The Indirect Toxicity Of Single Carbon Nanotubes To Cruor-fibrinolysis System Induced By Immunotoxicity In Lung

Due to their special structure and physicochemical properties, nanomaterials(NMs) are widely used in the chemical, pharmaceutical, and environmental protection fields and electronics. An an emerging class of environmental pollutants, NMs pose a real threat to human health. To date, many countries have implemented a large number of projects and plans with the aim of controlling the risks of nano-technologies. Biological safety researches of NMs and establishment of key evaluation techniques for nano-technologies are necessary to promote the rational use of NMs. Cardiovascular disease(CVD) is a serious danger to mankind. Respiratory tract exposure is one of the main avenues for NMs to enter the human body. Epidemiological studies have demonstrated that ultrafine particles(nanometre particles) in polluted air can increase the risk of cardiovascular disease(CVD), which is dangerous to mankind. However, little is known regarding indirect toxic effects on the cardiovascular system of respiratory tract exposure to nanometre particles. As a typical nanomaterial, single-walled carbon nanotube(SWCNT) have gained enormous popularity because of their unique properties. At present, most studies have focused on the direct damages to tissues, organs or cells after treatment with SWCNT, and little is known about the indirect toxic effects on the cardiovascular system of respiratory tract exposure to SWCNT. Objectives1. To study the changes of cruor-fibrinolysis system after exposure to SWCNT by respiratory tract, and clarify the changes of lung immune toxicity and the relevance and the possible biological mechanism.2. To investigate the indirect toxic effects of SWCNT exposure induced pulmonary toxicity of immune mediated cruor-fibrinolysis system changes and the possible mechanism. MethodsA rat intratracheal instillation model was used in the whole animal experiment. Forty-eight healthy Wistar rats were divided randomly into four groups. Rats were anaesthetised with ether and exposed to the SWCNT suspensions through intratracheal instillation every other day for sixty days. The experiment were divided into 30 days and 60 days of the two batch. After the rats were euthanised, the left base of the lung and the aorta pectoralis blood vessel were embedded in paraffin and thin-sectioned coronally. The sections were then stained with hematoxylin-eosin for examination by light microscopy. The levels of interleukin-1(IL-1α), interleukin-6(IL-6) and tumour necrosis factor alpha(TNF-α) in lung and blood tissues were detected using enzyme linked immunosorbent assay(ELISA) kit. Several molecular markers related to coagulation and the fibrinolysis system in plasma were analysed using ELISA kits. The markers included tissue plasminogen activator(t-PA), plasminogen activator inhibitor 1(PAI-?), D-dimer, antithrombin-III(AT-III), endothelin-1(ET-1), nitrogen oxide(NO) and von Willebrand factor(v WF). The thoracic aortas of the rats were observed by TEM. Immunohistochemical staining of t-PA, v WF and AT-III antigen in rat vascular intima were performed by the streptavidin-biotin complex(SABC) method. Total RNA was extracted from arterious peripheral blood and RT-PCR was performed. In vitro experiments, rat alveolar macrophage cell line and primary rat vascular endothelial cells were cultured. Also, cell models of inflammatory cytokines-stimulated and indirect exposure were established. Morphological changes were observed after treatment of SWCNT. Cells viability were detected by CCK-8. Levels of inflammatory factors and coagulation fibrinolytic activation factors in cell culture supernatants were detected using ELISA assay. ROS levels were measured by flow cytometry. The expression of related protein in vascular endothelial cells were detected by Western blot. Results1 The whole animal experiments1.1 Immunotoxicity damages in lungs of rats. After exposure to SWCNT through intratracheal instillation for 30 and 60 days, the lungs of rats in the exposure groups showed pathological changes. The lung tissues showed brownish black particle deposition and mild to moderate alveolar and local interstitial inflammation. The levels of IL-1α, IL-6 and TNF-α in lungs were significantly increased as compared to those in the control groups, which indicated that the inflammatory immune response in lung tissues.1.2 Changes in blood system1.2.1 After exposure to SWCNT for 30 days and 60 days, the rats appeared different degrees of systemic inflammation. Up-regulated levels of cytokines and increases in white blood cells, platelets and fibrinogen were detected in the plasma. These changes were followed by increased blood viscosity in the high dose SWCNT exposure group.1.2.2 Changes in coagulation and fibrinolysis activating factors were detected in the plasma of the rats. Lower expression of t-PA and higher expression of von v WF were observed in the vascular intima. Moreover, the plasma levels of v WF and ET-1 in the high dosage SWCNT group were elevated significantly. All these results indicated the dysfunction and damage of Vascular endothelial cells(VECs)in rats.1.3 damage of vascular endothelial layer1.3.1 Damage of the ultrastructure of the vascular intima in the rats were observed. After exposure for 60 days, nuclear malformation occurred in the vascular endothelial layer of rats exposed to SWCNT at 3.5 mg/kg b.w. The vascular endothelial layer of rats in the 10.5 mg/kg b.w SWCNT group showed mitochondrial swelling and cytoplasmic shrinkage. Mitochondrial vacuolisation was observed in the 17.5 mg/kg b.w SWCNT group.1.3.2 Lower expression of t-PA were observed in the vascular intima of rats exposed to SWCNT at 3.5, 10.5 and 17.5 mg/kg b.w for 30 days and 60 days, and higher expression of v WF were observed in the vascular intima of rats exposed to SWCNT at 10.5 and 17.5 mg/kg b.w for 30 days and 60 days. These results further validated the dysfunction of VECs in rats caused by SWCNT exposure.1.4 Gene expression in peripheral blood of ratsDecreased expression of the t-PA gene and increased expression of TM, p38 MAPK and PAI-1 genes were observed in the peripheral blood of the rats, suggesting that t-PA and TM gene expression could be sensitive biomarkers for respiratory tract exposure to SWCNT. The p38 MAPK signalling pathways may be involved in the mechanism of action of changes in coagulation and fibrinolysis activating factor levels in the body.2 In vitro cell tests2.1 Effects of SWCNT on alveolar macrophages in rats2.1.1 After exposure for 48 h, the nano-particles were swallowed by the alveolar macrophage, and the black particles appeared in cytoplasm. With the increase of exposure dose, the alveolar macrophage aggregated and the number of cells decreased significantly.2.1.2 With the extension of exposure dose of SWCNT and the increase of exposure time, the activity of alveolar macrophages decreased. Compared with the normal control group, the cell viability in groups of 50, 100, and 200 μg/m L SWCNT exposure were significantly decreased at 24 h, 48 h and 72 h, and the differences were statistically significant.2.1.3 After exposure to SWCNT 75 and 100 μg/m L for 96 h, the reactive oxygen species(ROS) levels of alveolar macrophages were higher than the normal control group. The ROS level in SWCNT 100 μg/m L group was increased significantly compared with the control group, the difference was statistically significant.2.1.4 After stimulated by SWCNT, the rat alveolar macrophages secreted IL-1α, IL-6 and TNF-α continuously. With the increase of the exposure time and dose, the secretion of inflammatory factors of IL-1α increased. After exposure for 24 h, 48 h and 96 h, the IL-1α levels in SWCNT 50, 75 and 100 μg/m L groups were significantly higher than those in normal control groups. After exposure for 24 h and 48 h, the IL-6 levels in SWCNT 50, 75 and 100 μg/m L groups were significantly higher than those of the control groups. After exposure for 48 h and 96 h, the TNF-α levels in SWCNT 75 and 100 μg/m L groups were significantly higher than those in normal control groups. The differences were all statistically significant.2.2 Influence of inflammatory factor to the function of VECsThere were changes for coagulation and fibrinolysis factors secreted by VECs after stimulated by TNF-α, which showed the decreased of AT-Ш and t-PA, and the increased v WF, TF and PAI-1. All these suggested the dysfunction of VECs.2.3 Influence of indirect exposure to SWCNT mediated by alveolar macrophages to the function of VECs and the related signaling pathways2.3.1 After treated by the indirect exposure, the levels of coagulation and fibrinolysis factors secreted by VECs changed, which showed the decreased of AT-Ш and t-PA, and the increased PAI-1 and TF. The results suggestd that alveolar macrophages had a role in mediating VECs, and could lead to changes of its function.2.3.2 The results of Western blot test showed that the expressions of t-PA protein in indirect exposure groups of vascular endothelial cells were significantly lower than those in normal control group. When the exposure doses of SWCNT were 75 and 100 μg/m L, the expressions of JNK protein in endothelial cells were significantly higher than those in normal control group. When exposed to 100 μg/m L SWCNT, the expressions of phosphorylated and non phosphorylated p38 MAPK in endothelial expression were significantly increased compared with normal control groups. These suggested that the p38 MAPK and JNK signal pathway may be the main pathway which involved in regulation of the coagulation factors. Conclusion1. SWCNT exposure by respiratory tract could lead to inflammatory injury of rat lung and release of inflammatory factors. At the same time, the cruor-f ibrinolysis system of the organism was occurred disorder.2. There were toxicity effects of SWCNT on alveolar macrophages in vitr o. The exposure to SWCNT could cause the generation of ROS and sustained secretion of proinflammatory cytokines. Additionally, TNF-α could induce chang es of the fibrinolytic related factors secreted by VECs.3. Compared with the direct exposure to SWCNT, the indirect effects med iated by alveolar macrophages on the vascular endothelial dysfunction may be more significant. p38 MAPK and JNK pathway may be involved in the regulati on of the indirect effects. The indirect effects of SWCNT to cruor-fibrinolysis system may be an important mechanism which is different from the direct effe cts considered as before.4. The immune toxicity of pulmonary plays an important role in mediatingthe changes of cruor-fibrinolysis system caused by SWCNT. One of the mech anisms may be: Through the release of inflammatory cytokines by alveolar ma crophages, SWCNT could indirectly mediated vascular endothelial dysfunction, r esulting in the changes of coagulation and fibrinolysis factors and disfunction o f cruor-fibrinolysis system.