[Co-exposure of carbon black and cadmium induces autophagy and inflammation in human bronchial epithelial cells via PERK 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.

Antioxidant effect of glutathione on promoting 2-keto-l-gulonic acid production in vitamin C fermentation system.

AIMS: Oxidative stress limited the growth of cells and 2-keto-l-gulonic acid (2-KGA) production in vitamin C (Vc) fermentation system. The study aims to investigate the antioxidant effect of glutathione on promoting 2-KGA in Vc fermentation system using Ketogulonicigenium vulgare 25B-1 and Bacillus endophyticus ST-1 as the co-culturing microbes. METHODS AND RESULTS: The activities of antioxidant-related enzymes and qPCR were used to study the antioxidant effect of glutathione addition in Vc fermentation system. The addition of GSH and GSH/GSSG increased 2-KGA production and decreased fermentation time, and the highest 2-KGA production increased by 40·63% and the lowest fermentation time shortened to 60 h when the addition of optimal concentration ratio of GSH/GSSG was 50 : 1. Moreover, the increased production of 2-KGA was accompanied by up-regulated the activities of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), catalase (CAT) and over-expressed oxidative stress-related genes sod, gst, gr, zwf, gp, which resulted in scavenging reactive oxygen species to reduce oxidative stress in Vc fermentation system. CONCLUSIONS: Glutathione showed a significant effect on increasing 2-KGA production and decreasing fermentation time in Vc fermentation system. GSH/GSSG could maintain a dynamic balance with two forms of glutathione and the optimal concentration ratio of GSH/GSSG was 50 : 1. SIGNIFICANCE AND IMPACT OF THE STUDY: Glutathione is proved to be effective to relieve oxidative stress. The promotion effects of GSSG and GSH on 2-KGA production could help to further explore the optimization of co-culture fermentation process for Vc industrial production.