AIM:To evaluate the effect of epigallocatechin gallate(EGCG) in preventing lens opacity and the aggregation of lens αB-crystallin in model rats of diabetes mellitus(DM).METHODS:This experimental study included Wistar...AIM:To evaluate the effect of epigallocatechin gallate(EGCG) in preventing lens opacity and the aggregation of lens αB-crystallin in model rats of diabetes mellitus(DM).METHODS:This experimental study included Wistar rats for DM as in vivo models and divided into 5 groups.The treatment groups were administered EGCG by orally for 20d and were then assessed for their degree of lens opacity with binocular microscope and lens αB-crystallin expression from Western blot analyze.RESULTS:Pearson correlation test and regression analysis on EGCG exposure and final random blood sugar(RBS) obtained a significance level of P<0.05.EGCG exposure can significantly lower RBS with an R~2 of 0.5634(56.34%).The same analysis on EGCG exposure and the degree of lens opacity obtained a significance level of P<0.05 and increased exposure to EGCG can significantly lower the degree of lens opacity with an R~2 of 0.8577(85.77%).Correlation analysis between EGCG and the expression of lens αB-crystallin can be concluded that the higher the EGCG exposure administered,the higher the native lens αB-crystallin expression and the lower the aggregate lens αB-crystallin expression.There was also significant effect in which every 1 mg/kg body weight dose of EGCG can increase the native lens αB-crystallin expression by 0.0063 and decrease the aggregate lens αB-crystallin expression by 0.0076.CONCLUSION:The administration of EGCG at a dose of 300,600,and 1200 mg shows a significant effect on preventing lens opacity and aggregation of αB-crystallin in diabetic rat models and this research could be a biomolecular prevention of cataract.展开更多
目的探讨αB-crystallin基因在喉癌组织中的表达及其与喉癌的关系。方法收集2008年3月~2014年10月南京医科大学第二附属医院及皖南医学院弋矶山医院手术切除的喉癌标本54例及13例癌旁正常黏膜标本,采用实时荧光定量PCR（Real-time PCR...目的探讨αB-crystallin基因在喉癌组织中的表达及其与喉癌的关系。方法收集2008年3月~2014年10月南京医科大学第二附属医院及皖南医学院弋矶山医院手术切除的喉癌标本54例及13例癌旁正常黏膜标本,采用实时荧光定量PCR（Real-time PCR）检测8例喉癌组织及其癌旁正常组织中αB-crystallin基因的表达,免疫组化法检测54例癌组织及13例癌旁正常组织中αB-crystallin蛋白的表达,比较不同组织中αB-crystallin阳性表达率。结果喉癌组织中的αB-crystallin m RNA表达量及αB-crystallin蛋白阳性表达率均明显高于癌旁正常组织,差异均有高度统计学意义（P〈0.01）;不同TMN分期及淋巴结转移情况的喉癌组织中αB-crystallin蛋白的阳性表达率比较差异有统计学意义（P〈0.05）,不同性别、年龄、肿瘤分型及分化程度的喉癌组织中αBcrystallin蛋白的阳性表达率比较差异无统计学意义（P〉0.05）。结论αB-crystallin基因的高表达与喉癌发生发展及转移有关,可能为喉癌患者的预后提供一个潜在的风险评估指标。展开更多
AIM: To evaluate αB-crystallin malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) changes in X-ray irradiated rat lens. METHODS: Eight-week-old Sprague-Dawley male rats received X-ray ...AIM: To evaluate αB-crystallin malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) changes in X-ray irradiated rat lens. METHODS: Eight-week-old Sprague-Dawley male rats received X-ray irradiation to the head with rest of the body protected. The exposure dose ranged from 2 to 25 Grays (Gy). The cataract status were examined by slit lamp and rated with 'four-grade systems' post-irradiation. The lens MDA level, and the activities of SOD and GPx were measured in a short-term experiment post-irradiation, and αB-crystallin protein levels were quantified. RESULTS: The lenses of normal control and the X-ray irradiated groups with the dose up to 10 Gy remained transparent throughout the experiment. The lens first appeared tiny scatters, and even lamellar opacities in the posterior capsule 45 days post-irradiation with the dose of 15 Gy, and progressed slowly to the advance stage of cataract; while, for the higher dose (25 Gy), the opacity of lens appeared much earlier, and progressed more rapidly to mature stage of cataract within 1 month. At the end of the observation (90 days post-irradiation), almost all lenses became complete opacity with the higher dose (25 Gy). The degree of lens opacity was rated accordingly. The lens MDA level was increased, and SOD and GPx activities were decreased with a dose-dependent manner post-irradiation. The αB-crystallin protein level was decreased dose-dependently at the end point of observation. CONCLUSION: Oxidative events and αB-crystallin may play important roles in the pathogenesis of cataract in X-ray irradiated rat lens.展开更多
Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocu...Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocular hypertension model in the right eye of Sprague-Dawley rats. Fluorogold retrograde tracing and immunofluorescence methods showed that the number of retinal ganglion cells decreased in the right eyes and caspase-3 expression increased following acute ocular hypertension. Intravitreal injection of aB-crystallin in the right eye increased the number of retinal ganglion cells and reduced caspase-3 expression. Results demonstrated that exogenous αB-crystallin protein inhibited caspase-3 expression and improved retinal ganglion cell survival following acute ocular hypertension.展开更多
Background:Parkinson’s disease(PD)is characterized by a chronic loss of dopaminergic neurons and the presence of proteinaceous inclusions(Lewy bodies)within some remaining neurons in the substantia nigra.Recently,ast...Background:Parkinson’s disease(PD)is characterized by a chronic loss of dopaminergic neurons and the presence of proteinaceous inclusions(Lewy bodies)within some remaining neurons in the substantia nigra.Recently,astroglial inclusion body has also been found in some neurodegenerative diseases including PD.However,the underlying molecular mechanisms of how astroglial protein aggregation forms remain largely unknown.Here,we investigated the contribution ofαB-crystallin(CRYAB),a small heat shock protein,inα-synuclein inclusion formation in astrocytes.Methods:Small interfering RNA(siRNA)-mediated CRYAB(siCRYAB)knockdown or CRYAB overexpression was performed to investigate the impact of CRYAB on the autophagy in human glioblastoma cell line U251 cells.Coimmunoprecipitation(co-IP)and immunoblotting were used to dissect the interaction among multiple proteins.The clearance ofα-synuclein in vitro was evaluated by immunocytochemistry.CRYAB transgenic mice and transgenic mice overexpressing A30P mutant form of humanα-synuclein were used to examine the influence of CRYAB toα-synuclein accumulation in vivo.Results:We found that knockdown of CRYAB in U251 cells or primary cultured astrocytes resulted in a marked augmentation of autophagy activity.In contrast,exogenous CRYAB disrupted the assembly of the BAG3-HSPB8-HSC70 complex via binding with BAG3,thereby suppressing the autophagy activity.Furthermore,CRYAB-regulated autophagy has relevance to PD pathogenesis.Knockdown of CRYAB remarkably promoted cytoplasmic clearance ofα-synuclein preformed fibrils(PFFs).Conversely,selective overexpression of CRYAB in astrocytes markedly suppressed autophagy leading to the accumulation of α-synuclein aggregates in the brain of transgenic mice expressing humanα-synuclein A30P mutant.Conclusions:This study reveals a novel function for CRYAB as a natural inhibitor of astrocytic autophagy and shows that knockdown of CYRAB may provide a therapeutic target against proteinopathies such as synucleinopathies.展开更多
AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning t...AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.展开更多
Protein misfolding and aggregation are crucial pathogenic factors for cataracts,which are the leading cause of visual impairment worldwide.α-crystallin,as a small molecular chaperone,is involved in preventing protein...Protein misfolding and aggregation are crucial pathogenic factors for cataracts,which are the leading cause of visual impairment worldwide.α-crystallin,as a small molecular chaperone,is involved in preventing protein misfolding and maintaining lens transparency.The chaperone activity of α-crystallin depends on its oligomeric state.Our previous work identified a natural compound,celastrol,which could regulate the oligomeric state of αB-crystallin.In this work,based on the UNcle and SEC analysis,we found that celastrol induced𝛼αB-crystallin to form large oligomers.Large oligomer formation enhanced the chaperone activity of αB-crystallin and prevented aggregation of the cataract-causing mutant αA3-G91del.The interactions between𝛼αB-crystallin and celastrol were detected by the FRET(Fluorescence Resonance Energy Transfer)technique,and verified by molecular docking.At least 9 binding patterns were recognized,and some binding sites covered the groove structure of αB-crystallin.Interestingly,αB-R120G,a cataract-causing mutation located at the groove structure,and celastrol can decrease the aggregates of αB-R120G.Overall,our results suggested celastrol not only promoted the formation of large αB-crystallin oligomers,which enhanced its chaperone activity,but also bound to the groove structure of its α-crystallin domain to maintain its structural stability.Celastrol might serve as a chemical and pharmacological chaperone for cataract treatment.展开更多
文摘AIM:To evaluate the effect of epigallocatechin gallate(EGCG) in preventing lens opacity and the aggregation of lens αB-crystallin in model rats of diabetes mellitus(DM).METHODS:This experimental study included Wistar rats for DM as in vivo models and divided into 5 groups.The treatment groups were administered EGCG by orally for 20d and were then assessed for their degree of lens opacity with binocular microscope and lens αB-crystallin expression from Western blot analyze.RESULTS:Pearson correlation test and regression analysis on EGCG exposure and final random blood sugar(RBS) obtained a significance level of P<0.05.EGCG exposure can significantly lower RBS with an R~2 of 0.5634(56.34%).The same analysis on EGCG exposure and the degree of lens opacity obtained a significance level of P<0.05 and increased exposure to EGCG can significantly lower the degree of lens opacity with an R~2 of 0.8577(85.77%).Correlation analysis between EGCG and the expression of lens αB-crystallin can be concluded that the higher the EGCG exposure administered,the higher the native lens αB-crystallin expression and the lower the aggregate lens αB-crystallin expression.There was also significant effect in which every 1 mg/kg body weight dose of EGCG can increase the native lens αB-crystallin expression by 0.0063 and decrease the aggregate lens αB-crystallin expression by 0.0076.CONCLUSION:The administration of EGCG at a dose of 300,600,and 1200 mg shows a significant effect on preventing lens opacity and aggregation of αB-crystallin in diabetic rat models and this research could be a biomolecular prevention of cataract.
文摘目的探讨αB-crystallin基因在喉癌组织中的表达及其与喉癌的关系。方法收集2008年3月~2014年10月南京医科大学第二附属医院及皖南医学院弋矶山医院手术切除的喉癌标本54例及13例癌旁正常黏膜标本,采用实时荧光定量PCR（Real-time PCR）检测8例喉癌组织及其癌旁正常组织中αB-crystallin基因的表达,免疫组化法检测54例癌组织及13例癌旁正常组织中αB-crystallin蛋白的表达,比较不同组织中αB-crystallin阳性表达率。结果喉癌组织中的αB-crystallin m RNA表达量及αB-crystallin蛋白阳性表达率均明显高于癌旁正常组织,差异均有高度统计学意义（P〈0.01）;不同TMN分期及淋巴结转移情况的喉癌组织中αB-crystallin蛋白的阳性表达率比较差异有统计学意义（P〈0.05）,不同性别、年龄、肿瘤分型及分化程度的喉癌组织中αBcrystallin蛋白的阳性表达率比较差异无统计学意义（P〉0.05）。结论αB-crystallin基因的高表达与喉癌发生发展及转移有关,可能为喉癌患者的预后提供一个潜在的风险评估指标。
基金Scientific Research Foundation for Returned Scholars, the Second Hospital Affiliated to Soochow University (No.SDFEY-2007-10)National Natural Science Foundation of China (No.81000383)+2 种基金Research Fund for the Doctoral Program of Higher Education of China (No.20100072120051)Program of Tongji University (No.1500219024 No.2010QH04 and No. 2010YF02)
文摘AIM: To evaluate αB-crystallin malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) changes in X-ray irradiated rat lens. METHODS: Eight-week-old Sprague-Dawley male rats received X-ray irradiation to the head with rest of the body protected. The exposure dose ranged from 2 to 25 Grays (Gy). The cataract status were examined by slit lamp and rated with 'four-grade systems' post-irradiation. The lens MDA level, and the activities of SOD and GPx were measured in a short-term experiment post-irradiation, and αB-crystallin protein levels were quantified. RESULTS: The lenses of normal control and the X-ray irradiated groups with the dose up to 10 Gy remained transparent throughout the experiment. The lens first appeared tiny scatters, and even lamellar opacities in the posterior capsule 45 days post-irradiation with the dose of 15 Gy, and progressed slowly to the advance stage of cataract; while, for the higher dose (25 Gy), the opacity of lens appeared much earlier, and progressed more rapidly to mature stage of cataract within 1 month. At the end of the observation (90 days post-irradiation), almost all lenses became complete opacity with the higher dose (25 Gy). The degree of lens opacity was rated accordingly. The lens MDA level was increased, and SOD and GPx activities were decreased with a dose-dependent manner post-irradiation. The αB-crystallin protein level was decreased dose-dependently at the end point of observation. CONCLUSION: Oxidative events and αB-crystallin may play important roles in the pathogenesis of cataract in X-ray irradiated rat lens.
基金supported by the China Post-doctoral Foundation(The protective effect of αB-crystallin on retinal ganglial cells in glaucoma),No.20080430444
文摘Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocular hypertension model in the right eye of Sprague-Dawley rats. Fluorogold retrograde tracing and immunofluorescence methods showed that the number of retinal ganglion cells decreased in the right eyes and caspase-3 expression increased following acute ocular hypertension. Intravitreal injection of aB-crystallin in the right eye increased the number of retinal ganglion cells and reduced caspase-3 expression. Results demonstrated that exogenous αB-crystallin protein inhibited caspase-3 expression and improved retinal ganglion cell survival following acute ocular hypertension.
基金This work was supported by grants from the Natural Science Foundation of China(31430036,91742116,U1801681)National Key Basic Research Program of China(2015CB553500)+3 种基金Key Research Program of Frontier Sciences(QYZDJ-SSW-SMC002)Strategic Priority Research Program of Chinese Academy of Science(XDB32020100)Shanghai Municipal Science and Technology Major Project(2018SHZDZX05)the Shanghai Municipal Science and Technology Commission(17ZR1435300 to SZZ).
文摘Background:Parkinson’s disease(PD)is characterized by a chronic loss of dopaminergic neurons and the presence of proteinaceous inclusions(Lewy bodies)within some remaining neurons in the substantia nigra.Recently,astroglial inclusion body has also been found in some neurodegenerative diseases including PD.However,the underlying molecular mechanisms of how astroglial protein aggregation forms remain largely unknown.Here,we investigated the contribution ofαB-crystallin(CRYAB),a small heat shock protein,inα-synuclein inclusion formation in astrocytes.Methods:Small interfering RNA(siRNA)-mediated CRYAB(siCRYAB)knockdown or CRYAB overexpression was performed to investigate the impact of CRYAB on the autophagy in human glioblastoma cell line U251 cells.Coimmunoprecipitation(co-IP)and immunoblotting were used to dissect the interaction among multiple proteins.The clearance ofα-synuclein in vitro was evaluated by immunocytochemistry.CRYAB transgenic mice and transgenic mice overexpressing A30P mutant form of humanα-synuclein were used to examine the influence of CRYAB toα-synuclein accumulation in vivo.Results:We found that knockdown of CRYAB in U251 cells or primary cultured astrocytes resulted in a marked augmentation of autophagy activity.In contrast,exogenous CRYAB disrupted the assembly of the BAG3-HSPB8-HSC70 complex via binding with BAG3,thereby suppressing the autophagy activity.Furthermore,CRYAB-regulated autophagy has relevance to PD pathogenesis.Knockdown of CRYAB remarkably promoted cytoplasmic clearance ofα-synuclein preformed fibrils(PFFs).Conversely,selective overexpression of CRYAB in astrocytes markedly suppressed autophagy leading to the accumulation of α-synuclein aggregates in the brain of transgenic mice expressing humanα-synuclein A30P mutant.Conclusions:This study reveals a novel function for CRYAB as a natural inhibitor of astrocytic autophagy and shows that knockdown of CYRAB may provide a therapeutic target against proteinopathies such as synucleinopathies.
基金Supported by National Natural Science Foundation of China Grant (No.81270996)Science and Technology Project Foundation of Hainan Province (No.ZDYF201631)Health Science and Technology Innovation Project Foundation of Sanya (No.2016YW22)
文摘AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.
基金supported by the National Natural Science Foundation of China(31872724,and 81900837)the Natural Science Foundation of Zhejiang Province(LR21H120001).
文摘Protein misfolding and aggregation are crucial pathogenic factors for cataracts,which are the leading cause of visual impairment worldwide.α-crystallin,as a small molecular chaperone,is involved in preventing protein misfolding and maintaining lens transparency.The chaperone activity of α-crystallin depends on its oligomeric state.Our previous work identified a natural compound,celastrol,which could regulate the oligomeric state of αB-crystallin.In this work,based on the UNcle and SEC analysis,we found that celastrol induced𝛼αB-crystallin to form large oligomers.Large oligomer formation enhanced the chaperone activity of αB-crystallin and prevented aggregation of the cataract-causing mutant αA3-G91del.The interactions between𝛼αB-crystallin and celastrol were detected by the FRET(Fluorescence Resonance Energy Transfer)technique,and verified by molecular docking.At least 9 binding patterns were recognized,and some binding sites covered the groove structure of αB-crystallin.Interestingly,αB-R120G,a cataract-causing mutation located at the groove structure,and celastrol can decrease the aggregates of αB-R120G.Overall,our results suggested celastrol not only promoted the formation of large αB-crystallin oligomers,which enhanced its chaperone activity,but also bound to the groove structure of its α-crystallin domain to maintain its structural stability.Celastrol might serve as a chemical and pharmacological chaperone for cataract treatment.
