血红素加氧酶-1降低RAW264.7巨噬细胞炎症因子表达的线粒体机制研究

Mitochondrial mechanism of heme oxygenase-1 in reducing expression of inflammatory factors in RAW264.7 macrophage

目的探讨血红素加氧酶-1(HO-1)抑制RAW264.7巨噬细胞肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、活性氧(ROS)合成的线粒体机制。方法体外培养RAW264.7巨噬细胞,将其随机分为空白对照组(C组)、内毒素脂多糖(LPS)造模组(CL组)、血红素(Hemin,HO-1诱导剂)+LPS组(HL组)、锌原卟啉-IX(ZnPP-IX,HO-1拮抗剂)+LPS组(ZL组)、3-甲基腺嘌呤(3-MA,线粒体自噬抑制剂)+LPS组(ML组)、雷帕霉素(RAPA,线粒体自噬促进剂)+LPS组(RL组)共6组。采用蛋白质印迹法(Western blot)检测HO-1、TNF-α、IL-1β、微管相关蛋白1A/1B轻链3B(LC3B)、受体裂变蛋白1(FIS1)、线粒体动力蛋白1(DRP1)相对表达量,并使用ROS检测试剂盒检测ROS含量。结果与C组比较,另5组的HO-1、LC3B、ROS表达量均增加,DRP1表达量均降低,差异有统计学意义(P<0.05);与C组比较,CL组、ZL组和ML组的FIS1表达量均增加,HL组、RL组的FIS1表达量均降低,差异有统计学意义(P<0.05);与C组比较,HL组的IL-1β、TNF-α表达量均降低,差异有统计学意义(P<0.05)。与CL组比较,HL组的LC3B表达量降低,ZL组、ML组和RL组的LC3B表达量均增加,差异有统计学意义(P<0.05);与CL组比较,HL组的TNF-α、IL-1β表达量均降低,RL组的IL-1β表达量增加,TNF-α表达量降低,ML组的TNF-α、IL-1β表达量增加,差异有统计学意义(P<0.05);与CL组比较,HL组、RL组的ROS表达量降低,ZL组、ML组的ROS表达量增加,差异有统计学意义(P<0.05)。HL组的TNF-α、IL-1β、DRP1表达量低于另5组,HO-1表达量高于另5组,差异有统计学意义(P<0.05);RL组的FIS1表达量低于HL组,LC3B表达量高于HL组,差异有统计学意义(P<0.05)。RL组的IL-1β、TNF-α、FIS1、DRP1、ROS表达量低于ML组,LC3B表达量高于ML组,差异有统计学意义(P<0.05)。结论HO-1可抑制RAW264.7巨噬细胞线粒体分裂而降低炎症因子表达,且效果优于线粒体自噬。

Objective To evaluate mitochondrial mechanism of heme oxygenase-1(HO-1)in hibiting the synthesis of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and reactive oxygen species(ROS)in RAW264.7 macrophages.Methods RAW264.7 macrophages were cultured in vitro and randomly divided into six groups including blank control group(group C),lipopolysaccharide(LPS)model group(CL group),hemin(HO-1 inducer)+LPS group(HL group),zinc proporphyrin-IX(ZnPP-IX,HO-1 antagonist)+LPS group(ZL group),3-methyladenine(3-MA,mitochondrial autophagy inhibitor)+LPS group(ML group),rapamycin(RAPA,mitochondrial autophagy promoter)+LPS group(RL group).Western blot was used to detect the relative expression levels of HO-1,TNF-α,IL-1β,microtubule-associated protein 1A/1B light chain 3B(LC3B),mitochondrial fission protein 1(FIS1)and relative quantity dynamin-related protein 1(DRP1),and ROS assay kit was used to detect the ROS content.Results Compared with the group C,the expression levels of HO-1,LC3B and ROS in the other five groups were increased,and the expression level of DRP1 decreased(P<0.05).Compared with the group C,FIS1 expressions in the CL group,ZL group and ML group increased,while FIS1 expression in the HL group and RL group decreased,the differences were statistically significant(P<0.05).Compared with the C group,the expression levels of IL-1β and TNF-α in the HL group were decreased,the differences were statistically significant(P<0.05).Compared with the CL group,LC3B expression in the HL group was decreased,while LC3B expressions in the ZL group,ML group and RL group were increased,the differences were statistically significant(P<0.05).Compared with the CL group,the expression levels of TNF-αand IL-1β in the HL group were decreased,the expression level of IL-1β in the RL group was increased and level of TNF-α was decreased,while the expression levels of TNF-α and IL-1β in the ML group were increased,the differences were statistically significant(P<0.05).Compared with the CL group,ROS expression levels in the HL and RL groups were decreased,while were increased in the ZL and ML groups,the differences were statistically significant(P<0.05).The expression levels of TNF-α,IL-1β and DRP1 in the HL group were lower than those in the other five groups,while the expression level of HO-1 was higher than that in the other five groups,the differences were statistically significant(P<0.05).The expression level of FIS1 in the RL group was lower than that in the HL group,and the expression level of LC3B was higher than that in the HL group(P<0.05).The expression levels of IL-1β,TNF-α,FIS1,DRP1 and ROS in the RL group were lower than those in the ML group,while the expression level of LC3B was higher than those in the ML group,the differences were statistically significant(P<0.05).Conclusion HO-1 can inhibit the mitochondrial division of RAW264.7 macrophages thereby reducing the expression of inflammatory factors,and the effect is better than that of mitochondrial autophagy.