK-8 assay showed that Ang Ⅱ had the least effect on cell activity when its concentration was 10-6 mol/L.(2)Ang Ⅱ can induce the polarization of mononuclear macrophages of RAW264.7 mice to M1 type.The frequency of expression of the M1 marker CD86 and the M2 marker CD206 was examined by flow cytometry.After 24 hours of intervention,the expression frequency of M1 marker CD86 in Ang Ⅱ group was significantly higher than that in control group(P < 0.01).The expression frequency of M2 marker CD206 in Ang Ⅱ group was not significantly different from that in control group.Western-Blot results showed that the expression of macrophage M1 markers CD86 and i NOS was time-dependent,and reached the peak after 12 hours of Ang Ⅱ intervention(P < 0.01),then the expression of CD86 and i NOS decreased.The expression of M2 markers CD206 had no significant difference compared with control group.(3)Ang Ⅱ can up-regulate Cx43 on RAW264.7 mouse mononuclear macrophages.The expression and localization of Cx43 on nucleated macrophages of RAW264.7 mice were detected by immunofluorescence assay.Cx43 was localized in the cell membrane.The expression of Cx43 protein in RAW264.7 mouse mononuclear macrophages was time-dependent by Western-Blot technique.The protein expression reached the peak after 12 h of Ang Ⅱ intervention(P < 0.01).(4)After pretreatment with Gap26/Gap19,a specific blocker of Cx43,the M1-type related indicators i NOS,TNF-α,IL-1β,IL-6,and CD86 were inhibited.The results of q RT-PCR showed that the m RNA of M1 type marker on mononuclear macrophages of RAW264.7 mice was significantly increased(P<0.01)after treatment with Ang Ⅱ for 12 h,and Gap26 was pretreated for 30 min.Post-M1 polarization was inhibited(P<0.01);after 30 min of Gap19 pretreatment,macrophage polarization was also inhibited to M1(P<0.01).Western-Blot and immunofluorescence assays were used to detect mononuclear giant mice.Localization and expression of phagocyte M1 marker i NOS and CD86,immunofluorescence localization of i NOS was expressed in whole cells,CD86 was mainly expressed on cell membrane.ELISA was used to detect cytokine TNF-α in RAW264.7 mouse mononuclear macrophage supernatant.,IL-1β,IL-6 protein levels,Ang Ⅱ group TNF-α,IL-1β,IL-6 protein levels were significantly higher than the control group(P <0.01);Gap26,Gap19 pretreatment After that,M1 type polarization was suppressed(P<0.01).(5)Ang Ⅱ activates Cx43-mediated NF-κB(p65)signaling pathway.Compared with the control group,the p65 phosphorylation protein p-p65 protein in the Ang Ⅱ group was significantly up-regulated(P< 0.01),indicating that the NF-κB(p65)signaling pathway was activated by Ang Ⅱ.To investigate the relationship between Cx43 and NF-κB(p65)signaling pathway,we used Cx43-specific blocker Gap26/Gap19 for 30 min,and then used Ang Ⅱ to intervene in macrophages.Western-Blot results showed Ang Ⅱ+Gap26 and Ang Ⅱ.The level of p65 phosphorylation protein in the +Gap19 group was significantly lower than that in the Ang Ⅱ group(P< 0.01),demonstrating that Ang Ⅱ-activated NF-κB(p65)signaling pathway is dependent on Cx43.(6)Ang Ⅱ induced RAW264.7 mouse monocyte macrophages to polarize M1 by activating NF-κB(p65)signaling pathway,and NF-κB(p65)signaling pathway inhibitor BAY117082 can inhibit M1 polarization..The m RNA expressions of M1 markers i NOS,TNF-α,IL-1β,IL-6and CD86 in mouse monocyte macrophages were detected by q RT-PCR.The Ang Ⅱ group was significantly higher than the control group(P<0.01),M1 type polarization-related index was significantly inhibited after pretreatment with inhibitor BAY117082(P<0.01).The protein expressions of M1 markers i NOS and CD86 in mouse monocyte macrophages were detected by Western-Blot assay.The M1 markers TNF-α and IL-1β in MAL264.7 mouse monocyte macrophage supernatant were detected by ELISA.The protein level of IL-6 was changed,and the M1 type index was inhibited after pretreatment with the inhibitor BAY117082(P<0.01).Conclusion: 1.Ang Ⅱ induces macrophage M1 polarization by up-regulating Cx43-activated NF-k B(p65)signaling pathway;2.Gap26/Gap19,a specific blocker of Cx43,inhibits Ang Ⅱ-induced M1 polarization..