S-亚硝基-N-乙酰-DL-青霉胺对RAW264.7巨噬细胞亚型分化的影响

目的:探讨S-亚硝基-N-乙酰-DL-青霉胺(SNAP)对巨噬细胞亚型分化的影响及其机制。方法:以RAW264.7巨噬细胞为研究对象,分为空白对照组、SNAP组、SNAP+PBA(4-苯基丁酸)组,采用不同浓度(30、100、300、400、500μmol/L)的SNAP或300μmol/L SNAP+20 mmol/L PBA对巨噬细胞进行干预24 h,应用RT-PCR法检测RAW264.7巨噬细胞亚型分化标志物M1(iNOS,CD86)、M2(Arg-I,MR)及CHOP mRNA的表达,应用Western blot技术检测iNOS及ERS通路中相关蛋白CHOP、P-PERK的表达。结果:与空白对照组比较,SNAP组iNOS、CD86、CHOPmRNA的表达均明显降低(P0.05),Arg-ImRNA表达明显升高(P0.05),而MR mRNA表达升高,但差异无统计学意义(P0.05);与300μmol/L SNAP组比较,300μmol/L+PBA组iNOS、CHOP mRNA均无明显变化(P0.05),CD86 mRNA升高,Arg-I、MR mRNA均明显降低(P0.05)。SNAP组CHOP、iNOS、p-PERK蛋白表达均明显低于对照组(P0.05),300μmol/LSNAP+20 mmol/LPBA组与300μmol/LSNAP组比较iNOS蛋白、p-PERK、CHOP蛋白表达升高(P0.05)。结论:NO可能通过内质网应激机制抑制巨噬细胞向M1亚型分化。

Effect of S-nitroso-N-acetyl-DL-penicillamine (SNAP) on the Subtype Differentiation of RAW264.7 Macrophages

Objective:To investigate the effect and mechanism of SNAP on the subtype differentiation of RAW264.7 macrophages.Methods:RAW264.7 macrophages were plated in 12 wells plate as 106/ml for 24 h before intervention of SNAP in different concentration (30, 100, 300, 400 and 500 umol/L) or 300 umol/L SNAP+20 mmol/L PBA. Total RNA of cells were extracted after intervention for 24 hours. The mRNA expression of phonetype marker iNOS, CD86 (as M1 phenotypes markers), MR, and Arginase-I (Arg-I) (as M2 phenotypes markers) were detected respectively by real time PCR. The protein expression of CHOP, p-PERK were detected by western blotting.Results:Compared with the control group, the iNOS, CD86 and CHOP mRNA expression significantly decreased(P<0.05), Arg-I mRNA expression increased, but no significant difference was found(P>0.05). Compared with 300 umol/L SNAP group, no remarkable difference was found in iNOS, CHOP mRNA expression of 300 umol/L+PBA group, but Arg-I, MR mRNA expression both significantly decreased (P<0.05). The protein expression of CHOP, iNOS and p-PERK of SNAP group were all significantly lower than those of the control group (P<0.05), compared with 300 umol/L SNAP group, the protein expression of CHOP, p-PERK, iNOS increased.Conclusion:SNAP could suppress macrophage differentiation to M1 subtype through endoplasmic reticulum stress (ERS).