2型髓系细胞触发受体在小鼠肺缺血/再灌注损伤中的作用及调控机制

Role and regulatory mechanism of triggering receptor expressed on myeloid cells 2 in mice lung ischemia/reperfusion injury

目的探讨2型髓系细胞触发受体(TREM2)在小鼠肺缺血/再灌注损伤(LIRI)中的作用及其可能的调控机制。方法将36只健康雄性C57BL/6小鼠按照随机数字表法分成正常对照组及LIRI 2、6、12、24、48 h组,每组6只。采用开胸夹闭左肺肺门法建立LIRI模型。取各组小鼠左肺组织,测定肺湿/干质量比值(W/D),苏木素-伊红(HE)染色后光镜下观察肺组织病理学改变,透射电镜下观察肺组织超微结构改变;用酶联免疫吸附试验(ELISA)检测肺组织白细胞介素(IL-1β、IL-18)水平;用聚合酶链反应(PCR)检测肺组织TREM2和天冬氨酸特异性半胱氨酸蛋白酶1(caspase-1)的mRNA表达;用蛋白质免疫印迹试验(Western blotting)检测肺组织TREM2、caspase-1、细胞焦亡标志成孔蛋白Gasdermin-D(GSDMD)的蛋白表达。结果LIRI后2 h模型小鼠肺组织开始出现损伤,肺超微结构改变,肺组织病理学评分增加;6 h时肺损伤程度最为严重;12 h后肺损伤逐渐减轻,肺组织病理学评分逐渐降低。与正常对照组比较,LIRI 2、6、12、24、48 h组肺W/D比值和肺组织病理学评分均明显增高,差异均有统计学意义〔肺W/D比值:7.06±0.52、8.34±0.17、6.42±0.35、5.34±0.25、5.59±0.45比4.69±0.23,肺组织病理学评分(分):5.50±0.54、9.75±0.89、5.88±0.84、3.63±0.74、4.13±0.64比1.13±0.35,均P<0.05〕。与正常对照组比较,LIRI后2 h肺组织IL-1β、IL-18水平即明显升高,于6 h达峰值〔IL-1β(ng/L):502.76±12.25比56.50±8.07,IL-18(ng/L):414.02±10.75比81.63±5.29,均P<0.05〕,随后逐渐降低,48 h时仍显著高于正常对照组。PCR和Western blotting结果显示,LIRI后2 h模型小鼠TREM2表达即明显低于正常对照组,于6 h达谷值〔TREM2 mRNA(2^(-ΔΔCt)):0.47±0.05比1.02±0.05,TREM2/GAPDH:0.23±0.13比0.48±0.17,均P<0.05〕,随后逐渐升高,于24 h达峰值〔TREM2 mRNA(2^(-ΔΔCt)):3.98±0.15比1.02±0.05,TREM2/GAPDH:0.71±0.17比0.48±0.17,均P<0.05〕;而caspase-1和GSDMD的表达趋势与TREM2相反,呈先升高后降低趋势,均于再灌注后6 h达到高峰〔caspase-1 mRNA(2^(-ΔΔCt)):2.20±0.13比1.01±0.02,caspase-1/GAPDH:0.64±0.02比0.20±0.06,GSDMD/GAPDH:1.23±0.01比0.87±0.02,均P<0.05〕。结论TREM2可能参与了小鼠LIRI的发生,其机制可能与TREM2影响caspase-1介导的肺内细胞焦亡有关。

Objective To investigate the role and regulatory mechanism of triggering receptor expressed on myeloid cell 2(TREM2)in mice lung ischemia/reperfusion injury(LIRI).Methods Thirty-six healthy male C57BL/6 mice were divided into six groups according to the random number method(n=6):normal control group,and LIRI 2,6,12,24,48 hours group.Mice LIRI models were established by clamping the left hilum.The wet/dry weight ratio(W/D)of left lung tissue was measured.Lung injury was observed and evaluated by hematoxylin-eosin(HE)staining and electron microscopy.The levels of interleukins(IL-1β,IL-18)in lung tissue were detected by enzyme linked immunosorbent assay(ELISA).The mRNA expressions of TREM2 and caspase-1 were determined by polymerase chain reaction(PCR).The protein expressions of TREM2,caspase-1,Gasdermin-D(GSDMD)were determined by Western blotting.Results At 2 hours after LIRI,lung injury began to appear,the lung ultrastructure changed,and the lung injury score increased;at 6 hours,the degree of lung injury was the most serious;after 12 hours,the lung injury gradually reduced and the lung injury score gradually decreased.Compared with the normal control group,lung W/D ratio and lung injury score of LIRI 2,6,12,24,48 hours groups were significantly higher,the differences were statistically significant(lung W/D ratio:7.06±0.52,8.34±0.17,6.42±0.35,5.34±0.25,5.59±0.45 vs.4.69±0.23;lung injury score:5.50±0.54,9.75±0.89,5.88±0.84,3.63±0.74,4.13±0.64 vs.1.13±0.35,all P<0.05).Compared with the normal control group,the levels of IL-1βand IL-18 in lung tissue were significantly increased at 2 hours after LIRI,reached a peak at 6 hours[IL-1β(ng/L):502.76±12.25 vs.56.50±8.07,IL-18(ng/L):414.02±10.75 vs.81.63±5.29,both P<0.05],then decreased gradually,and were still significantly higher than the normal control group at 48 hours.The PCR and Western blotting showed that the expression of TREM2 was significantly lower than that in the normal control group at 2 hours after LIRI,and reached a valley at 6 hours[TREM2 mRNA(2^(-ΔΔCt)):0.47±0.05 vs.1.02±0.05,TREM2/GAPDH:0.23±0.13 vs.0.48±0.17,both P<0.05],then gradually increased,and reached the peak at 24 hours[TREM2 mRNA(2^(-ΔΔCt)):3.98±0.15 vs.1.02±0.05,TREM2/GAPDH:0.71±0.17 vs.0.48±0.17,both P<0.05].The trend of expression of caspase-1 and GSDMD were opposite to that of TREM2,which increased at first and then decreased,and reached a peak at 6 hours after reperfusion[caspase-1 mRNA(2^(-ΔΔCt)):2.20±0.13 vs.1.01±0.02,caspase-1/GAPDH:0.64±0.02 vs.0.20±0.06,GSDMD/GAPDH:1.23±0.01 vs.0.87±0.02,all P<0.05].Conclusions TREM2 might be involved in LIRI in mice.The mechanism may be related to the effect of TREM2 on caspase-1-mediated pyroptosis.