The Function Of Pannexin-1 Channel In LPS-induced THP-1-derived Macrophage ATP Release And Lipid Deposition

Objective:Atherosclerosis(AS)is a multifactorial chronic inflammatory diseases of the vessel wall of medium to large-sized arteries.The diseases seriously affect on people's health and quality of life.It has become the leading cause of cardiovascular disease.Channel proteins as important elements to maintaining normal cell life activity,It is closely related to the development and progression of atherosclerosis.Pannexins(Panx)family of proteins is a membrane channel protein,where Pannexin-1 protein is the the main form of expression in tissues and cells.Studies suggest that Pannexin-1 oligomers form membrane channels.When channels were opened,which allows molecular weight of less than 1kDa ions or molecules to pass through,including adenosine triphosphate(ATP)and some other small molecules.ATP is an important cell signaling molecules,it was also plays a vital role in the development and progression of atherosclerosis,such as macrophages for release inflammatory cytokines,regulating macrophages for lipid transport,impacting foam cell formation process.However,Pannexin-1 channel involved lipopolysaccharide(LPS)-induced macrophage ATP release has not been stated.Therefore,this project aims to investigate whether Pannexin-1 channel could regulate the release of ATP from LPS-induced macrophage and the involvement of macrophage cells uptake oxidized low density lipoproteins(ox-LDL)in the release mechanism in order to develop a new ideas for the prevention and treatment of atherosclerotic disease.Method:(1)To change the differentiation of THP-1 monocytes into THP-1-derived macrophages,cells were incubated with Phorbol 12-myristate 13-acetate(PMA).To further ensure that PMA was sufficient for the differentiation process,we observed of morphological changes and analyzed CD 14 mRNA expression levels.(2)Using RT-PCR and qPCR detection the expression levels of Pannexin-1 mRNA in THP-1 cell and THP-1-derived macrophages,and further analyzed the effect of LPS stimulating on the expression of Pannexin-1 mRNA.(3)Using fluorescein enzyme assay for determining cells release ATP content after LPS stimulation and Pannexin-1 channel inhibitor.(4)Cellular uptake of oxLDL was measured by using fluorescent microscope,and image analysis software(Image-pro plus v6.0)was used to analyze the fluorescence images,calculate the average fluorescence intensity of the corresponding picture,and further to assess the capacity of the macrophages binding to Dil-oxLDL.Results:(1)THP-1 cell morphology is round and suspended growth.Then the cells incubated with PMA for 24 hours.Cells cultured into a spindle or spindle-shaped,and has a large number of pseudopodia to appear,was suspended and attached growth.Genetic testing showed that CD 14 mRNA expression levels of THP-1-derived macrophage were significantly raised,there was significant difference compared with the THP-1 cell(**P<0.01).RT-PCR and qPCR showed that a large number of Pannexin-1 mRNA expression in THP-1 cells.After cells were incubated with PMA,Pannexin-1 mRNA expression of THP-1-derived macrophages was increased significantly compared with the control group(**P<0.01).Futhermore,after cells induced by 1?g/mL LPS stimulation for 4 h,Pannexin-1 mRNA expression was further increased by LPS stimulation(##P<0.01).(2)THP-1-derived macrophages were stimulated with LPS(1?g/mL)for the indicated times(Omin?5min?10min?15min?20min?25min?30min).LPS stimulation group,at each time point,cells releasing ATP content was increased significantly compared with the control group(**P<0.01)and was peaked at LPS stimulation for 5 minutes;When pretreatment of cells with the Pannexin-1 channel blocker carbenoxolone(CBX;100?M)for 30 minutes,and then cells was stimulated with LPS for 5 minutes.ATP releasing content was detected.Results showed that ATP releasing content using CBX cells was significantly reduced after LPS stimulation(**P<0.01).Similarly,the specific blocker of Pannexin-1 channel(10Panx1;200 ?m)pretreatment of cells for 30 minutes,the results showed that 10Panx1 significantly inhibited ATP release from LPS stimulation,there was a statistically significant difference(##P<0.01).(3)Macrophage phagocytosis experiment results showed that 1?g/mL LPS stimulation of THP-1-derived macrophages for 4 hours,can greatly promote the cells uptake of Dil-ox-LDL,there was a statistically significant difference compared with the control group(**P<0.01);When Pannexin-1 channel blockers CBX(100?M)or lOPanxl(200?M)pretreatment of cells for 30 minutes,then cells were stimulated with LPS for 4 hours.The results showed that CBX(100?M)or lOPanxl(200?M)can weaken cell uptake of Dil-ox-LDL,with significant differences compared to LPS stimulation group(##P<0.05).Conclusion:(1)Pannexin-1 involved in the regulation of LPS-induced THP-1-derived macrophage ATP release process;(2)Pannexin-1 channel adjustable LPS induced macrophage uptake ox-LDL.