S1P/S1PR1通过上调TAM受体和吞噬相关蛋白表达增强巨噬细胞胞葬作用OA

Aim To investigate whether sphingosine-1-phosphate(S1P)promotes LC3-associated phagocytosis via its receptor S1PR1,thereby enhancing the efferocytosis of THP-1-derived macrophages.Methods THP-1 mono-cytes were induced to differentiate into macrophages using 80 μg/L phorbol ester;254 nm ultraviolet radiation was used to induce early apoptosis in Jurkat cells for cell phagocytosis experiments.Macrophages were treated with 0,0.5,1.0 and 1.5 μmol/L S1P for 12 h,or treated with 1.5 μmol/L S1P alone or in combination with S1PR1 antagonist W146,S1PR2 antagonist JTE-013,S1PR3 antagonist CAY10444 and ATG4B inhibitor NSC185058.Western blot was used to detect the expression levels of MerTK,Axl,Tyro3,LC3B and LAMP1,fluorescence-labeled apoptotic cells were applied in phagocy-tosis assays to evaluate phagocytic efficiency,ELISA was employed to measure the levels of pro-inflammatory factors(TNF-α,IL-6 and IL-1β)and anti-inflammatory factors(TGF-β,IL-4 and IL-10)in the supernatant.Results S1P could significantly enhance the phagocytic capacity of macrophages for apoptotic cells,and this effect was concentration-de-pendent.Specifically,the phagocytic efficiency of macrophages for apoptotic cells was significantly improved(P<0.01);the expression of cell burial receptors MerTK,Axl and Tyro3 was markedly upregulated(P<0.01);at the same time,levels of anti-inflammatory factors(TGF-β,IL-4 and IL-10)increased(P<0.01),while pro-inflammatory factors(TNF-α,IL-6 and IL-1β)decreased(P<0.01).The results of the S1P blockade experiment showed that blocking S1PR1 with W146 could reverse the efferocytosis of S1P(P<0.05),while blocking S1PR2 or S1PR3 had no significant effect.S1P treatment significantly increased the gene and protein expression levels of LC3B and LAMP1 in macrophages(P<0.01)and promoted lysosome maturation.After NSC185058 treatment,the phagocytic efficiency and anti-inflammatory factor secretion of macrophages decreased(P<0.05).In addition,the inhibitory effect of blocking S1PR1 and LC3-related phagocytosis on efferocytosis was more significant(P<0.01).Conclusion S1P significantly enhances the phagocytic clearance ability of THP-1-derived macrophages towards apoptotic cells through S1PR1-mediated LC3-related phagocytosis,and promotes the transformation of macrophages into anti-inflammatory phenotypes.