BACKGROUND Massive rotator cuff tears(RCTs)result in impaired shoulder function and quality of life.These tears lead to structural changes in the rotator cuff muscles,which compromise recovery after repair and increas...BACKGROUND Massive rotator cuff tears(RCTs)result in impaired shoulder function and quality of life.These tears lead to structural changes in the rotator cuff muscles,which compromise recovery after repair and increase re-tear rates.AIM To investigate the potential inhibitory effects of alpha-tocopherol(vitamin E)and OTR-4131 on muscle atrophy,fatty infiltration,and fibrosis in rotator cuff muscles following a massive RCT using a Wistar rat model,and establish a standardized methodology for evaluating potential therapeutic agents.METHODS This protocol outlines a controlled animal study using 40 male Wistar rats,randomized into five groups.The experimental groups will receive either systemic administration of alpha-tocopherol or local administration of OTR-4131 via intramuscular injection into the supraspinatus and infraspinatus muscles.Two sham groups will receive systemic and local saline injections respectively,while a control group will undergo no intervention.The interventions will be administered after surgical transection of the supraspinatus and infraspinatus tendons.Outcomes will be assessed via wet muscle weight measurements,muscle fiber diameter,fatty infiltration percentage,and fibrosis evaluation using histological methods.RESULTS The study anticipates that alpha-tocopherol and OTR-4131 will reduce muscle atrophy,fatty infiltration,and fibrosis compared to control and sham groups,supporting their potential protective role in rotator cuff muscle degeneration.CONCLUSION The results are expected to improve the understanding on the role of alpha-tocopherol and OTR-4131 in rotator cuff muscle protection after massive RCT and may serve as a foundation for further preclinical and clinical research aimed at improving rotator cuff repair outcomes.展开更多
为研究东阿阿胶对小鼠皮肤屏障损伤的修复作用。采用胶带剥离法构建小鼠皮肤屏障损伤模型,并使用阿胶对小鼠进行灌胃给药。造模后,每天检测小鼠受损皮肤处的经皮失水量(Transepidermal water loss,TEWL)。并于造模第4天和第7天后,通过...为研究东阿阿胶对小鼠皮肤屏障损伤的修复作用。采用胶带剥离法构建小鼠皮肤屏障损伤模型,并使用阿胶对小鼠进行灌胃给药。造模后,每天检测小鼠受损皮肤处的经皮失水量(Transepidermal water loss,TEWL)。并于造模第4天和第7天后,通过光学活体成像观察皮肤屏障损伤的修复效果。通过RT-qPCT检测丝聚蛋白(Filaggrin,FLG)、内披蛋白(Involucrin,IVL)、紧密连接蛋白闭锁小带蛋白1(Zonula occludens-1,ZO-1)与炎症因子白介素-1α(Interleukin-1α,IL-1α)、白介素-1β(Interleukin-1β,IL-1β)等的mRNA水平。通过免疫组化检测皮肤屏障相关蛋白的蛋白含量,免疫荧光检测胸腺基质淋巴生成素(Thymic stromal lymphopoietin,TSLP)的表达。结果显示,东阿阿胶显著降低胶带剥离小鼠的经皮失水量,降低皮肤厚度并改善表皮结构紊乱,上调FLG,IVL和ZO-1的mRNA相对表达量与蛋白表达,抑制TSLP的蛋白表达与炎症因子的mRNA相对表达量。结果表明,东阿阿胶通过调控屏障蛋白与炎症因子的表达实现皮肤屏障修复,为其在皮肤健康领域的应用提供理论依据。展开更多
RNA binding proteins(RBPs) are a crucial class of proteins that interact with RNA and play a key role in various biological process.Deficiencies or abnormalities of RBPs are closely linked to the occurrence and progre...RNA binding proteins(RBPs) are a crucial class of proteins that interact with RNA and play a key role in various biological process.Deficiencies or abnormalities of RBPs are closely linked to the occurrence and progression of numerous diseases,making RBPs potential therapeutic targets.However,the limited tissue penetration of 254 nm UV irradiation makes it difficult to efficiently crosslink weak and dynamic RNA-protein interactions in mammal tissues.Additionally,RNA degradation in metal catalyzed click reaction further hinders the enrichment of RNA-protein complexes(RPCs).Due to these inherent limitations,globally profiling the RNA binding proteome in mammal organs has long been a challenge.Herein,we proposed a novel method,which utilized a dual crosslinking with formaldehyde and 254 nm UV irradiation,metabolic labeling and metal-free thiol-yne click reaction to enable large-scale enrichment and identification of RBPs in mouse liver,called FTYc_UV.In this method,formaldehyde is first used to crosslink the crude RNA-protein complexes(cRPCs) in situ to address the problem of poor tissue penetration of 254 nm UV irradiation.Furthermore,this method integrates metabolic labeling with a metal-free thiol-yne click reaction to achieve non-destructive RNA tagging.After specifically RNA-RBPs crosslinking by 254 nm UV irradiation in tissue lysates,formaldehyde decrosslinking is employed to remove non-specific proteins,leading to effective enrichment of RPCs from mouse liver and thereby overcoming the poor specificity of formaldehyde crosslinking.Application of FTYc_UV in mouse liver successfully identified over 1600 RBPs covering approximately 75 % of previously reported RBPs.Furthermore,420 candidate RBPs,including 151metabolic enzymes,were also obtained,demonstrating the sensitivity of FTYc_UV and the potential of this method for in-depth exploration of RNA-protein interactions in biological and clinical research.展开更多
Ischemic stroke remains a leading cause of disability and death,with mesenchymal stem cell-derived exosomes emerging as a promising therapeutic avenue.However,the optimal timing and underlying therapeutic mechanisms o...Ischemic stroke remains a leading cause of disability and death,with mesenchymal stem cell-derived exosomes emerging as a promising therapeutic avenue.However,the optimal timing and underlying therapeutic mechanisms of exosome treatment require further elucidation.In this study,we used a murine model of middle cerebral artery occlusion to investigate the therapeutic efficacy of human umbilical cord mesenchymal stem cell-derived exosomes administered intravenously at an early(6 hours)or delayed(3 days)time point post-ischemia.Compared with delayed treatment,early administration of exosomes resulted in significantly superior efficacy,as evidenced by improved neurological function scores and reduced infarct volumes.Transcriptomic analysis of brain tissues from mice receiving early exosome treatment revealed marked downregulation of inflammation-related genes,including Ccl2,Ccl5,Cxcl10,Il-1β,Il-6,Itgam,Itgax,and Tnf-α.Metabolomic profiling of these brain tissues further identified modulation of key metabolites,including trimethylamine N-oxide,glutathione,1-stearoyl-rac-glycerol,and phosphatidylcholine,suggesting that alteration of metabolic pathways contributes to the therapeutic effect.Integrated transcriptomic and metabolomic analysis pinpointed significant modulation of pathways involving metabolism of eicosapentaenoic acid,lysine,propanoate,and tyrosine.These findings suggest that umbilical cord mesenchymal stem cell-derived exosomes,particularly when administered early post-ischemia,exert their neuroprotective effects by broadly suppressing inflammatory pathways and modulating key metabolic processes in the ischemic brain,highlighting their potential as a therapeutic intervention for ischemic stroke.展开更多
The major aim of stroke therapy is to stimulate brain repair and improve behavioral recovery after cerebral ischemia.One option is to stimulate endogenous neurogenesis in the subventricular zone and direct the newly f...The major aim of stroke therapy is to stimulate brain repair and improve behavioral recovery after cerebral ischemia.One option is to stimulate endogenous neurogenesis in the subventricular zone and direct the newly formed neurons to the damaged area.However,only a small percentage of these neurons survive,and many do not reach the damaged area,possibly because the corpus callosum impedes the migration of subventricular zone-derived stem cells into the lesioned cortex.A second major obstacle to stem cell therapy is the strong inflammatory reaction induced by cerebral ischemia,whereby the associated phagocytic activity of brain macrophages removes both therapeutic cells and/or cell-based drug carriers.To address these issues,neurogenesis was electrically stimulated in the subventricular zone,followed by isolation of proliferating cells,including newly formed neurons,which were subsequently mixed with a nutritional hydrogel.This mixture was then transferred to the stroke cavity of day 14 post-stroke mice.We found that the performance of the treated animals improved in behavioral tests,including novel object,open field,hole board,grooming,and“time-to-feel”adhesive tape tests.Furthermore,immunostaining revealed that the stem cell marker nestin,the neuroepithelial marker Mash1,and the immature neuronal marker doublecortin-positive cells survived in the transplanted area for 2 weeks,possibly due to reduced phagocytic activity and supportive angiogenesis.These results clearly indicate that the transplantation of committed subventricular zone stem cells combined with a protective nutritional gel directly into the infarct cavity after the peak of stroke-induced neuroinflammation represents a feasible approach to improve neurorestoration after cerebral ischemia.展开更多
基金thank the staff of the accredited animal facility of the laboratory of anatomy,Histology and Embryology of Aristotle University of Thessaloniki’s veterinary school for their assistance in animal handling and care.
文摘BACKGROUND Massive rotator cuff tears(RCTs)result in impaired shoulder function and quality of life.These tears lead to structural changes in the rotator cuff muscles,which compromise recovery after repair and increase re-tear rates.AIM To investigate the potential inhibitory effects of alpha-tocopherol(vitamin E)and OTR-4131 on muscle atrophy,fatty infiltration,and fibrosis in rotator cuff muscles following a massive RCT using a Wistar rat model,and establish a standardized methodology for evaluating potential therapeutic agents.METHODS This protocol outlines a controlled animal study using 40 male Wistar rats,randomized into five groups.The experimental groups will receive either systemic administration of alpha-tocopherol or local administration of OTR-4131 via intramuscular injection into the supraspinatus and infraspinatus muscles.Two sham groups will receive systemic and local saline injections respectively,while a control group will undergo no intervention.The interventions will be administered after surgical transection of the supraspinatus and infraspinatus tendons.Outcomes will be assessed via wet muscle weight measurements,muscle fiber diameter,fatty infiltration percentage,and fibrosis evaluation using histological methods.RESULTS The study anticipates that alpha-tocopherol and OTR-4131 will reduce muscle atrophy,fatty infiltration,and fibrosis compared to control and sham groups,supporting their potential protective role in rotator cuff muscle degeneration.CONCLUSION The results are expected to improve the understanding on the role of alpha-tocopherol and OTR-4131 in rotator cuff muscle protection after massive RCT and may serve as a foundation for further preclinical and clinical research aimed at improving rotator cuff repair outcomes.
文摘脊髓损伤(spinal cord injury,SCI)是一种高致残性中枢神经系统创伤,其病理进程复杂且多机制交织,涉及神经免疫炎症过度激活、氧化应激损伤、神经细胞凋亡、自噬紊乱及能量代谢失衡等关键环节,严重破坏脊髓神经功能完整性,显著降低患者生活质量。目前临床针对SCI的神经修复策略疗效有限,难以实现多病理环节的协同干预,因此探索新型核心治疗靶点与精准干预方案成为该领域的迫切需求。沉默信息调节因子(silence information regulator,Sirtuin,SIRT)家族(SIRT1~SIRT7)作为NAD+依赖的去乙酰化酶,在细胞代谢调控、免疫稳态维持、应激损伤修复等关键生物学过程中发挥核心作用,已被证实是神经系统疾病的潜在干预靶点。本文系统梳理了Sirtuins家族各成员的细胞定位与核心生物学功能,重点综述其在SCI病理进程中的调控作用及分子机制:SIRT1、3、5、6通过去乙酰化修饰抑制核因子κB(NF-κB)通路过度激活、阻断NLR家族pyrin结构域包含蛋白3(NLRP3)炎症小体组装,参与SCI后神经免疫炎症的调节,同时通过激活核因子E2相关因子2(Nrf2)抗氧化通路、提升超氧化物歧化酶(SOD)、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)等抗氧化酶活性,减轻脊髓组织氧化应激损伤,形成“抗炎-抗氧化”协同保护;SIRT7通过促进DNA损伤修复、抑制凋亡信号通路,延缓神经细胞凋亡;SIRT3与SIRT5则靶向线粒体功能,通过调控三羧酸循环、氧化磷酸化相关酶的修饰状态,改善线粒体能量代谢,同时调节叉头盒O3a(FOXO3a)、AMP依赖的蛋白激酶(AMPK)的乙酰化水平,恢复自噬稳态,为神经修复提供代谢支撑。总结发现,多种天然中药成分(如白藜芦醇、苦参碱)及合成化合物(如SRT1720、AGK2)可通过靶向调控Sirtuins家族成员,影响SCI病理进展。因此,以Sirtuins为靶点的联合治疗策略(如联合干细胞移植、神经因子补充、抗氧化剂干预等)有望突破单一疗法的局限,通过多机制协同作用提升SCI的修复效果。综上,Sirtuins家族在SCI的病理生理进程中具有关键作用机制及潜在干预价值,本文总结并展望了靶向Sirtuins的新型治疗策略,以期为该领域的基础研究与临床转化提供新思路。
基金financial support from the National Key R&D Program of China (No.2021YFA1302604)Scientific and technological innovation project of China Academy of Chinese Medical Sciences (No.CI2021B017)China Postdoctoral Science Foundation (No.2023T160727)。
文摘RNA binding proteins(RBPs) are a crucial class of proteins that interact with RNA and play a key role in various biological process.Deficiencies or abnormalities of RBPs are closely linked to the occurrence and progression of numerous diseases,making RBPs potential therapeutic targets.However,the limited tissue penetration of 254 nm UV irradiation makes it difficult to efficiently crosslink weak and dynamic RNA-protein interactions in mammal tissues.Additionally,RNA degradation in metal catalyzed click reaction further hinders the enrichment of RNA-protein complexes(RPCs).Due to these inherent limitations,globally profiling the RNA binding proteome in mammal organs has long been a challenge.Herein,we proposed a novel method,which utilized a dual crosslinking with formaldehyde and 254 nm UV irradiation,metabolic labeling and metal-free thiol-yne click reaction to enable large-scale enrichment and identification of RBPs in mouse liver,called FTYc_UV.In this method,formaldehyde is first used to crosslink the crude RNA-protein complexes(cRPCs) in situ to address the problem of poor tissue penetration of 254 nm UV irradiation.Furthermore,this method integrates metabolic labeling with a metal-free thiol-yne click reaction to achieve non-destructive RNA tagging.After specifically RNA-RBPs crosslinking by 254 nm UV irradiation in tissue lysates,formaldehyde decrosslinking is employed to remove non-specific proteins,leading to effective enrichment of RPCs from mouse liver and thereby overcoming the poor specificity of formaldehyde crosslinking.Application of FTYc_UV in mouse liver successfully identified over 1600 RBPs covering approximately 75 % of previously reported RBPs.Furthermore,420 candidate RBPs,including 151metabolic enzymes,were also obtained,demonstrating the sensitivity of FTYc_UV and the potential of this method for in-depth exploration of RNA-protein interactions in biological and clinical research.
基金supported by the National Key R&D Program of China,Nos.2021YFA1101703/2021YFA1101700(to YD).
文摘Ischemic stroke remains a leading cause of disability and death,with mesenchymal stem cell-derived exosomes emerging as a promising therapeutic avenue.However,the optimal timing and underlying therapeutic mechanisms of exosome treatment require further elucidation.In this study,we used a murine model of middle cerebral artery occlusion to investigate the therapeutic efficacy of human umbilical cord mesenchymal stem cell-derived exosomes administered intravenously at an early(6 hours)or delayed(3 days)time point post-ischemia.Compared with delayed treatment,early administration of exosomes resulted in significantly superior efficacy,as evidenced by improved neurological function scores and reduced infarct volumes.Transcriptomic analysis of brain tissues from mice receiving early exosome treatment revealed marked downregulation of inflammation-related genes,including Ccl2,Ccl5,Cxcl10,Il-1β,Il-6,Itgam,Itgax,and Tnf-α.Metabolomic profiling of these brain tissues further identified modulation of key metabolites,including trimethylamine N-oxide,glutathione,1-stearoyl-rac-glycerol,and phosphatidylcholine,suggesting that alteration of metabolic pathways contributes to the therapeutic effect.Integrated transcriptomic and metabolomic analysis pinpointed significant modulation of pathways involving metabolism of eicosapentaenoic acid,lysine,propanoate,and tyrosine.These findings suggest that umbilical cord mesenchymal stem cell-derived exosomes,particularly when administered early post-ischemia,exert their neuroprotective effects by broadly suppressing inflammatory pathways and modulating key metabolic processes in the ischemic brain,highlighting their potential as a therapeutic intervention for ischemic stroke.
基金supported by European Union Funding Programme,PNRR,No. 760058(to DMH)the UEFISCDI Project,No. PN-III-P4-IDPCE-2020-059(to APW)
文摘The major aim of stroke therapy is to stimulate brain repair and improve behavioral recovery after cerebral ischemia.One option is to stimulate endogenous neurogenesis in the subventricular zone and direct the newly formed neurons to the damaged area.However,only a small percentage of these neurons survive,and many do not reach the damaged area,possibly because the corpus callosum impedes the migration of subventricular zone-derived stem cells into the lesioned cortex.A second major obstacle to stem cell therapy is the strong inflammatory reaction induced by cerebral ischemia,whereby the associated phagocytic activity of brain macrophages removes both therapeutic cells and/or cell-based drug carriers.To address these issues,neurogenesis was electrically stimulated in the subventricular zone,followed by isolation of proliferating cells,including newly formed neurons,which were subsequently mixed with a nutritional hydrogel.This mixture was then transferred to the stroke cavity of day 14 post-stroke mice.We found that the performance of the treated animals improved in behavioral tests,including novel object,open field,hole board,grooming,and“time-to-feel”adhesive tape tests.Furthermore,immunostaining revealed that the stem cell marker nestin,the neuroepithelial marker Mash1,and the immature neuronal marker doublecortin-positive cells survived in the transplanted area for 2 weeks,possibly due to reduced phagocytic activity and supportive angiogenesis.These results clearly indicate that the transplantation of committed subventricular zone stem cells combined with a protective nutritional gel directly into the infarct cavity after the peak of stroke-induced neuroinflammation represents a feasible approach to improve neurorestoration after cerebral ischemia.
