ATP6v0d2 Inhibits Lipopolysaccharide-induced IL-1β Expression And Cell Adhesion In Human Endothelial Cells

BACKGROUNDAtherosclerosis is a complicated disease involving multiple risk factors.According to the REPORT ON CARDIOVASCMLAR DISEASES IN CHINA(2018),the prevalence of cardiovascular disease(CVD)in China is continuously rising,it is estimated that about 290 million patients suffer from cardiovascular disease.At present,CVD is the leading cause of death in both urban and rural Chinese population.Numerous studies have suggested that atherosclerosis is associated with chronic inflammatory,activation and dysregulation of inflammatory pathways have been took part in the different stages of atherosclerosis.When the vascular endothelial is stimulated by inflammation,the activated endothelial cells can recruitment of monocytes,and promote monocyte-endothelium adhesion,this is the starting point of atherosclerosis.Interleukin-1β(IL-1β),a multifunctional cytokine,can act on immune cells such as monocytes and dendritic cells as well as nonimmune cell types,including endothelial and smooth muscle cells,which can stimulate the production of colony stimulating factor,platelet growth factor and other cytokines.Hence it can play an important role in cell adhesion and tissue repair.Studies have been reported that lipopolysaccharide(LPS)can upregulate the expression of IL-1β and promote the monocyte-endothelium adhesion.However,the stimuli and cellular mechanisms for such inflammatory remain poorly defined.The vacuolar(H+)ATPase(V-ATPase)is a membrane proton pump,which plays an important role in maintaining homeostasis of internal environment.The d2 isoform of the vacuolar(H+)ATPase V0 domain(ATP6v0d2)is a significant part of the protein.The results show that ATP6v0d2 is mainly distributed in osteoclasts,kidney cells and other tissue cells.It can affect the maturation of osteoclasts by regulating the pH value of osteoclast microenvironment.Meanwhile,Other studies showed that ATP6v0d2 can promote the completion of autophagy via promotion of autophagosome-lysosome fusion through its interaction with STX17 and VAMP8.However,ATP6v0d2 has not been reported to participate in the development of atherosclerosis.Whether ATP6v0d2 can participate in the pathological process of atherosclerosis by mediating LPS induced chronic inflammation,and the possible specific mechanism still need further study.MATERIALS AND METHODSHuman umbilical vein endothelial cells(HUVECs)and the human monocytic THP-1 cells were purchased from the American Type Culture Collection(ATCC).1.HUVECs were treated with 0,0.1,0.5 and 1μg/mL of LPS.After 24 hours,qPCR and Western blot were used to detect the expression levels of ATP6v0d2 and IL-1β.2.Using 1μg/mL of LPS to treat HUVECs at 0,3,6,12 and 24h time points.The mRNA and protein were extracted to detect the expression levels of ATP6v0d2 and IL-1β.3.HUVECs were transfected with ATP6v0d2 small interference fragment,with or without LPS treatment,qPCR and WB were used to detect the transfection efficiency of ATP6v0d2,and the expression levels of IL-1β were detected as well.4.HUVECs were transfected with ATP6v0d2 small interference fragment for 24h,with or without LPS treatment for another 24h,incubating 5h with CFSE green fluorescent labeled THP-1 mononuclear cells,after phosphate buffer saline(PBS)washing,photographing with inverted fluorescence microscope.Using image-j software to count the number of fluorescence points,which was the number of THP-1 mononuclear cells adhered to endothelial cells.RESULTS1.HUVECs treated with different concentrations of LPS,the results showed that LPS could downregulate the expression of ATP6v0d2 in a dose manner.1μg/mL of LPS was used to treat endothelial cells for 0,3,6,12,24h respectively.The results showed that the levels of ATP6v0d2 mRNA and protein expression could be downregulated by LPS in a time dependent manner.2.Different concentrations of LPS were treated to HUVECs,and IL-1β mRNA and protein levels were upregulated in a dose dependent manner.Meanwhile,the levels of mRNA and protein of IL-1β expression were upregulated in a time dependent manner after 0,3,6,12,24h treatment to HUVECs with 1μg/mL LPS.3.Treatment HUVECs with ATP6v0d2 small interfering fragment,the expression of ATP6v0d2 decreased,while the expression of IL-1β increased,and the expression of IL-1β induced by LPS increased more significantly.It is suggested that ATP6v0d2 may inhibit the expression of IL-1β induced by LPS.4.Treatment HUVECs with ATP6v0d2 small interfering fragment could enhance THP-1 monocytes adhesion to endothelial cells,which means ATP6v0d2 can inhibit this effect.CONCLUSIONHerein,we provide reliable evidence that LPS can inhibit the expression of ATP6v0d2.In addition,LPS can induce the expression of IL-1β and the adhesion of monocytes to endothelial cells,which may be inhibited by ATP6v0d2.This study provides a new way to explain the molecular mechanism of LPS induced adhesion between monocytes and endothelial cells.Therefore,ATP6v0d2 may be a potential target used to protect against inflammation-induced adhesion of monocytes to endothelial cells.