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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.
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Aim To evaluate the influence of METH on MMP, mitochondrial ultrastructure, and the expression levels of mitochondrial proteins, Mfnland Fisl, in human neuroblastoma SH-SY5Y cells in vitro. Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentrations of METH(0. 0, 1. 0, 1. 5 and 2. 0 mmol L-1), and for various periods of exposure for 3, 6, 12, 24 h, the MMP of SH-SY5Y cells was stained by MMP assay kit (JC-1) , the mitochondrial ultrastructure of SH-SY5Y cells exposed to METH was observed by transmission electron microscope, and the expression levels of Mfnl and Fisl proteins were detected by Western blot. Results Compared with control group for various periods of exposure(3,6,12,24 h), the red/green fluorescence ratios of MMP and the expression levels of Mfn1 protein decreased significantly in METH groups (P<0. 05) , while the expression levels of Fisl pro-tein increased significantly (P <0.05). SH-SY5Y cells were treated with METH for 24 h prior to observation under transmission electron microscope ( TEM ). The mitochondria of SH-SY5Y cells in unprocessed group showed the oval, rodlike and double-layer membrane structure, along with clear normal mitochondrial cristae. However, the oval and rodlike structure of mitochondria in SH-SY5Y cells of METH treatment groups had been split into small ball structures. Moreover , mitochondrial autophagosome and autophagic iyso-some could also be found. Conclusions METH could induce a decrease in MMP, mitochondrial ultrastruc-tural changes, and changes in the expression levels of Mfnl and Fisl in SH-SY5Y cells, which might be associated with nerve cell damage caused by METH.
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Objective To investigate the influence of inhibited gene expression of fisson 1 (Fis1) gene on the level of Fis1,mitofusin 1 (Mfn1) and mitochondrial membrane potential in SH-SY5Y cells with fluorine,to study the role of mitochondrial dynamic balance in the pathogenesis of chronic fluorosis.Methods SH-SY5Y cells were cultured in vitro,when adherent cells entered the logarithmic phase,using a group design,they were divided into four groups:blank control group (control),fluoride group [2 mmol/L sodium fluoride (NaF)],fluoride negative control group (2 mmol/L NaF + non-specific siRNA) and the gene-silencing group (2 mmol/L NaF + specific siRNA-Fis1).The protein expression levels of Fis1 and Mfn1 were measured by Western blotting;the mRNA expression levels of Fis1 and Mfn1 were measured by Real-time PCR;and the levels of the mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit.Results Compared with control (1.37 ± 0.18,1.00 ± 0.04;1.57 ± 0.19,1.00 ± 0.04;1.00 ± 0.10),the expression levels of Fisl protein (1.72 ± 0.04) and mRNA (1.48 ± 0.13) in fluoride group were increased,the expression levels of Mfn1 protein (0.87 ± 0.02) and mRNA (0.69 ± 0.07) in fluoride group were decreased,the level of mitochondrial membrane potential (0.76 ± 0.13) was decreased (P ＜ 0.05).Compared with control,the expression levels of Fis1 protein (0.79 ± 0.07) and mRNA (0.06 ± 0.03) in gene-silencing group were decreased,the expression levels of Mfn1 protein (1.71 ± 0.04) and mRNA (1.52 ± 0.05) in gene-silencing group were increased (P ＜ 0.05),the level of mitochondrial membrane potential (0.94 ± 0.01) was decreased.Compared with fluoride group,the expression levels of Fis1 protein and mRNA in gene-silencing group were decreased,the expression levels of Mfn1 protein and mRNA in gene-silencing group were increased,the level of mitochondrial membrane potential in gene-silencing group was increased (P ＜ 0.05).Conclusion Gene expression inhibition of Fis1 gene can reduce the mitochondrial division and damage of mitochondrial membrane potential in SH-SY5Y cells induced by fluoride.
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Objective To evaluate the influence of fluoride on mitochondrial membrane potential of neuroblastoma SH-SY5Y cells,and on the expression levels of mitochondrial proteins mitofusion 1 (Mfn1) and fission 1 (Fis1).Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentration of sodium fluoride [0.0 (control),0.4,2.0 and 4.0 mmol/L],and various periods exposure of 6,12,24,48 h;the mitochondrial membrane potential of SH-SY5Y cells was detected by mitochondrial membrane potential assay kit (JC-1);and the expression levels of Mfn1 and Fis1 proteins were detected by Western blotting.Results Compared with the control group (1.63 ± 0.18,1.13 ± 0.15,1.30 ± 0.02) for various periods exposure (6,12,48 h),the red/green fluorescence ratios of the mitochondrial membrane potential of SH-SY5Y cells exposed to 2.0 and 4.0 mmol/L of sodium fluoride were decreased significantly (1.01 ± 0.10,0.80 ± 0.04;0.75 ± 0.13,0.62 ± 0.10;0.82 ± 0.01,0.56 ± 0.04,P ＜ 0.05);compared with the control group (0.93 ± 0.03,1.05 ± 0.07,1.17 ± 0.04) for various periods exposure,the expression levels of mitochondrial Mfn1 protein were decreased significantly in 0.4,2.0,4.0 mmol/L sodium fluoride groups (6,12,48 h:0.75 ± 0.02,0.65 ± 0.05,0.57 ± 0.06;0.83 ± 0.06,0.79 ± 0.06,0.69 ±0.06;0.98 ± 0.05,0.73 ± 0.07,0.62 ± 0.09,P ＜ 0.05).Compared with the control group (0.90 ± 0.05) for exposure time 12 h,the expression levels of Fis1 protein were increased significantly in 2.0,4.0 mmol/L sodium fluoride groups (1.14 ± 0.06,1.23 ± 0.06,P ＜ 0.05).Conclusions The mitochondrial membrane potential and the expression levels of mitofusion 1 and fission 1 of SH-SY5Y cells treated with fluoride are abnormal,which might be associated with the theory of nerve cell damage from high oxidative stress.
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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.
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Purpose: The aim of this study is to preliminarily investigate the expression of mitochondrial fusion protein 1 (MFN1) in a lens‑induced animal myopia (LIM) model and to explore the relationship between MFN1 and the visual development. Materials and Methods: MFN1 gene expression in guinea pigs was examined during the development of minus LIM, 15 tri‑colored guinea pigs were obtained, and one eye of each pig was randomly selected and treated with −7.00D lenses. Ocular refraction and axial length were collected before intervention and 1, 2, and 3 weeks after intervention. After the refraction and axial length measurements at 1, 2, and 3 weeks of lens intervention, five guinea pigs were randomly selected. MFN1 expression in the retina of both eyes was tested by immunohistochemistry technique. Results: MFN1‑positive cells could be observed in the retina of both eyes. The positive cells in the LIM eyes were staining deeper, and much more positive cells could be observed. Furthermore, MFN1‑positive expression could be seen mainly in ganglion cells after 1 week of minus lens intervention, and with time extension, more and more positive cells appeared in the rod‑cone cell and bipolar cell layer, and this phenomenon could not be found in the normal control eyes. Conclusion: This study suggested that MFN1 might be correlated to the development of myopia.