目的:高迁移率族蛋白B1(high-mobility group protein 1,HMGB1)在肺动脉高压患者血浆中水平显著升高,但其生物学意义尚不明确。抗增殖蛋白1(prohibitin 1,PHB1)是维持血管细胞稳态的重要蛋白质。本研究旨在探讨HMGB1对肺动脉内皮细胞的...目的:高迁移率族蛋白B1(high-mobility group protein 1,HMGB1)在肺动脉高压患者血浆中水平显著升高,但其生物学意义尚不明确。抗增殖蛋白1(prohibitin 1,PHB1)是维持血管细胞稳态的重要蛋白质。本研究旨在探讨HMGB1对肺动脉内皮细胞的影响以及PHB在其中的作用和机制。方法:体内实验:构建野百合碱(monocrotaline,MCT)诱导的肺动脉高压大鼠模型,采用右心室收缩压(right ventricular systolic pressure,RVSP)以及右心室与左心室加室间隔重量和之比评判造模是否成功,ELISA检测动物血浆中HMGB1水平,蛋白质印迹法检测动物肺组织中PHB1水平,CD31免疫荧光检测肺血管内皮的完整性。体外实验:以HMGB1孵育肺动脉内皮细胞观察HMGB1对PAEC损伤的影响,分别过表达和沉默PHB1,通过检测活性氧水平、细胞色素c(cytochrome c,cyto-c)含量和caspase-3的活化研究PHB1在其中的作用。结果:与对照组相比,肺动脉高压大鼠血浆中HMGB1水平显著升高(P<0.05),肺组织中PHB1表达下降同时伴有肺动脉内皮细胞减少(P<0.05);HMGB1干预可损伤肺动脉内皮细胞,并导致PHB1表达降低(P<0.05);而过表达PHB1能减轻HMGB1诱导的肺动脉内皮细胞损伤和氧化应激(P<0.05)。PHB1通过抑制氧化应激减少HMGB1诱导的cyto-c的表达以及caspase-3的剪切(P<0.05)。结论:PHB1表达下调介导了HMGB1诱导的肺动脉内皮细胞损伤,其机制与诱导氧化应激、增加cyto-c释放以及促进caspase-3剪切有关。展开更多
Prohibitin(PHB),an evoluti on arily con served mitochondrial inner membra ne protein,is highly expressed in cells that require strong mitoch on drial function.Recently,we dem on strated that the deleti on of Phb in sp...Prohibitin(PHB),an evoluti on arily con served mitochondrial inner membra ne protein,is highly expressed in cells that require strong mitoch on drial function.Recently,we dem on strated that the deleti on of Phb in spermatocytes results in impaired mitochondrial function.In addition,PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels,but a positive one with mitochondrial membrane potential and sperm motility.These results suggest that mitochondrial PHB expression plays a role in sperm motility.However,the mechanism of PHB-mediated regulation of sperm motility remai ns unk nown.Here,we dem on strate for the first time that PHB interacts with protei n kinase B(AKT)and exists in a complex with phospho-PHB(pT258)and phospho-AKT in the mitochondrial sheath of murine sperm,as determined using colocalization and coimmunoprecipitation assays.After blocking AKT activity using wortmannin(a phosphatidylinositol 3-kinase[PI3K]inhibitor),murine sperm have significantly(P<0.05)decreased levels of phospho-PHB(pT258)and the total and progressive motility.Furthermore,significantly(P<0.05)lower levels of phospho-PI3K P85 subunit a+γ(pY199 and pY46)and phospho-AKT(pS473;pT308)are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with no rmospermic subjects,which suggest a reduced activity of the PI3K/AKT pathway in these in fertile subjects.Importantly,these sperm from infertile subjects also have a significantly(P<0.05)lower level of phospho-PHB(pT258).Collectively,our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility,where PHB phosphorylation(pT258)level and PI3K/AKT activity are key regulatory factors.展开更多
BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis...BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis,cell cycle regulation,prolifera-tion,and survival.Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma(HCC).However,the role of PHB1 in HCC is controversial.AIM To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro.METHODS HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria;then,PHB1 levels in the sera and liver tissues of these participates were determined using ELISA,RT-PCR,and immunohistoche-mistry.Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA(shRNA-PHB1)for 24-72 h.Cell prolif-eration was analysed with an MTT assay.Cell cycle progression and apoptosis were analysed using flow cytometry(FACS).The mRNA and protein expression levels of the cell cycle-related molecules p21,Cyclin A2,Cyclin E1,and CDK2 and the cell apoptosis-related molecules cytochrome C(Cyt C),p53,Bcl-2,Bax,caspase 3,and caspase 9 were measured by real-time PCR and Western blot,respectively.RESULTS Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals,and decreased PHB1 was positively correlated with low differentiation,TNM stage III-IV,and alpha-fetoprotein≥400μg/L.Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner.FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis.The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells.The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41%±1.06%,which was significantly greater than that of apoptotic control cells(3.65%±0.85%,P<0.01)and empty vector-transfected cells(4.21%±0.52%,P<0.01).Similar results were obtained with SMMC-7721 cells.Furthermore,the mRNA and protein expression levels of p53,p21,Bax,caspase 3,and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2,Cy-clin E1,CDK2,and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells.However,when PHB1 was upregulated in human HCC cells,Cyt C expression levels were increased in the cytosol and decreased in the mitochondria,which indicated that Cyt C had been released into the cytosol.Conversely,these effects were reversed when PHB1 was knocked down.CONCLUSION PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.展开更多
BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatecto...BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatectomy(PHx),as determined by subcellular proteomic analysis.AIM To investigate the potential role of Phb1 during LR.METHODS We examined changes in Phb1 mRNA and protein levels,subcellular distribution,and abundance in rat liver during LR following 70%PHx.We also evaluated mitochondrial changes and apoptosis using electron microscopy and flow cytometry.RNA-interference-mediated knockdown of Phb1(PHBi)was performed in BRL-3A cells.RESULTS Compared with sham-operation control groups,Phb1 mRNA and protein levels in 70%PHx test groups were downregulated at 24 h,then upregulated at 72 and 168 h.Phb1 was mainly located in mitochondria,showed a reduced abundance at 24 h,significantly increased at 72 h,and almost recovered to normal at 168 h.Phb1 was also present in nuclei,with continuous increase in abundance observed 72 and 168 h after 70%PHx.The altered ultrastructure and reduced mass of mitochondria during LR had almost completely recovered to normal at 168 h.PHBi in BRL-3A cells resulted in increased S-phase entry,a higher number of apoptotic cells,and disruption of mitochondrial membrane potential.CONCLUSION Phb1 may contribute to maintaining mitochondrial stability and could play a role in regulating cell proliferation and apoptosis of rat liver cells during LR.展开更多
文摘目的:高迁移率族蛋白B1(high-mobility group protein 1,HMGB1)在肺动脉高压患者血浆中水平显著升高,但其生物学意义尚不明确。抗增殖蛋白1(prohibitin 1,PHB1)是维持血管细胞稳态的重要蛋白质。本研究旨在探讨HMGB1对肺动脉内皮细胞的影响以及PHB在其中的作用和机制。方法:体内实验:构建野百合碱(monocrotaline,MCT)诱导的肺动脉高压大鼠模型,采用右心室收缩压(right ventricular systolic pressure,RVSP)以及右心室与左心室加室间隔重量和之比评判造模是否成功,ELISA检测动物血浆中HMGB1水平,蛋白质印迹法检测动物肺组织中PHB1水平,CD31免疫荧光检测肺血管内皮的完整性。体外实验:以HMGB1孵育肺动脉内皮细胞观察HMGB1对PAEC损伤的影响,分别过表达和沉默PHB1,通过检测活性氧水平、细胞色素c(cytochrome c,cyto-c)含量和caspase-3的活化研究PHB1在其中的作用。结果:与对照组相比,肺动脉高压大鼠血浆中HMGB1水平显著升高(P<0.05),肺组织中PHB1表达下降同时伴有肺动脉内皮细胞减少(P<0.05);HMGB1干预可损伤肺动脉内皮细胞,并导致PHB1表达降低(P<0.05);而过表达PHB1能减轻HMGB1诱导的肺动脉内皮细胞损伤和氧化应激(P<0.05)。PHB1通过抑制氧化应激减少HMGB1诱导的cyto-c的表达以及caspase-3的剪切(P<0.05)。结论:PHB1表达下调介导了HMGB1诱导的肺动脉内皮细胞损伤,其机制与诱导氧化应激、增加cyto-c释放以及促进caspase-3剪切有关。
基金This project was funded by grants from the National Natural Science Foundation of China(No.81270738)and the Major State Basic Research Development Program of China(No.2014CB943104).
文摘Prohibitin(PHB),an evoluti on arily con served mitochondrial inner membra ne protein,is highly expressed in cells that require strong mitoch on drial function.Recently,we dem on strated that the deleti on of Phb in spermatocytes results in impaired mitochondrial function.In addition,PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels,but a positive one with mitochondrial membrane potential and sperm motility.These results suggest that mitochondrial PHB expression plays a role in sperm motility.However,the mechanism of PHB-mediated regulation of sperm motility remai ns unk nown.Here,we dem on strate for the first time that PHB interacts with protei n kinase B(AKT)and exists in a complex with phospho-PHB(pT258)and phospho-AKT in the mitochondrial sheath of murine sperm,as determined using colocalization and coimmunoprecipitation assays.After blocking AKT activity using wortmannin(a phosphatidylinositol 3-kinase[PI3K]inhibitor),murine sperm have significantly(P<0.05)decreased levels of phospho-PHB(pT258)and the total and progressive motility.Furthermore,significantly(P<0.05)lower levels of phospho-PI3K P85 subunit a+γ(pY199 and pY46)and phospho-AKT(pS473;pT308)are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with no rmospermic subjects,which suggest a reduced activity of the PI3K/AKT pathway in these in fertile subjects.Importantly,these sperm from infertile subjects also have a significantly(P<0.05)lower level of phospho-PHB(pT258).Collectively,our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility,where PHB phosphorylation(pT258)level and PI3K/AKT activity are key regulatory factors.
基金the Key Research and Development Program of Shaanxi,No.2021SF-227 and No.2020SF-297the Natural Science Basic Research Program of Shaanxi,No.2023-JC-YB-770。
文摘BACKGROUND Prohibitin 1(PHB1)has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed,and it participates in a variety of essential cellular functions,including apoptosis,cell cycle regulation,prolifera-tion,and survival.Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma(HCC).However,the role of PHB1 in HCC is controversial.AIM To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro.METHODS HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria;then,PHB1 levels in the sera and liver tissues of these participates were determined using ELISA,RT-PCR,and immunohistoche-mistry.Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA(shRNA-PHB1)for 24-72 h.Cell prolif-eration was analysed with an MTT assay.Cell cycle progression and apoptosis were analysed using flow cytometry(FACS).The mRNA and protein expression levels of the cell cycle-related molecules p21,Cyclin A2,Cyclin E1,and CDK2 and the cell apoptosis-related molecules cytochrome C(Cyt C),p53,Bcl-2,Bax,caspase 3,and caspase 9 were measured by real-time PCR and Western blot,respectively.RESULTS Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals,and decreased PHB1 was positively correlated with low differentiation,TNM stage III-IV,and alpha-fetoprotein≥400μg/L.Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner.FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis.The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells.The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41%±1.06%,which was significantly greater than that of apoptotic control cells(3.65%±0.85%,P<0.01)and empty vector-transfected cells(4.21%±0.52%,P<0.01).Similar results were obtained with SMMC-7721 cells.Furthermore,the mRNA and protein expression levels of p53,p21,Bax,caspase 3,and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2,Cy-clin E1,CDK2,and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells.However,when PHB1 was upregulated in human HCC cells,Cyt C expression levels were increased in the cytosol and decreased in the mitochondria,which indicated that Cyt C had been released into the cytosol.Conversely,these effects were reversed when PHB1 was knocked down.CONCLUSION PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.
文摘BACKGROUND The function of prohibitin 1(Phb1)during liver regeneration(LR)remains relatively unexplored.Our previous research identified downregulation of Phb1 in rat liver mitochondria 24 h after 70%partial hepatectomy(PHx),as determined by subcellular proteomic analysis.AIM To investigate the potential role of Phb1 during LR.METHODS We examined changes in Phb1 mRNA and protein levels,subcellular distribution,and abundance in rat liver during LR following 70%PHx.We also evaluated mitochondrial changes and apoptosis using electron microscopy and flow cytometry.RNA-interference-mediated knockdown of Phb1(PHBi)was performed in BRL-3A cells.RESULTS Compared with sham-operation control groups,Phb1 mRNA and protein levels in 70%PHx test groups were downregulated at 24 h,then upregulated at 72 and 168 h.Phb1 was mainly located in mitochondria,showed a reduced abundance at 24 h,significantly increased at 72 h,and almost recovered to normal at 168 h.Phb1 was also present in nuclei,with continuous increase in abundance observed 72 and 168 h after 70%PHx.The altered ultrastructure and reduced mass of mitochondria during LR had almost completely recovered to normal at 168 h.PHBi in BRL-3A cells resulted in increased S-phase entry,a higher number of apoptotic cells,and disruption of mitochondrial membrane potential.CONCLUSION Phb1 may contribute to maintaining mitochondrial stability and could play a role in regulating cell proliferation and apoptosis of rat liver cells during LR.
