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
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
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
目的:探讨阿托伐他汀对大鼠蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)的缓解效果及对线粒体融合蛋白2(Mitofusin-2)、脑源性神经营养因子(BDNF)表达的影响,为SAH后CVS的防治提供实验依据和新方法。方法:选取雄性SD大鼠30只,随机数字表法随...目的:探讨阿托伐他汀对大鼠蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)的缓解效果及对线粒体融合蛋白2(Mitofusin-2)、脑源性神经营养因子(BDNF)表达的影响,为SAH后CVS的防治提供实验依据和新方法。方法:选取雄性SD大鼠30只,随机数字表法随机分为假手术组、模型组和治疗组,每组10只。模型组和治疗组使用枕大池二次注血法制作蛛网膜下腔出血模型,假手术组同样操作方式注入生理盐水。治疗组予阿托伐他汀20 mg/kg溶于2 mL蒸馏水灌胃。假手术组与模型组给与蒸馏水2 mL灌胃。观察干预5 d后各组大鼠的体重、死亡率、神经功能缺失情况、基底动脉血管内径、管壁厚度以及平滑肌细胞凋亡情况,观察各组Mitofusin-2、BDNF表达水平。结果: 3组大鼠的体重由低到高分别为假手术组、治疗组、模型组,差异具有统计学意义( P <0.05),假手术组和治疗组各有1只大鼠死亡,模型组有2只大鼠死亡,3组死亡率无显著差异( P >0.05)。3组神经功能评分由低到高分别为模型组、治疗组和假手术组,差异具有统计学意义( P <0.05)。3组血管内径由小到大分别为模型组、治疗组、假手术组,差异具有统计学意义( P <0.05),而血管壁的厚度由小到大分别为假手术组、治疗组、模型组,差异具有统计学意义( P <0.05)。3组血管内皮细胞凋亡率由低到高分别为模型组、治疗组和假手术组,差异具有统计学意义( P <0.05)。Mitofusin-2的表达水平由低到高分别为假手术组、模型组和治疗组,差异具有统计学意义( P <0.05),BDNF的表达水平由低到高分别为假手术组、治疗组和模型组,差异具有统计学意义( P <0.05)。结论:阿托伐他汀能够减轻SAH后CVS的发生,减轻脑组织的损伤,且其机制可能通过上调Mitofusin-2的表达相关。展开更多
Objective:To investigate the mitigation effect of atorvastatin on cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) in rats and its effect on mitochondrial fusion protein 2 (Mitofusin-2) and brain-derived n...Objective:To investigate the mitigation effect of atorvastatin on cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) in rats and its effect on mitochondrial fusion protein 2 (Mitofusin-2) and brain-derived neurotrophic factor (BDNF), which provides an experimental basis and a new method for the prevention and treatment of CVS after SAH.Methods:30 male SD rats were randomly divided into the sham operation group, the model group and the treatment group, with 10 rats in each group. In the model group and the treatment group, the subarachnoid hemorrhage model was made by the double injection of blood in the occipital cistern, and the sham operation group was injected with physiological saline in the same manner. The treatment group was given atorvastatin 20 mg/kg, which was dissolved in 2 mL of distilled water. The sham operation group and the model group were given 2 mL of distilled water. The body weight, mortality, neurological deficit, basilar artery inner diameter, wall thickness and smooth muscle cell apoptosis were observed in the rats 5 d after intervention. The expression levels of Mitofusin-2 and BDNF in each group were observed.Results:The body weight of the three groups was from low to high in the sham operation group, the treatment group and the model group, and the difference was statistically significant. One rat died in the sham operation group and the treatment group, respectively, 2 rats died in the model group and there was no significant difference in mortality between the three groups. The scores of the three groups of neurological function were from low to high among the model group, treatment group and sham operation group, and the difference was statistically significant. The diameter of the three groups of blood vessels was from small to large among the model group, treatment group and sham operation group, and the difference was statistically significant. The apoptotic rate of the three groups of vascular endothelial cells was from small to large among the model group, treatment group and sham operation group, and the difference was statistically significant. The expression levels of Mitofusin-2 were from low to high among the sham operation group, model group and treatment group, respectively.Conclusion:Atorvastatin can alleviate the occurrence of CVS after SAH and alleviate brain tissue damage, and its mechanism may be related to up-regulation of Mitofusin-2 expression.展开更多
芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛...芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛选法(磁珠-SELEX)开展10轮筛选,经由高通量测序获得6条候补序列(S1~S6),并进行家族性、同源性分析及二级结构预测。结果表明,6条候选核酸适体的重复率可达46.38%,其自由能在-9.02到-2.47 kcal·moL^(-1)之间,根据自由能能量稳定原则,S1和S5吉布斯自由能最低最稳定,分别为-6.70和-9.02 kcal·moL^(-1)。利用ELISA试验进行亲和力测试,结果表明核酸适体S1和S2的亲和能力较强,S1:KD=67.02 nmol·L^(-1),R2=0.925 8,S2:KD=97.65 nmol·L^(-1),R2=0.795 1。核酸适体S1与过敏原Ses i 2的结合力和其他过敏原蛋白相比有显著差异,可视为具有特异性。本研究最终获得一条兼具良好亲和力和特异性的核酸适体S1,为芝麻过敏原快速检测提供了技术支撑。展开更多
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.
文摘目的:探讨阿托伐他汀对大鼠蛛网膜下腔出血(SAH)后脑血管痉挛(CVS)的缓解效果及对线粒体融合蛋白2(Mitofusin-2)、脑源性神经营养因子(BDNF)表达的影响,为SAH后CVS的防治提供实验依据和新方法。方法:选取雄性SD大鼠30只,随机数字表法随机分为假手术组、模型组和治疗组,每组10只。模型组和治疗组使用枕大池二次注血法制作蛛网膜下腔出血模型,假手术组同样操作方式注入生理盐水。治疗组予阿托伐他汀20 mg/kg溶于2 mL蒸馏水灌胃。假手术组与模型组给与蒸馏水2 mL灌胃。观察干预5 d后各组大鼠的体重、死亡率、神经功能缺失情况、基底动脉血管内径、管壁厚度以及平滑肌细胞凋亡情况,观察各组Mitofusin-2、BDNF表达水平。结果: 3组大鼠的体重由低到高分别为假手术组、治疗组、模型组,差异具有统计学意义( P <0.05),假手术组和治疗组各有1只大鼠死亡,模型组有2只大鼠死亡,3组死亡率无显著差异( P >0.05)。3组神经功能评分由低到高分别为模型组、治疗组和假手术组,差异具有统计学意义( P <0.05)。3组血管内径由小到大分别为模型组、治疗组、假手术组,差异具有统计学意义( P <0.05),而血管壁的厚度由小到大分别为假手术组、治疗组、模型组,差异具有统计学意义( P <0.05)。3组血管内皮细胞凋亡率由低到高分别为模型组、治疗组和假手术组,差异具有统计学意义( P <0.05)。Mitofusin-2的表达水平由低到高分别为假手术组、模型组和治疗组,差异具有统计学意义( P <0.05),BDNF的表达水平由低到高分别为假手术组、治疗组和模型组,差异具有统计学意义( P <0.05)。结论:阿托伐他汀能够减轻SAH后CVS的发生,减轻脑组织的损伤,且其机制可能通过上调Mitofusin-2的表达相关。
文摘Objective:To investigate the mitigation effect of atorvastatin on cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH) in rats and its effect on mitochondrial fusion protein 2 (Mitofusin-2) and brain-derived neurotrophic factor (BDNF), which provides an experimental basis and a new method for the prevention and treatment of CVS after SAH.Methods:30 male SD rats were randomly divided into the sham operation group, the model group and the treatment group, with 10 rats in each group. In the model group and the treatment group, the subarachnoid hemorrhage model was made by the double injection of blood in the occipital cistern, and the sham operation group was injected with physiological saline in the same manner. The treatment group was given atorvastatin 20 mg/kg, which was dissolved in 2 mL of distilled water. The sham operation group and the model group were given 2 mL of distilled water. The body weight, mortality, neurological deficit, basilar artery inner diameter, wall thickness and smooth muscle cell apoptosis were observed in the rats 5 d after intervention. The expression levels of Mitofusin-2 and BDNF in each group were observed.Results:The body weight of the three groups was from low to high in the sham operation group, the treatment group and the model group, and the difference was statistically significant. One rat died in the sham operation group and the treatment group, respectively, 2 rats died in the model group and there was no significant difference in mortality between the three groups. The scores of the three groups of neurological function were from low to high among the model group, treatment group and sham operation group, and the difference was statistically significant. The diameter of the three groups of blood vessels was from small to large among the model group, treatment group and sham operation group, and the difference was statistically significant. The apoptotic rate of the three groups of vascular endothelial cells was from small to large among the model group, treatment group and sham operation group, and the difference was statistically significant. The expression levels of Mitofusin-2 were from low to high among the sham operation group, model group and treatment group, respectively.Conclusion:Atorvastatin can alleviate the occurrence of CVS after SAH and alleviate brain tissue damage, and its mechanism may be related to up-regulation of Mitofusin-2 expression.
文摘芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛选法(磁珠-SELEX)开展10轮筛选,经由高通量测序获得6条候补序列(S1~S6),并进行家族性、同源性分析及二级结构预测。结果表明,6条候选核酸适体的重复率可达46.38%,其自由能在-9.02到-2.47 kcal·moL^(-1)之间,根据自由能能量稳定原则,S1和S5吉布斯自由能最低最稳定,分别为-6.70和-9.02 kcal·moL^(-1)。利用ELISA试验进行亲和力测试,结果表明核酸适体S1和S2的亲和能力较强,S1:KD=67.02 nmol·L^(-1),R2=0.925 8,S2:KD=97.65 nmol·L^(-1),R2=0.795 1。核酸适体S1与过敏原Ses i 2的结合力和其他过敏原蛋白相比有显著差异,可视为具有特异性。本研究最终获得一条兼具良好亲和力和特异性的核酸适体S1,为芝麻过敏原快速检测提供了技术支撑。
