时亚欣, 王晓艳, 陈颖, 寇杰, 宋瑞瑞, 李清钊, 沈福海, 金玉兰. 三甲基腺嘌呤和雷帕霉素对染尘大鼠肺巨噬细胞自噬及凋亡的影响[J]. 环境与职业医学, 2015, 32(10): 914-920. DOI: 10.13213/j.cnki.jeom.2015.14743
引用本文: 时亚欣, 王晓艳, 陈颖, 寇杰, 宋瑞瑞, 李清钊, 沈福海, 金玉兰. 三甲基腺嘌呤和雷帕霉素对染尘大鼠肺巨噬细胞自噬及凋亡的影响[J]. 环境与职业医学, 2015, 32(10): 914-920. DOI: 10.13213/j.cnki.jeom.2015.14743
SHI Ya-xin , WANG Xiao-yan , CHEN Ying , KOU Jie , SONG Rui-rui , LI Qing-zhao , SHEN Fu-hai , JIN Yu-lan . Effects on Autophagy and Apoptosis of Lung Macrophages in Dust Exposed Rats of 3-Methyladenine and Rapamycin[J]. Journal of Environmental and Occupational Medicine, 2015, 32(10): 914-920. DOI: 10.13213/j.cnki.jeom.2015.14743
Citation: SHI Ya-xin , WANG Xiao-yan , CHEN Ying , KOU Jie , SONG Rui-rui , LI Qing-zhao , SHEN Fu-hai , JIN Yu-lan . Effects on Autophagy and Apoptosis of Lung Macrophages in Dust Exposed Rats of 3-Methyladenine and Rapamycin[J]. Journal of Environmental and Occupational Medicine, 2015, 32(10): 914-920. DOI: 10.13213/j.cnki.jeom.2015.14743

三甲基腺嘌呤和雷帕霉素对染尘大鼠肺巨噬细胞自噬及凋亡的影响

Effects on Autophagy and Apoptosis of Lung Macrophages in Dust Exposed Rats of 3-Methyladenine and Rapamycin

  • 摘要: 目的 观察三甲基腺嘌呤(3-MA)和雷帕霉素对染尘大鼠肺巨噬细胞自噬活动的调控以及对细胞凋亡的影响。

    方法 120 只雄性Wistar 大鼠均采取非暴露气管滴注的方法一次性灌注50 mg/mL SiO2 粉尘进行染毒, 然后随机分为3 组:对照组、3-MA组和雷帕霉素组。其中后两组以1.5 mg/(kg& #183;d)和2 μg/(kg& #183;d)的剂量腹腔注射3-MA或雷帕霉素, 隔日给药持续28 d, 3-MA组和雷帕霉素组大鼠分别在染毒7、14、28、60、90 d 处死, 收集其肺灌洗液, 离心获取肺巨噬细胞, 测定其凋亡率。采用蛋白免疫印迹方法检测自噬标志性蛋白微管相关蛋白1 轻链3(LC3)及自噬调控蛋白Beclin1。

    结果 雷帕霉素组大鼠染尘不同时间LC3 蛋白表达均增加且比对照组高;5 个观察时点大鼠肺巨噬细胞凋亡率分别为(1.74& #177;0.73)%、(1.25& #177;0.35)%、(1.44& #177;1.24)%、(3.70& #177;1.51)%、(36.98& #177;11.91)%与对照组(5.63& #177;1.01)%、(4.95& #177;0.63)%、(4.10& #177;0.43)%、(6.75& #177;4.19)%、(30.23& #177;11.05%)相比, 除90 d 高于对照组低(P < 0.05), 其他各时期均比对照组低;3-MA组在给药期间LC3 蛋白表达比对照组低, 停药后蛋白表达有所增高, 90 d 时蛋白表达水平与对照组接近;而该组肺巨噬细胞的凋亡率为(2.86& #177;1.13)%、(8.80& #177;0.28)%、(6.84& #177;1.29)%、(2.54& #177;1.65)%、(37.22& #177;16.22)%, 7 d 时凋亡率低于其他时期;14 d 和28 d 时凋亡率增加且比同时期对照组高(P < 0.05);60 d 时凋亡率再次降低;90 d 时凋亡率与对照组无明显差异。

    结论 3-MA和雷帕霉素干预后, 染尘大鼠自噬活动强弱不同, 且与肺巨噬细胞的凋亡率呈负相关。

     

    Abstract: Objective To observe the effects of 3-methyladenine (3-MA) and rapamycin on autophagy activity and cell apoptosis of dust exposed rat pulmonary macrophages.

    Methods A total of 120 male Wistar rats were administered with 50 mg/mL SiO2 dust by indotracheal intubation and divided into control, 3-MA, and rapamycin groups. The 3-MA and rapamycin groups were intraperitoneally injected with 1.5 mg/(kg& #183;d) 3-MA and 2 μg/(kg& #183;d) rapamycin every other day, for 28 d. Then the rats were neutralized on day 7, 14, 28, 60, and 90 to collect lung lavage followed by determining apoptosis rate of macrophages after centrifugation. Western blotting was used to detect the expression of autophagy protein light chain 3 (LC3) and autophagy regulatory protein Beclin1.

    Results In the rapamycin group, the LC3 protein expressions at various time points were highter as compared with the control group; the early apoptosis rates of pulmonary macrophage sampled at five selected time points were lower for the former four time points but higher for the latter time point than the corresponding values in the control group (1.74& #177;0.73)%, (1.25& #177; 0.35)%, (1.44& #177;1.24)%, (3.70& #177;1.51)% and (36.98& #177;11.91)% vs.(5.63& #177;1.01)%, (4.95& #177;0.63)%, (4.10& #177;0.43)%, (6.75& #177;4.19)%, and (30.23& #177;11.05)%, respectively, all Ps < 0.0.5 except for day 90. In the 3-MA group, the LC3 protein expression levels were lower than those of the control group during exposure, but increased when the drug stopped, and returned to the level of the control group on day 90. In the 3-MA group, the early apoptosis rates of pulmonary macrophage were (2.86& #177;1.13)%, (8.80& #177;0.28)%, (6.84& #177;1.29)%, (2.54& #177;1.65)%, and (37.22& #177;16.22)%, the lowest value was found on day 7; the apoptosis rates were higher than those of the control group on day 14 and 28 (P < 0.05), lowered again on day 60, and showed no significant difference from the control group on day 90.

    Conclusion The findings suggest that 3-MA or rapamycin interventions could induce pulmonary macrophage autophagy activity to varied degrees and in negative association with the apoptosis rate of pulmonary macrophages.

     

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