Objective To observe the expression of S100A8 and S100A9 in alveolar macrophages (AMs) of chronic obstructive pulmonary disease (COPD) rats, and explore the effect on the release of inflammatory mediators from AMs in COPD rats. Methods Twelve adult male Wistar rats were randomly divided into a normal control group and a COPD group. The COPD model was established by exposing the rats to cigarette smoke and intratracheal injection of endotoxin for 1 month. The pathological changes of lung tissue of rats were observed under light microscope. Total cells counts and the number of AMs, lymphocytes, neutrophils in bronchoalveolar lavage fluid (BALF) of two groups were examined by Wright's staining methods. Rat AMs from the control group and the COPD group were isolated and cultured, and then treated with different doses of S100A8 and S100A9 for 6 hours and 12 hours. The levels of interleukin (IL)-8, IL-6 and tumour necrosis factor-α (TNF-α) in the AMs supernatants were measured by enzyme linked immunosorbent assay. The expression of S100A8 and S100A9 mRNA in AMs of rats were observed by in situ hybridization. The immunohistochemical method was used to observed the expression of S100A8/A9 protein of AMs. Results After cigarette smoking combined with intratracheal injection of endotoxin for 1 month, the lung tissue of rats showed typical pathological changes of COPD. Total cell counts and the number of AMs, lymphocytes, neutrophils in BALF of the COPD rats were significantly higher than those of the normal rats (P<0.05). Among them, the increase in the number of AMs was the most obvious. Compared with the control group, the expression of S100A8 mRNA, S100A9 mRNA and S100A8/A9 protein in AMs of the COPD group were up-regulated significantly (P<0.05). After the AMs of COPD rats were treated with S100A8 and S100A9, the contents of IL-8, IL-6 and TNF-α in AMs supernatants increased significantly in a time- and dose-dependent manner. When the AMs were treated with the same dose of S100A8 and S100A9 for the same time, the levels of IL-8, IL-6 and TNF-α in the AMs supernatant of the COPD group were higher than those of the normal control group. Conclusions The expression of S100A8 and S100A9 in cultured COPD rat AMs is significantly increased. S100A8 and S100A9 can promote the secretion and release of inflammatory factors IL-6, IL-8 and TNF-α from AMs of COPD rats in a time and dose-dependent manner. The effects of S100A8 and S100A9 on the secretion of IL-6, IL-8 and TNF-α in AM of COPD rats are significantly enhanced compared with those of normal rats.
ObjectiveTo investigate the effect of succinate induced polarization of MH-S murine alveolar macrophage cells on hyperoxia-induced epithelial-mesenchymal transition (EMT) of MLE-12 mouse alveolar epithelial cells. Methods Determine the exposure time: MLE-12 cells was cultured in an incubator with 95%O2 for different time to establish a cell model of acute hyperoxia-induced lung injury. The relative expression of EMT-related proteins (E-cadherin, N-cadherin, vimentin) was determined by Western blotting. Co-culture of MLE-12 and MH-S to explore the influence of MH-S on EMT: MLE-12 was divided into hyperoxia group for 0h, hyperoxia group for 48h and co-cultured with MH-S hyperoxia group for 48h (Co). The relative expression of EMT-related proteins was determined by Western blotting. Determination of succinate concentration and its effect on MH-S polarization and succinate receptor GPR91: MLE-12 was cultured in different concentrations of succinate medium for 24h, and the cell viability was determined by CCK-8. MH-S was divided into control group (C) and succinate group (S). Group C was cultured for 24h, and group S was added with succinate at the above concentration. The relative expression of GPR91 and polarization-related factor mRNA in MH-S was measured by RT-qPCR, and the expression of macrophage polarization-related proteins (CD11b, CD206, CD86) was measured by flow cytometry. Study on the effect of succinate on EMT by cell co-culture: MLE-12 and MH-S were co-cultured in a Transwell chamber and divided into control group (Co), succinate group (SUC) and GPR91 inhibitor group (I). Results Expression of EMT-related proteins in four groups of MLE-12 at different times: Compared with 0h, the expression of vimentin and N-cadherin in 24h and 48h increased, while the expression of E-cadherin in 48 h and 72 h decreased (P<0.05), and there was no significant difference in other groups. The follow-up experiment was conducted under hyperoxia conditions for 48h. Influence of MH-S on EMT: The expression of vimentin and N-cadherin in Co group was higher than that in 48h, and the expression of E-cadherin was lower than that in 48h (P<0.05). After 24 h of intervention with different concentrations of succinate on MLE-12, compared with the 0mmol/L, the cell viability of 2.5mmol/L, 1mmol/L and 500 μmol/L increased (P<0.05), and there was no significant difference in other groups, so the 1mmol/L succinate concentration was selected for subsequent experiment. Compared with group C, the expression of GPR91 mRNA in group S increased, and the expression of iNOS and CD86 mRNA in group S increased (P<0.05), but there was no significant difference in other groups. The analysis of flow cytometry showed that 1mmol/L succinate could increase the number and proportion of CD86+CD206– alveolar macrophages. Compared with Co group, the expression of vimentin and N-cadherin in SUC group increased, while the expression of E-cadherin decreased. Compared with SUC group, the expression of vimentin and N-cadherin in group I decreased, while the expression of E-cadherin increased (P<0.05). Conclusion Succinate can induce mouse alveolar macrophages polarization to M1 through GPR91, enhance EMT of mouse alveolar epithelial cell injury model under hyperoxia, and promote the formation of pulmonary fibrosis.