广藿香醇通过下调CD47表达促进巨噬细胞对人胃癌细胞的吞噬作用OA

Objective To investigate the effect of patchouli alcohol(PA)on the phagocytosis of human gastric cancer cells by macro-phages and its underlying mechanism.Methods The mRNA and protein expression levels of CD47 were first assessed in the human normal gastric mucosal epithelial cell line GES-1 and a panel of gastric cancer cell lines(including MKN45,SNU-1,HGC-27,and MKN74)using RT-PCR and Western blot analysis.Next,MKN45 cell lines were selected as the research subject and its proliferative activity treated with different concentrations of PA(0,0.1,0.25,0.5,1,10,20 mmol/L)was evaluated by CCK-8 assay.Subse-quently,MKN45 cells were treated with different concentrations of PA(0,0.25,0.5,1 mmol/L)for 24 h,and the mRNA and protein expression levels of CD47 were detected by RT-PCR and Western blot.M1-type macrophages were generated by differentiating human monocytic THP-1 cells,and then the expression of the M1 macrophage surface markers CD86 and inducible nitric oxide synthase(iNOS)was analyzed by flow cytometry.After transfecting MKN45 cells with a CD47 overexpression plasmid(oe-CD47)or its negative control plasmid(vector),cells were divided into blank,vector,and oe-CD47 groups.Transfection efficiency was verified by detecting CD47 mRNA and protein expression levels via RT-PCR and Western blot 48 hours post-transfection.To assess the effect of PA on phagocytosis,MKN45 cells were first treated with 1 mmol/L PA and then labeled with carboxyfluorescein diacetate succinimidyl ester(CFSE).The labeled cells were co-cultured with M1-type macrophages for 6 hours.The experiment included four groups:control group,PA group,PA+vector group,and PA+oe-CD47 group.Phagocytosis was quantified by flow cytometry,measuring the proportion of double-positive(CD11b+/CFSE+)cells.Results Compared with the GES-1 cell line,the mRNA and protein expression levels of CD47 were significantly elevated in gastric cancer cell lines MKN45,SNU-1,HGC-27,and MKN74,with the highest expression observed in MKN45 cell line(all P<0.05).The proliferation of MKN45 cells was significantly inhibited by PA in a dose-dependent manner(all P<0.05 compared to the untreated control).Compared with 0 mmol/L PA group,the mRNA and protein expression levels of CD47 were significantly decreased in 0.25,0.5,1 mmol/L PA groups(all P<0.05),especially in 1 mmol/L group.After induction,the positive rates for CD86 and iNOS in THP-1 cells were 96.54%and 97.46%,respectively,confirming successful induction of M1-type macrophages.Compared with blank group,CD47 mRNA and protein expression levels were significantly increased in oe-CD47 group(both P<0.05),while no significant difference was observed in vector group(P>0.05),confirming the successful construction of the CD47 overexpression cell model(transfection efficiency=82%).Compared with control group,the CD11b+/CFSE+double-positive rate was significantly increased in PA group(P<0.05).Compared with PA group,the CD11b+/CFSE+double-positive rate was significantly decreased in PA+oe-CD47 group(P<0.05),while no significant difference was found in PA+vector group(P>0.05).Conclusion Patchouli alcohol can enhance macrophage phagocytosis of gastric cancer cells by down-regulating CD47 expression.