目的:探究TRIM2通过泛素化修饰PKM2调控肝癌细胞糖酵解的机制。方法:TCGA和CPTAC数据库分析TRIM2基因及蛋白表达水平,并结合病人生存状态进行相关性分析。构建HepG2-shNC和HepG2-shTRIM2细胞株,细胞能量代谢仪检测两组细胞胞外酸化率和...目的:探究TRIM2通过泛素化修饰PKM2调控肝癌细胞糖酵解的机制。方法:TCGA和CPTAC数据库分析TRIM2基因及蛋白表达水平,并结合病人生存状态进行相关性分析。构建HepG2-shNC和HepG2-shTRIM2细胞株,细胞能量代谢仪检测两组细胞胞外酸化率和耗氧率,并测定细胞葡萄糖吸收率、乳酸产生量、ATP水平。免疫共沉淀验证TRIM2和PKM2在HepG2、HEK293T细胞中相互作用,免疫荧光检测PKM2和TRIM2共定位。免疫共沉淀检测在HepG2/HEK293T细胞中敲低/过表达TRIM2后PKM2的泛素化修饰水平,体外酶催化反应检测在HepG2/HEK293T细胞中敲低/过表达TRIM2后PKM2酶活性。Transwell小室法检测HepG2-shNC和HepG2-shTRIM2细胞侵袭和迁移能力。皮下移植瘤实验检测HepG2-shNC和HepG2-shTRIM2细胞体内成瘤能力。结果:TCGA和CPTAC数据库分析发现TRIM2在肝癌组织中mRNA及蛋白表达水平均升高,高TRIM2表达预示差的患者生存状态。TRIM2表达水平与糖酵解通路相关性高,敲低TRIM2可抑制HepG2胞外酸化率,提高耗氧率,细胞葡萄糖吸收率[(100.0000±2.0000)%vs(46.6667±8.1445)%]、乳酸产生量[(99.0000±3.6055)%vs(43.6667±10.01665)%]、ATP水平[(97.0000±3.6055)%vs(57.3333±6.6583)%]均显著下调。TRIM2与PKM2在HepG2细胞中相互作用且蛋白共定位。TRIM2抑制后HepG2细胞中PKM2泛素化水平升高,PKM2酶活性降低;在HEK293T细胞中表达Myc-TRIM2,下调PKM2蛋白泛素化修饰水平,提高PKM2酶活性。敲低TRIM2抑制HepG2细胞侵袭能力(1 vs 0.5067±0.1168)、迁移能力(1 vs 0.5133±0.08622),抑制细胞体内成瘤体积[(529.3333±29.28026)mm 3 vs(281.6667±25.6969)mm 3]和重量[(360.3333±42.7161)mg vs(172.1667±50.2849)mg]。结论:TRIM2在肝癌组织中高表达,通过去泛素化修饰PKM2促进其酶活性,进而调控肿瘤细胞糖酵解能力。展开更多
Epidemiological and animal studies indicate that pre-existing diabetes increases the risk of Parkinson's disease(PD).However,the mechanisms underlying this association remain unclear.In the present study,we found ...Epidemiological and animal studies indicate that pre-existing diabetes increases the risk of Parkinson's disease(PD).However,the mechanisms underlying this association remain unclear.In the present study,we found that high glucose(HG)levels in the cerebrospinal fluid(CSF)of diabetic rats might enhance the effect of a subthreshold dose of the neurotoxin 6-hydroxydopamine(6-OHDA)on the development of motor disorders,and the damage to the nigrostriatal dopaminergic neuronal pathway.In vitro,HG promoted the 6-OHDA-induced apoptosis in PC12 cells differentiated to neurons with nerve growth factor(NGF)(NGF-PC12).Metabolomics showed that HG promoted hyperglycolysis in neurons and impaired tricarboxylic acid cycle(TCA cycle)activity,which was closely related to abnormal mitochondrial fusion,thus resulting in mitochondrial loss.Interestingly,HG-induced upregulation of pyruvate kinase M2(PKM2)combined with 6-OHDA exposure not only mediated glycolysis but also promoted abnormal mitochondrial fusion by upregulating the expression of MFN2 in NGF-PC12 cells.In addition,we found that PKM2 knockdown rescued the abnormal mitochondrial fusion and cell apoptosis induced by HGþ6-OHDA.Furthermore,we found that shikonin(SK),an inhibitor of PKM2,restored the mitochondrial number,promoted TCA cycle activity,reversed hyperglycolysis,enhanced the tolerance of cultured neurons to 6-OHDA,and reduced the risk of PD in diabetic rats.Overall,our results indicate that diabetes promotes hyperglycolysis and abnormal mitochondrial fusion in neurons through the upregulation of PKM2,leading to an increase in the vulnerability of dopaminergic neurons to 6-OHDA.Thus,the inhibition of PKM2 and restoration of mitochondrial metabolic homeostasis/pathways may prevent the occurrence and development of diabetic PD.展开更多
Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was u...Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was used to analyze the safety and efficacy concentration of TF‑SB on MPC‑5 cells. Then, MPC‑5 was then divided into the control group, model group and TF‑SB group. In addition to the control group, model group and TF‑SB group were induced by high glucose to establish MPC‑5 cell injury model. The effects of TF‑SB on ATP, apoptosis and ROS levels of MPC‑5 cells were detected respectively. The contents of IL‑1β, TNF‑α, and MCP‑1 were determined by ELISA, the expression abundance of glycolytic genes (GLU1, PFK1 and HK1) were detected by RT‑PCR. Western blot method was used to detect the expression level of related proteins in Smad4/PKM2/HIF‑1α pathway. Results: Compared with the blank group, ATP content, GLU1, PKF1 and HK1 expression abundance of MPC‑5 cells in the model group decreased significantly, apoptosis, ROS level and IL‑1 β、 TNF‑ α And MCP‑1 significantly increased (P<0.01);Compared with model group, ATP content, GLU1, PKF1 and HK1 expression abundance, apoptosis, ROS level and IL‑1β in TF‑SB group were significantly increased , TNF‑ α The contents of MCP‑1 and MCP‑1 decreased significantly (P<0.01). In addition, compared with the blank group, the model group Smad4 and HIF‑1 α The protein expression and PKM2 expression in nucleus were significantly increased, while PKM2 expression in cytoplasm was significantly decreased (P<0.01);Compared with model group, TF‑SB group Smad4, HIF‑1 α The expression of PKM2 in the nucleus and expression of PKM2 were significantly decreased, while the expression of PKM2 in the cytoplasm was significantly increased (P<0.01). Conclusion: TF‑SB promotes the mitochondrial activity of MPC‑5 cells to induce glycolysis, and then inhibits the secretion of inflammation, which may play a role in treating diabetes nephropathy by inhibiting Smad4/PKM2/HIF‑1α signaling pathway.展开更多
文摘目的:探究TRIM2通过泛素化修饰PKM2调控肝癌细胞糖酵解的机制。方法:TCGA和CPTAC数据库分析TRIM2基因及蛋白表达水平,并结合病人生存状态进行相关性分析。构建HepG2-shNC和HepG2-shTRIM2细胞株,细胞能量代谢仪检测两组细胞胞外酸化率和耗氧率,并测定细胞葡萄糖吸收率、乳酸产生量、ATP水平。免疫共沉淀验证TRIM2和PKM2在HepG2、HEK293T细胞中相互作用,免疫荧光检测PKM2和TRIM2共定位。免疫共沉淀检测在HepG2/HEK293T细胞中敲低/过表达TRIM2后PKM2的泛素化修饰水平,体外酶催化反应检测在HepG2/HEK293T细胞中敲低/过表达TRIM2后PKM2酶活性。Transwell小室法检测HepG2-shNC和HepG2-shTRIM2细胞侵袭和迁移能力。皮下移植瘤实验检测HepG2-shNC和HepG2-shTRIM2细胞体内成瘤能力。结果:TCGA和CPTAC数据库分析发现TRIM2在肝癌组织中mRNA及蛋白表达水平均升高,高TRIM2表达预示差的患者生存状态。TRIM2表达水平与糖酵解通路相关性高,敲低TRIM2可抑制HepG2胞外酸化率,提高耗氧率,细胞葡萄糖吸收率[(100.0000±2.0000)%vs(46.6667±8.1445)%]、乳酸产生量[(99.0000±3.6055)%vs(43.6667±10.01665)%]、ATP水平[(97.0000±3.6055)%vs(57.3333±6.6583)%]均显著下调。TRIM2与PKM2在HepG2细胞中相互作用且蛋白共定位。TRIM2抑制后HepG2细胞中PKM2泛素化水平升高,PKM2酶活性降低;在HEK293T细胞中表达Myc-TRIM2,下调PKM2蛋白泛素化修饰水平,提高PKM2酶活性。敲低TRIM2抑制HepG2细胞侵袭能力(1 vs 0.5067±0.1168)、迁移能力(1 vs 0.5133±0.08622),抑制细胞体内成瘤体积[(529.3333±29.28026)mm 3 vs(281.6667±25.6969)mm 3]和重量[(360.3333±42.7161)mg vs(172.1667±50.2849)mg]。结论:TRIM2在肝癌组织中高表达,通过去泛素化修饰PKM2促进其酶活性,进而调控肿瘤细胞糖酵解能力。
基金the National Natural Science Foundation of China(Grant Nos.:82074039 and 82204584).
文摘Epidemiological and animal studies indicate that pre-existing diabetes increases the risk of Parkinson's disease(PD).However,the mechanisms underlying this association remain unclear.In the present study,we found that high glucose(HG)levels in the cerebrospinal fluid(CSF)of diabetic rats might enhance the effect of a subthreshold dose of the neurotoxin 6-hydroxydopamine(6-OHDA)on the development of motor disorders,and the damage to the nigrostriatal dopaminergic neuronal pathway.In vitro,HG promoted the 6-OHDA-induced apoptosis in PC12 cells differentiated to neurons with nerve growth factor(NGF)(NGF-PC12).Metabolomics showed that HG promoted hyperglycolysis in neurons and impaired tricarboxylic acid cycle(TCA cycle)activity,which was closely related to abnormal mitochondrial fusion,thus resulting in mitochondrial loss.Interestingly,HG-induced upregulation of pyruvate kinase M2(PKM2)combined with 6-OHDA exposure not only mediated glycolysis but also promoted abnormal mitochondrial fusion by upregulating the expression of MFN2 in NGF-PC12 cells.In addition,we found that PKM2 knockdown rescued the abnormal mitochondrial fusion and cell apoptosis induced by HGþ6-OHDA.Furthermore,we found that shikonin(SK),an inhibitor of PKM2,restored the mitochondrial number,promoted TCA cycle activity,reversed hyperglycolysis,enhanced the tolerance of cultured neurons to 6-OHDA,and reduced the risk of PD in diabetic rats.Overall,our results indicate that diabetes promotes hyperglycolysis and abnormal mitochondrial fusion in neurons through the upregulation of PKM2,leading to an increase in the vulnerability of dopaminergic neurons to 6-OHDA.Thus,the inhibition of PKM2 and restoration of mitochondrial metabolic homeostasis/pathways may prevent the occurrence and development of diabetic PD.
基金Heilongjiang Traditional Chinese Medicine Research Project (No.ZHY19?058)。
文摘Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was used to analyze the safety and efficacy concentration of TF‑SB on MPC‑5 cells. Then, MPC‑5 was then divided into the control group, model group and TF‑SB group. In addition to the control group, model group and TF‑SB group were induced by high glucose to establish MPC‑5 cell injury model. The effects of TF‑SB on ATP, apoptosis and ROS levels of MPC‑5 cells were detected respectively. The contents of IL‑1β, TNF‑α, and MCP‑1 were determined by ELISA, the expression abundance of glycolytic genes (GLU1, PFK1 and HK1) were detected by RT‑PCR. Western blot method was used to detect the expression level of related proteins in Smad4/PKM2/HIF‑1α pathway. Results: Compared with the blank group, ATP content, GLU1, PKF1 and HK1 expression abundance of MPC‑5 cells in the model group decreased significantly, apoptosis, ROS level and IL‑1 β、 TNF‑ α And MCP‑1 significantly increased (P<0.01);Compared with model group, ATP content, GLU1, PKF1 and HK1 expression abundance, apoptosis, ROS level and IL‑1β in TF‑SB group were significantly increased , TNF‑ α The contents of MCP‑1 and MCP‑1 decreased significantly (P<0.01). In addition, compared with the blank group, the model group Smad4 and HIF‑1 α The protein expression and PKM2 expression in nucleus were significantly increased, while PKM2 expression in cytoplasm was significantly decreased (P<0.01);Compared with model group, TF‑SB group Smad4, HIF‑1 α The expression of PKM2 in the nucleus and expression of PKM2 were significantly decreased, while the expression of PKM2 in the cytoplasm was significantly increased (P<0.01). Conclusion: TF‑SB promotes the mitochondrial activity of MPC‑5 cells to induce glycolysis, and then inhibits the secretion of inflammation, which may play a role in treating diabetes nephropathy by inhibiting Smad4/PKM2/HIF‑1α signaling pathway.
