A microgravity environment has been shown to cause ocular damage and affect visual acuity,but the underlying mechanisms remain unclear.Therefore,we established an animal model of weightlessness via tail suspension to ...A microgravity environment has been shown to cause ocular damage and affect visual acuity,but the underlying mechanisms remain unclear.Therefore,we established an animal model of weightlessness via tail suspension to examine the pathological changes and molecular mechanisms of retinal damage under microgravity.After 4 weeks of tail suspension,there were no notable alterations in retinal function and morphology,while after 8 weeks of tail suspension,significant reductions in retinal function were observed,and the outer nuclear layer was thinner,with abundant apoptotic cells.To investigate the mechanism underlying the degenerative changes that occurred in the outer nuclear layer of the retina,proteomics was used to analyze differentially expressed proteins in rat retinas after 8 weeks of tail suspension.The results showed that the expression levels of fibroblast growth factor 2(also known as basic fibroblast growth factor)and glial fibrillary acidic protein,which are closely related to Müller cell activation,were significantly upregulated.In addition,Müller cell regeneration and Müller cell gliosis were observed after 4 and 8 weeks,respectively,of simulated weightlessness.These findings indicate that Müller cells play an important regulatory role in retinal outer nuclear layer degeneration during weightlessness.展开更多
Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells,which is involved in retinal ganglion cell apopt...Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells,which is involved in retinal ganglion cell apoptosis in glaucoma.Modulation of Kir4.1 expression in Müller cells may therefore be a potential strategy for attenuating retinal ganglion cell damage in glaucoma.In this study,we identified seven predicted phosphorylation sites in Kir4.1 and constructed lentiviral expression systems expressing Kir4.1 mutated at each site to prevent phosphorylation.Following this,we treated Müller glial cells in vitro and in vivo with the m Glu R I agonist DHPG to induce Kir4.1 or Kir4.1 Tyr^(9)Asp overexpression.We found that both Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression inhibited activation of Müller glial cells.Subsequently,we established a rat model of chronic ocular hypertension by injecting microbeads into the anterior chamber and overexpressed Kir4.1 or Kir4.1 Tyr^(9)Asp in the eye,and observed similar results in Müller cells in vivo as those seen in vitro.Both Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression inhibited Müller cell activation,regulated the balance of Bax/Bcl-2,and reduced the m RNA and protein levels of pro-inflammatory factors,including interleukin-1βand tumor necrosis factor-α.Furthermore,we investigated the regulatory effects of Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression on the release of pro-inflammatory factors in a co-culture system of Müller glial cells and microglia.In this co-culture system,we observed elevated adenosine triphosphate concentrations in activated Müller cells,increased levels of translocator protein(a marker of microglial activation),and elevated interleukin-1βm RNA and protein levels in microglia induced by activated Müller cells.These changes could be reversed by Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression in Müller cells.Kir4.1 overexpression,but not Kir4.1 Tyr^(9)Asp overexpression,reduced the number of proliferative and migratory microglia induced by activated Müller cells.Collectively,these results suggest that the tyrosine residue at position nine in Kir4.1 may serve as a functional modulation site in the retina in an experimental model of glaucoma.Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression attenuated Müller cell activation,reduced ATP/P2X receptor–mediated interactions between glial cells,inhibited microglial activation,and decreased the synthesis and release of pro-inflammatory factors,consequently ameliorating retinal ganglion cell apoptosis in glaucoma.展开更多
Müller glia,as prominent glial cells within the retina,plays a significant role in maintaining retinal homeostasis in both healthy and diseased states.In lower vertebrates like zebrafish,these cells assume respon...Müller glia,as prominent glial cells within the retina,plays a significant role in maintaining retinal homeostasis in both healthy and diseased states.In lower vertebrates like zebrafish,these cells assume responsibility for spontaneous retinal regeneration,wherein endogenous Müller glia undergo proliferation,transform into Müller glia-derived progenitor cells,and subsequently regenerate the entire retina with restored functionality.Conversely,Müller glia in the mouse and human retina exhibit limited neural reprogramming.Müller glia reprogramming is thus a promising strategy for treating neurodegenerative ocular disorders.Müller glia reprogramming in mice has been accomplished with remarkable success,through various technologies.Advancements in molecular,genetic,epigenetic,morphological,and physiological evaluations have made it easier to document and investigate the Müller glia programming process in mice.Nevertheless,there remain issues that hinder improving reprogramming efficiency and maturity.Thus,understanding the reprogramming mechanism is crucial toward exploring factors that will improve Müller glia reprogramming efficiency,and for developing novel Müller glia reprogramming strategies.This review describes recent progress in relatively successful Müller glia reprogramming strategies.It also provides a basis for developing new Müller glia reprogramming strategies in mice,including epigenetic remodeling,metabolic modulation,immune regulation,chemical small-molecules regulation,extracellular matrix remodeling,and cell-cell fusion,to achieve Müller glia reprogramming in mice.展开更多
Inflammation plays a crucial role in the regeneration of fish and avian retinas.However,how inflammation regulates Müller glia(MG)reprogramming remains unclear.Here,we used single-cell RNA sequencing to investiga...Inflammation plays a crucial role in the regeneration of fish and avian retinas.However,how inflammation regulates Müller glia(MG)reprogramming remains unclear.Here,we used single-cell RNA sequencing to investigate the cell heterogeneity and interactions of MG and immune cells in the regenerating zebrafish retina.We first showed that two types of quiescent MG(resting MG1 and MG2)reside in the uninjured retina.Following retinal injury,resting MG1 transitioned into an activated state expressing known reprogramming genes,while resting MG2 gave rise to rod progenitors.We further showed that retinal microglia can be categorized into three subtypes(microglia-1,microglia-2,and proliferative)and pseudotime analysis demonstrated dynamic changes in microglial status following retinal injury.Analysis of cell–cell interactions indicated extensive crosstalk between immune cells and MG,with many interactions shared among different immune cell types.Finally,we showed that inflammation activated Jak1–Stat3 signaling in MG,promoting their transition from a resting to an activated state.Our study reveals the cell heterogeneity and crosstalk of immune cells and MG in zebrafish retinal repair,and may provide valuable insights into future mammalian retina regeneration.展开更多
AIM:To investigate the effects and the underlying mechanism(s)of conbercept on the phagocytosis of hard exudates(HEs)by Müller glia in diabetic retinopathy(DR).METHODS:Twenty-one eyes from 17 patients with diabet...AIM:To investigate the effects and the underlying mechanism(s)of conbercept on the phagocytosis of hard exudates(HEs)by Müller glia in diabetic retinopathy(DR).METHODS:Twenty-one eyes from 17 patients with diabetic macular edema(DME)underwent optical coherence tomography(OCT)imaging to examine the changes of HEs before and after intravitreal conbercept injection(IVC).In vitro,rat retinal Müller cell line(rMC-1)was cultured under high glucose and treated with oxidized low-density lipoprotein(Ox-LDL)with or without conbercept.Phagocytosis was analysed with immunofluorescence,flow cytometry,and Western blot.Expressions of scavenger receptors(LOX-1,CD36)were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR).Conbercept’s effects on vascular endothelial growth factor A(VEGF-A),VEGFR2,inflammation(NF-κB,IL-6,iNOS),and oxidative stress(ROS)were evaluated with Western blot and immunofluorescence.RESULTS:The area of HEs showed minimal change after the first IVC(1.39±1.41 to 1.38±1.3 mm2,P=0.938),but significantly decreased after the third IVC(0.45±0.66 mm2,P=0.002).In vitro,conbercept enhanced the phagocytosis of Ox-LDL by rMC-1 cells under high glucose condition.Conbercept reduced ROS and inflammation(NF-κB,IL-6,iNOS)in high glucose-treated rMC-1 cells by suppression of VEGF/VEGFR2 pathway.The inhibition of NF-κB by conbercept further activated PPARγ-CD36 axis,increasing CD36 expression and promoting Ox-LDL uptake,thereby facilitating the clearance of HEs.CONCLUSION:Conbercept reduces HEs in DR by enhancing Müller glia phagocytosis possibly through activating PPARγ-CD36 axis,which is mediated by inhibition of VEGF signaling.Modulation of Müller glia phagocytic capacity might provide a novel therapeutic strategy to treat DR and DME.展开更多
目的研究羟苯磺酸钙在高糖诱导的环境下对Müller细胞氧化损伤的保护作用及其机制。方法通过高糖诱导建立Müller细胞氧化损伤模型,并将细胞分为4组,即对照组(正常培养)、高糖组(35mmol/L葡萄糖培养基)、对照+羟苯磺酸钙组(常...目的研究羟苯磺酸钙在高糖诱导的环境下对Müller细胞氧化损伤的保护作用及其机制。方法通过高糖诱导建立Müller细胞氧化损伤模型,并将细胞分为4组,即对照组(正常培养)、高糖组(35mmol/L葡萄糖培养基)、对照+羟苯磺酸钙组(常规培养基础上加入0.5μmol/L羟苯磺酸钙)和高糖+羟苯磺酸钙组(高糖基础上加入0.5μmol/L羟苯磺酸钙)。使用CCK-8评估细胞增殖,流式细胞术检测细胞凋亡,试剂盒检测氧化应激指标,蛋白印迹技术检测内向整流钾离子通道4.1(inwardly rectifying K channel 4.1,Kir4.1)和水通道蛋白4(aquaporin-4,AQP4)蛋白水平。结果与对照组比较,高糖组Müller细胞增殖活性降低且凋亡率升高,细胞发生氧化应激,AQP4蛋白表达水平升高而Kir4.1蛋白表达水平降低(P<0.05)。与高糖组比较,高糖+羟苯磺酸钙组细胞增殖活性增加且凋亡率降低,细胞氧化应激损伤减轻,AQP4蛋白表达水平降低而Kir4.1蛋白表达水平升高(P<0.05)。结论羟苯磺酸钙可能通过调节AQP4/Kir4.1轴抑制高糖诱导的Müller细胞氧化损伤。展开更多
基金supported by the Army Laboratory Animal Foundation of China,No.SYDW[2020]22(to TC)the Shaanxi Provincial Key R&D Plan General Project of China,No.2022SF-236(to YM)the National Natural Science Foundation of China,No.82202070(to TC)。
文摘A microgravity environment has been shown to cause ocular damage and affect visual acuity,but the underlying mechanisms remain unclear.Therefore,we established an animal model of weightlessness via tail suspension to examine the pathological changes and molecular mechanisms of retinal damage under microgravity.After 4 weeks of tail suspension,there were no notable alterations in retinal function and morphology,while after 8 weeks of tail suspension,significant reductions in retinal function were observed,and the outer nuclear layer was thinner,with abundant apoptotic cells.To investigate the mechanism underlying the degenerative changes that occurred in the outer nuclear layer of the retina,proteomics was used to analyze differentially expressed proteins in rat retinas after 8 weeks of tail suspension.The results showed that the expression levels of fibroblast growth factor 2(also known as basic fibroblast growth factor)and glial fibrillary acidic protein,which are closely related to Müller cell activation,were significantly upregulated.In addition,Müller cell regeneration and Müller cell gliosis were observed after 4 and 8 weeks,respectively,of simulated weightlessness.These findings indicate that Müller cells play an important regulatory role in retinal outer nuclear layer degeneration during weightlessness.
基金supported by the National Natural Science Foundation of China,Nos.32271043(to ZW)and 82171047(to YM)the both Science and Technology Major Project of Shanghai,No.2018SHZDZX01 and ZJLabShanghai Center for Brain Science and Brain-Inspired Technology(to ZW)。
文摘Downregulation of the inwardly rectifying potassium channel Kir4.1 is a key step for inducing retinal Müller cell activation and interaction with other glial cells,which is involved in retinal ganglion cell apoptosis in glaucoma.Modulation of Kir4.1 expression in Müller cells may therefore be a potential strategy for attenuating retinal ganglion cell damage in glaucoma.In this study,we identified seven predicted phosphorylation sites in Kir4.1 and constructed lentiviral expression systems expressing Kir4.1 mutated at each site to prevent phosphorylation.Following this,we treated Müller glial cells in vitro and in vivo with the m Glu R I agonist DHPG to induce Kir4.1 or Kir4.1 Tyr^(9)Asp overexpression.We found that both Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression inhibited activation of Müller glial cells.Subsequently,we established a rat model of chronic ocular hypertension by injecting microbeads into the anterior chamber and overexpressed Kir4.1 or Kir4.1 Tyr^(9)Asp in the eye,and observed similar results in Müller cells in vivo as those seen in vitro.Both Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression inhibited Müller cell activation,regulated the balance of Bax/Bcl-2,and reduced the m RNA and protein levels of pro-inflammatory factors,including interleukin-1βand tumor necrosis factor-α.Furthermore,we investigated the regulatory effects of Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression on the release of pro-inflammatory factors in a co-culture system of Müller glial cells and microglia.In this co-culture system,we observed elevated adenosine triphosphate concentrations in activated Müller cells,increased levels of translocator protein(a marker of microglial activation),and elevated interleukin-1βm RNA and protein levels in microglia induced by activated Müller cells.These changes could be reversed by Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression in Müller cells.Kir4.1 overexpression,but not Kir4.1 Tyr^(9)Asp overexpression,reduced the number of proliferative and migratory microglia induced by activated Müller cells.Collectively,these results suggest that the tyrosine residue at position nine in Kir4.1 may serve as a functional modulation site in the retina in an experimental model of glaucoma.Kir4.1 and Kir4.1 Tyr^(9)Asp overexpression attenuated Müller cell activation,reduced ATP/P2X receptor–mediated interactions between glial cells,inhibited microglial activation,and decreased the synthesis and release of pro-inflammatory factors,consequently ameliorating retinal ganglion cell apoptosis in glaucoma.
基金supported by the National Natural Science Foundation of China,No.31930068National Key Research and Development Program of China,Nos.2018YFA0107302 and 2021YFA1101203(all to HX).
文摘Müller glia,as prominent glial cells within the retina,plays a significant role in maintaining retinal homeostasis in both healthy and diseased states.In lower vertebrates like zebrafish,these cells assume responsibility for spontaneous retinal regeneration,wherein endogenous Müller glia undergo proliferation,transform into Müller glia-derived progenitor cells,and subsequently regenerate the entire retina with restored functionality.Conversely,Müller glia in the mouse and human retina exhibit limited neural reprogramming.Müller glia reprogramming is thus a promising strategy for treating neurodegenerative ocular disorders.Müller glia reprogramming in mice has been accomplished with remarkable success,through various technologies.Advancements in molecular,genetic,epigenetic,morphological,and physiological evaluations have made it easier to document and investigate the Müller glia programming process in mice.Nevertheless,there remain issues that hinder improving reprogramming efficiency and maturity.Thus,understanding the reprogramming mechanism is crucial toward exploring factors that will improve Müller glia reprogramming efficiency,and for developing novel Müller glia reprogramming strategies.This review describes recent progress in relatively successful Müller glia reprogramming strategies.It also provides a basis for developing new Müller glia reprogramming strategies in mice,including epigenetic remodeling,metabolic modulation,immune regulation,chemical small-molecules regulation,extracellular matrix remodeling,and cell-cell fusion,to achieve Müller glia reprogramming in mice.
基金supported by the National Natural Science Foundation of China,Nos.81970820(to HX),31771644(to JL),31930068(to JL),82371176(to JL),81801331(to LC)National Key Research and Development Project of China.Nos.2017YFA0104100(to JL),2017YFA0701304(to HX)+1 种基金Shanghai Yangzhi Rehabilitation Hospital(Shanghai Sunshine Rehabilitation Center)Talent Introduction Plan,No.KYPT202204(to LC)the Fundamental Research Funds for the Central Universities,No.22120230292(to JL)。
文摘Inflammation plays a crucial role in the regeneration of fish and avian retinas.However,how inflammation regulates Müller glia(MG)reprogramming remains unclear.Here,we used single-cell RNA sequencing to investigate the cell heterogeneity and interactions of MG and immune cells in the regenerating zebrafish retina.We first showed that two types of quiescent MG(resting MG1 and MG2)reside in the uninjured retina.Following retinal injury,resting MG1 transitioned into an activated state expressing known reprogramming genes,while resting MG2 gave rise to rod progenitors.We further showed that retinal microglia can be categorized into three subtypes(microglia-1,microglia-2,and proliferative)and pseudotime analysis demonstrated dynamic changes in microglial status following retinal injury.Analysis of cell–cell interactions indicated extensive crosstalk between immune cells and MG,with many interactions shared among different immune cell types.Finally,we showed that inflammation activated Jak1–Stat3 signaling in MG,promoting their transition from a resting to an activated state.Our study reveals the cell heterogeneity and crosstalk of immune cells and MG in zebrafish retinal repair,and may provide valuable insights into future mammalian retina regeneration.
基金Supported by the National Natural Science Foundation of China(No.82171062,No.82301222,No.32201244).
文摘AIM:To investigate the effects and the underlying mechanism(s)of conbercept on the phagocytosis of hard exudates(HEs)by Müller glia in diabetic retinopathy(DR).METHODS:Twenty-one eyes from 17 patients with diabetic macular edema(DME)underwent optical coherence tomography(OCT)imaging to examine the changes of HEs before and after intravitreal conbercept injection(IVC).In vitro,rat retinal Müller cell line(rMC-1)was cultured under high glucose and treated with oxidized low-density lipoprotein(Ox-LDL)with or without conbercept.Phagocytosis was analysed with immunofluorescence,flow cytometry,and Western blot.Expressions of scavenger receptors(LOX-1,CD36)were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR).Conbercept’s effects on vascular endothelial growth factor A(VEGF-A),VEGFR2,inflammation(NF-κB,IL-6,iNOS),and oxidative stress(ROS)were evaluated with Western blot and immunofluorescence.RESULTS:The area of HEs showed minimal change after the first IVC(1.39±1.41 to 1.38±1.3 mm2,P=0.938),but significantly decreased after the third IVC(0.45±0.66 mm2,P=0.002).In vitro,conbercept enhanced the phagocytosis of Ox-LDL by rMC-1 cells under high glucose condition.Conbercept reduced ROS and inflammation(NF-κB,IL-6,iNOS)in high glucose-treated rMC-1 cells by suppression of VEGF/VEGFR2 pathway.The inhibition of NF-κB by conbercept further activated PPARγ-CD36 axis,increasing CD36 expression and promoting Ox-LDL uptake,thereby facilitating the clearance of HEs.CONCLUSION:Conbercept reduces HEs in DR by enhancing Müller glia phagocytosis possibly through activating PPARγ-CD36 axis,which is mediated by inhibition of VEGF signaling.Modulation of Müller glia phagocytic capacity might provide a novel therapeutic strategy to treat DR and DME.
文摘目的研究羟苯磺酸钙在高糖诱导的环境下对Müller细胞氧化损伤的保护作用及其机制。方法通过高糖诱导建立Müller细胞氧化损伤模型,并将细胞分为4组,即对照组(正常培养)、高糖组(35mmol/L葡萄糖培养基)、对照+羟苯磺酸钙组(常规培养基础上加入0.5μmol/L羟苯磺酸钙)和高糖+羟苯磺酸钙组(高糖基础上加入0.5μmol/L羟苯磺酸钙)。使用CCK-8评估细胞增殖,流式细胞术检测细胞凋亡,试剂盒检测氧化应激指标,蛋白印迹技术检测内向整流钾离子通道4.1(inwardly rectifying K channel 4.1,Kir4.1)和水通道蛋白4(aquaporin-4,AQP4)蛋白水平。结果与对照组比较,高糖组Müller细胞增殖活性降低且凋亡率升高,细胞发生氧化应激,AQP4蛋白表达水平升高而Kir4.1蛋白表达水平降低(P<0.05)。与高糖组比较,高糖+羟苯磺酸钙组细胞增殖活性增加且凋亡率降低,细胞氧化应激损伤减轻,AQP4蛋白表达水平降低而Kir4.1蛋白表达水平升高(P<0.05)。结论羟苯磺酸钙可能通过调节AQP4/Kir4.1轴抑制高糖诱导的Müller细胞氧化损伤。
