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
目的观察重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)对缺血/再灌注损伤大鼠心肌细胞Mitofusin2(Mfn2)蛋白表达的影响及其抗心肌细胞凋亡的作用。方法选取成年SD大鼠35只,随机分为正常组(Normal),假手术组(Sham),缺...目的观察重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)对缺血/再灌注损伤大鼠心肌细胞Mitofusin2(Mfn2)蛋白表达的影响及其抗心肌细胞凋亡的作用。方法选取成年SD大鼠35只,随机分为正常组(Normal),假手术组(Sham),缺血再灌注组(I/R),缺血再灌注EPO治疗组(I/R+EPO)。各组分别于再灌注3h和24h后,剪取心脏缺血/再灌注损伤区域,用脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测心肌细胞凋亡,免疫组化法检测Mfn2蛋白的表达。结果再灌注3h和24h后,与正常组和假手术组相比,I/R组Mfn2蛋白的表达和心肌细胞凋亡均显著增加;与I/R组相比,I/R+EPO组Mfn2蛋白的表达和心肌细胞凋亡均显著降低。结论EPO可以下调缺血再灌注损伤后心肌细胞Mfn2蛋白的表达,抑制心肌细胞的凋亡。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
Objective:To identify the molecular mechanisms of the effects of the Buyin Qianzheng formula(BYQZF)on the mitochondrial dynamics in a Parkin overexpression Parkinson's disease(PD)cell model.Methods:First,a stable ...Objective:To identify the molecular mechanisms of the effects of the Buyin Qianzheng formula(BYQZF)on the mitochondrial dynamics in a Parkin overexpression Parkinson's disease(PD)cell model.Methods:First,a stable Parkin overexpression cell model was constructed using plasmid transfection.Then,we examined the protective effect of BYQZF on the mitochondrial dysfunction of the Parkin overexpression PD cell model induced by neurotoxin 1-methyl-4-phenylpyridinium ion(MPPþ).The mRNA expression level of Parkin was evaluated using real-time quantitative PCR.The cell survival rate was detected using the Cell Counting Kit-8 assay.We evaluated the cellular adenosine triphosphate(ATP)levels using luciferase assays.A laser scanning confocal microscope was used to observe the mitochondrial morphology,activity,and mitochondrial membrane potential(DJm).Western blot was conducted to evaluate the levels of the fusion proteins mitofusin1,mitofusin2,optic atrophy 1,dynaminrelated protein 1,and mitochondrial fission protein 1.Results:Parkin overexpression attenuated MPPþ-induced mitochondrial damage,increased mitochondrial activity and DJm.BYQZF increased the survival of MPPþ-induced cells that overexpressed Parkin and upregulated the mitochondrial form factor and activity.It also inhibited a decrease in the DJm and ATP levels.These findings suggested that BYQZF protected against MPPþ-induced mitochondrial dysfunction and enhanced the protective effect of Parkin overexpression.Furthermore,the formula upregulated the expression of the fusion proteins mitofusin1,mitofusin2,and optic atrophy 1(closely related to mitochondrial quality remodeling),and reduced the expression of the fission protein dynamicrelated protein 1,as well as mitochondrial fission protein 1.Conclusion:The mechanism by which BYQZF increased the mitochondrial protective effect of Parkin gene overexpression in MPPþ-induced cells may be related to improving mitochondrial function and regulating the balance of mitochondrial division and fusion proteins.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
目的探讨姜黄素对脓毒症小鼠急性肺损伤的保护作用及机制。方法取120只清洁级BALB/c雄性小鼠随机(随机数字法)分成8组,即假手术组、脓毒症组、姜黄素对照组、姜黄素干预组、阴性病毒-脓毒症组、阴性病毒-姜黄素干预组、线粒体融合蛋白(M...目的探讨姜黄素对脓毒症小鼠急性肺损伤的保护作用及机制。方法取120只清洁级BALB/c雄性小鼠随机(随机数字法)分成8组,即假手术组、脓毒症组、姜黄素对照组、姜黄素干预组、阴性病毒-脓毒症组、阴性病毒-姜黄素干预组、线粒体融合蛋白(Mfn2)干扰-脓毒症组、Mfn2干扰-姜黄素干预组,每组15只。脓毒症组及姜黄素干预组通过盲肠结扎穿孔术(cecal ligation and puncture,CLP)造模,姜黄素干预组及姜黄素对照组予姜黄素200 mg/(kg·d)灌胃1周,阴性病毒-脓毒症组及阴性病毒-姜黄素干预组通过尾静脉注射阴性腺相关病毒来建立,Mfn2干扰-脓毒症组及Mfn2干扰-姜黄素干预组通过尾静脉注射携带Mfn2干扰序列的腺相关病毒建立模型,各组均于24 h后处死小鼠。通过肺湿干比和病理学检查反映肺损伤程度,ELISA法检测肺泡灌洗液的炎症因子TNF-α和IL-6,Western blot检测细胞凋亡的关键分子caspase-3的活化,并通过TUNEL检测细胞凋亡。采用SPSS 22.0软件进行统计分析,计数资料比较采用χ^2检验,计量资料组间比较采用单因素方差分析。结果与假手术组比较,脓毒症组小鼠肺组织湿干重比显著增加(71.11±3.78 vs 31.11±5.61,P=0.002);与假手术组相比,脓毒症组织病理评分明显增加(P=0.006),炎症因子TNF-α(P=0.001)和IL-6(P=0.012)显著增高,肺组织细胞凋亡及凋亡相关蛋白caspase-3 cleaved表达同样明显增加(P=0.001)。与脓毒症组比较,姜黄素干预组小鼠肺组织湿干重比明显降低(32.84±6.15 vs 71.11±3.78,P=0.004),组织病理评分显著降低(P=0.004),炎症因子TNF-α(P=0.013)和IL-6(P=0.003)均明显降低,肺组织细胞凋亡及凋亡相关蛋白caspase-3 cleaved表达同样显著减少(P=0.012)。在下调Mfn2后,Mfn2干扰-姜黄素干预组与Mfn2干扰-脓毒症组相比,小鼠肺组织干湿重比无明显降低,组织病理评分也无显著降低,炎症因子TNF-α和IL-6均无明显降低,肺组织细胞凋亡及凋亡相关蛋白caspase-3 cleaved表达无显著减少。结论姜黄素可能通过上调线粒体融合蛋白2的表达减轻脓毒症急性肺损伤。展开更多
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.
文摘目的观察重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)对缺血/再灌注损伤大鼠心肌细胞Mitofusin2(Mfn2)蛋白表达的影响及其抗心肌细胞凋亡的作用。方法选取成年SD大鼠35只,随机分为正常组(Normal),假手术组(Sham),缺血再灌注组(I/R),缺血再灌注EPO治疗组(I/R+EPO)。各组分别于再灌注3h和24h后,剪取心脏缺血/再灌注损伤区域,用脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测心肌细胞凋亡,免疫组化法检测Mfn2蛋白的表达。结果再灌注3h和24h后,与正常组和假手术组相比,I/R组Mfn2蛋白的表达和心肌细胞凋亡均显著增加;与I/R组相比,I/R+EPO组Mfn2蛋白的表达和心肌细胞凋亡均显著降低。结论EPO可以下调缺血再灌注损伤后心肌细胞Mfn2蛋白的表达,抑制心肌细胞的凋亡。
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This work is supported by the National Natural Science Foundation of China(81774110 and 81573773).
文摘Objective:To identify the molecular mechanisms of the effects of the Buyin Qianzheng formula(BYQZF)on the mitochondrial dynamics in a Parkin overexpression Parkinson's disease(PD)cell model.Methods:First,a stable Parkin overexpression cell model was constructed using plasmid transfection.Then,we examined the protective effect of BYQZF on the mitochondrial dysfunction of the Parkin overexpression PD cell model induced by neurotoxin 1-methyl-4-phenylpyridinium ion(MPPþ).The mRNA expression level of Parkin was evaluated using real-time quantitative PCR.The cell survival rate was detected using the Cell Counting Kit-8 assay.We evaluated the cellular adenosine triphosphate(ATP)levels using luciferase assays.A laser scanning confocal microscope was used to observe the mitochondrial morphology,activity,and mitochondrial membrane potential(DJm).Western blot was conducted to evaluate the levels of the fusion proteins mitofusin1,mitofusin2,optic atrophy 1,dynaminrelated protein 1,and mitochondrial fission protein 1.Results:Parkin overexpression attenuated MPPþ-induced mitochondrial damage,increased mitochondrial activity and DJm.BYQZF increased the survival of MPPþ-induced cells that overexpressed Parkin and upregulated the mitochondrial form factor and activity.It also inhibited a decrease in the DJm and ATP levels.These findings suggested that BYQZF protected against MPPþ-induced mitochondrial dysfunction and enhanced the protective effect of Parkin overexpression.Furthermore,the formula upregulated the expression of the fusion proteins mitofusin1,mitofusin2,and optic atrophy 1(closely related to mitochondrial quality remodeling),and reduced the expression of the fission protein dynamicrelated protein 1,as well as mitochondrial fission protein 1.Conclusion:The mechanism by which BYQZF increased the mitochondrial protective effect of Parkin gene overexpression in MPPþ-induced cells may be related to improving mitochondrial function and regulating the balance of mitochondrial division and fusion proteins.
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.
文摘目的探讨姜黄素对脓毒症小鼠急性肺损伤的保护作用及机制。方法取120只清洁级BALB/c雄性小鼠随机(随机数字法)分成8组,即假手术组、脓毒症组、姜黄素对照组、姜黄素干预组、阴性病毒-脓毒症组、阴性病毒-姜黄素干预组、线粒体融合蛋白(Mfn2)干扰-脓毒症组、Mfn2干扰-姜黄素干预组,每组15只。脓毒症组及姜黄素干预组通过盲肠结扎穿孔术(cecal ligation and puncture,CLP)造模,姜黄素干预组及姜黄素对照组予姜黄素200 mg/(kg·d)灌胃1周,阴性病毒-脓毒症组及阴性病毒-姜黄素干预组通过尾静脉注射阴性腺相关病毒来建立,Mfn2干扰-脓毒症组及Mfn2干扰-姜黄素干预组通过尾静脉注射携带Mfn2干扰序列的腺相关病毒建立模型,各组均于24 h后处死小鼠。通过肺湿干比和病理学检查反映肺损伤程度,ELISA法检测肺泡灌洗液的炎症因子TNF-α和IL-6,Western blot检测细胞凋亡的关键分子caspase-3的活化,并通过TUNEL检测细胞凋亡。采用SPSS 22.0软件进行统计分析,计数资料比较采用χ^2检验,计量资料组间比较采用单因素方差分析。结果与假手术组比较,脓毒症组小鼠肺组织湿干重比显著增加(71.11±3.78 vs 31.11±5.61,P=0.002);与假手术组相比,脓毒症组织病理评分明显增加(P=0.006),炎症因子TNF-α(P=0.001)和IL-6(P=0.012)显著增高,肺组织细胞凋亡及凋亡相关蛋白caspase-3 cleaved表达同样明显增加(P=0.001)。与脓毒症组比较,姜黄素干预组小鼠肺组织湿干重比明显降低(32.84±6.15 vs 71.11±3.78,P=0.004),组织病理评分显著降低(P=0.004),炎症因子TNF-α(P=0.013)和IL-6(P=0.003)均明显降低,肺组织细胞凋亡及凋亡相关蛋白caspase-3 cleaved表达同样显著减少(P=0.012)。在下调Mfn2后,Mfn2干扰-姜黄素干预组与Mfn2干扰-脓毒症组相比,小鼠肺组织干湿重比无明显降低,组织病理评分也无显著降低,炎症因子TNF-α和IL-6均无明显降低,肺组织细胞凋亡及凋亡相关蛋白caspase-3 cleaved表达无显著减少。结论姜黄素可能通过上调线粒体融合蛋白2的表达减轻脓毒症急性肺损伤。
