Tooth germ injury can lead to abnormal tooth development and even tooth loss,affecting various aspects of the stomatognathic system including form,function,and appearance.However,the research about tooth germ injury m...Tooth germ injury can lead to abnormal tooth development and even tooth loss,affecting various aspects of the stomatognathic system including form,function,and appearance.However,the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited.Therefore,it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model.Here,we constructed a Tg(dlx2b:Dendra2-NTR)transgenic line that labeled tooth germ specifically.Taking advantage of the NTR/Mtz system,the dlx2b+tooth germ cells were depleted by Mtz effectively.The process of tooth germ repair was evaluated by antibody staining,in situ hybridization,Ed U staining and alizarin red staining.The severely injured tooth germ was repaired in several days after Mtz treatment was stopped.In the early stage of tooth germ repair,the expression of phosphorylated 4E-BP1 was increased,indicating that mTORC1 is activated.Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ.Normally,mouse incisors were repaired after damage,but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress.Overall,we are the first to construct a stable and repeatable repair model of severe tooth germ injury,and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair,providing a potential target for clinical treatment of tooth germ injury.展开更多
Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autop...Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.展开更多
For healthspan and lifespan,ERK,AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark1-7.Here we examined whether IL-11,a pro-inflammatory cytokine of the IL-6 family,has a neg...For healthspan and lifespan,ERK,AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark1-7.Here we examined whether IL-11,a pro-inflammatory cytokine of the IL-6 family,has a negative effect on age-associated disease and lifespan.As mice age,IL-11 is upregulated across cell types and tissues to regulate an ERK-AMPK-mTORC1 axis to modulate cellular,tissue-and organismal-level ageing pathologies.Deletion of Il11 or Il11ra1 protects against metabolic decline,multi-morbidity and frailty in old age.展开更多
目的观察银盏心脉滴丸对大鼠心肌细胞(H9C2)mTORC1/4EBP1信号通路的影响以探讨其对线粒体功能的机制。方法制备银盏心脉滴丸含药血清。将细胞分为对照组、模型组(缺氧/复氧)、空白血清组及银盏心脉滴丸含药血清组。制备缺氧/复氧细胞损...目的观察银盏心脉滴丸对大鼠心肌细胞(H9C2)mTORC1/4EBP1信号通路的影响以探讨其对线粒体功能的机制。方法制备银盏心脉滴丸含药血清。将细胞分为对照组、模型组(缺氧/复氧)、空白血清组及银盏心脉滴丸含药血清组。制备缺氧/复氧细胞损伤模型,利用激光共聚焦检测线粒体膜电位(ΔΨm),利用流式细胞仪检测活性氧(reactive oxygen species, ROS),运用Western Blot技术检测细胞雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)、真核翻译起始因子4E结合蛋白(eukaryotic translation initiation factor 4E-binding proteins,4E-BP)、mTOR复合体蛋白(regulatory-associated protein of mammalian target of rapamycin,Raptor)蛋白表达,运用q-PCR方法检测mTORC1、4EBP1、Raptor的mRNA含量。结果与对照组相比,模型组细胞内绿色荧光增强,ΔΨm显著高于对照组(P<0.05);而与模型组相比,银盏心脉滴丸组红色荧光增强(P<0.05),说明银盏心脉保护ΔΨm稳定,保护线粒体膜结构;与对照组相比,模型组的ROS合成较对照组升高(P<0.05);而与模型组相比,银盏心脉滴丸组细胞内ROS产生明显降低(P<0.05);与对照组相比,模型组mTOR、Raptor基因mRNA水平下调(P<0.05),4ebp1基因mRNA水平上调,差异有统计学意义(P<0.05)。与模型组比较,银盏心脉滴丸组mTOR、Raptor基因mRNA水平上调(P<0.05),4ebp1基因mRNA水平下调(P<0.05)。结论银盏心脉滴丸改善缺氧复氧损伤的机制可能是通过激活mTORC1/4EBP1信号通路,从而抑制线粒体膜电位,改善活性氧,保护线粒体功能。展开更多
基金supported by the National Natural Science Foundation of China(NFSC)(No.31371473 to D.Y.,No.32270888 to D.Y.and No.31970783 to D.Y.)program for Top talent Distinguished Professor from Chongqing Medical University[No.(2021)215 to D.Y.]program for Youth Innovation in Future Medicine from Chongqing Medical University(No.W0060 to D.Y.)。
文摘Tooth germ injury can lead to abnormal tooth development and even tooth loss,affecting various aspects of the stomatognathic system including form,function,and appearance.However,the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited.Therefore,it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model.Here,we constructed a Tg(dlx2b:Dendra2-NTR)transgenic line that labeled tooth germ specifically.Taking advantage of the NTR/Mtz system,the dlx2b+tooth germ cells were depleted by Mtz effectively.The process of tooth germ repair was evaluated by antibody staining,in situ hybridization,Ed U staining and alizarin red staining.The severely injured tooth germ was repaired in several days after Mtz treatment was stopped.In the early stage of tooth germ repair,the expression of phosphorylated 4E-BP1 was increased,indicating that mTORC1 is activated.Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ.Normally,mouse incisors were repaired after damage,but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress.Overall,we are the first to construct a stable and repeatable repair model of severe tooth germ injury,and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair,providing a potential target for clinical treatment of tooth germ injury.
基金supported by grants from the Natural Science Foundation of China(91949205,31730035,81721005)the Science and Technology Committee of China(2016YFC1305800)+1 种基金the Special Project of Technological Innovation of Hubei Province(2018ACA142)Guangdong Provincial Key S&T Program(2018B030336001)。
文摘Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.
文摘For healthspan and lifespan,ERK,AMPK and mTORC1 represent critical pathways and inflammation is a centrally important hallmark1-7.Here we examined whether IL-11,a pro-inflammatory cytokine of the IL-6 family,has a negative effect on age-associated disease and lifespan.As mice age,IL-11 is upregulated across cell types and tissues to regulate an ERK-AMPK-mTORC1 axis to modulate cellular,tissue-and organismal-level ageing pathologies.Deletion of Il11 or Il11ra1 protects against metabolic decline,multi-morbidity and frailty in old age.
文摘目的观察银盏心脉滴丸对大鼠心肌细胞(H9C2)mTORC1/4EBP1信号通路的影响以探讨其对线粒体功能的机制。方法制备银盏心脉滴丸含药血清。将细胞分为对照组、模型组(缺氧/复氧)、空白血清组及银盏心脉滴丸含药血清组。制备缺氧/复氧细胞损伤模型,利用激光共聚焦检测线粒体膜电位(ΔΨm),利用流式细胞仪检测活性氧(reactive oxygen species, ROS),运用Western Blot技术检测细胞雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)、真核翻译起始因子4E结合蛋白(eukaryotic translation initiation factor 4E-binding proteins,4E-BP)、mTOR复合体蛋白(regulatory-associated protein of mammalian target of rapamycin,Raptor)蛋白表达,运用q-PCR方法检测mTORC1、4EBP1、Raptor的mRNA含量。结果与对照组相比,模型组细胞内绿色荧光增强,ΔΨm显著高于对照组(P<0.05);而与模型组相比,银盏心脉滴丸组红色荧光增强(P<0.05),说明银盏心脉保护ΔΨm稳定,保护线粒体膜结构;与对照组相比,模型组的ROS合成较对照组升高(P<0.05);而与模型组相比,银盏心脉滴丸组细胞内ROS产生明显降低(P<0.05);与对照组相比,模型组mTOR、Raptor基因mRNA水平下调(P<0.05),4ebp1基因mRNA水平上调,差异有统计学意义(P<0.05)。与模型组比较,银盏心脉滴丸组mTOR、Raptor基因mRNA水平上调(P<0.05),4ebp1基因mRNA水平下调(P<0.05)。结论银盏心脉滴丸改善缺氧复氧损伤的机制可能是通过激活mTORC1/4EBP1信号通路,从而抑制线粒体膜电位,改善活性氧,保护线粒体功能。
