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.

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.

Objective:To study the infection of human rhinovirus, respiratory syncytial virus and human adenovirus in Jinan from April 2018 to March 2019, and analyze epidemiological characteristics of human adenovirus.Methods:All of 1969 nasopharyngeal swabs were collected from hospitalized patients with acute respiratory tract infections in The Fourth People’s Hospital of Jinan, Qilu Children′s Hospital of Shandong University, Peoples Hospital of Zhangqiu District from April 2018 to March 2019, fluorescence quantitative PCR was used to detect the positive rate of human rhinovirus, respiratory syncytial virus and human adenovirus. Seven adenovirus positive samples were isolated and examined by sequencing, then we determined adenovirus type, constructed gene phylogenetic trees for analysis.Results:Of the 1969 samples, 242 were positive, the total positive rate was 12.30% (242/1969), the positive rate was 3.00% (59/1969) for rhinovirus, 6.30% (124/1969) for respiratory syncytial virus (RSV), and 3.86% (76/1969) for adenovirus. There was no significant difference in the detective rate of rhinovirus in different age groups (Fisher′s exact test value =8.376, P=0.720), the positive rates of RSV and adenovirus in different age groups was statistically significant ( χ2=19.28, 12.16; P=0.001, 0.016). There was a statistically significant difference in the positive rate of adenovirus between different sexes ( χ2=14.33, P<0.001), and there was no statistically significant difference in the positive rate of rhinovirus and RSV between males and females ( χ2=0.30, 2.90, P=0.862, 0.089). Comparing the positive rates of viral nucleic acid in different months, we found that the positive rate of rhinovirus, RSV and adenovirus separately reached the highest in October, December and November (8.61%, 26.50% and 8.84%). We constructed a gene phylogenetic tree after seven positive samples of adenoviruses were sequenced, by the molecular typing method we detected that seven adenovirus-positive samples were all HAdV-2 type. Conclusions:By comparing the epidemiological trends of human rhinovirus, RSV and human adenovirus in Jinan from April 2018 to March 2019 in different ages, genders, and months, providing reference basis for the early prevention and clinical treatment of acute respiratory tract infection.