Pannexin-1-NLRP3 Pathway Mediates Apelin-13/APJ Promoting Atherosclerosis

Background and objective:Atherosclerosis?AS?is a complex pathological process,and its pathogenesis is still not completely clear.Studies showed that Apelin/APJ system was closely related to the formation of AS.However the specific role and mechanism of Apelin/APJ system in the process of AS are still unclear.The pannexin-1 half-channel-NLRP3 pathway plays an important role in cell proliferation,migration,inflammatory response,oxidative stress and is involved in the formation of AS.The purpose of this study was to investigate whether Apelin-13 mediates vascular smooth muscle proliferation,macrophage inflammatory response,vascular endothelial oxidative stress,monocyteendothelial cell adhesion through the Pannexin-1 half-channel-NLRP3 pathway,and whether Apelin-13 has an effect on the formation of atherosclerosis in ApoE^-/-mice and the role of Pannexin-1 half-channel-NLRP3 pathway in this process.It provides a new research targets for the treatment of AS.Part1 Apelin-13/APJ stimulates the proliferation of Human aortic vascular smooth muscle cells?HA-VSMCs?through the pannexin-1 half-channel-NLRP3 pathwayObjective:Vascular smooth muscle cell?VSMC?proliferation is thought to be closely related to atherosclerosis?AS?.The purpose of this study is to investigate whether Apelin-13 can promote VSMC proliferation and whether pannexin-1 half-channel-NLRP3 pathway mediates the effect of Apelin-13/APJ on HAVSMCs proliferation.Methods:1.HA-VSMCs were treated with Apelin-13 in different concentrations and for different time,and then the proliferation of HA-VSMCs was detected by MTT assay and Brud analysis.2.q RT-PCR and Western Blot were used to detect the expressions of pannexin-1 m RNA and protein after different concentrations Apelin-13 stimulated HA-VSMCs.Immunofluorescence was used to detect the effect of Apelin-13 on the expression of pannexin-1 in HA-VSMCs.q RT-PCR and Western Blot were used to detect the expressions of NLRP3 m RNA and protein after Apelin-13 stimulated HA-VSMCs.3.HA-VSMCs were treated with Apelin-13 in different concentrations,and then the release of ATP was tested by ATP biofluorescence kit.4.HA-VSMCs were pretreated with Probenecid,the effect of Apelin-13 on the expression of pannexin-1,NLRP3 protein in HA-VSMCs was tested by West Blot.5.HA-VSMCs were pretreated with Probenecid,a blocking agent of pannexin-1 half channel,and then the effect of Apelin-13 on the release of ATPin HA-VSMCs was tested by ATP biofluorescence kit and the proliferation of HA-VSMCs were tested by MTT assay and Brud analysis.6.The effect of Apelin-13 on the proliferation of HA-VSMCs was tested after NLRP3 being silenced by si RNA-NLRP3.Results:1.The results of MTT assay and Brud analysis showed that Apelin-13 can stimulate HA-VSMCs proliferation in a concentration?0-10?M?and time?0-72h?dependent manner.2.The results of q RT-PCR and Western Blot showed that Apelin-13 promoted the expression of pannexin-1 m RNA and protein in HA-VSMCs in a concentration-dependent manner?0-10?M?.Immunofluorescence assay showed that Apelin-13 promoted the expression of Pannexin-1 protein in HAVSMCs.3.ATP biofluorescence detection showed that Apelin-13 promoted ATP release in HA-VSMCs in a concentration-dependent manner?0-10?M?.4.The results of q RT-PCR and Western Blot showed that Apelin-13 promoted the expression of NLRP3 m RNA and protein in HA-VSMCs in a concentration-dependent manner?0-10?M?.5.The pretreatment of HA-VSMCs with Probenecid significantly inhibited the expression of pannexin-1 and NLRP3 protein induced by Apelin-13.6.The pretreatment of HA-VSMCs with Probenecid significantly inhibited the release of ATP induced by Apelin-13,and the proliferation of HA-VSMCswas promoted by Apelin-13.7.si RNA-NLRP3 transfection significantly inhibited the expression of NLRP3 protein and the proliferation of HA-VSMCs induced by Apelin-13.Conclusion:Apelin-13 promoted the proliferation of HA-VSMCs.The Pannexin-1 half-channel-NLRP3 pathway mediated the proliferation of HA-VSMCs induced by Apelin-13.After blocking the Pannexin-1 half-channel-NLRP3 pathway,the proliferation of HA-VSMCs induced by Apelin-13 was significantly inhibited.Part2 Apelin-13/APJ stimulates the inflammation reaction of THP-1 through the pannexin-1 half-channel-NLRP3 pathwayObjective: The inflammation reaction induced by macrophage infiltration and the release of inflammatory cytokines plays an important role in the development of AS.The purpose of this study is to investigate whether Apelin-13 promotes the inflammation reaction of THP-1 macrophages,and explore whether the pannexin-1 half-channel-NLRP3 pathway mediates the inflammation reaction.Methods:1.THP-1 was treated with Apelin-13 in different concentrations,and then ELISA was used to detect the secretion of TNF-?,IL-1? and IL-6 in THP-1.2.q RT-PCR and Western Blot were used to detect the expression of pannexin-1 m RNA and protein in THP-1 stimulated by Apelin-13.Immunofluorescence was used to detect the expression of pannexin-1 protein in THP-1 stimulated by Apelin-13.q RT-PCR and Western Blot were used to detect the expressions of NLRP3 m RNA and protein in THP-1 stimulated by Apelin-13.3.THP-1 were treated with Apelin-13 in different concentrations,and then the release of ATP was tested by ATP biofluorescence kit.4.THP-1 were pretreated with Probenecid,the effects of Apelin-13 on the expressions of pannexin-1 and NLRP3 protein in THP-1 were detected by Western Blot.5.THP-1 were pretreated with Probenecid,the effects of Apelin-13 on the secretions of TNF-?,IL-1? and IL-6 were detected,and the influence of Apelin-13 on the release of ATP in THP-1 was detected.6.THP-1 were pretreated with Probenecid,the effects of Apelin-13 on the expressions of pannexin-1 and NLRP3 protein in THP-1 were detected by Western Blot.7.After NLRP3 was silenced by si RNA-NLRP3,the effects of Apelin-13 on the secretions of TNF-?,IL-1? and IL-6 in THP-1 were detected by ELISA.Results:1.The results of ELISA showed that Apelin-13 promoted the secretions of TNF-?,IL-1? and IL-6 in THP-1 in a concentration-dependent manner?0-10?M?.2.The results of q RT-PCR and Western Blot showed that Apelin-13 promoted the expression of pannexin-1 m RNA and protein in THP-1 in a concentration-dependent manner?0-10?M?.Immunofluorescence assay showed that Apelin-13 promoted the expression of Pannexin-1 protein in THP-1.3.ATP biofluorescence detection showed that Apelin-13 promoted ATP release in THP-1 in a concentration-dependent manner?0-10?M?.4.The results of q RT-PCR and Western Blot showed that Apelin-13 promoted the expression of NLRP3 m RNA and protein in THP-1 in a concentration-dependent manner?0-10?M?.5.Probenecid significantly inhibited the expression of pannexin-1 and NLRP3 protein induced by Apelin-13 in THP-1.6.Probenecid significantly inhibited the release of ATP and the secretions of TNF-?,IL-1? and IL-6 in THP-1 induced by Apelin-13.7.Si RNA-NLRP3 transfection significantly inhibited the secretions of TNF-?,IL-1? and IL-6 in THP-1 induced by Apelin-13.Conclusion:Apelin-13 can promote the secretion of IL-1?,IL-6,TNF-? in THP-1 macrophages,and the Pannexin-1 half-channel-NLRP3 pathway is involved in this process.After blocking the pannexin-1 half-channel NLRP3 pathway,inflammatory response of THP-1 macrophages induced by Apelin-13 was significantly inhibited.Part3 Apelin-13/APJ stimulates oxidative stress in endothelial cell and promotes monocyte-endothelial cell adhesion through the pannexin-1 halfchannel-NLRP3 pathwayObjective: Oxidative stress in endothelial cell and monocyte-endothelial cell adhesion play an important role in the process of AS.The purpose of the research is to investigate whether Apelin-13 can stimulate oxidative stress in endothelial cell and promote monocyte-endothelial cell adhesion,and further explore whether the pannexin-1 half-channel-NLRP3 pathway mediates the oxidative stress and monocyte-endothelial cell adhesion induced by Apelin-13/APJ.Methods:1.HUVECs were treated with Apelin-13 in different concentrations,then ROS levels in HUVECs were assayed with DCFH-DA fluorescent probe through flow cytometry.2.q RT-PCR and Western Blot were used to detect the expressions of pannexin-1,NLRP3 m RNA and protein after Apelin-13 stimulated HUVECs.3.HUVECs were treated with Apelin-13 in different concentrations,and then the release of ATP was tested by ATP biofluorescence kit.4.HUVECs were pretreated with Probenecid,then the effect of Apelin-13 on the expressions of pannexin-1 and NLRP3 protein in HUVECs were detected by Western Blot,the effect of Apelin-13 on ROS levels in HUVECs was tested by flow cytometry,the influence of Apelin-13 on the release of ATP in HUVECs was tested by ATP biofluorescence kit.5.After NLRP3 was silenced by si RNA-NLRP3,the effect of Apelin-13 on ROS levels in HUVECs was assayed with DCFH-DA fluorescent probe through flow cytometry6.Monocytes were labeled by Calcein AM,Multiscan Spectrum was used to measure monocyte-endothelial cell adhesion induced by Apelin-13.After pretreatment with Probenecid,monocyte-endothelial cell adhesion induced by Apelin-13 was detected.After NLRP3 was silenced by si RNA-NLRP3,monocyte-endothelial cell adhesion induced by Apelin-13 was detected.Results:1.The results of flow cytometry showed that Apelin-13 promoted ROS production in HUVECs in a concentration-dependent manner?0-10?M?and time-dependent?0-72h?manner.2.The results of q RT-PCR and Western Blot showed that Apelin-13 promoted the expression of pannexin-1 m RNA and protein in HUVECs in a concentration-dependent manner?0-10?M?.3.ATP biofluorescence detection showed that Apelin-13 promoted the release of ATP in HUVECs in a concentration-dependent manner?0-10?M?.4.The results of q RT-PCR and Western Blot showed that Apelin-13promoted the expressions of NLRP3 m RNA and protein in HUVECs in a concentration-dependent manner?0-10?M?.5.The pretreatment of HUVECs with Probenecid significantly inhibited the expressions of pannexin-1 and NLRP3 protein induced by Apelin-13,inhibited ROS production and the release of ATP.6.Si RNA-NLRP3 transfection significantly inhibited ROS production in HUVECs induced by Apelin-13.7.Apelin-13 significantly promoted monocyte-endothelial cell adhesion;Probenecid significantly inhibited monocyte-endothelial cell adhesion induced by Apelin-13;Si RNA-NLRP3 transfection significantly inhibited monocyteendothelial cell adhesion induced by Apelin-13.Conclusion:Apelin-13 can promote the production of ROS in HUVECs and promote the adhesion of monocytes to HUVECs.The Pannexin-1 half-channel-NLRP3 pathway is involved in this process.After blocking the pannexin-1 half-channel NLRP3 pathway,ROS production and monocyte-HUVECs adhesion in HUVECs induced by Apelin-13 was significantly inhibited.Part 4 Apelin 13/APJ promotes ApoE^-/-atherosclerosis in mice through pannexin-1 half-channel-NLRP3 pathwayObjective: To establish an animal model of atherosclerosis?AS?through feeding high-fat diet to ApoE^-/-mice.To observe the effect of different concentrations of Apelin-13 on the formation of AS in ApoE^-/-mice,and further explore whether pannexin-1 half-channel-NLRP3 pathway is involved in Apelin-13 promoting AS in ApoE^-/-mice.Methods:1.Model and experimental groups: male ApoE^-/-mice were randomly divided into 3 groups: model group,low-dose Apelin-13 group?1mg/kg?and high-dose Apelin-13 group?5mg/kg?.2.Movat staining was performed on the paraffin sections of the aortic root of mice in each group,and tissue sections were observed under a biological microscope to calculate the area of AS plaques in each group.3.Verhoeff-Van Gieson staining was performed on the paraffin sections of aortic arch branches and aortic roots of mice in each group,and the staining of plaques was observed under the microscope to calculate the rupture rate and rupture area of AS plaques in each group.4.Immunohistochemistry was used to detect ?-actin and MAC-3 in aorta AS plaques of ApoE^-/-mice.5.Sirius red staining was used to detect collagen fibers in aorta AS plaques.6.Western blot was used to detect the expressions of pannexin-1 and NLRP3 protein in the aorta of ApoE^-/-mice.Results:1.Movat staining showed that Apelin-13 promoted the formation of AS in ApoE^-/-mouse aorta.The damage area of AS in the high-dose Apelin-13 mice was significantly greater than that in the model group?P<0.05?.2.Verhoeff-Van Gieson staining showed that high dose Apelin-13 increased the plaque rupture rate and average rupture area in the aortic root of ApoE^-/-mice?P<0.05?,but had no effect on the rupture rate and average rupture area in the aortic arch branch.3.The results of immunohistochemistry showed that there was no significant difference in the level of ?-actin in the atherosclerotic plaque in each group,and the area of MAC-3 positive reaction area increased in the highdose Apelin-13 group,which was significantly different from the model group and the low-dose Apelin-13 group?P<0.05?.High dose of exogenous Apelin-13 can significantly promote the invasion of macrophages and aggravate the formation of AS plaques in mice.4.The results of sirius red staining showed that there was no significant difference in collagen content in aortic atherosclerotic plaque of each group..5.The results of Western blot showed that Apelin-13 significantly increased the expressions of pannexin-1 and NLRP3 protein in the aorta of ApoE^-/-mice.Conclusion:Apelin-13 promoted the development of AS in ApoE^-/-mouse,significantly increased macrophage infiltration in AS plaques,and The pannexin-1 half-channel NLRP3 pathway may be involved in this process.The General conclusion:Apelin-13/APJ stimulates the proliferation of HA-VSMCs,the inflammation reaction of THP-1,and oxidative stress in endothelial cell and promotes monocyte-endothelial cell adhesion through the pannexin-1 halfchannel-NLRP3 pathway.Apelin-13/APJ may promotes atherosclerosis in ApoE^-/-mice through pannexin-1 half-channel-NLRP3 pathway.