Objective: To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells. Methods: In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 μg/ml Cd, 100 μg/ml CBNPs, 100 μg/ml CBNPs+2 μg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results: There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined (P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group (P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression (P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group (P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group (P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene (P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 (P<0.05), but the interaction between the two chemicals had no statistical significance (P>0.05) . Conclusion: CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.
目的： 探究碳黑与镉（cadmium，Cd）复合暴露对人支气管上皮细胞株16HBE细胞蛋白激酶R样内质网激酶（protein kinase R-like endoplasmic reticulum kinase，PERK）通路的影响，及其所致自噬和炎症反应的作用机制。 方法： 于2022年1月，复苏培养人支气管上皮16HBE细胞。将碳黑纳米颗粒（carbon black nanoparticles，CBNPs）氧化后吸附Cd(2+)构建CBNPs-Cd复合物。CCK-8试验检测CBNPs与Cd不同浓度和时间组合暴露对16HBE细胞活力的影响。以2 μg/ml Cd，100 μg/ml CBNPs，100 μg/ml CBNPs+2 μg/ml Cd联合暴露24 h作为后续剂量分组。分别应用透射电镜检测自噬体和自噬溶酶体数量。蛋白免疫印迹（Western blotting）法检测PERK通路蛋白PERK、真核翻译起始因子2α（eukaryotic initiation factor 2α，eIf2α）、激活转录因子4（activating transcription factor 4，ATF4）、自噬相关蛋白螯合体1（sequestosome 1，SQSTM1）/P62和微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3，MAlPILC3，简称LC3）的表达。以基因沉默技术抑制PERK基因后，检测自噬标志蛋白P62、LC3的变化，使用荧光定量PCR技术检测炎症因子白细胞介素-6 （interleukin-6，IL6）、白细胞介素-8 （interleukin-8，IL8）的表达。多组比较采用单因素方差分析，组内两两之间的比较采用LSD检验；多因素的成分分析采用析因分析。 结果： CBNPs与Cd单独/复合暴露24 h后，16HBE细胞活力变化均无统计学意义（P>0.05）。与对照组比较，CBNPs与Cd单独/复合暴露组16HBE细胞P62和LC3表达均明显升高（P<0.05），且复合暴露组较其他组别的自噬体和自噬溶酶体数量增加。与对照组比较，CBNPs和Cd单独暴露对p-PERK/PERK、p-eIf2α/eIf2α蛋白表达影响均无统计学意义（P>0.05），但复合暴露组p-PERK/PERK、p-eIf2α/eIf2α、ATF4蛋白的表达均升高（P<0.05），且CBNPs与Cd单独/复合暴露组16HBE细胞IL6、IL8水平均明显高于对照组（P<0.05）。CBNPs-Cd复合暴露组敲低PERK基因后，LC3蛋白和IL6、IL8水平均减少（P<0.05）。析因分析结果显示，CBNPs和Cd单独暴露对P62、LC3和IL6的表达发挥明显作用（P<0.05），但两化学物之间交互作用无统计学意义（P>0.05）。 结论： CBNPs-Cd复合暴露可能通过激活PERK-eIf2α-ATF4通路，致人支气管上皮细胞自噬受阻、炎症反应增加。.
Keywords: Autophagy; Cadmium; Carbon black nanoparticles; Epithelial cells; Inflammation; Protein kinase R-like endoplasmic reticulum kinase-eukaryotic initiation factor 2α-activating transcription factor 4 (PERK-eIf2α-ATF4) pathway.