Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of a...Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.展开更多
目的观察磷脂酰肌醇-3激酶(PI3K)-蛋白质丝氨酸/苏氨酸激酶(AKt)-内皮型一氧化氮合酶(e NOS)信号转导通路在硫化氢(H2S)抑制内皮素-1(endothelin-1,ET-1)诱导心肌肥大过程中的作用。方法体外培养原代心肌细胞,将其随机分为6组,每组4孔,...目的观察磷脂酰肌醇-3激酶(PI3K)-蛋白质丝氨酸/苏氨酸激酶(AKt)-内皮型一氧化氮合酶(e NOS)信号转导通路在硫化氢(H2S)抑制内皮素-1(endothelin-1,ET-1)诱导心肌肥大过程中的作用。方法体外培养原代心肌细胞,将其随机分为6组,每组4孔,1对照组:加入等体积无血清的DMEM培养基;2肥大(ET-1)组:加入终浓度为10-8 mol/L的ET-1;剩余4组为实验组,各组分别加入不同终浓度的H2S供体-Na HS:310-15 M Na HS组:加入10-15 mol/L Na HS+10-8 mol/l ET-1;410-14 M Na HS组:加入10-14 mol/L Na HS+10-8 mol/L ET-1;510-13 M Na HS组:加入10-13 mol/L Na HS+10-8 mol/L ET-1;610-12 M Na HS组:加入10-12 mol/L Na HS+10-8 mol/L ET-1。上述各组药物分别刺激24 h后测定心肌细胞表面积、细胞总蛋白含量、培养液NO含量,RT-PCR检测心肌细胞心房利钠肽(atrial natriuretic peptide,ANP)、脑钠肽(B-type natriuretic peptide,BNP)、磷脂酰肌醇-3激酶(phosphatidylinositol-3-kinase,PI3K)、蛋白激酶B(protein kinase B,PKB/AKt)、e NOS m RNA水平,Western Blot技术检测总AKt和磷酸化AKt蛋白表达含量。结果肥大(ET-1)组的心肌细胞表面积(1933.80±143.06)和细胞总蛋白含量(367.51±25.9)均高于对照组(787.27±107.66,218.55±21.28,P<0.05),ANP及BNP m RNA的表达量也明显增加(P<0.05),但PI3K、AKt、e NOS m RNA表达水平,磷酸化AKt程度和NO的释放量(4.60±0.73)低于对照组(8.63±0.30,P<0.05),各实验组给予不同浓度Na HS刺激后能够浓度依赖性的抑制这种肥大效应(P<0.05),同时上调了PI3K/AKt/e NOS通路各信号分子的表达量(P<0.05)。结论 H2S对ET-1诱导的心肌肥大有一定的抑制作用,这种作用可能与激活PI3K-AKt-e NOS信号通路有关。展开更多
Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regu-lation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS ac-tivity via phosphorylation modification but...Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regu-lation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS ac-tivity via phosphorylation modification but the mechanisms involved vary with stimuli applied. We have demonstrated that cytochrome P450 (CYP) epoxygenase-dependent metabolites of ara-chidonic acid, epoxyeicosatrienoic acids (EETs), can robustly up-regulate eNOS expression and its activity, however the relevant signaling pathways responsible for activity regulation are not well known. In this study, we explored the role of PI3 kinase (PI3K)/protein kinase B (Akt) sig-naling pathway in eNOS expression and its phosphorylation in response to EETs via direct addi-tion of EETs into cultured bovine aorta endothelial cells (BAECs) and recombinant adeno- asso-ciated virus-mediated transfection of CYP epoxygenase genes CYPF87V and CYP2C11 to pro-duce endogenous EETs followed by co-treatment with PI3K or Akt inhibitor. Results show that both exogenous and endogenous EETs could remarkably enhance eNOS expression and its phosphorylation at Ser1179 and Thr497 residues; PI3K inhibitor LY294002 could inhibit EETs-induced increase in eNOS-Ser(P)1179 but had no effect on the change of eNOS-Thr(P)497, while Akt inhibitor could attenuate the increase in phosphor-eNOS at both residues; both of the two inhibitors could block EETs-enhanced eNOS expression. These results lead to conclusions: (i) EETs-mediated regulation of eNOS activity may be related with the changes of phosphoryla-tion level at eNOS-Ser1179 via PI3K/Akt and eNOS-Thr497 via Akt; (ii) PI3K/Akt signaling pathway is involved in the up-regulation of eNOS expression by EETs.展开更多
基金supported by the Natural Nature Science Foundation of China,Nos.82030071,81874004the Science and Technology Major Project of Changsha,No.kh2103008(all to JZH).
文摘Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.
文摘目的观察磷脂酰肌醇-3激酶(PI3K)-蛋白质丝氨酸/苏氨酸激酶(AKt)-内皮型一氧化氮合酶(e NOS)信号转导通路在硫化氢(H2S)抑制内皮素-1(endothelin-1,ET-1)诱导心肌肥大过程中的作用。方法体外培养原代心肌细胞,将其随机分为6组,每组4孔,1对照组:加入等体积无血清的DMEM培养基;2肥大(ET-1)组:加入终浓度为10-8 mol/L的ET-1;剩余4组为实验组,各组分别加入不同终浓度的H2S供体-Na HS:310-15 M Na HS组:加入10-15 mol/L Na HS+10-8 mol/l ET-1;410-14 M Na HS组:加入10-14 mol/L Na HS+10-8 mol/L ET-1;510-13 M Na HS组:加入10-13 mol/L Na HS+10-8 mol/L ET-1;610-12 M Na HS组:加入10-12 mol/L Na HS+10-8 mol/L ET-1。上述各组药物分别刺激24 h后测定心肌细胞表面积、细胞总蛋白含量、培养液NO含量,RT-PCR检测心肌细胞心房利钠肽(atrial natriuretic peptide,ANP)、脑钠肽(B-type natriuretic peptide,BNP)、磷脂酰肌醇-3激酶(phosphatidylinositol-3-kinase,PI3K)、蛋白激酶B(protein kinase B,PKB/AKt)、e NOS m RNA水平,Western Blot技术检测总AKt和磷酸化AKt蛋白表达含量。结果肥大(ET-1)组的心肌细胞表面积(1933.80±143.06)和细胞总蛋白含量(367.51±25.9)均高于对照组(787.27±107.66,218.55±21.28,P<0.05),ANP及BNP m RNA的表达量也明显增加(P<0.05),但PI3K、AKt、e NOS m RNA表达水平,磷酸化AKt程度和NO的释放量(4.60±0.73)低于对照组(8.63±0.30,P<0.05),各实验组给予不同浓度Na HS刺激后能够浓度依赖性的抑制这种肥大效应(P<0.05),同时上调了PI3K/AKt/e NOS通路各信号分子的表达量(P<0.05)。结论 H2S对ET-1诱导的心肌肥大有一定的抑制作用,这种作用可能与激活PI3K-AKt-e NOS信号通路有关。
基金This work was supported by the National Natural Science Foundation(Grant Nos.39870307,30270561&30430320).
文摘Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regu-lation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS ac-tivity via phosphorylation modification but the mechanisms involved vary with stimuli applied. We have demonstrated that cytochrome P450 (CYP) epoxygenase-dependent metabolites of ara-chidonic acid, epoxyeicosatrienoic acids (EETs), can robustly up-regulate eNOS expression and its activity, however the relevant signaling pathways responsible for activity regulation are not well known. In this study, we explored the role of PI3 kinase (PI3K)/protein kinase B (Akt) sig-naling pathway in eNOS expression and its phosphorylation in response to EETs via direct addi-tion of EETs into cultured bovine aorta endothelial cells (BAECs) and recombinant adeno- asso-ciated virus-mediated transfection of CYP epoxygenase genes CYPF87V and CYP2C11 to pro-duce endogenous EETs followed by co-treatment with PI3K or Akt inhibitor. Results show that both exogenous and endogenous EETs could remarkably enhance eNOS expression and its phosphorylation at Ser1179 and Thr497 residues; PI3K inhibitor LY294002 could inhibit EETs-induced increase in eNOS-Ser(P)1179 but had no effect on the change of eNOS-Thr(P)497, while Akt inhibitor could attenuate the increase in phosphor-eNOS at both residues; both of the two inhibitors could block EETs-enhanced eNOS expression. These results lead to conclusions: (i) EETs-mediated regulation of eNOS activity may be related with the changes of phosphoryla-tion level at eNOS-Ser1179 via PI3K/Akt and eNOS-Thr497 via Akt; (ii) PI3K/Akt signaling pathway is involved in the up-regulation of eNOS expression by EETs.
