Objective:The neurotoxicity of carbon monoxide(CO)to the central nervous system is a key pathogenesis of delayed encephalopathy after acute carbon monoxide poisoning(DEACMP).Our previous study found that retinoic acid...Objective:The neurotoxicity of carbon monoxide(CO)to the central nervous system is a key pathogenesis of delayed encephalopathy after acute carbon monoxide poisoning(DEACMP).Our previous study found that retinoic acid(RA)can suppress the neurotoxic effects of CO.This study further explores,in vivo and in vitro,the molecular mechanisms by which RA alleviates CO-induced central nervous system damage.Methods:A cytotoxic model was established using the mouse hippocampal neuronal cell line HT22 and primary oligodendrocytes exposed to CO,and a DEACMP animal model was established in adult Kunming mice.Cell viability and apoptosis of hippocampal neurons and oligodendrocytes were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay and Annexin V/propidium iodide(PI)double staining.The transcriptional and protein expression of each gene was detected using real time fluorescence quantitative PCR(RT-qPCR)and Western blotting.Long noncoding RNA(lncRNA)SNHG15 and LINGO-1 were knocked down or overexpressed to observe changes in neurons and oligodendrocytes.In DEACMP mice,SNHG15 or LINGO-1 were knocked down to assess changes in central nervous tissue and downstream protein expression.Results:RA at 10 and 20μmol/L significantly reversed CO-induced apoptosis of hippocampal neurons and oligodendrocytes,downregulation of SNHG15 and LINGO-1,and upregulation of brain-derived neurotrophic factor(BDNF)and tyrosine kinase receptor B(TrkB)(all P<0.05).Overexpression of SNHG15 or LINGO-1 weakened the protective effect of RA against CO-induced cytotoxicity(all P<0.05).Knockdown of SNHG15 or LINGO-1 alleviated CO-induced apoptosis of hippocampal neurons and oligodendrocytes and upregulated BDNF and TrkB expression levels(all P<0.05).Experiments in DEACMP model mice showed that knockdown of SNHG15 or LINGO-1 mitigated central nervous system injury in DEACMP(all P<0.05).Conclusion:RA alleviates CO-induced apoptosis of hippocampal neurons and oligodendrocytes,thereby reducing central nervous system injury and exerting neuroprotective effects.LncRNA SNHG15 and LINGO-1 are key molecules mediating RA induced inhibition of neuronal apoptosis and are associated with the BDNF/TrkB pathway.These findings provide a theoretical framework for optimizing the clinical treatment of DEACMP and lay an experimental foundation for elucidating its molecular mechanisms.展开更多
目的表达和纯化带多聚组氨酸(6×His)标签的人LINGO-1胞外段(hLINGO-1aa76-319)融合蛋白,并制备兔源性抗hLINGO-1aa76-319的多克隆抗体(pAb)。方法利用PCR从pCMV-SPORT6获得hLINGO-1aa76-319编码序列,将其亚克隆至原核表达载体pET30...目的表达和纯化带多聚组氨酸(6×His)标签的人LINGO-1胞外段(hLINGO-1aa76-319)融合蛋白,并制备兔源性抗hLINGO-1aa76-319的多克隆抗体(pAb)。方法利用PCR从pCMV-SPORT6获得hLINGO-1aa76-319编码序列,将其亚克隆至原核表达载体pET30a(+),构建重组表达质粒pET30a(+)-hLINGO-1aa76-319;将阳性重组质粒转化大肠杆菌,IPTG诱导表达6×His-hLINGO-1aa76-319融合蛋白,经Ni-NTA螯合树脂纯化,纯化蛋白免疫新西兰大白兔制备多克隆抗血清,Protein A Sepharose柱纯化获得多抗,ELISA法检测抗体效价,Western blot法检测抗体特异性。结果成功构建了pET30a(+)-hLINGO-1aa76-319原核表达载体,原核蛋白hLINGO-1aa76-319以包涵体形式在大肠杆菌高水平表达,通过复性与亲和层析获得纯度在90%以上的hLINGO-1aa76-319蛋白,蛋白浓度为4600mg/L,制备的抗hLINGO-1aa76-319多抗效价高达1:1.6×106,Western blotting鉴定其具有良好的特异性。结论获得高纯度hLINGO-1aa76-319蛋白并成功制备特异性pAb,为进一步研究LINGO-1的生物学功能提供实验基础。展开更多
目的:研究沉默海马含亮氨酸重复序列和免疫球蛋白结构域的蛋白-1(LINGO-1)对APP/PS1小鼠空间学习和记忆能力以及海马有髓神经纤维的影响。方法:随机选取18只雄性APP/PS1小鼠,随机分为对照组和rAAV-LINGO-1-shRNA组,通过海马立体定位注...目的:研究沉默海马含亮氨酸重复序列和免疫球蛋白结构域的蛋白-1(LINGO-1)对APP/PS1小鼠空间学习和记忆能力以及海马有髓神经纤维的影响。方法:随机选取18只雄性APP/PS1小鼠,随机分为对照组和rAAV-LINGO-1-shRNA组,通过海马立体定位注射表达LINGO-1-shRNA的重组腺相关病毒rAAV-LINGO-1-shRNA,运用Morris水迷宫方法测试小鼠的空间学习和记忆能力;运用免疫荧光染色和real time RT-PCR分析小鼠海马内LINGO-1表达水平;运用real time RT-PCR分析小鼠海马内LINGO-1下游分子RhoA和ROCK的表达水平;运用无偏体视学方法结合透射电子显微镜技术对小鼠海马(CA1~3和齿状回)的体积,海马内有髓神经纤维的长度、髓鞘体积及其损伤情况进行定量研究。结果:与对照组小鼠相比,rAAV-LINGO-1-shRNA组小鼠的逃避潜伏期显著性缩短,穿台次数显著增多,而目标象限游泳时间及目标象限游泳路程比无显著性改变;与对照组相比,rAAV-LINGO-1-shRNA组海马内LINGO-1显著下调,同时其下游RhoA和ROCK的表达水平降低;对照组和rAAV-LINGO-1-shRNA组海马体积无显著性差异;与对照组相比,rAAV-LINGO-1-shRNA组海马内有髓神经纤维的长度和正常髓鞘的体积显著性增加,受损的有髓神经纤维占比和受损的髓鞘占比均显著降低。结论:沉默海马LINGO-1能够有效改善APP/PS1小鼠海马依赖的空间学习和记忆能力,抑制RhoA/ROCK的表达,减轻海马内有髓神经纤维及其髓鞘的损伤,这将为阿尔茨海默病的发病及治疗提供新的方向。展开更多
基金supported by the Natural Science Foundation of Hunan Province(2021JJ31089)the Scientific Research Project of Health Commission of Hunan Province(202203104548),China。
文摘Objective:The neurotoxicity of carbon monoxide(CO)to the central nervous system is a key pathogenesis of delayed encephalopathy after acute carbon monoxide poisoning(DEACMP).Our previous study found that retinoic acid(RA)can suppress the neurotoxic effects of CO.This study further explores,in vivo and in vitro,the molecular mechanisms by which RA alleviates CO-induced central nervous system damage.Methods:A cytotoxic model was established using the mouse hippocampal neuronal cell line HT22 and primary oligodendrocytes exposed to CO,and a DEACMP animal model was established in adult Kunming mice.Cell viability and apoptosis of hippocampal neurons and oligodendrocytes were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay and Annexin V/propidium iodide(PI)double staining.The transcriptional and protein expression of each gene was detected using real time fluorescence quantitative PCR(RT-qPCR)and Western blotting.Long noncoding RNA(lncRNA)SNHG15 and LINGO-1 were knocked down or overexpressed to observe changes in neurons and oligodendrocytes.In DEACMP mice,SNHG15 or LINGO-1 were knocked down to assess changes in central nervous tissue and downstream protein expression.Results:RA at 10 and 20μmol/L significantly reversed CO-induced apoptosis of hippocampal neurons and oligodendrocytes,downregulation of SNHG15 and LINGO-1,and upregulation of brain-derived neurotrophic factor(BDNF)and tyrosine kinase receptor B(TrkB)(all P<0.05).Overexpression of SNHG15 or LINGO-1 weakened the protective effect of RA against CO-induced cytotoxicity(all P<0.05).Knockdown of SNHG15 or LINGO-1 alleviated CO-induced apoptosis of hippocampal neurons and oligodendrocytes and upregulated BDNF and TrkB expression levels(all P<0.05).Experiments in DEACMP model mice showed that knockdown of SNHG15 or LINGO-1 mitigated central nervous system injury in DEACMP(all P<0.05).Conclusion:RA alleviates CO-induced apoptosis of hippocampal neurons and oligodendrocytes,thereby reducing central nervous system injury and exerting neuroprotective effects.LncRNA SNHG15 and LINGO-1 are key molecules mediating RA induced inhibition of neuronal apoptosis and are associated with the BDNF/TrkB pathway.These findings provide a theoretical framework for optimizing the clinical treatment of DEACMP and lay an experimental foundation for elucidating its molecular mechanisms.
文摘目的表达和纯化带多聚组氨酸(6×His)标签的人LINGO-1胞外段(hLINGO-1aa76-319)融合蛋白,并制备兔源性抗hLINGO-1aa76-319的多克隆抗体(pAb)。方法利用PCR从pCMV-SPORT6获得hLINGO-1aa76-319编码序列,将其亚克隆至原核表达载体pET30a(+),构建重组表达质粒pET30a(+)-hLINGO-1aa76-319;将阳性重组质粒转化大肠杆菌,IPTG诱导表达6×His-hLINGO-1aa76-319融合蛋白,经Ni-NTA螯合树脂纯化,纯化蛋白免疫新西兰大白兔制备多克隆抗血清,Protein A Sepharose柱纯化获得多抗,ELISA法检测抗体效价,Western blot法检测抗体特异性。结果成功构建了pET30a(+)-hLINGO-1aa76-319原核表达载体,原核蛋白hLINGO-1aa76-319以包涵体形式在大肠杆菌高水平表达,通过复性与亲和层析获得纯度在90%以上的hLINGO-1aa76-319蛋白,蛋白浓度为4600mg/L,制备的抗hLINGO-1aa76-319多抗效价高达1:1.6×106,Western blotting鉴定其具有良好的特异性。结论获得高纯度hLINGO-1aa76-319蛋白并成功制备特异性pAb,为进一步研究LINGO-1的生物学功能提供实验基础。
文摘目的:研究沉默海马含亮氨酸重复序列和免疫球蛋白结构域的蛋白-1(LINGO-1)对APP/PS1小鼠空间学习和记忆能力以及海马有髓神经纤维的影响。方法:随机选取18只雄性APP/PS1小鼠,随机分为对照组和rAAV-LINGO-1-shRNA组,通过海马立体定位注射表达LINGO-1-shRNA的重组腺相关病毒rAAV-LINGO-1-shRNA,运用Morris水迷宫方法测试小鼠的空间学习和记忆能力;运用免疫荧光染色和real time RT-PCR分析小鼠海马内LINGO-1表达水平;运用real time RT-PCR分析小鼠海马内LINGO-1下游分子RhoA和ROCK的表达水平;运用无偏体视学方法结合透射电子显微镜技术对小鼠海马(CA1~3和齿状回)的体积,海马内有髓神经纤维的长度、髓鞘体积及其损伤情况进行定量研究。结果:与对照组小鼠相比,rAAV-LINGO-1-shRNA组小鼠的逃避潜伏期显著性缩短,穿台次数显著增多,而目标象限游泳时间及目标象限游泳路程比无显著性改变;与对照组相比,rAAV-LINGO-1-shRNA组海马内LINGO-1显著下调,同时其下游RhoA和ROCK的表达水平降低;对照组和rAAV-LINGO-1-shRNA组海马体积无显著性差异;与对照组相比,rAAV-LINGO-1-shRNA组海马内有髓神经纤维的长度和正常髓鞘的体积显著性增加,受损的有髓神经纤维占比和受损的髓鞘占比均显著降低。结论:沉默海马LINGO-1能够有效改善APP/PS1小鼠海马依赖的空间学习和记忆能力,抑制RhoA/ROCK的表达,减轻海马内有髓神经纤维及其髓鞘的损伤,这将为阿尔茨海默病的发病及治疗提供新的方向。
