吸入炭黑气溶胶对小鼠心血管系统的损害作用
Cardiovascular damage in mice induced by inhalation of carbon black aerosols
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摘要:
目的 探讨反复吸入纳米级炭黑气溶胶对小鼠心血管系统的损害作用及可能的作用机制。
方法 8周龄雄性C57BL/6小鼠分为对照组（n=24）、染毒组（n=12）和恢复组（n=12）。染毒组小鼠全身暴露于30.16 mg/m3炭黑气溶胶中6 h/d，连续染毒28 d；恢复组于染毒28 d后静养28 d；对照组给予过滤空气。每天监测染毒柜内炭黑颗粒的浓度和粒径。末次染毒或恢复28 d后的24 h，每组取9只小鼠，麻醉后进行心电图测定；采血后测定血清心肌酶谱肌酸激酶（CK）、同工酶（CK-MB）、乳酸脱氢酶（LDH）、谷丙转氨酶（ALT）、谷草转氨酶（AST）和炎性细胞因子白介素-1β（IL-1β）、白介素-6（IL-6）、白介素-8（IL-8）、肿瘤坏死因子-α（TNF-α）；取小鼠心脏，测定心脏组织中炎性细胞因子（IL-1β、IL-6、IL-8、TNF-α）和氧化应激指标超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）。每组取3只小鼠进行心脏病理组织学检查。
结果 炭黑颗粒平均质量浓度为（30.16±3.52）mg/m3，粒径小于400 nm的颗粒物占82.53%，粒径小于800 nm的颗粒物占98.13%。小鼠吸入炭黑气溶胶7、14、21、28 d及恢复期，染毒组和对照组小鼠体重差异无统计学意义。与对照组相比，染毒组及恢复组小鼠心电图S-T段抬高且PR间期延长（P < 0.05），其他心电图指标与对照组相比差异无统计学意义。与对照组相比，染毒组小鼠血清CK和CK-MB活性增加（P < 0.05），分别为对照组的1.42、1.24倍；而恢复组小鼠CK和CK-MB活性降至正常水平。染毒组小鼠血清中IL-6、IL-8和TNF-α水平高于对照组（P < 0.05），分别为1.79、1.89、1.70倍；而心脏组织匀浆中仅IL-6水平高于对照组（P < 0.05），为1.40倍。恢复组小鼠血清IL-6和IL-8水平均有所下降，但是仍高于对照组（P < 0.05），而心脏组织中IL-1β、IL-6、IL-8和TNF-α水平均未见改变。与对照组相比，染毒组小鼠心脏组织中SOD水平降低且MDA水平增加（P < 0.05），而恢复组小鼠SOD水平和MDA水平与对照组相比差异无统计学意义。病理组织学检查可见，对照组心肌纤维排列整齐；小鼠吸入炭黑气溶胶后，在心脏组织中未观察到黑色炭黑颗粒沉积，心肌纤维排列紊乱，并可见心肌细胞水肿及炎性细胞渗出；在恢复期，小鼠心脏组织中也可以见到少量的炎性细胞渗出。
结论 反复吸入30.16 mg/m3纳米级炭黑气溶胶可以造成小鼠心肌缺血性损伤，其机制可能与炭黑颗粒所致的炎性损伤和氧化应激有关。
Abstract:Objective To explore the injury of cardiovascular system and its potential mechanism in mice induced by repeated inhalation of nanoscaled carbon black (CB) aerosols.
Methods Eight-week-old male C57BL/6 mice were divided into control group (n=24), exposure group (n=12), and recovery group (n=12). The exposure mice were exposed to CB aerosols in an inhalation chamber at 30.16 mg/m3 for 6 h/d for a continuous inhalation of 28d; the recovery mice were exposed to CB aerosols for 28 d and then recovered for another 28 d; the control mice were exposed to filtered air. The concentration and aerodynamic diameter of CB particles were daily monitored during the exposure. Nine mice in each group were randomly selected and the cardiac function parameters were determined using electrocardiography (ECG) under anesthesia at 24 h after the last exposure or recovery. The serum samples were collected for the determination of myocardial enzymesincluding creatine kinase (CK), creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) and inflammatory cytokinesincluding interleukin-1β (IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), and tumor necrosis factor-α (TNF-α). Heart tissue samples were collected for the detection of inflammatory cytokines (IL-1β, IL-6, IL-8, and TNF-α) and oxidative stress indicessuch as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA). The histopathological evaluation on heart tissues of three mice in each group was conducted after HE staining.
Results The average concentration of CB particles was (30.16±3.52) mg/m3, the particles with aerodynamic diameter less than 400 nm accounted for 82.53%, and the particles with aerodynamic diameter less than 800 nm accounted for 98.13%. On exposure day 7, 14, 21, 28 and during the recovery, there was no difference in body weight between the exposure mice and the control mice. Compared with the control group, S-T segment and PR interval of ECG in the exposure group and the recovery group increased significantly (P < 0.05), but no difference was observed in other ECG indices (P > 0.05). The activities of CK and CK-MB in serum of mice in the exposure group were 1.42 and 1.24 times as high as those of the control group (P < 0.05), and the two indicators of mice in the recovery group decreased to normal levels. The serum levels of IL-6, IL-8, and TNF-α in the exposure group were 1.79, 1.89, and 1.70 times as high as those of the control group (P < 0.05), and only the IL-6 level in heart tissues was 1.40 times higher. Although the serum levels of IL-6 and IL-8 had slight reductions in the recovery period, their levels were still higher than those in the control group (P < 0.05), and there was no difference in the levels of IL-1β, IL-6, IL-8, and TNF-α in heart tissues between the control group and the recovery group. SOD activity decreased and MDA level increased in heart tissues of mice after CB inhalation (P < 0.05), and no differences were observed in the two indicators between the recovery group and the control group. Histopathological examination results showed that myocardial fibers were well arranged in the control group; CB particles were not observed in heart tissues of mice after CB inhalation, but myocardial fibers were disarranged, and myocardial cell edema and inflammatory cells were observed; in the recovery group, a small amount of inflammatory cells were also seen in the heart tissue samples of mice.
Conclusion Ischemic injury in heart of mice is induced following repeated inhalation of 30.16 mg/m3 CB nanoscaled aerosols, and the mechanism might be related to inflammatory injury and oxidative stress induced by CB particles.
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