Airway epithelial cell death contributes to the pathogenesis of lung fibrosis. Polyhexamethylene guanidine phosphate (PHMG-p), commonly used as a disinfectant, has been shown to be strongly associated with lung fibrosis in epidemiological and toxicological studies. However, the molecular mechanism underlying PHMG-p-induced epithelial cell death is currently unclear. We synthesized a PHMG-p–fluorescein isothiocyanate (FITC) conjugate and assessed its uptake into lung epithelial A549 cells. To examine intracellular localization, the cells were treated with PHMG-p–FITC; then, the cytoplasmic organelles were counterstained and observed with confocal microscopy. Additionally, the organelle-specific cell death pathway was investigated in cells treated with PHMG-p. PHMG-p–FITC co-localized with the endoplasmic reticulum (ER), and PHMG-p induced ER stress in A549 cells and mice. The ER stress inhibitor tauroursodeoxycholic acid (TUDCA) was used as a pre-treatment to verify the role of ER stress in PHMG-p-induced cytotoxicity. The cells treated with PHMG-p showed apoptosis, which was inhibited by TUDCA. Our results indicate that PHMG-p is rapidly located in the ER and causes ER-stress-mediated apoptosis, which is an initial step in PHMG-p-induced lung fibrosis.

Over the decades, guanidine-based oligomer groups have been one of the most widely used antimicrobial agents. Reportedly, these cationic oligomers cause serious damage to microorganisms but have low toxicity to humans. However, public concerns regarding the guanidine group have rapidly grown after the fatal misuse of these oligomers as humidifier disinfectants, which resulted in thousands of fatalities in South Korea. Herein, we investigated liposome leakage and cellular permeability changes caused by polyhexamethylene guanidine (PHMG) and polyhexamethylene biguanide (PHMB), both representative guanidine-based oligomers. The leakage of zwitterionic liposomes, induced by cationic oligomers, was more extensive than that of negative liposomes, indicating that oligomer adsorption onto lipid head groups via electrostatic interaction cannot fully explain the induced lipid membrane damage. Furthermore, lipid packing parameters, including intrinsic curvature, cholesterol content, and lipid phases, affected liposome leakage, particularly for PHMG. Cellular permeability tests were performed using an A549 cell monolayer model and a respiratory 3D tissue model, revealing that PHMG and PHMB damaged cell membranes and reduced cell barrier function. Furthermore, liposome leakage induced by PHMG and PHMB was higher in human lung surfactant-mimicking liposomes than that observed in Escherichia coli-mimicking liposomes. These results indicated that human cells are susceptible to guanidine-based oligomers. Considering that the interaction of oligomers and cell membranes is a major mechanism of toxicity initiation, this study provides crucial insights into the action of these disinfectants on mammalian cells.

Hepatic fibrosis results from chronic liver damage and is characterized by excessive accumulation of extracellular matrix (ECM). In this study, we showed that dendropanoxide (DPX), isolated from Dendropanax morbifera, had anti-fibrotic effects on hepatic fibrosis by inhibiting hepatic stellate cell (HSC) activation. DPX suppressed mRNA and protein expression of α-SMA, fibronectin, and collagen in activated HSCs. Moreover, DPX (40 mg/kg) treatment significantly lowered levels of liver injury markers (aspartate aminotransferase and alanine transaminase), expression of fibrotic markers, and deposition of ECM in a carbon tetrachloride-induced mouse model. Anti-fibrotic effects of DPX were comparable to those of silymarin in a hepatic fibrosis mouse model. As a possible mechanism of anti-fibrotic effects, we showed that DPX inhibited autophagosome formation (LC3B-II) and degradation of p62, which have important roles in HSC activation. These findings suggest that DPX inhibits HSC activation by inhibiting autophagy and can be utilized in hepatic fibrosis therapy.

The adverse outcome pathway (AOP) was introduced as an alternative method to avoid unnecessary animal tests. Under the AOP framework, an in silico methods, molecular initiating event (MIE) modeling is used based on the ligand-receptor interaction. Recently, the intersecting AOPs (AOP 347), including two MIEs, namely peroxisome proliferator-activated receptor-gamma (PPAR-γ) and toll-like receptor 4 (TLR4), associated with pulmonary fibrosis was proposed. Based on the AOP 347, this study developed two novel quantitative structure-activity relationship (QSAR) models for the two MIEs. The prediction performances of different MIE modeling methods (e.g., molecular dynamics, pharmacophore model, and QSAR) were compared and validated with in vitro test data. Results showed that the QSAR method had high accuracy compared with other modeling methods, and the QSAR method is suitable for the MIE modeling in the AOP 347. Therefore, the two QSAR models based on the AOP 347 can be powerful models to screen biocidal mixture related to pulmonary fibrosis.