ABSTRACT
Objective:To investigate the regulatory effects of mitofusin 1 (MFN1) on lipopolysaccharide (LPS)-induced Raw264.7 mouse macrophages pyroptosis and to provide reference for further study on the prevention of inflammation and fibrosis caused by macrophage dysfunction.Methods:Raw264.7 mouse macrophages were cultured in vitro and used to construct a model of LPS-induced pyroptosis. CCK-8 staining, PI staining, LDH release assay and Western blot were used to verify the Raw264.7 pyroptosis induced by LPS. MFN1 expression was detected by Western blot. DCFH-DA probe was used to detect the synthesis of total reactive oxygen species (ROS); Mito-SOX was used to detect mitochondrial ROS; JC-1 mitochondrial membrane potential was detected by fluorescence probe to reflect mitochondrial damage. Based on Ubibrowser database, it was predicted that MFN1 could bind to a variety of E3 ubiquitin ligases. Then, immunofluorescence and co-immunoprecipitation (CO-IP) were used to analyze MFN1 ubiquitination. An overexpression plasmid for MFN1 was constructed and transfected into Raw264.7 cells to detect the changes in pyroptosis and mitochondrial function. Results:LPS could induce the pyroptosis of Raw264.7 cells and mitochondrial dysfunction. MFN1 expression was decreased after LPS stimulation. Ubiquitinated MFN1 was detected by CO-IP. Ubiquitination inhibitor MG-132 inhibited LPS-induced expression of pyroptosis-related proteins including NLRP3, Pro-caspase-1, Caspase-1, IL-1β and IL-18 and improved mitochondrial function. MFN1 overexpression relieved the mitochondrial dysfunction and pyroptosis of Raw264.7 cells induced by LPS.Conclusions:The ubiquitination of MFN1 induced by LPS was involved in mitochondrial dysfunction and macrophage pyroptosis, suggesting that MFN1 was a potential target for the treatment of macrophage-induced inflammation and related diseases.
ABSTRACT
Objective: To detect the expression of mitofusion-1(Mfn1) in periodontal ligament stem cells (PDLSCs) isolated from healthy and periodontitis tissue and to study the effect of Mfn1 on the osteogenic differentiation of PDLSCs. Methods: PDLSCs were isolated from the healthy and periodontitis human samples(H-PDLSCs and P-PDLSCs). IL-1β was applied to mimic the inflammation microenvironment(H-PDLSCs + IL-1β). RT-PCR was used to detect the expression of Mfn1 in HPDLSCs, P-PDLSCs and H-PDLSCs + IL-1β. The expression of Mfn1 in P-PDLSCs was down-regulated by siRNA of Mfn1 (siMfn1). The osteogenic differentiation of the cells was examined by RT-PCR, alizarin red staining and cetyl pyridine chloride quantitative analysis. Results: The expression level of Mfn1 in P-PDLSCs and H-PDLSCs + IL-1β (5 μg/ml) groups was higher than that in H-PDLSCs(P＜ 0. 05). When the expression of Mfn1 in P-PDLSCs was down-regulated by siMfn1 the osteogenic differentiation ability of P-PDSLCs was restored(P＜ 0. 05). Conclusion: Inflammation may promote Mfn1 expression in PDLSCs and inhibite the osteogenic differentiation of P-PDLSCs.