Effect Of Pannexin1 On LXR Mediated Macrophage Phagocytosis Of Ox-LDL And Its First Exploration Of Molecular Mechanism

Atherosclerosis(Atherosclerosis,AS)is a multi-factor induced by chronic vascular inflammatory disease.In the pathogenesis of AS,macrophages turn into foam cells,foam cell cause the formation of atherosclerosis plaque.Plaque instability and platelet accumulation can lead to ischemic stroke or myocardial infarction,plaque rupture and thrombosis,cause the interruption of blood flow,which is a serious threat to human health.This suggests that lipid metabolism plays an important role in the occurrence of AS.It has been found that the activation of liver X receptor(LXR)can reduce the formation of AS plaque,the activation of LXR can induce cells to release adenosine triphosphate(ATP),but the specific mechanism is not clear.Pannexin1 membrane channel protein oligomers formed allowing small molecules to pass through after the opening,which ATP released through the channel.But the Pannexin1 channel is involved in macrophage lipid formation of foam cells has not been reported.Therefore,this paper investigates whether Pannexin1 channels are involved THP-1 derived macrophage uptake of oxidized low density lipoprotein(oxidized low density lipoprotein,ox-LDL)process induced by LXR and clarify its mechanism,which is expected to open up new idea for the prevention and treatment of atherosclerosis.Methods:1.By phorbol ester(Phorbol 12-myristate,13-acetate,PMA)THP-1 as thedifferentiation of THP-1 derived macrophages,and the expression of cell morphology and CD14 m RNA on macrophages were identified.2.The expression of Pannexin-1 and LXR m RNA in THP-1 derived macrophages was detected by RT-PCR technique.3.Using the Western Blot technique to detect the expression of LXR? and LXR?before and after GW3965 stimulation.4.The fluorescence microscope with fluorescence images was taken before and after the GW3965 Pannexin1 channel inhibitor THP-1 derived macrophages by uptake of Dil-ox-LDL,and using image analysis software(Image-pro plus V6.0)on the fluorescence image analysis,the number of cells was calculated corresponding to the picture,with changes to measure the cellular uptake amount of Dil-ox-LDL.5.Using the luciferase method and RT-PCR technique to detect the ATP release and the expression of ABCA1 before and after GW3965 stimulation and the application of Pannexin-1 inhibitors.Results:1.THP-1 induced differentiation of PMA cells treated with 160 n M after 24 hours,the cell morphology by differentiation of the previous round,suspended growth into the shuttle or spindle type,and a large number of pseudopodia,adherent growth.The results show the expression of CD14 m RNA was up-regulated in THP-1 derived macrophages,THP-1 cells compared with significant difference(p<0.001).2.The activation of LXR can induce the expression of LXR alpha and beta,when using GW3965 to stimulate THP-1 derived macrophages,it can be found that the expression of LXR alpha and beta can be significantly increased,the difference was statistically significant(p<0.001).3.THP-1 derived macrophages was stimulated by GW3965 in different time(0min,5min,10 min,20min,30min),ATP content in GW3965 stimulation group ateach time point of the release of cells increased significantly compared with the control group(p<0.001),and GW3965 reached the peak at 10 minutes.when cells were pretreated with Pannexin1 antagonist 10Panx1(200?M)for 30 minutes,The results show that the release amount of ATP was significantly decreased compared with GW3965 stimulation groupd,the difference was statistically significant(p<0.01).4.The activation of LXR can induce the expression of ABCA1,when cells were pretreated with Pannexin1 antagonist 10Panx1(200?M)for 30 minutes,It found that10Panx1 can significantly inhibit the expression of ABCA1 induced by GW3965,the difference was statistically significant(p<0.01).5.The activation of LXR can induce the expression of LXR??LXR?,when cells were pretreated with Pannexin1 inhibitor 10Panx1(200?M)for 30 minutes,It found that 10Panx1 can significantly inhibit the expression of LXR? ? LXR? induced by GW3965,the difference was statistically significant(p<0.01).Conclusion:1 Pannexin1 channel inhibitor(10Panx1)can inhibit the release of ATP by LXR agonist(GW3965)in THP-1 macrophages.2 Pannexin1 channel inhibitor(10Panx1)inhibited the ability of LXR agonist(GW3965)to induce macrophage uptake of ox-LDL.3 Pannexin1 channel inhibitor(10Panx1)inhibited the expression of ABCA1 induced by LXR(GW3965)in macrophage...