Background:Mastitis seriously affects the mammary health of humans and animals.Studies have found that inflammation and oxidative stress play key roles in the occur-rence and development of mastitis.Therefore,in-depth...Background:Mastitis seriously affects the mammary health of humans and animals.Studies have found that inflammation and oxidative stress play key roles in the occur-rence and development of mastitis.Therefore,in-depth research on related molecular mechanisms is of great significance.Methods:Postpartum mice were anesthetized with pentobarbital and administered lipopolysaccharide to develop the mouse mastitis model.Proteomic analysis was per-formed to compare protein expression in mitochondria-associated endoplasmic retic-ulum membranes(MAM)from two mouse mammary gland groups.Western blot was used to detect the expression of MAM-related proteins in mitochondria.AlphaFold3 was used to predict the molecular structures of phosphofurin acidic cluster sorting protein 2(PACS2)and mitofusin 2(MFN2)and their interaction levels.The MFN2-PACS2 interaction was investigated using co-immunoprecipitation and small interfer-ing RNA.Results:The results showed that the inflammation level in the mammary gland tissue of mice with mastitis significantly increased,the total antioxidant capacity decreased,and the expression of MAM-related proteins MFN2 and PACS2 was significantly downregulated.In cell experiments,overexpression of MFN2 can inhibit inflamma-tion and oxidative stress responses,and promote the interaction between MFN2 and PACS2 to affect the formation of MAMs.Conclusion:In summary,this study suggests that mastitis can alter the expression of MAM-related proteins in mouse breast tissue.The interaction between MFN2 and PACS2 regulates the formation of MAMs.Overexpression of MFN2 can promote the formation of MAMs and inhibit inflammation and oxidative stress response in mam-mary epithelial cells.Our results provided a new theoretical basis and potential thera-peutic targets for the prevention and treatment of mastitis.展开更多
OBJECTIVE:To investigate the effect of mitofusin 2 (Mfn2) and its downstream signaling pathway on glomerular mesangial cells (GMCs) proliferation in IgA nephropathy (IgAN),as well as the mechanism of action of Jixueca...OBJECTIVE:To investigate the effect of mitofusin 2 (Mfn2) and its downstream signaling pathway on glomerular mesangial cells (GMCs) proliferation in IgA nephropathy (IgAN),as well as the mechanism of action of Jixuecao (Herba Centellae Asiaticae,HCA) in the treatment of IgAN.METHODS:Adenovirus-mediated Mfn2 gene transfection and Mfn2 expression were analyzed by real-time polymerase chain reaction (PCR) and Western blotting.IgA1 induced the proliferation of GMCs,which were then treated with HCA.Cell proliferation was detected with cell counting kit-8 (CCK-8),and Mfn2 expression was analyzed by real-time PCR and western blotting.An IgAN animal model was also established and treated with HCA.GMCs proliferation was detected by hematoxylin-eosin staining,mitochondrial structure was analyzed by electron microscopy,mitochondrial function was determined by the Clark oxygen electrode method,and the expression of Mfn2,Phospho-extracellular regulated protein kinases1/2 (P-ERK1/2),Cyclin-dependent kinase 2 (CDK2),Phospho-p27 (p-p27),and cyclin A was analyzed by Western blotting.RESULTS:In vitro,HCA inhibited GMCs in a concentration-dependent manner in association with the upregulation of Mfn2 expression.The overexpression of Mfn2 inhibited IgA1-induced GMCs proliferation and elevated the effect of HCA.In vivo,treatment with HCA could alleviate albuminuria and creatinine and GMCs proliferation.These effects were related to the upregulation of Mfn2,p-p27 and inhibition of p-ERK1/2,CDK2,and cyclinA.Mitochondrial swelling,vacuolar degeneration,and reduction of respiratory control rate were identified in IgAN,but HCA could improve the mitochondrial structure and function.CONCLUSION:HCA inhibited GMCs proliferation via the upregulation Mfn2 and the inhibition of Ras-Raf-ERK/MAPK.We revealed that changes of mitochondrial structure and function are associated with IgAN,but that HCA can improve these mitochondrial features.展开更多
AIM:To investigate the effects of mitofusin-2(MFN2) on insulin sensitivity and its potential targets in the liver of rats fed with a high-fat diet(HFD).METHODS:Rats were fed with a control or HFD for 4 or 8 wk,and wer...AIM:To investigate the effects of mitofusin-2(MFN2) on insulin sensitivity and its potential targets in the liver of rats fed with a high-fat diet(HFD).METHODS:Rats were fed with a control or HFD for 4 or 8 wk,and were then infected with a control or an MFN2 expressing adenovirus once a week for 3wk starting from the 9th wk.Blood glucose(BG),plasma insulin and insulin sensitivity of rats were determined at end of the 4th and 8th wk,and after treatment with different amounts of MFN2 expressing adenovirus(108,109 or 1010 vp/kg body weight).BG levels were measured by Accu-chek Active Meter.Plasma insulin levels were analyzed by using a Rat insulin enzymelinked immunosorbent assay kit.Insulin resistance was evaluated by measuring the glucose infusion rate(GIR) using a hyperinsulinemic euglycemic clamp technique.The expression or phosphorylation levels of MFN2 and essential molecules in the insulin signaling pathway,such as insulin receptor(INSR),insulin receptor substrate 2(IRS2),phosphoinositide-3-kinase(PI3K),protein kinase beta(AKT2) and glucose transporter type 2(GLUT2) was assayed by quantitative real-time polymerase chain reaction and Western-blotting.RESULTS:After the end of 8wk,the body weight of rats receiving the normal control diet(ND) and the HFD was not significantly different(P>0.05).Compared with the ND group,GIR in the HFD group was significantly decreased(P<0.01),while the levels of BG,triglycerides(TG),total cholesterol(TC) and insulin in the HFD group were significantly higher than those in the ND group(P<0.05).Expression of MFN2 mRNA and protein in liver of rats was significantly downregulated in the HFD group(P<0.01) after 8 wk of HFD feeding.The expression of INSR,IRS2 and GLUT2 were down-regulated markedly(P<0.01).Although there were no changes in PI3K-P85 and AKT2 expression,their phosphorylation levels were decreased significantly(P<0.01).After intervention with MFN2 expressing adenovirus for 3wk,the expression of MFN2 mRNA and protein levels were up-regulated(P<0.01).There was no difference in body weight of rats between the groups.The levels of BG,TG,TC and insulin in rats were lower than those in the Ad group(P<0.05),but GIR in rats infected with Ad-MFN2 was significantly increased(P<0.01),compared with the Ad group.The expression of INSR,IRS2 and GLUT2 was increased,while phosphorylation levels of PI3K-P85 and AKT2 were increased(P<0.01),compared with the Ad group.CONCLUSION:HFDs induce insulin resistance,and this can be reversed by MFN2 over-expression targeting the insulin signaling pathway.展开更多
Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferatio...Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANGⅡ by cell counting and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway pro-tein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and upregulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANGⅡ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.展开更多
In order to evaluate the effect of mitofusin-2 gene （mfn2） on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene...In order to evaluate the effect of mitofusin-2 gene （mfn2） on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-Ⅴ/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively （P〈0.05）. The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin （CAMP） followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively （P〈0.05）. It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.展开更多
OBJECTIVE:To evaluate the protective effects of Chang'an decoction(肠安方,CAD)on colitis,and investigate the potential mechanisms underlying these effects from the perspectives of endoplasmic reticulum(ER)stress i...OBJECTIVE:To evaluate the protective effects of Chang'an decoction(肠安方,CAD)on colitis,and investigate the potential mechanisms underlying these effects from the perspectives of endoplasmic reticulum(ER)stress induced by mitofusin 2(MFN2).METHODS:The composition of CAD was identified by liquid chromatography-mass spectrometry technology.A mice model of dextran sulfate sodium(DSS)induced colitis was established and therapeutic effects of CAD were determined by detecting body weight,disease activity index,colon length and histopathological changes.Then,the expression levels of MFN2,ER stress markers and Nucleotide-binding domain and leucine-rich repeat protein3(NLRP3)relevant proteins were detected by polymerase chain reaction(PCR),Western blot,immunohistochemistry and immunofluorescence staining.Subsequently,knockdown and overexpression cell model were constructed to further investigate the underlying mechanism of MFN2 mediating ER stress and energy metabolism by PCR,Western blot,electron microscopy and reactive oxygen species(ROS)staining.Finally,inflammatory indicator and tight junction proteins were measured by PCR and immunofluorescence staining to evaluate the protective effects of CAD.RESULTS:Results showed that the indispensable regulatory role of MFN2 in mediating ER stress and mitochondrial damage was involved in the protective effects of CAD on colitis in mice fed with DSS.Network pharmacology analysis also revealed CAD may play a protective effect on colitis by affecting mitochondrial function.In addition,our data also suggested a causative role for MFN2 in the development of inflammatory responses and energy metabolic alterations by constructing a knockdown and overexpression cell model whereby alter proper ER-mitochondria interaction in Caco-2 cells.Furthermore,relative expression analyses of ER stress markers and NLRP3 inflammasome showed the onset of ER stress and activation of NLRP3 inflammasome,which is consistent with the above findings.In contrast,intervention of CAD could improve the mucosal barrier integrity and colonic inflammatory response effectively through inhibiting ER stress response mediated by MFN2.CONCLUSION:CAD could alleviate ER stress by regulating MFN2 to exert therapeutic effects on DSS-induced colitis,which might provide an effective natural therapeutic approach for the treatment of ulcerative colitis.展开更多
Mitofusin2 （Mfn2） plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells （VSMCs）. The purpose of this study was to investigate the effects of Mfn2 on the traffick- ing of intracell...Mitofusin2 （Mfn2） plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells （VSMCs）. The purpose of this study was to investigate the effects of Mfn2 on the traffick- ing of intracellular cholesterol in the foam ceils derived from rat VSMCs （rVSMCs） and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 （ABCA1）, adenosine triphosphate-binding cassette subfamily G member 1 （ABCG1） and peroxisome proliferator-activated receptor gamma （PPARy）. The rVSMCs were co-cultured with oxi- dized low density lipoprotein （LDL, 80 ~tg/mL） to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers （20, 40 and 60 pfu/cell） of recombinant adenovirus containing Mfn2 gene （Adv-Mfn2） were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group （P〈0.05）, and the ex- pression levels significantly increased when the titer of Adv-Mfn2 increased （P〈0.05）. At 24 or 48 h af- ter oxidized LDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased （P〈0.05）, but it was sig- nificantly decreased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）, and it also significantly decreased when the titer of Adv-Mfn2 increased （P〈0.05）. The mRNA and pro- tein expression levels of ABCA1 and ABCG1 were significantly increased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）. Though the mRNA and protein expression levels of PPARy was not significantly increased （P〉0.05）, the phosporylation levels of PPARy were signifi- cantly decreased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）. These results suggest that the transfection of Adv-Mfn2 can significantly reduce intracellular cholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPAR＇/phosporylation and then increasing pro- tein expression levels of ABCAI and ABCG1, which may be helpful to suppress the formation of foam cells.展开更多
AIM To investigate the protective mechanism of mitofusin-2 (Mfn2) in rat remote ischemic perconditioning (RIC) models and revalidate it in alpha mouse liver-12 (AML-12) hypoxia cell lines. METHODS Sprague-Dawley rats ...AIM To investigate the protective mechanism of mitofusin-2 (Mfn2) in rat remote ischemic perconditioning (RIC) models and revalidate it in alpha mouse liver-12 (AML-12) hypoxia cell lines. METHODS Sprague-Dawley rats were divided into three groups (n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting (WB) and quantitative real-time (qRT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture (hypoxia) and anoxic incubator tank culture with Mfn2 knockdown (hypoxia + Si), and data of qRT-PCR, WB, mitochondrial membrane potential (Delta psi m), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected. RESULTS Both sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group (P < 0.05). qRTPCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2 (MICUs) axis was changed (P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of..m and apoptosis (P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group (P < 0.005). Finally, qRT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups (P < 0.005). CONCLUSION Mfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.展开更多
Objective To investigate the inhibitory effects of mitofusin 2(MFN2)on hepatic stellate cell(HSC)activation and liver fibrosis progression in nonalcoholic fatty liver disease(NAFLD)through the inhibition ofβ-catenin ...Objective To investigate the inhibitory effects of mitofusin 2(MFN2)on hepatic stellate cell(HSC)activation and liver fibrosis progression in nonalcoholic fatty liver disease(NAFLD)through the inhibition ofβ-catenin nuclear translocation.Methods In vitro,primary mouse HSCs were treated with palmitic acid(PA),and MFN2 expression was modulated using lentiviral overexpression or knockdown.Fibrotic markers andβ-catenin localization were analyzed via Western blot,cellular fractionation,and immunofluorescence.In vivo,liver fibrosis was induced in C57BL/6 J mice using a high-fat diet(HFD)combined with CCl4 injections.MFN2 was systemically overexpressed or silenced via AAV2 vectors delivered through tail vein injection.Liver tissues were examined histologically and biochemically for fibrosis progression.Results PA treatment markedly downregulated MFN2 and upregulated fibrotic markers in HSCs.Overexpression of MFN2 strongly suppressed HSC activation,reducedα-SMA and N-cadherin levels,and significantly inhibitedβ-catenin nuclear accumulation.Conversely,MFN2 knockdown exacerbated fibrotic responses and promotedβ-catenin translocation.In mice,MFN2 overexpression substantially attenuated collagen deposition and improved liver histology,while MFN2 silencing significantly aggravated fibrosis and enhancedβ-catenin signaling.Conclusion MFN2 inhibits HSC activation and liver fibrosis by suppressingβ-catenin nuclear translocation,making it a promising therapeutic target for NAFLD-related fibrosis and associated complications,such as hepatocellular carcinoma.展开更多
Recent studies have shown that fibrotic scar formation following cerebral ischemic injury has varying effects depending on the microenvironment.However,little is known about how fibrosis is induced and regulated after...Recent studies have shown that fibrotic scar formation following cerebral ischemic injury has varying effects depending on the microenvironment.However,little is known about how fibrosis is induced and regulated after cerebral ischemic injury.Sonic hedgehog signaling participates in fibrosis in the heart,liver,lung,and kidney.Whether Shh signaling modulates fibrotic scar formation after cerebral ischemic stroke and the underlying mechanisms are unclear.In this study,we found that Sonic Hedgehog expression was upregulated in patients with acute ischemic stroke and in a middle cerebral artery occlusion/reperfusion injury rat model.Both Sonic hedgehog and Mitofusin 2 showed increased expression in the middle cerebral artery occlusion rat model and in vitro fibrosis cell model induced by transforming growth factor-beta 1.Activation of the Sonic hedgehog signaling pathway enhanced the expression of phosphorylated Smad 3 and Mitofusin 2 proteins,promoted the formation of fibrotic scars,protected synapses or promoted synaptogenesis,alleviated neurological deficits following middle cerebral artery occlusion/reperfusion injury,reduced cell apoptosis,facilitated the transformation of meninges fibroblasts into myofibroblasts,and enhanced the proliferation and migration of meninges fibroblasts.The Smad3 phosphorylation inhibitor SIS3 reversed the effects induced by Sonic hedgehog signaling pathway activation.Bioinformatics analysis revealed significant correlations between Sonic hedgehog and Smad3,between Sonic hedgehog and Mitofusin 2,and between Smad3 and Mitofusin 2.These findings suggest that Sonic hedgehog signaling may influence Mitofusin 2 expression by regulating Smad3 phosphorylation,thereby modulating the formation of early fibrotic scars following cerebral ischemic stroke and affecting prognosis.The Sonic Hedgehog signaling pathway may serve as a new therapeutic target for stroke treatment.展开更多
Background：Both Mitofusin 2 （Mfn2） and pelvic organ prolapse （POP） are related to aging.The aim of the present study was to investigate the variations of Mfn2 expression in the uterosacral ligaments of patients w...Background：Both Mitofusin 2 （Mfn2） and pelvic organ prolapse （POP） are related to aging.The aim of the present study was to investigate the variations of Mfn2 expression in the uterosacral ligaments of patients with and/or without POP and their correlations with the expression of procollagen.Methods：Fibroblasts were cultured using tissue specimens that were harvested from the uterosacral ligaments of POP and non-POP （NPOP） patients （n =10 for each group） from September 2016 to December 2016.The Cell Counting Kit-8 （CCK-8） assay was used to compare the differences in cell proliferation between the two groups.Relative quantitative reverse transcription-polymerase chain reaction and Western blotting assays were employed to assess the differences in the mRNA and protein expression levels of Mfn2 and procollagen 1A1/1A2/3A1 between the two groups.The changes in procollagen expression were assessed following the downregulation of Mfn2 in the POP group using RNAi.The data were assessed with independent sample t-test or general linear model univariate analysis using the SPSS 13.0 software.Results：The results from CCK-8 assay indicated that cell viability in the POP group was significantly lower compared with that of the NPOP group （td5,7,9,11=-5.925,-6.851,-9.129,and-9.661,respectively,all P 〈 0.001,from D5 to D 11）.The mRNA and protein expression levels of Mfn2 in the cultured fibroblasts of the POP group were significantly higher compared with those of the NPOP group （mRNA：t =2.425,P =0.032;protein：t =2.392,P =0.037,respectively）,whereas only the expression levels of procollagen 1A1/1A2/3A1 were significantly higher in the NPOP group （mRNA：t =-2.165,P1A1 =0.041;t =-2.741,P1A2 =0.026;t =-2.147,P3A1 =0.045,respectively;protein：t =-2.418,P1A1 =0.029;t =-2.405,P1A2 =0.033;t =-2.470,P3A1 =0.012,respectively）.The expression levels of procollagen in the POP group increased following the downregulation of Mfn2.Conclusions：The proliferation rate and cell viability of the fibroblasts in the POP group were significantly lower compared with those in the NPOP group.In the POP fibroblasts,Mfn2 expression was increased,while procollagen expression was decreased.展开更多
Hypertonia is a neurological dysfunction associated with a number of central nervous system disorders, including cerebral palsy, Parkinson＇s disease, dystonia, and epilepsy. Genetic studies have identified a homozygo...Hypertonia is a neurological dysfunction associated with a number of central nervous system disorders, including cerebral palsy, Parkinson＇s disease, dystonia, and epilepsy. Genetic studies have identified a homozygous truncation mutation in Trakl that causes hypertonia in mice. Moreover, elevated Trakl protein expression is associated with several types of cancers and variants in Trakl are linked to childhood absence epilepsy in humans. Despite the importance of Trakl in health and disease, the mechanisms of Trakl action remain unclear and the pathogenic effects of Trakl mutation are unknown. Here we report that Trakl has a crucial function in regulation of mitochondrial fusion. Depletion of Trakl inhibits mitochondrial fusion, result- ing in mitochondrial fragmentation, whereas overex- pression of Trakl elongates and enlarges mitochondria. Our analyses revealed that Trakl interacts and colocal- izes with mitofusins on the outer mitochondrial mem- brane and functions with mitofusins to promote mitochondrial tethering and fusion. Furthermore, Trakl is required for stress-induced mitochondrial hyperfu- sion and pro-survival response. We found that hyper- tonia-associated mutation impairs Trakl mitochondrial localization and its ability to facilitate mitochondrial tethering and fusion. Our findings uncover a novel function of Trakl as a regulator of mitochondrial fusion and provide evidence linking dysregulated mitochon- drial dynamics to hypertonia pathogenesis.展开更多
Charcot-Marie-Tooth disease （CMT）, also known as hereditary motor and sensory neuropathies,comprises a genetically heterogeneous group of inherited peripheral neuropathies. Clinically it is characterized by progress...Charcot-Marie-Tooth disease （CMT）, also known as hereditary motor and sensory neuropathies,comprises a genetically heterogeneous group of inherited peripheral neuropathies. Clinically it is characterized by progressive distal weakness, muscle atrophy, distal sensory loss and loss of deep tendon reflexes. Following electrophysiological criteria, CMT is divided into two main forms：展开更多
基金National Natural Science Foundation of China,Grant/Award Number:32302826 and 32372961Jilin Provincial Special Project for Health Research Talents,Grant/Award Number:2020SCZ40China Postdoctoral Science Foundation,Grant/Award Number:2023M740623。
文摘Background:Mastitis seriously affects the mammary health of humans and animals.Studies have found that inflammation and oxidative stress play key roles in the occur-rence and development of mastitis.Therefore,in-depth research on related molecular mechanisms is of great significance.Methods:Postpartum mice were anesthetized with pentobarbital and administered lipopolysaccharide to develop the mouse mastitis model.Proteomic analysis was per-formed to compare protein expression in mitochondria-associated endoplasmic retic-ulum membranes(MAM)from two mouse mammary gland groups.Western blot was used to detect the expression of MAM-related proteins in mitochondria.AlphaFold3 was used to predict the molecular structures of phosphofurin acidic cluster sorting protein 2(PACS2)and mitofusin 2(MFN2)and their interaction levels.The MFN2-PACS2 interaction was investigated using co-immunoprecipitation and small interfer-ing RNA.Results:The results showed that the inflammation level in the mammary gland tissue of mice with mastitis significantly increased,the total antioxidant capacity decreased,and the expression of MAM-related proteins MFN2 and PACS2 was significantly downregulated.In cell experiments,overexpression of MFN2 can inhibit inflamma-tion and oxidative stress responses,and promote the interaction between MFN2 and PACS2 to affect the formation of MAMs.Conclusion:In summary,this study suggests that mastitis can alter the expression of MAM-related proteins in mouse breast tissue.The interaction between MFN2 and PACS2 regulates the formation of MAMs.Overexpression of MFN2 can promote the formation of MAMs and inhibit inflammation and oxidative stress response in mam-mary epithelial cells.Our results provided a new theoretical basis and potential thera-peutic targets for the prevention and treatment of mastitis.
基金Supported by Hangzhou Health Technology Project:Study of the Relationship Between Mitofusin 2 And Cyclosporine A Nephropathy(No.2018A52)National Natural Science Foundation of China:Study on Mechanism of Centella Asiatica Compound in the Treatment of Iga Nephropathy by Regulating Mesangial Cell Proliferation via Mitofusin 2(No.81373631)
文摘OBJECTIVE:To investigate the effect of mitofusin 2 (Mfn2) and its downstream signaling pathway on glomerular mesangial cells (GMCs) proliferation in IgA nephropathy (IgAN),as well as the mechanism of action of Jixuecao (Herba Centellae Asiaticae,HCA) in the treatment of IgAN.METHODS:Adenovirus-mediated Mfn2 gene transfection and Mfn2 expression were analyzed by real-time polymerase chain reaction (PCR) and Western blotting.IgA1 induced the proliferation of GMCs,which were then treated with HCA.Cell proliferation was detected with cell counting kit-8 (CCK-8),and Mfn2 expression was analyzed by real-time PCR and western blotting.An IgAN animal model was also established and treated with HCA.GMCs proliferation was detected by hematoxylin-eosin staining,mitochondrial structure was analyzed by electron microscopy,mitochondrial function was determined by the Clark oxygen electrode method,and the expression of Mfn2,Phospho-extracellular regulated protein kinases1/2 (P-ERK1/2),Cyclin-dependent kinase 2 (CDK2),Phospho-p27 (p-p27),and cyclin A was analyzed by Western blotting.RESULTS:In vitro,HCA inhibited GMCs in a concentration-dependent manner in association with the upregulation of Mfn2 expression.The overexpression of Mfn2 inhibited IgA1-induced GMCs proliferation and elevated the effect of HCA.In vivo,treatment with HCA could alleviate albuminuria and creatinine and GMCs proliferation.These effects were related to the upregulation of Mfn2,p-p27 and inhibition of p-ERK1/2,CDK2,and cyclinA.Mitochondrial swelling,vacuolar degeneration,and reduction of respiratory control rate were identified in IgAN,but HCA could improve the mitochondrial structure and function.CONCLUSION:HCA inhibited GMCs proliferation via the upregulation Mfn2 and the inhibition of Ras-Raf-ERK/MAPK.We revealed that changes of mitochondrial structure and function are associated with IgAN,but that HCA can improve these mitochondrial features.
文摘AIM:To investigate the effects of mitofusin-2(MFN2) on insulin sensitivity and its potential targets in the liver of rats fed with a high-fat diet(HFD).METHODS:Rats were fed with a control or HFD for 4 or 8 wk,and were then infected with a control or an MFN2 expressing adenovirus once a week for 3wk starting from the 9th wk.Blood glucose(BG),plasma insulin and insulin sensitivity of rats were determined at end of the 4th and 8th wk,and after treatment with different amounts of MFN2 expressing adenovirus(108,109 or 1010 vp/kg body weight).BG levels were measured by Accu-chek Active Meter.Plasma insulin levels were analyzed by using a Rat insulin enzymelinked immunosorbent assay kit.Insulin resistance was evaluated by measuring the glucose infusion rate(GIR) using a hyperinsulinemic euglycemic clamp technique.The expression or phosphorylation levels of MFN2 and essential molecules in the insulin signaling pathway,such as insulin receptor(INSR),insulin receptor substrate 2(IRS2),phosphoinositide-3-kinase(PI3K),protein kinase beta(AKT2) and glucose transporter type 2(GLUT2) was assayed by quantitative real-time polymerase chain reaction and Western-blotting.RESULTS:After the end of 8wk,the body weight of rats receiving the normal control diet(ND) and the HFD was not significantly different(P>0.05).Compared with the ND group,GIR in the HFD group was significantly decreased(P<0.01),while the levels of BG,triglycerides(TG),total cholesterol(TC) and insulin in the HFD group were significantly higher than those in the ND group(P<0.05).Expression of MFN2 mRNA and protein in liver of rats was significantly downregulated in the HFD group(P<0.01) after 8 wk of HFD feeding.The expression of INSR,IRS2 and GLUT2 were down-regulated markedly(P<0.01).Although there were no changes in PI3K-P85 and AKT2 expression,their phosphorylation levels were decreased significantly(P<0.01).After intervention with MFN2 expressing adenovirus for 3wk,the expression of MFN2 mRNA and protein levels were up-regulated(P<0.01).There was no difference in body weight of rats between the groups.The levels of BG,TG,TC and insulin in rats were lower than those in the Ad group(P<0.05),but GIR in rats infected with Ad-MFN2 was significantly increased(P<0.01),compared with the Ad group.The expression of INSR,IRS2 and GLUT2 was increased,while phosphorylation levels of PI3K-P85 and AKT2 were increased(P<0.01),compared with the Ad group.CONCLUSION:HFDs induce insulin resistance,and this can be reversed by MFN2 over-expression targeting the insulin signaling pathway.
基金supported by grants from the National Natural Science Foundation of China (No. 30872714 and No.30971244)
文摘Angiotensin Ⅱ (ANGⅡ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANGⅡ by cell counting and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway pro-tein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and upregulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANGⅡ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.
基金a grant of the Clinical Key Subject Foundation from Ministry of Health of China (No. 2004CB518705)
文摘In order to evaluate the effect of mitofusin-2 gene （mfn2） on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-Ⅴ/PI double labeling method was employed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 cDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the percentage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively （P〈0.05）. The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin （CAMP） followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively （P〈0.05）. It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and promote their sensitivity to CAMP with a synergic effect.
基金the National Natural Science Foundation of China Youth Fund:Study on the Mechanism of Chang'an Decoction Regulating the Endoplasmic Reticulum Stress Response via Mfn2 in the Treatment of Ulcerative Colitis(No.82004305)and the National Natural Science Foundation of China Youth Fund:Study on Protective Mechanisms of Longqi Jiangzhi Decoction against Non-alcoholic Steatohepatitis based on Th17 Cells Differentiation Induced by SPP1(No.82104549)。
文摘OBJECTIVE:To evaluate the protective effects of Chang'an decoction(肠安方,CAD)on colitis,and investigate the potential mechanisms underlying these effects from the perspectives of endoplasmic reticulum(ER)stress induced by mitofusin 2(MFN2).METHODS:The composition of CAD was identified by liquid chromatography-mass spectrometry technology.A mice model of dextran sulfate sodium(DSS)induced colitis was established and therapeutic effects of CAD were determined by detecting body weight,disease activity index,colon length and histopathological changes.Then,the expression levels of MFN2,ER stress markers and Nucleotide-binding domain and leucine-rich repeat protein3(NLRP3)relevant proteins were detected by polymerase chain reaction(PCR),Western blot,immunohistochemistry and immunofluorescence staining.Subsequently,knockdown and overexpression cell model were constructed to further investigate the underlying mechanism of MFN2 mediating ER stress and energy metabolism by PCR,Western blot,electron microscopy and reactive oxygen species(ROS)staining.Finally,inflammatory indicator and tight junction proteins were measured by PCR and immunofluorescence staining to evaluate the protective effects of CAD.RESULTS:Results showed that the indispensable regulatory role of MFN2 in mediating ER stress and mitochondrial damage was involved in the protective effects of CAD on colitis in mice fed with DSS.Network pharmacology analysis also revealed CAD may play a protective effect on colitis by affecting mitochondrial function.In addition,our data also suggested a causative role for MFN2 in the development of inflammatory responses and energy metabolic alterations by constructing a knockdown and overexpression cell model whereby alter proper ER-mitochondria interaction in Caco-2 cells.Furthermore,relative expression analyses of ER stress markers and NLRP3 inflammasome showed the onset of ER stress and activation of NLRP3 inflammasome,which is consistent with the above findings.In contrast,intervention of CAD could improve the mucosal barrier integrity and colonic inflammatory response effectively through inhibiting ER stress response mediated by MFN2.CONCLUSION:CAD could alleviate ER stress by regulating MFN2 to exert therapeutic effects on DSS-induced colitis,which might provide an effective natural therapeutic approach for the treatment of ulcerative colitis.
基金supported by the National Natural Science Foundation of China(No.30971244)
文摘Mitofusin2 （Mfn2） plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells （VSMCs）. The purpose of this study was to investigate the effects of Mfn2 on the traffick- ing of intracellular cholesterol in the foam ceils derived from rat VSMCs （rVSMCs） and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 （ABCA1）, adenosine triphosphate-binding cassette subfamily G member 1 （ABCG1） and peroxisome proliferator-activated receptor gamma （PPARy）. The rVSMCs were co-cultured with oxi- dized low density lipoprotein （LDL, 80 ~tg/mL） to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers （20, 40 and 60 pfu/cell） of recombinant adenovirus containing Mfn2 gene （Adv-Mfn2） were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group （P〈0.05）, and the ex- pression levels significantly increased when the titer of Adv-Mfn2 increased （P〈0.05）. At 24 or 48 h af- ter oxidized LDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased （P〈0.05）, but it was sig- nificantly decreased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）, and it also significantly decreased when the titer of Adv-Mfn2 increased （P〈0.05）. The mRNA and pro- tein expression levels of ABCA1 and ABCG1 were significantly increased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）. Though the mRNA and protein expression levels of PPARy was not significantly increased （P〉0.05）, the phosporylation levels of PPARy were signifi- cantly decreased in Adv-Mfn2-transfected group as compared with untransfected group （P〈0.05）. These results suggest that the transfection of Adv-Mfn2 can significantly reduce intracellular cholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPAR＇/phosporylation and then increasing pro- tein expression levels of ABCAI and ABCG1, which may be helpful to suppress the formation of foam cells.
基金Supported by Science and Technology Innovation Talents Support Plan,Department of Education,Henan Province,China,No.17HASTIT044China Postdoctoral Science Foundation,No.2017M610374
文摘AIM To investigate the protective mechanism of mitofusin-2 (Mfn2) in rat remote ischemic perconditioning (RIC) models and revalidate it in alpha mouse liver-12 (AML-12) hypoxia cell lines. METHODS Sprague-Dawley rats were divided into three groups (n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting (WB) and quantitative real-time (qRT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture (hypoxia) and anoxic incubator tank culture with Mfn2 knockdown (hypoxia + Si), and data of qRT-PCR, WB, mitochondrial membrane potential (Delta psi m), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected. RESULTS Both sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group (P < 0.05). qRTPCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2 (MICUs) axis was changed (P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of..m and apoptosis (P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group (P < 0.005). Finally, qRT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups (P < 0.005). CONCLUSION Mfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.
基金supported by Startup Fund for Scientific Research,Fujian Medical University(No.2019QH1146)the Natural Science Foundation of Fujian Province(general program)(No.2020J011063)+2 种基金the Joint Funds for the Innovation of Science and Technology,Fujian Province(No.2024Y9028)the Guided Project of Social Development in Fujian Province(No.2025Y0006)the Zhejiang Provincial Traditional Chinese Medicine Science and Technology Program Project(No.2025ZL118).
文摘Objective To investigate the inhibitory effects of mitofusin 2(MFN2)on hepatic stellate cell(HSC)activation and liver fibrosis progression in nonalcoholic fatty liver disease(NAFLD)through the inhibition ofβ-catenin nuclear translocation.Methods In vitro,primary mouse HSCs were treated with palmitic acid(PA),and MFN2 expression was modulated using lentiviral overexpression or knockdown.Fibrotic markers andβ-catenin localization were analyzed via Western blot,cellular fractionation,and immunofluorescence.In vivo,liver fibrosis was induced in C57BL/6 J mice using a high-fat diet(HFD)combined with CCl4 injections.MFN2 was systemically overexpressed or silenced via AAV2 vectors delivered through tail vein injection.Liver tissues were examined histologically and biochemically for fibrosis progression.Results PA treatment markedly downregulated MFN2 and upregulated fibrotic markers in HSCs.Overexpression of MFN2 strongly suppressed HSC activation,reducedα-SMA and N-cadherin levels,and significantly inhibitedβ-catenin nuclear accumulation.Conversely,MFN2 knockdown exacerbated fibrotic responses and promotedβ-catenin translocation.In mice,MFN2 overexpression substantially attenuated collagen deposition and improved liver histology,while MFN2 silencing significantly aggravated fibrosis and enhancedβ-catenin signaling.Conclusion MFN2 inhibits HSC activation and liver fibrosis by suppressingβ-catenin nuclear translocation,making it a promising therapeutic target for NAFLD-related fibrosis and associated complications,such as hepatocellular carcinoma.
基金supported by the National Natural Science Foundation of China,Nos.82171456(to QY)and 81971229(to QY)the Natural Science Foundation of Chongqing,Nos.CSTC2021JCYJ-MSXMX0263(to QY)and CSTB2023NSCQ-MSX1015(to XL)Doctoral Innovation Project of The First Affiliated Hospital of Chongqing Medical University,Nos.CYYY-BSYJSCXXM-202318(to JW)and CYYY-BSYJSCXXM-202327(to HT).
文摘Recent studies have shown that fibrotic scar formation following cerebral ischemic injury has varying effects depending on the microenvironment.However,little is known about how fibrosis is induced and regulated after cerebral ischemic injury.Sonic hedgehog signaling participates in fibrosis in the heart,liver,lung,and kidney.Whether Shh signaling modulates fibrotic scar formation after cerebral ischemic stroke and the underlying mechanisms are unclear.In this study,we found that Sonic Hedgehog expression was upregulated in patients with acute ischemic stroke and in a middle cerebral artery occlusion/reperfusion injury rat model.Both Sonic hedgehog and Mitofusin 2 showed increased expression in the middle cerebral artery occlusion rat model and in vitro fibrosis cell model induced by transforming growth factor-beta 1.Activation of the Sonic hedgehog signaling pathway enhanced the expression of phosphorylated Smad 3 and Mitofusin 2 proteins,promoted the formation of fibrotic scars,protected synapses or promoted synaptogenesis,alleviated neurological deficits following middle cerebral artery occlusion/reperfusion injury,reduced cell apoptosis,facilitated the transformation of meninges fibroblasts into myofibroblasts,and enhanced the proliferation and migration of meninges fibroblasts.The Smad3 phosphorylation inhibitor SIS3 reversed the effects induced by Sonic hedgehog signaling pathway activation.Bioinformatics analysis revealed significant correlations between Sonic hedgehog and Smad3,between Sonic hedgehog and Mitofusin 2,and between Smad3 and Mitofusin 2.These findings suggest that Sonic hedgehog signaling may influence Mitofusin 2 expression by regulating Smad3 phosphorylation,thereby modulating the formation of early fibrotic scars following cerebral ischemic stroke and affecting prognosis.The Sonic Hedgehog signaling pathway may serve as a new therapeutic target for stroke treatment.
基金This work was supported by a grant from the National Natural Science Foundation of China （No. 81401185）.
文摘Background：Both Mitofusin 2 （Mfn2） and pelvic organ prolapse （POP） are related to aging.The aim of the present study was to investigate the variations of Mfn2 expression in the uterosacral ligaments of patients with and/or without POP and their correlations with the expression of procollagen.Methods：Fibroblasts were cultured using tissue specimens that were harvested from the uterosacral ligaments of POP and non-POP （NPOP） patients （n =10 for each group） from September 2016 to December 2016.The Cell Counting Kit-8 （CCK-8） assay was used to compare the differences in cell proliferation between the two groups.Relative quantitative reverse transcription-polymerase chain reaction and Western blotting assays were employed to assess the differences in the mRNA and protein expression levels of Mfn2 and procollagen 1A1/1A2/3A1 between the two groups.The changes in procollagen expression were assessed following the downregulation of Mfn2 in the POP group using RNAi.The data were assessed with independent sample t-test or general linear model univariate analysis using the SPSS 13.0 software.Results：The results from CCK-8 assay indicated that cell viability in the POP group was significantly lower compared with that of the NPOP group （td5,7,9,11=-5.925,-6.851,-9.129,and-9.661,respectively,all P 〈 0.001,from D5 to D 11）.The mRNA and protein expression levels of Mfn2 in the cultured fibroblasts of the POP group were significantly higher compared with those of the NPOP group （mRNA：t =2.425,P =0.032;protein：t =2.392,P =0.037,respectively）,whereas only the expression levels of procollagen 1A1/1A2/3A1 were significantly higher in the NPOP group （mRNA：t =-2.165,P1A1 =0.041;t =-2.741,P1A2 =0.026;t =-2.147,P3A1 =0.045,respectively;protein：t =-2.418,P1A1 =0.029;t =-2.405,P1A2 =0.033;t =-2.470,P3A1 =0.012,respectively）.The expression levels of procollagen in the POP group increased following the downregulation of Mfn2.Conclusions：The proliferation rate and cell viability of the fibroblasts in the POP group were significantly lower compared with those in the NPOP group.In the POP fibroblasts,Mfn2 expression was increased,while procollagen expression was decreased.
文摘Hypertonia is a neurological dysfunction associated with a number of central nervous system disorders, including cerebral palsy, Parkinson＇s disease, dystonia, and epilepsy. Genetic studies have identified a homozygous truncation mutation in Trakl that causes hypertonia in mice. Moreover, elevated Trakl protein expression is associated with several types of cancers and variants in Trakl are linked to childhood absence epilepsy in humans. Despite the importance of Trakl in health and disease, the mechanisms of Trakl action remain unclear and the pathogenic effects of Trakl mutation are unknown. Here we report that Trakl has a crucial function in regulation of mitochondrial fusion. Depletion of Trakl inhibits mitochondrial fusion, result- ing in mitochondrial fragmentation, whereas overex- pression of Trakl elongates and enlarges mitochondria. Our analyses revealed that Trakl interacts and colocal- izes with mitofusins on the outer mitochondrial mem- brane and functions with mitofusins to promote mitochondrial tethering and fusion. Furthermore, Trakl is required for stress-induced mitochondrial hyperfu- sion and pro-survival response. We found that hyper- tonia-associated mutation impairs Trakl mitochondrial localization and its ability to facilitate mitochondrial tethering and fusion. Our findings uncover a novel function of Trakl as a regulator of mitochondrial fusion and provide evidence linking dysregulated mitochon- drial dynamics to hypertonia pathogenesis.
文摘Charcot-Marie-Tooth disease （CMT）, also known as hereditary motor and sensory neuropathies,comprises a genetically heterogeneous group of inherited peripheral neuropathies. Clinically it is characterized by progressive distal weakness, muscle atrophy, distal sensory loss and loss of deep tendon reflexes. Following electrophysiological criteria, CMT is divided into two main forms：
