Single-cell transcriptomes reveal heterogeneity of chlorine-induced mice acute lung injury and the inhibitory effect of pentoxifylline on ferroptosis.

To investigate the effect of pentoxifylline (PTX) on Chlorine (Cl2)-induced acute lung injury (ALI) by single-cell RNA sequencing (scRNA-seq). Female BALB/c mice were exposed to Cl2 at 400 ppm for 15 min. H&E staining was used to observe the degree of lung injury. scRNA-seq was conducted to analysis of normal and Cl2-exposed mice lung tissues. Immunofluorescence was used to observe genes of interest. Thirty-two mice were randomly divided into four groups: Control, Cl2, Cl2+Fer-1, Cl2+PTX. TEM, WB and ELISA were used to detect ferroptosis-related indicators. The 5, 8, 10, 12, 16, 20 clusters were epithelial cells and 4, 15, 18, 19, 21 clusters were endothelial cells. Pseudo-time analysis revealed the differentiation trajectory of epithelial cells and key regulatory genes (Gclc, Bpifa1, Dnah5 and Dnah9) during the process of injury. Cell-cell communication analysis identified several important receptor-ligand complexes (Nrp1-Vegfa, Nrp2-Vegfa, Flt1-Vegfa and Flt4-Vegfa). Ferroptosis were found up-regulated in epithelial and endothelial cells by GSVA analysis. Highly expressed genes to which closely related ferroptosis were found by SCENIC analysis. PTX could significantly decrease the levels of MDA and abnormal high expression of solute carrier family 7 member 11 (SLC7A11, the key transporter of cystine) as well as increase the expression of GSH/GSSG and glutathione peroxidase 4 (GPX4) (p < 0.05). This study revealed novel molecular features of Cl2-induced ALI. PTX may be a potential specific drug by inhibiting the process of ferroptosis in epithelial and endothelial cells.

Diffusion simulation and risk assessment model establishment of chlorine gas leakage based on terrain conditions.

This study researches the impact of terrain factors on chlorine gas diffusion processes based on SLAB model. Simulating the law of wind speed changing with altitude by calculating the real-time speed with vertical height combing actual terrain data, and integrating the influence of terrain on wind speed by using Reynolds Average Navier-Stokes (RANS) algorithm, K-turbulence model, and standard wall functions, then plotting the gas diffusion range in the map with terrain data according to the Gaussian-Cruger projection algorithm and dividing the hazardous areas according to the public exposure guidelines (PEG). The accidental chlorine gas releases near Lishan Mountain, Xi'an City, were simulated by the improved SLAB model. The results show that there are obvious differences analyzing contrastively the endpoint distance and area of chlorine gas dispersion under real terrain condition and ideal condition at different times; it can be found that the endpoint distance of the real terrain conditions is 1.34 km shorter than that of the ideal conditions at 300 s with terrain factors, and also the thermal area is 3,768,026m2 less than that of the ideal conditions. In addition, it can predict the specific number of casualties within different levels of harm at 2 min after chlorine gas dispersion, and casualties are constantly changing over time. The fusion of terrain factors can be used to optimize the SLAB model, which is expected to provide an important reference for effective rescue.

Protective effects of pentoxifylline against chlorine-induced acute lung injury in rats.

OBJECTIVE: Chlorine is a chemical threat agent that can be harmful to humans. Inhalation of high levels of chlorine can lead to acute lung injury (ALI). Currently, there is no satisfactory treatment, and effective antidote is urgently needed. Pentoxifylline (PTX), a methylxanthine derivative and nonspecific phosphodiesterase inhibitor, is widely used for the treatment of vascular disorders. The present study was aimed to investigate the inhibitory effects of PTX on chlorine-induced ALI in rats. METHODS: Adult male Sprague-Dawley rats were exposed to 400 ppm Cl2 for 5 min. The histopathological examination was carried out and intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. Subsequently, to evaluate the effect of PTX, a dose of 100 mg/kg was administered. The activities of superoxide dismutase (SOD) and the contents of malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG) and lactate dehydrogenase (LDH) were determined by using commercial kits according to the manufacturer's instructions. Western blot assay was used to detect the protein expressions of SOD1, SOD2, catalase (CAT), hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), occludin, E-cadherin, bcl-xl, LC 3, Beclin 1, PTEN-induced putative kinase 1 (PINK 1) and Parkin. RESULTS: The histopathological examination demonstrated that chlorine could destroy the lung structure with hemorrhage, alveolar collapse, and inflammatory infiltration. ROS accumulation was significantly higher in the lungs of rats suffering from inhaling chlorine (P<0.05). PTX markedly reduced concentrations of MAD and GSSG, while increased GSH (P<0.05). The protein expression levels of SOD1 and CAT also decreased (P<0.05). Furthermore, the activity of LDH in rats treated with PTX was significantly decreased compared to those of non-treated group (P<0.05). Additionally, the results also showed that PTX exerted an inhibition effect on protein expressions of HIF-1α, VEGF and occludin, and increased the level of E-cadherin (P<0.05). While the up-regulation of Beclin 1, LC 3II/I, Bcl-xl, and Parkin both in the lung tissues and mitochondria, were found in PTX treated rats (P<0.05). The other protein levels were decreased when treated with PTX (P<0.05). CONCLUSION: PTX could ameliorate chlorine-induced lung injury via inhibition effects on oxidative stress, hypoxia and autophagy, thus suggesting that PTX could serve as a potential therapeutic approach for ALI.

Heterogeneity of T cells and macrophages in chlorine-induced acute lung injury in mice using single-cell RNA sequencing.

OBJECTIVE: Chlorine (Cl2), as an asphyxiant toxicant, induced poisoning incidents and acute lung injury (ALI) occur frequently. The specific pathogenesis of Cl2-induced ALI remains unclear. Immune cells play an important role in the process of lung damage. We used single-cell RNA sequencing (scRNA-seq) technology to explore T cells and macrophages molecular mechanism. METHODS: Female BALB/c mice were exposed to 400 ppm Cl2 for 15 min. scRNA-seq technology was used to observe the heterogeneity of T cells and macrophages. Hematoxylin-eosin (H&E) staining was used to evaluate the degree of lung injury. Immunofluorescence was used to verify the highly expressed genes of our interest. RESULTS: A total of 5316 to 7742 cells were classified into eight different cell types. Several new highly expressed anti-inflammatory and pro-inflammatory genes were found in T cells and macrophages, which were further verified in vitro. Through the pseudotime analysis of macrophages, it was found that the expression of pro-inflammatory and anti-inflammatory genes showed opposite trends in the development of Cl2-induced ALI. This study also mapped T cells-macrophage communication and identified the development of several important receptor-ligand complexes in Cl2-induced ALI. CONCLUSIONS: These findings are worthy of further exploration and provide new resources and directions for the study of Cl2-induced ALI in mice, especially in immune and inflammation mechanisms.