Mitogen-activated protein kinase(MAPK)cascades are key signaling modules downstream of receptors/sensors that perceive either endogenously produced stimuli such as peptide ligands and damage-associated molecular patte...Mitogen-activated protein kinase(MAPK)cascades are key signaling modules downstream of receptors/sensors that perceive either endogenously produced stimuli such as peptide ligands and damage-associated molecular patterns(DAMPs)or exogenously originated stimuli such as pathogen/microbe-associated molecular patterns(P/MAMPs),pathogen-derived effectors,and environmental factors.In this review,we provide a historic view of plant MAPK research and summarize recent advances in the establishment of MAPK cascades as essential components in plant immunity,response to environmental stresses,and normal growth and development.Each tier of the MAPK cascades is encoded by a small gene family,and multi ple members can function redundantly in an MAPK cascade.Yet,they carry out a diverse array of biological functions in plants.How the signaling specificity is achieved has become an interesting topic of MAPK research.Future investigations into the molecular mechanism(s)underlying the regulation of MAPK activation including the activation kinetics and magnitude in response to a stimulus,the spatiotemporal expression patterns of all the components in the signaling pathway,and functional characterization of novel MAPK substrates are central to our understanding of MAPK functions and signaling specificity in plants.展开更多
The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple st...The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.展开更多
Objective: To investigate the attenuating effect of Hydroxysafflor yellow A(HSYA) on inflammatory injury in chronic obstructive pulmonary disease(COPD). Methods: Rats were randomly assigned to 7 groups according to bo...Objective: To investigate the attenuating effect of Hydroxysafflor yellow A(HSYA) on inflammatory injury in chronic obstructive pulmonary disease(COPD). Methods: Rats were randomly assigned to 7 groups according to body weight including normal control group, HSYA blank group(76.8 mg/kg), COPD group, COPD+HSYA(30, 48, 76.8 mg/kg) groups and COPD+dexamethasone(2 mg/kg), 10 in each group. Passive cigarette smoke and intratracheal instil ation of lipopolysaccharides were used to establish a COPD model in rats. Hematoxylin and eosin staining of lung tissue sections was used, real-time polymerase chain reaction(PCR) was used to assay m RNA levels of some cytokines in lung tissues, the cytokines in bronchoalveolar lavage fluid(BALF) were measured by enzyme-linked immunosorbent assay(ELISA), Western blot analysis was used to determine phosphorylated p38 mitogen-activated protein kinase(MAPK) levels in lung tissues, and nuclear factor-κB(NF-κB) p65 protein levels in lung tissues were detected by immunohistochemistry. Results: Lung alveolar septa destruction, alveolus fusion, inflammatory cel infiltration, and bronchiole exudation were observed. These pathological changes were al eviated in the COPD+HSYA group. The m RNA expression of inflammatory factors were significantly increased in lung tissues from COPD rats(all P<0.01) and were inhibited by HSYA. Levels of inflammatory cytokines in BALF of COPD rats were significantly increased(all P<0.01) which were inhibited by HSYA(all P<0.01, 48, 76.8 mg/kg). The levels of p38 MAPK phosphorylation and p65 in lung tissues of COPD rats were significantly increased(al P<0.01) and were suppressed by HSYA(all P<0.01, 48, 76.8 mg/kg). Conclusions: HSYA could alleviate inflammatory cell infiltration and other pathological changes in the lungs of COPD rats. HSYA could inhibit inflammatory cytokine expression, and increase phosphorylation of p38 MAPK and NF-κB p65 in the lungs of COPD rats. The protective mechanism of HSYA to inhibit COPD inflammation might be by attenuating NF-κB an展开更多
Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB le...Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BB展开更多
Background:Wound management of diabetic foot ulcers(DFUs)is a complex and challenging task,and existing strategies fail to meet clinical needs.Therefore,it is important to develop novel drug candidates and discover ne...Background:Wound management of diabetic foot ulcers(DFUs)is a complex and challenging task,and existing strategies fail to meet clinical needs.Therefore,it is important to develop novel drug candidates and discover new therapeutic targets.However,reports on peptides as molecular probes for resolving issues related to DFUs remain rare.This study utilized peptide RL-QN15 as an exogenous molecular probe to investigate the underlying mechanism of endogenous non-coding RNA in DFU wound healing.The aim was to generate novel insights for the clinical management of DFUs and identify potential drug targets.Methods:We investigated the wound-healing efficiency of peptide RL-QN15 under diabetic con-ditions using in vitro and in vivo experimental models.RNA sequencing,in vitro transfection,quantitative real-time polymerase chain reaction,western blotting,dual luciferase reporter gene detection,in vitro cell scratches,and cell proliferation and migration assays were performed to explore the potential mechanism underlying the promoting effects of RL-QN15 on DFU repair.Results:Peptide RL-QN15 enhanced the migration and proliferation of human immortalized keratinocytes(HaCaT cells)in a high-glucose environment and accelerated wound healing in a DFU rat model.Based on results from RNA sequencing,we defined a new microRNA(miR-4482-3p)related to the promotion of wound healing.The bioactivity of miR-4482-3p was verified by inhibiting and overexpressing miR-4482-3p.Inhibition of miR-4482-3p enhanced the migration and proliferation ability of HaCaT cells as well as the expression of vascular endothelial growth factor B(VEGFB).RLQN15 also promoted the migration and proliferation ability of HaCaT cells,and VEGFB expression was mediated via inhibition of miR-4482-3p expression by the p38 mitogen-activated protein kinase(p38MAPK)and smad3 signaling pathways.Conclusions:RL-QN15 is an effective molecule for the treatment of DFUs,with the underlying mechanism related to the inhibition of miR-4482-3p expression via the p38MAPK and smad3 signal展开更多
AIM： To address the possibility that insulin-like growth factor （IGF）-Ⅱ is a growth factor and its signaling pathway so as to develop a molecular therapy for hepatoblastoma. METHODS： Huh-6 and HepG2, human hepato...AIM： To address the possibility that insulin-like growth factor （IGF）-Ⅱ is a growth factor and its signaling pathway so as to develop a molecular therapy for hepatoblastoma. METHODS： Huh-6 and HepG2, human hepatoblastoma cell lines, were used. IGF-Ⅱ was added to the medium deprived of serum. Western blot analysis was performed to clarify the expression of IGF-Ⅰ receptor （IGF-IR）. Inhibitors of IGF-IR （piclopodophyllin, PPP）, phosphatidyl-inositol （PI） 3-kinase （LY294002 and Wortmannin）, or mitogen-activated protein （MAP） kinase （PD98059） were added to unveil the signaling pathway of IGF-Ⅱ. Cells were analyzed morphologically with hematoxylin-eosin staining to reveal the mechanism of suppression of cell proliferation. RESULTS： IGF-Ⅱ stimulated cells proliferated to 2.7 （269% ± 76%） （mean ± SD） （Huh-6） and 2.1 （211% ± 85%） times （HepG2）. IGF-IR was expressed in Huh-6 and HepG2. PPP suppressed the cell number to 44% ± 11% （Huh-6） and 39% ± 5% （HepG2）. LY294002 and Wortmannin suppressed the cell number to 30% ± 5% （Huh-6）, 44% ± 0.4% （HepG2）, 49% ± 1.0% （Huh-6） and 46% ± 1.1% （HepG2）, respectively. PD98059 suppressed the cell number to 33% ± 11% for HepG2 but not for Huh-6. When cell proliferation was prohibited, many Huh-6 and HepG2 cells were dead with pyknotic or fragmented nuclei, suggesting apoptosis. CONCLUSION： IGF- Ⅱ was shown to be a growth factor of hepatoblastoma via IGF-Ⅰ receptor and PI3 kinase which were good candidates for target of molecular therapy.展开更多
Transcription factors can be used to engineer plants for enhanced productivity.However,the mechanism(s)by which the C2H2-type zinc fi nger transcription factor enhances pathogen resistance in cells is not fully unders...Transcription factors can be used to engineer plants for enhanced productivity.However,the mechanism(s)by which the C2H2-type zinc fi nger transcription factor enhances pathogen resistance in cells is not fully understood.Here,Agrobacterium tumefaciens carrying the gene for Arabidopsis thaliana cysteine2/histidine2-type transcription factor 6(ZAT6)was used to engineer rice(Oryza sativa L.),cotton(Gossypium hirsutum L.),and slash pine(Pinus elliottii Engelm.)to generate transgenic cell lines.Transgenic cells were then inoculated with the pathogenic bacterium Pseudomonas syringae.Compared to the control,cell viability of transgenic cells increased 39–47%and growth rate increased 9–15%by 7 days after inoculation in rice,cotton and slash pine.Acid phosphatase activity and alkaline phosphatase activity and transcript levels of Ca 2+-dependent protein kinase genes OsCPK1,OsCPK2,OsCPK6,and OsCPK8 and mitogen-activated protein kinase genes OsMAPK1,OsMAPK2,OsMAPK3,and OsMAPK8 increased signifi cantly in transgenic rice cells by 3 day after inoculation,and extracellular pH had decreased by 10–14%by 96 min after inoculation in transgenic rice,cotton and slash pine cells.These results suggest that ZAT6 enhances P.syringae resistance in plant cells by modulating transcription of CPK and MAPK and oxidase activity.展开更多
目的:探索补肾中药淫羊藿有效成分淫羊藿苷对间充质干细胞株C3H10T1/2的促成骨分化作用,及该作用与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的相关性。方法:体外培养间充质干细胞株C3H10T1/2,给予淫羊藿苷(0...目的:探索补肾中药淫羊藿有效成分淫羊藿苷对间充质干细胞株C3H10T1/2的促成骨分化作用,及该作用与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的相关性。方法:体外培养间充质干细胞株C3H10T1/2,给予淫羊藿苷(0、10-7、10-6、10-5和10-4mol/L)联合成骨诱导液干预26d后,碱性磷酸酶(alkaline phosphatase,ALP)染色观察成骨分化情况。10-5mol/L淫羊藿苷干预C3H10T1/2细胞2、8、24与48h后,实时荧光定量聚合酶链式反应法(polymerase chain reaction,PCR)检测p38、细胞外调节蛋白激酶(extracellular signal-regulated protein kinase,p42/44)mRNA的表达。10-5mol/L淫羊藿苷干预C3H10T1/2细胞10、30、60和120min后,蛋白质印迹法检测p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38)、p42和p44,以及翼式转录因子氨基末端激酶(c-Jun N-terminal kinase,JNK)及其磷酸化产物的蛋白表达情况。结果:10-5mol/L淫羊藿苷联合成骨诱导液的促进C3H10T1/2细胞成骨分化能力最强。PCR结果显示,淫羊藿苷作用2h后p38基因表达显著下调(P<0.01);p42和p44mRNA水平在淫羊藿苷干预2h后均显著下调(P<0.01),而在淫羊藿苷干预48h后表达均上调(P<0.05,P<0.01);其他时间点各通路基因表达情况均无显著变化(P>0.05)。10-5mol/L淫羊藿苷作用10min后,磷酸化p42/44蛋白表达减弱,磷酸化p38蛋白表达增强,并持续到30min,但对JNK蛋白的表达无明显影响。结论:淫羊藿苷促进间充质干细胞株C3H10T1/2向成骨细胞分化,其作用可能与激活p38和抑制ERK蛋白的表达有关。展开更多
基金supported by the Zhongshan Young Principal Investigator award from Nanjing Agricultural University and a grant from Jiangsu Provincial Post-doctoral Research Funding Program(2020Z325)。
文摘Mitogen-activated protein kinase(MAPK)cascades are key signaling modules downstream of receptors/sensors that perceive either endogenously produced stimuli such as peptide ligands and damage-associated molecular patterns(DAMPs)or exogenously originated stimuli such as pathogen/microbe-associated molecular patterns(P/MAMPs),pathogen-derived effectors,and environmental factors.In this review,we provide a historic view of plant MAPK research and summarize recent advances in the establishment of MAPK cascades as essential components in plant immunity,response to environmental stresses,and normal growth and development.Each tier of the MAPK cascades is encoded by a small gene family,and multi ple members can function redundantly in an MAPK cascade.Yet,they carry out a diverse array of biological functions in plants.How the signaling specificity is achieved has become an interesting topic of MAPK research.Future investigations into the molecular mechanism(s)underlying the regulation of MAPK activation including the activation kinetics and magnitude in response to a stimulus,the spatiotemporal expression patterns of all the components in the signaling pathway,and functional characterization of novel MAPK substrates are central to our understanding of MAPK functions and signaling specificity in plants.
文摘The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.
基金Supported by the National Natural Science Foundation of China(No.81270103)the Natural Science Foundation of Beijing(No.7132047)the Project of Integrated Traditional Chinese Medicine-Western Medicine Institute of Heart Lung and Blood Vessel Diseases
文摘Objective: To investigate the attenuating effect of Hydroxysafflor yellow A(HSYA) on inflammatory injury in chronic obstructive pulmonary disease(COPD). Methods: Rats were randomly assigned to 7 groups according to body weight including normal control group, HSYA blank group(76.8 mg/kg), COPD group, COPD+HSYA(30, 48, 76.8 mg/kg) groups and COPD+dexamethasone(2 mg/kg), 10 in each group. Passive cigarette smoke and intratracheal instil ation of lipopolysaccharides were used to establish a COPD model in rats. Hematoxylin and eosin staining of lung tissue sections was used, real-time polymerase chain reaction(PCR) was used to assay m RNA levels of some cytokines in lung tissues, the cytokines in bronchoalveolar lavage fluid(BALF) were measured by enzyme-linked immunosorbent assay(ELISA), Western blot analysis was used to determine phosphorylated p38 mitogen-activated protein kinase(MAPK) levels in lung tissues, and nuclear factor-κB(NF-κB) p65 protein levels in lung tissues were detected by immunohistochemistry. Results: Lung alveolar septa destruction, alveolus fusion, inflammatory cel infiltration, and bronchiole exudation were observed. These pathological changes were al eviated in the COPD+HSYA group. The m RNA expression of inflammatory factors were significantly increased in lung tissues from COPD rats(all P<0.01) and were inhibited by HSYA. Levels of inflammatory cytokines in BALF of COPD rats were significantly increased(all P<0.01) which were inhibited by HSYA(all P<0.01, 48, 76.8 mg/kg). The levels of p38 MAPK phosphorylation and p65 in lung tissues of COPD rats were significantly increased(al P<0.01) and were suppressed by HSYA(all P<0.01, 48, 76.8 mg/kg). Conclusions: HSYA could alleviate inflammatory cell infiltration and other pathological changes in the lungs of COPD rats. HSYA could inhibit inflammatory cytokine expression, and increase phosphorylation of p38 MAPK and NF-κB p65 in the lungs of COPD rats. The protective mechanism of HSYA to inhibit COPD inflammation might be by attenuating NF-κB an
基金supported by Research Start-up Funding of Shenzhen Traditional Chinese Medicine Hospital,No.2021-07(to FB)Sanming Project of Medicine in Shenzhen,No.SZZYSM 202111011(to XDQ and FB)+1 种基金Key Discipline Established by Zhejiang Province,Jiaxing City Jointly-Pain Medicine,No.2019-ss-ttyx(to LSX)Jiaxing Key Laboratory of Neurology and Pain Medicine,No.[2014]81(to LSX)。
文摘Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BB
基金supported by grants from the National Natural Science Foundation of China(32360138,32060212,32301054 and 81760648)Key Program of Yunnan Fundamental Research Project(202301AS070036)+1 种基金Outstanding Youth Program of Yunnan Applied Basic Research Project-Kunming Medical University Union Foundation(202301AY070001-301)Project of Yunnan Applied Basic Research Project-Kunming Medical University Union Foundation(202101AY070001-006 and 202101AY070001-036).
文摘Background:Wound management of diabetic foot ulcers(DFUs)is a complex and challenging task,and existing strategies fail to meet clinical needs.Therefore,it is important to develop novel drug candidates and discover new therapeutic targets.However,reports on peptides as molecular probes for resolving issues related to DFUs remain rare.This study utilized peptide RL-QN15 as an exogenous molecular probe to investigate the underlying mechanism of endogenous non-coding RNA in DFU wound healing.The aim was to generate novel insights for the clinical management of DFUs and identify potential drug targets.Methods:We investigated the wound-healing efficiency of peptide RL-QN15 under diabetic con-ditions using in vitro and in vivo experimental models.RNA sequencing,in vitro transfection,quantitative real-time polymerase chain reaction,western blotting,dual luciferase reporter gene detection,in vitro cell scratches,and cell proliferation and migration assays were performed to explore the potential mechanism underlying the promoting effects of RL-QN15 on DFU repair.Results:Peptide RL-QN15 enhanced the migration and proliferation of human immortalized keratinocytes(HaCaT cells)in a high-glucose environment and accelerated wound healing in a DFU rat model.Based on results from RNA sequencing,we defined a new microRNA(miR-4482-3p)related to the promotion of wound healing.The bioactivity of miR-4482-3p was verified by inhibiting and overexpressing miR-4482-3p.Inhibition of miR-4482-3p enhanced the migration and proliferation ability of HaCaT cells as well as the expression of vascular endothelial growth factor B(VEGFB).RLQN15 also promoted the migration and proliferation ability of HaCaT cells,and VEGFB expression was mediated via inhibition of miR-4482-3p expression by the p38 mitogen-activated protein kinase(p38MAPK)and smad3 signaling pathways.Conclusions:RL-QN15 is an effective molecule for the treatment of DFUs,with the underlying mechanism related to the inhibition of miR-4482-3p expression via the p38MAPK and smad3 signal
基金Supported by the Japan Society for the Promotion of Science (JSPS) (16590577) and the Kawano Masanori Memorial Foundation for Promotion of Pediatrics (15-6)
文摘AIM： To address the possibility that insulin-like growth factor （IGF）-Ⅱ is a growth factor and its signaling pathway so as to develop a molecular therapy for hepatoblastoma. METHODS： Huh-6 and HepG2, human hepatoblastoma cell lines, were used. IGF-Ⅱ was added to the medium deprived of serum. Western blot analysis was performed to clarify the expression of IGF-Ⅰ receptor （IGF-IR）. Inhibitors of IGF-IR （piclopodophyllin, PPP）, phosphatidyl-inositol （PI） 3-kinase （LY294002 and Wortmannin）, or mitogen-activated protein （MAP） kinase （PD98059） were added to unveil the signaling pathway of IGF-Ⅱ. Cells were analyzed morphologically with hematoxylin-eosin staining to reveal the mechanism of suppression of cell proliferation. RESULTS： IGF-Ⅱ stimulated cells proliferated to 2.7 （269% ± 76%） （mean ± SD） （Huh-6） and 2.1 （211% ± 85%） times （HepG2）. IGF-IR was expressed in Huh-6 and HepG2. PPP suppressed the cell number to 44% ± 11% （Huh-6） and 39% ± 5% （HepG2）. LY294002 and Wortmannin suppressed the cell number to 30% ± 5% （Huh-6）, 44% ± 0.4% （HepG2）, 49% ± 1.0% （Huh-6） and 46% ± 1.1% （HepG2）, respectively. PD98059 suppressed the cell number to 33% ± 11% for HepG2 but not for Huh-6. When cell proliferation was prohibited, many Huh-6 and HepG2 cells were dead with pyknotic or fragmented nuclei, suggesting apoptosis. CONCLUSION： IGF- Ⅱ was shown to be a growth factor of hepatoblastoma via IGF-Ⅰ receptor and PI3 kinase which were good candidates for target of molecular therapy.
文摘Transcription factors can be used to engineer plants for enhanced productivity.However,the mechanism(s)by which the C2H2-type zinc fi nger transcription factor enhances pathogen resistance in cells is not fully understood.Here,Agrobacterium tumefaciens carrying the gene for Arabidopsis thaliana cysteine2/histidine2-type transcription factor 6(ZAT6)was used to engineer rice(Oryza sativa L.),cotton(Gossypium hirsutum L.),and slash pine(Pinus elliottii Engelm.)to generate transgenic cell lines.Transgenic cells were then inoculated with the pathogenic bacterium Pseudomonas syringae.Compared to the control,cell viability of transgenic cells increased 39–47%and growth rate increased 9–15%by 7 days after inoculation in rice,cotton and slash pine.Acid phosphatase activity and alkaline phosphatase activity and transcript levels of Ca 2+-dependent protein kinase genes OsCPK1,OsCPK2,OsCPK6,and OsCPK8 and mitogen-activated protein kinase genes OsMAPK1,OsMAPK2,OsMAPK3,and OsMAPK8 increased signifi cantly in transgenic rice cells by 3 day after inoculation,and extracellular pH had decreased by 10–14%by 96 min after inoculation in transgenic rice,cotton and slash pine cells.These results suggest that ZAT6 enhances P.syringae resistance in plant cells by modulating transcription of CPK and MAPK and oxidase activity.
文摘目的:探索补肾中药淫羊藿有效成分淫羊藿苷对间充质干细胞株C3H10T1/2的促成骨分化作用,及该作用与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的相关性。方法:体外培养间充质干细胞株C3H10T1/2,给予淫羊藿苷(0、10-7、10-6、10-5和10-4mol/L)联合成骨诱导液干预26d后,碱性磷酸酶(alkaline phosphatase,ALP)染色观察成骨分化情况。10-5mol/L淫羊藿苷干预C3H10T1/2细胞2、8、24与48h后,实时荧光定量聚合酶链式反应法(polymerase chain reaction,PCR)检测p38、细胞外调节蛋白激酶(extracellular signal-regulated protein kinase,p42/44)mRNA的表达。10-5mol/L淫羊藿苷干预C3H10T1/2细胞10、30、60和120min后,蛋白质印迹法检测p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38)、p42和p44,以及翼式转录因子氨基末端激酶(c-Jun N-terminal kinase,JNK)及其磷酸化产物的蛋白表达情况。结果:10-5mol/L淫羊藿苷联合成骨诱导液的促进C3H10T1/2细胞成骨分化能力最强。PCR结果显示,淫羊藿苷作用2h后p38基因表达显著下调(P<0.01);p42和p44mRNA水平在淫羊藿苷干预2h后均显著下调(P<0.01),而在淫羊藿苷干预48h后表达均上调(P<0.05,P<0.01);其他时间点各通路基因表达情况均无显著变化(P>0.05)。10-5mol/L淫羊藿苷作用10min后,磷酸化p42/44蛋白表达减弱,磷酸化p38蛋白表达增强,并持续到30min,但对JNK蛋白的表达无明显影响。结论:淫羊藿苷促进间充质干细胞株C3H10T1/2向成骨细胞分化,其作用可能与激活p38和抑制ERK蛋白的表达有关。
