大麻素2型受体基因过表达与小鼠巨噬细胞焦亡的关系

Relationship between over-expression of endocannabinoid receptor 2 and macrophage pyroptosis in mice

目的评价大麻素2型受体(CB2R)基因过表达与小鼠巨噬细胞焦亡的关系。方法利用慢病毒转染小鼠骨髓源性巨噬细胞,制备稳定细胞系:慢病毒LV5阴性对照细胞系LV5-NC、慢病毒LV5CB2R基因过表达细胞系LV5-CB2R-OE(OE)。采用随机数字表法将2种细胞系分别分为3组(n=18):对照组(LV5-NC-C组、OE-C组)、LPS/ATP组(LV5-NC-LPS/ATP组、OE-LPS/ATP组)和CB2R特异性激动剂HU308组(LV5-NC-HU308组、OE-HU308组)。对照组细胞正常培养,LPS/ATP组先加入终浓度为0.5μg/ml LPS孵育5 h后,加入5 mmol/L的ATP孵育1 h。LPS/ATP+HU308组加入终浓度为0.5μg/ml的LPS和10μmol/L的HU308共孵育5 h,再给予浓度为5 mmol/L的ATP孵育1 h。采用RT-PCR法检测CB2R、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、caspase-1和消皮素D(GSDMD)的mRNA表达,采用Western blot法检测caspase-1表达,采用ELISA法检测培养液IL-18和IL-1β的浓度。结果2种细胞系中与对照组比较,LPS/ATP组NLRP3、caspase-1和GSDMD的mRNA表达上调,IL-18和IL-1β浓度升高(P<0.05);与LPS/ATP组比较,HU308组NLRP3、caspase-1和GSDMD的mRNA表达下调,IL-18和IL-1β浓度降低(P<0.05)。V5-NC-C组与OE-C组比较、LV5-NC-LPS/ATP组与OE-LPS/ATP组比较、LV5-NC-HU308组与OE-HU308组比较,上述指标差异无统计学意义(P>0.05)。结论CB2R基因过表达并不能有效抑制小鼠巨噬细胞焦亡的发生,只有激活CB2R才可抑制。

Objective To evaluate the relationship between the over-expression of endocannabinoid receptor 2(CB2R)and macrophage pyroptosis in mice.Methods Bone marrow-derived macrophages of mice were transfected by lentivirus vector and successfully screened out two stable cell lines:lentivirus LV5 negative control cells(LV5-NC)and lentivirus LV5CB2R overexpressing cells(OE).Two cell lines were respectively divided into 3 groups(n=18 each)using a random number table method:control group(LV5-NC-C group,OE-C group),LPS/ATP group(LV5-NC-LPS/ATP group,OE-LPS/ATP group)and CB2R specific agonist HU308 group(LV5-NC-HU308 group,OE-HU308 group).Cells in group C were commonly cultured.In LPS/ATP group,cells were incubated with LPS at a final concentration of 0.5μg/ml for 5 h,and then incubated with ATP at the final concentration of 5 mmol/L for 1 h.In group LPS/ATP+HU308,cells were incubated for 5 h with LPS at the final concentration of 0.5μg/ml and HU308 at the final concentration of 10μmol/L and then with ATP at the final concentration of 5 mmol/L for 1 h.The expression of CB2R,nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3(NLRP3),caspase-1,and gasdermin D(GSDMD)mRNA was detected by real-time polymerase chain reaction,the expression of caspase-1 was detected by Western blot,and the concentrations of interleukin-18(IL-18)and IL-1βin the culture medium were determined by enzyme-linked immunosorbent assay.Results In each cell line,compared with group C,the expression of NLRP3,caspase-1 and GSDMD was significantly up-regulated,and the concentrations of IL-18 and IL-1βwere increased in group LPS/ATP(P<0.05).Compared with group LPS/ATP,the expression of NLRP3,caspase-1 and GSDMD was significantly down-regulated,the concentrations of IL-18 and IL-lβwere decreased in group HU308(P<0.05).There was no significant differences in the indicators mentioned above between group V5-NC-C and group OE-C,between group LV5-NC-LPS/ATP and group OE-LPS/ATP,and between group LV5-NC-HU308 and OE-HU308(P>0.05).Conclusion Over-expression of CB2R gene cannot effectively inhibit the occurrence of macrophage pyroptosis,and only activation of CB2R can inhibit it in mice.