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find Keyword "Hyperoxia lung injury" 1 results
  • Effect of succinate-induced polarization of mouse alveolar macrophages on hyperoxia epithelial-mesenchymal transition

    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.

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