BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its d...BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its damage is an important indicator of DR.Receptor for activated C kinase 1(RACK1)activates protein kinase C-ε(PKC-ε)to promote the generation of reactive oxygen species(ROS)in RPE cells,leading to apoptosis.Therefore,we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS,thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS In this study,Sprague-Dawley rats and adult RPE cell line-19(ARPE-19)cells were used as in vivo and in vitro models,respectively,to explore the role of RACK1 in mediating PKC-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins in RPE cells following a prolonged modeling.Similarly,ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε,accompanied by an increases in ROS production,apoptosis rate,and monolayer permeability.However,silencing RACK1 significantly downregulated the expression of PKC-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.展开更多
AIM: To investigate the dynamic changes of activator protein 1(AP1) and collagen I expression in the sclera of form-deprivation myopic model in guinea pigs. METHODS: A form-deprivation myopic model in guinea pigs were...AIM: To investigate the dynamic changes of activator protein 1(AP1) and collagen I expression in the sclera of form-deprivation myopic model in guinea pigs. METHODS: A form-deprivation myopic model in guinea pigs were established with the left eye covered for 2 to 6 wk(FDM group). Normal control group(n=25) were untreated. Changes in refractive power and axial length(AL) were measured and recorded at different time points. Expressions of AP1 and collagen 1 of the sclera were measured with Western blotting and reverse transcription-polymerase chain reaction(RT-PCR). The relationship between AP1 and collagen I levels was analyzed. RESULTS: After 0, 2, 4, 6 wk, and 4/-1 wk of form-deprivation, the diopter in the FDM group was gradually changed(2.08±0.31,-1.23±0.68,-4.17±0.58,-7.07±0.55, and-2.67±0.59 D, respectively, P<0.05), and the AL was gradually increased(5.90±0.38, 6.62±0.37, 7.30±0.35, 7.99±0.31, and 6.97±0.32 mm, respectively, P<0.05). With the prolongation of covered time, the protein expressions of AP1 and collagen I in the FDM group were gradually down-regulated(all P<0.05);the mRNA expressions of them were also gradually down-regulated(all P<0.05);and there was positive correlation between them. The control group had no obvious change in each index(all P>0.05). CONCLUSION: AP1 may be an important transcription factor involved in the regulation of collagen I synthesis and degradation during myopic scleral remodeling.展开更多
Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient...Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.展开更多
AIM:To investigate the role of Ras association domain family protein 1 isoform A (RASSF1A) in gastric tumorigenesis. METHODS:Through over-expression of RASSF1A gene in the SGC7901 cell line which was induced by a lipo...AIM:To investigate the role of Ras association domain family protein 1 isoform A (RASSF1A) in gastric tumorigenesis. METHODS:Through over-expression of RASSF1A gene in the SGC7901 cell line which was induced by a lipofectamine-mediated gene transfer approach. Activator protein-1 (AP-1) DNA binding activity was measured by electrophoretic mobility shift assay (EMSA). RESULTS:Compared with the control clones, cells over- expressing RASSF1A exhibited significant inhibition of cell growth with G1 cell cycle arrest in vitro and in vivo. The over-expression of RASSF1A significantly inhibited AP-1 activity in SGC7901 cells (0.981±0.011 vs 0.354±0.053, P<0.001). In addition, both Western blot analysis and immunocytochemistry demonstrated that RASSF1A down-regulated the expression of c-Fos (0.975± 0.02 vs 0.095±0.024, P<0.001) but not c-Jun. CONCLUSION: Over-expression of RASSF1A inhibits the growth of SGC7901 cells by negatively regulating the AP-1 activity, the latter in turn negatively signals cell proliferation.展开更多
基金Supported by National Natural Science Foundation of China,No.82260211Key Research and Development Project in Jiangxi Province,No.20203BBG73058Chinese Medicine Science and Technology Project in Jiangxi Province,No.2020A0166.
文摘BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its damage is an important indicator of DR.Receptor for activated C kinase 1(RACK1)activates protein kinase C-ε(PKC-ε)to promote the generation of reactive oxygen species(ROS)in RPE cells,leading to apoptosis.Therefore,we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS,thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS In this study,Sprague-Dawley rats and adult RPE cell line-19(ARPE-19)cells were used as in vivo and in vitro models,respectively,to explore the role of RACK1 in mediating PKC-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins in RPE cells following a prolonged modeling.Similarly,ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε,accompanied by an increases in ROS production,apoptosis rate,and monolayer permeability.However,silencing RACK1 significantly downregulated the expression of PKC-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.
基金Supported by the Natural Science Foundation of Anhui Province(No.1508085MH188)Education and Research Project of Anhui Education Department(No.2016jyxm0546)
文摘AIM: To investigate the dynamic changes of activator protein 1(AP1) and collagen I expression in the sclera of form-deprivation myopic model in guinea pigs. METHODS: A form-deprivation myopic model in guinea pigs were established with the left eye covered for 2 to 6 wk(FDM group). Normal control group(n=25) were untreated. Changes in refractive power and axial length(AL) were measured and recorded at different time points. Expressions of AP1 and collagen 1 of the sclera were measured with Western blotting and reverse transcription-polymerase chain reaction(RT-PCR). The relationship between AP1 and collagen I levels was analyzed. RESULTS: After 0, 2, 4, 6 wk, and 4/-1 wk of form-deprivation, the diopter in the FDM group was gradually changed(2.08±0.31,-1.23±0.68,-4.17±0.58,-7.07±0.55, and-2.67±0.59 D, respectively, P<0.05), and the AL was gradually increased(5.90±0.38, 6.62±0.37, 7.30±0.35, 7.99±0.31, and 6.97±0.32 mm, respectively, P<0.05). With the prolongation of covered time, the protein expressions of AP1 and collagen I in the FDM group were gradually down-regulated(all P<0.05);the mRNA expressions of them were also gradually down-regulated(all P<0.05);and there was positive correlation between them. The control group had no obvious change in each index(all P>0.05). CONCLUSION: AP1 may be an important transcription factor involved in the regulation of collagen I synthesis and degradation during myopic scleral remodeling.
文摘Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.
文摘AIM:To investigate the role of Ras association domain family protein 1 isoform A (RASSF1A) in gastric tumorigenesis. METHODS:Through over-expression of RASSF1A gene in the SGC7901 cell line which was induced by a lipofectamine-mediated gene transfer approach. Activator protein-1 (AP-1) DNA binding activity was measured by electrophoretic mobility shift assay (EMSA). RESULTS:Compared with the control clones, cells over- expressing RASSF1A exhibited significant inhibition of cell growth with G1 cell cycle arrest in vitro and in vivo. The over-expression of RASSF1A significantly inhibited AP-1 activity in SGC7901 cells (0.981±0.011 vs 0.354±0.053, P<0.001). In addition, both Western blot analysis and immunocytochemistry demonstrated that RASSF1A down-regulated the expression of c-Fos (0.975± 0.02 vs 0.095±0.024, P<0.001) but not c-Jun. CONCLUSION: Over-expression of RASSF1A inhibits the growth of SGC7901 cells by negatively regulating the AP-1 activity, the latter in turn negatively signals cell proliferation.
