长度和化学修饰在多壁碳纳米管诱导内皮细胞活化中的作用

目的: 研究比较不同长度和化学修饰的多壁碳纳米管(multi-walled carbon nanotubes,MWCNTs)对内皮细胞的活化作用,并探讨核苷酸结合寡聚结构域样受体家族含pyrin结构域蛋白3(nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3,NLRP3)炎性小体的相关机制。方法: 采用动态光散射法对MWCNTs悬液进行表征,将不同长度[短MWCNT(short MWCNT, S-MWCNT) 0.5~2.0 μm或长MWCNT(long MWCNT, L-MWCNT) 10~30 μm]或相同长度(10~30 μm)下不同化学修饰的MWCNTs[未修饰(L-MWCNT)、羧基修饰(L-MWCNT-COOH)、氨基修饰(L-MWCNT-NH₂)和羟基修饰(L-MWCNT-OH)]作用于小鼠脑微血管内皮细胞系b.End3。分别采用细胞增殖检测(cell counting kit,CCK)-8试验和乳酸脱氢酶释放试验评价MWCNTs的细胞毒性,通过酶联免疫吸附试验测定胞外血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)含量并进一步使用人单核细胞系THP-1进行黏附力试验,评价不同种类MWCNTs的内皮细胞活化效应。进一步选择S-MWCNT、L-MWCNT和L-MWCNT-COOH探讨内皮细胞活化的炎症机制,采用免疫蛋白印迹法检测MWCNTs作用后细胞炎性小体蛋白NLRP3的表达水平。结果: 在较高浓度(125 μg/cm²)下作用24 h,不同种类的MWCNTs均可显著抑制b.End3的细胞活性并影响细胞膜完整性。在无明显细胞毒性的浓度下(6.25 μg/cm²),不同种类的MWCNTs作用12 h均可显著诱导b.End3细胞内皮细胞活化,表现为VCAM-1释放水平增加,以及b.End3对THP-1的黏附水平增强。相同浓度下,L-MWCNT诱导细胞内皮细胞活化的效应显著强于S-MWCNT;相同长度下,L-MWCNT和L-MWCNT-COOH对细胞内皮细胞活化的效应差异无统计学意义。在6.25 μg/cm²浓度下,S-MWCNT、L-MWCNT、L-MWCNT-COOH可时间依赖性地上调b.End3细胞的NLRP3蛋白表达水平。与相同浓度的S-MWCNT相比,L-MWCNT可显著上调细胞NLRP3的蛋白表达水平;在相同长度下,L-MWCNT和L-MWCNT-COOH作用后细胞的NLRP3蛋白表达差异无统计学意义。结论: 与化学修饰作用相比,MWCNTs长度增加更易诱导内皮细胞活化,其中间机制可能与NLRP3炎性小体的激活有关。MWCNTs的安全性评价应关注其理化特性对血管内皮生物效应的影响。

Effects of length and chemical modification on the activation of vascular endothelial cells induced by multi-walled carbon nanotubes

Objective: To investigate the effects of multi-walled carbon nanotubes (MWCNTs) with different length or chemical modification on endothelial cell activation and to explore the role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. Methods: MWCNTs were characterized by dynamic light scattering (DLS) after being suspended in culture medium. The immortalized mouse cerebral microvascular endothelial cell line b.End3 was treated with short MWCNTs (S-MWCNT, 0.5 to 2 μm), long MWCNTs (L-MWCNT, 10 to 30 μm) and the above long MWCNTs functionalized by carboxyl-(L-MWCNT-COOH), amino-(L-MWCNT-NH₂) or hydroxyl-(L-MWCNT-OH) modification. Cytotoxicity of MWCNTs in b.End3 cells was determined by cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release assay, and non-toxic low dose was selected for subsequent experiments. Effects of all types of MWCNTs on the endothelial activation of b.End3 were determined by the measurement of vascular cell adhesion molecule-1 (VCAM-1) concentration in cell supernatant and adhesion assay of human monocytic cell line THP-1 to b.End3.To further elucidate the mechanism involved, the protein expressions of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3) in cells treated with S-MWCNT, L-MWCNT and L-MWCNT-COOH were measured by Western blot. Results: At a higher concentration (125 μg/cm²) and treated for 24 h, all types of MWCNTs significantly inhibited viability of b.End3 cells. At a sub-toxic concentration (6.25 μg/cm²), all types of MWCNTs treated for 12 h significantly induced the activation of b.End3 cells, as evidenced by the elevated VCAM-1 release and THP-1 adhesion. Compared with S-MWCNT, L-MWCNT significantly promoted endothelial cell activation. L-MWCNT and L-MWCNT-COOH activated b.End3 cells to a similar extent. Furthermore, treatment with S-MWCNT, L-MWCNT and L-MWCNT-COOH increased NLRP3 expression in a time-dependent manner at 6.25 μg/cm². Compared with S-MWCNT, cells treated with L-MWCNT for 4 h and 12 h exhibited significantly increased protein expressions of NLRP3. However, no significant differences were detected in the level of NLRP3 protein in cells treated with L-MWCNT and L-MWCNT-COOH. Conclusion: Compared with the surface chemical modification, length changes of MWCNTs exerted more influence on endothelial cell activation, which may be related to the activation of NLRP3 inflammasome. Our study contributes further understanding of the impact of MWCNTs on endothelial cells, which may have implications for the improvement of safety evaluation of MWCNTs.