| [Background and objective]Atherosclerosis(AS)lesions are often accompanied with acidification(pH<7.0),characterized by tissue acidification within the intima area of the vessels.The range of pH in atherosclerotic lesions is connected with lipid accumulation,and the lowest pH is often occurred in the lipid core plaques.Acid-sensing ion channel 1(ASIC1)is a critical H~+receptor,which is responsible for the perception and transduction of extracellular acidity signals.Our previous studies found that extracellular acidification promotes foam cell formation by inhibiting cholesterol efflux from macrophages through activating ASIC1,but the mechanism remains to be further elucidated.In this study,we constructed a macrophage-derived lipid accumulation cell model induced by extracellular acidification to explore the effects of extracellular acidification on macrophage lipophagy and its mechanism,and further to confirm whether extracellular acidification inhibits macrophage-mediated lipophagy by activating the ASIC1/RIP1 pathway and promoting TFEB phosphorylation,thereby reducing intracellular cholesterol efflux and promoting lipid accumulation.This study will reveal a novel mechanism by which extracellular acidification facilitating foam cell formation from the view of ASIC1 regulating lipophagy of macrophges,and also provide a new potential target for preventing atherosclerotic diseases.[Methods]First,in this project,RAW 264.7 macrophages were incubated with ox-LDL 25μg/m L in an acidic medium of pH 6.5 for 24 h to build macrophage-derived foam cell models.Experimental grouping:pH 7.4,pH 6.5,pH 7.4+ox-LDL,pH 6.5+ox-LDL.Western blotting(WB)was used to detect ASIC1,RIP1,p-RIP1,p-TFEB,LC3,p62 protein expressions,immunofluorescence was used to detect ASIC1 membrane translocation;oil red O staining was used to detect intracellular lipid accumulation.Second,RAW264.7 cells were cultured in the medium with or without PcTx-1(ASIC1 specific inhibitor)or Nec-1(RIP1 inhibitor)for 24 h.Experimental groups:pH 7.4+ox-LDL,pH 6.5+ox-LDL,pH 6.5+ox-LDL+PcTx-1,pH 6.5+ox-LDL+Nec-1.The expressions of ASIC1,RIP1,p-RIP1,p-TFEB,LC3 and p62 were measured by WB.Intracellular lipid accumulation in the cells is observed by oil red O staining.The co-localization of lipids with autophagosomes and lysosome was evaluated by immunofluorescence.Morphological changes of lipophagy flux in the cells were observed by using transmission electron microscopy.ABCA1-mediated cholesterol efflux was determined by cholesterol fluorescence kits.[Results]1.Extracellular acidification promotes lipid accumulation in macrophages.The oil red O experiment results showed that compared with pH 7.4+ox-LDL group,the intracellular lipid accumulation in the pH 6.5+ox-LDL treatment group was significantly increased.These results indicate that extracellular acidification promotes foam cell formation.2.Extracellular acidification blocks the lipophagy flux of macrophages.WB results showed that the protein levels of LC3Ⅱ/LC3Ⅰin the acidified environment treatment group decreased significantly,and the expression of p62 increased.3.Extracellular acidification promotes ASIC1 expression and its membrane translocation.WB results showed that compared with the normal pH(pH 7.4)group,ASIC1 protein expression of macrophages was increased after cultured with pH 6.5 solution.The results of immunofluorescence staining showed that the ASIC1 protein in the normal pH group was mainly located in nucleus,and the ASIC1 protein in the pH 6.5 group mainly located in the plasma membrane of the cell.4.Extracellular acidification induces phosphorylation of RIP1 and phosphorylation of TFEB.WB results showed that RIP1 phosphorylation and TFEB phosphorylation levels increased in the acidified environment treatment groups.5.The phosphorylation of TFEB is promoted by activating ASIC1/RIP1pathway under extracellular acidification conditions.Intervention treatment with PcTx-1 and Nec-1 abolished ASIC1 membrane translocation,RIP1 and TFEB phosphorylation induced by extracellular acidification.WB results showed that the p-RIP1 and p-TFEB protein levels in the ASIC1 specific inhibitor PcTx-1 treatment group were significantly reduced;and the phosphorylation level of TFEB were decreased in the RIP1 inhibitor Nec-1treatment group.6.Intracellular lipid accumulation is significantly reduced after treating with PcTx-1 and Nec-1.The results of oil red O staining showed that the lipid accumulation of macrophages in the PcTx-1 and Nec-1 groups was significantly reduced as compared with the control group.These results indicate that inhibiting the ASIC1/RIP1 pathway can antagonize the effect of extracellular acidification on foam cell formation.7.The intervention of PcTx-1 and Nec-1 can significantly promote the lipophagy flux of macrophages induced by extracellular acidification.WB showed that compared with the control,the expression of LC3Ⅱ/LC3Ⅰis increased,and the expression of p62 protein is decreased after treatment with PcTx-1 and Nec-1.The results of transmission electron microscopy showed that compared with the control group,the number of autophagosomes and lysosomes containing lipid droplets in PcTx-1 and Nec-1 groups were increased,while the lipid droplets were significantly reduced.The results of immunofluorescence co-localization showed that the co-localization of lipid droplets with LC3 and LAMP1 both in the PcTx-1 and Nec-1 was increased as compared with the control group,respectively.These results indicate that inhibition of the ASIC1/RIP1 pathway can reverse the inhibition of macrophage lipophagy flux by extracellular acidification.8.PcTx-1or Nec-1 treatment significantly promotes cholesterol efflux from macrophage foam cells.The results of cholesterol fluorescence kits showed that ABCA1-mediated cholesterol efflux increased after treating with ASIC1inhibitor PcTx-1 or RIP1 inhibitor Nec-1.[Conclusions]1.Extracellular acidification inhibits lipophagy in macrophages.2.Extracellular acidification promotes phosphorylation of TFEB by activating ASIC1/RIP1 and inhibits macrophage lipophagy. |
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