BACKGROUND:Pulmonary fibrosis(PF)is one of the main causes of death in patients with paraquat(PQ)poisoning.This study aimed to evaluate the relationship between mitochondrial fi ssion and oxidative stress in PQ-induce...BACKGROUND:Pulmonary fibrosis(PF)is one of the main causes of death in patients with paraquat(PQ)poisoning.This study aimed to evaluate the relationship between mitochondrial fi ssion and oxidative stress in PQ-induced epithelial-mesenchymal transition(EMT)and PF.METHODS:C57BL/6 mice and MLE-12 cells were exposed to PQ to construct a PF model in vivo and in vitro.Histological changes in the lungs were examined by hematoxylin and eosin(H&E)staining.Mitochondrial morphology was detected by MitoTracker®Deep Red FM or transmission electron microscopy(TEM).Western blotting and immunofluorescence were used to determine the expression of protein.The migration ability of the cells was detected by the cell scratch test.Mitochondrial DNA(mtDNA)levels were assessed by real-time polymerase chain reaction(PCR).Enzyme-linked immunosorbent assay(ELISA)was applied to detect cytokine levels.Superoxide dismutase(SOD)activity and the levels of glutathione(GSH)and malondialdehyde(MDA)were detected by chemichromatometry.RESULTS:PQ exposure caused EMT and PF in vivo and in vitro.PQ destroyed mitochondrial structure and enhanced the expression of dynamin-related protein 1(Drp1),which were accompanied by oxidative stress.Inhibiting mitochondrial fission using mitochondrial division inhibitor-1(Mdivi-1),a selective inhibitor of Drp1,attenuated PQ-induced EMT and oxidative damage.Treatment with N-acetyl-L-cysteine(NAC),an antioxidant,reduced Drp1 expression,attenuated mitochondrial structure damage and inhibited PQ-induced EMT and PF.Both Mdivi-1 and NAC treatment markedly suppressed mtDNA release,the expression of Toll-like receptor 9(TLR9)and phosphorylation(P)-NF-κB p65 as well as cytokines(interleukin 6[IL-6],interleukin-1β[IL-1β],and tumor necrosis factor-α[TNF-α])production.CONCLUSION:Mutual promotion of mitochondrial fission and oxidative stress contributes to EMT in PQ-induced PF,which is associated with the mtDNA/TLR9/NF-κB pathway.展开更多
D-tryptophan is a special kind of nonprotein amino acid showing multiple physiological functions,but the detailed mechanisms are not fully revealed,impairing its further development and applications.This work was to i...D-tryptophan is a special kind of nonprotein amino acid showing multiple physiological functions,but the detailed mechanisms are not fully revealed,impairing its further development and applications.This work was to investigate D-tryptophan physiological function and demonstrate the underlying mechanisms.D-tryptophan suppressed HaCaT cell proliferation but increased cell migration.Specifically,D-tryptophan decreased E-cadherin and increased Snail,Twist,and Slug expression,resulting in the development of an epithelial-mesenchymal transitions(EMT)phenomenon.Moreover,D-tryptophan promoted the expression of transforming growth factor-β(TGF-β)1,and Smad4 knockout damages D-tryptophan’s ability.These results indicated that D-tryptophan stimulated HaCaT cells to produce TGF-β1 and thus activated the TGF-β/Samd pathway,resulting in the triggering of EMT.This study revealed the molecular mechanisms of D-tryptophan activity,provided D-tryptophan as a potential approach for cancer treatment,wound healing,organ development and other relevant applications.展开更多
Objective Transdifferentiation exists between stromal cells or between stromal cells and cancer cells.Evodiamine and berberine are predominant pharmacological components of Zuojin pill,a prescription of Traditional Ch...Objective Transdifferentiation exists between stromal cells or between stromal cells and cancer cells.Evodiamine and berberine are predominant pharmacological components of Zuojin pill,a prescription of Traditional Chinese Medicine,playing crucial functions in remolding of tumor microenvironment.This study aimed to explore the effect of combination of evodiamine with berberine(cBerEvo)on the phenotypic transition of colon epithelial cells induced by tumor-associated fibroblasts,as well as the involved mechanisms.Methods Human normal colon epithelial cell line HCoEpiC cells were treated with the prepared conditioned medium of CCD-18 Co,a human colon myofibroblast line,to induce epithelial-mesenchymal transition.Phase contrast microscope was used to observe the morphological changes.Epithelial-mesenchymal transition markers including E-cadherin,vimentin and alpha-smooth muscle actin(α-SMA)were observed with immunofluorescence microscopy.Migration was assessed by wound healing assay.Western blotting was used to detect the expressions of E-cadherin,vimentin,α-SMA,Snail,ZEB1 and Smads.Results In contrast to the control,the tumor-associated fibroblasts-like CCD-18 Co cells induced downregulation of E-cadherin and up-regulation of vimentin,α-SMA,Snail and ZEB1(P<0.05),and promoted migration of HCoEpiCs(P<0.05),with over expression of Smads including Smad2,p-Smad2,Smad3,p-Smad3 and Smad4(P<0.05),which were abolished by a transforming growth factor-β(TGF-β)receptor inhibitor LY364947 and by cBerEvo in a concentration dependent manner.In addition,cBerEvo-inhibited ratios of p-Smad2/Smad2 and p-Smad3/Smad3 were also dose dependent.Conclusion The above results suggest that cBerEvo can regulate the differentiation of colon epithelial cells induced by CCD-18 Co through suppressing activity of TGF-β/Smads signaling pathway.展开更多
目的 探讨Wnt/β-catenin信号通路对大鼠腹膜间皮细胞上皮-间充质转化(EMT)进程的影响作用。方法 选择SPF级雄性Wistar大鼠20只,体质量180~200 g。随机分为对照组、模型组。采用5/6肾切除法+高糖腹膜透析液+脂多糖(LPS)法制作腹膜纤维...目的 探讨Wnt/β-catenin信号通路对大鼠腹膜间皮细胞上皮-间充质转化(EMT)进程的影响作用。方法 选择SPF级雄性Wistar大鼠20只,体质量180~200 g。随机分为对照组、模型组。采用5/6肾切除法+高糖腹膜透析液+脂多糖(LPS)法制作腹膜纤维化模型;大鼠于4周末次腹膜透析后,留取腹膜组织,采用苏木精-伊红(HE)染色观察腹膜组织形态学变化并测量腹膜组织厚度;采用实时定量聚合酶链式反应(PCR)、Western blot和免疫组织化学等方法检测腹膜组织中E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、Collagen-Ⅰ、β-catenin、Wnt-1和淋巴增强因子1(LEF1)的蛋白和mRNA表达。结果 与对照组相比,模型组大鼠腹膜组织明显增厚。模型组大鼠腹膜组织中E-cadherin、α-SMA、Collagen-Ⅰ、β-catenin、Wnt-1和LEF1蛋白与对照组比较明显升高(0.21±0.13 vs 1.33±0.41,0.27±0.20 vs 1.22±0.06,0.26±0.08 vs 1.08±0.13,0.21±0.11 vs 1.11±0.46,0.24±0.17 vs 1.99±0.36,0.37±0.15 vs 1.49±0.46。均P <0.01);mRNA表达水平明显升高(1.00±0.00 vs 3.07±0.85,1.00±0.00 vs 9.36±1.25,1.00±0.00 vs 13.5±0.31,1.00±0.00 vs 7.89±0.63,1.00±0.00 vs 18.2±1.79,1.00±0.00 vs 11.8±0.55。均P <0.01)。结论 Wnt/β-catenin信号通路显著影响EMT和腹膜纤维化的发展进程。展开更多
基金supported by the Wenzhou Municipal Science and Technology Bureau(Y2020092)partly by the Key Specialty of Traditional Chinese Medicine of Zhejiang Province in the 13th Five-Year Plan period(Emergency Department).
文摘BACKGROUND:Pulmonary fibrosis(PF)is one of the main causes of death in patients with paraquat(PQ)poisoning.This study aimed to evaluate the relationship between mitochondrial fi ssion and oxidative stress in PQ-induced epithelial-mesenchymal transition(EMT)and PF.METHODS:C57BL/6 mice and MLE-12 cells were exposed to PQ to construct a PF model in vivo and in vitro.Histological changes in the lungs were examined by hematoxylin and eosin(H&E)staining.Mitochondrial morphology was detected by MitoTracker®Deep Red FM or transmission electron microscopy(TEM).Western blotting and immunofluorescence were used to determine the expression of protein.The migration ability of the cells was detected by the cell scratch test.Mitochondrial DNA(mtDNA)levels were assessed by real-time polymerase chain reaction(PCR).Enzyme-linked immunosorbent assay(ELISA)was applied to detect cytokine levels.Superoxide dismutase(SOD)activity and the levels of glutathione(GSH)and malondialdehyde(MDA)were detected by chemichromatometry.RESULTS:PQ exposure caused EMT and PF in vivo and in vitro.PQ destroyed mitochondrial structure and enhanced the expression of dynamin-related protein 1(Drp1),which were accompanied by oxidative stress.Inhibiting mitochondrial fission using mitochondrial division inhibitor-1(Mdivi-1),a selective inhibitor of Drp1,attenuated PQ-induced EMT and oxidative damage.Treatment with N-acetyl-L-cysteine(NAC),an antioxidant,reduced Drp1 expression,attenuated mitochondrial structure damage and inhibited PQ-induced EMT and PF.Both Mdivi-1 and NAC treatment markedly suppressed mtDNA release,the expression of Toll-like receptor 9(TLR9)and phosphorylation(P)-NF-κB p65 as well as cytokines(interleukin 6[IL-6],interleukin-1β[IL-1β],and tumor necrosis factor-α[TNF-α])production.CONCLUSION:Mutual promotion of mitochondrial fission and oxidative stress contributes to EMT in PQ-induced PF,which is associated with the mtDNA/TLR9/NF-κB pathway.
基金financially supported by the National Key R&D Program of China(2018YFD0401200)the National Natural Science Foundation of China(31801452)the Zhejiang Provincial Science and Technology Foundation of China(2018C02025 and 2019C02069)。
文摘D-tryptophan is a special kind of nonprotein amino acid showing multiple physiological functions,but the detailed mechanisms are not fully revealed,impairing its further development and applications.This work was to investigate D-tryptophan physiological function and demonstrate the underlying mechanisms.D-tryptophan suppressed HaCaT cell proliferation but increased cell migration.Specifically,D-tryptophan decreased E-cadherin and increased Snail,Twist,and Slug expression,resulting in the development of an epithelial-mesenchymal transitions(EMT)phenomenon.Moreover,D-tryptophan promoted the expression of transforming growth factor-β(TGF-β)1,and Smad4 knockout damages D-tryptophan’s ability.These results indicated that D-tryptophan stimulated HaCaT cells to produce TGF-β1 and thus activated the TGF-β/Samd pathway,resulting in the triggering of EMT.This study revealed the molecular mechanisms of D-tryptophan activity,provided D-tryptophan as a potential approach for cancer treatment,wound healing,organ development and other relevant applications.
基金supported by the National Natural Science Foundation of China(81903985,the running period is 2020.1-2022.12)Natural Science Foundation of Guangdong Province(2018A030310060,the running period is 2018.6-2021.5)Postdoctoral Research Foundation of China(2018M643353,the running period is 2018.9-2019.7)。
文摘Objective Transdifferentiation exists between stromal cells or between stromal cells and cancer cells.Evodiamine and berberine are predominant pharmacological components of Zuojin pill,a prescription of Traditional Chinese Medicine,playing crucial functions in remolding of tumor microenvironment.This study aimed to explore the effect of combination of evodiamine with berberine(cBerEvo)on the phenotypic transition of colon epithelial cells induced by tumor-associated fibroblasts,as well as the involved mechanisms.Methods Human normal colon epithelial cell line HCoEpiC cells were treated with the prepared conditioned medium of CCD-18 Co,a human colon myofibroblast line,to induce epithelial-mesenchymal transition.Phase contrast microscope was used to observe the morphological changes.Epithelial-mesenchymal transition markers including E-cadherin,vimentin and alpha-smooth muscle actin(α-SMA)were observed with immunofluorescence microscopy.Migration was assessed by wound healing assay.Western blotting was used to detect the expressions of E-cadherin,vimentin,α-SMA,Snail,ZEB1 and Smads.Results In contrast to the control,the tumor-associated fibroblasts-like CCD-18 Co cells induced downregulation of E-cadherin and up-regulation of vimentin,α-SMA,Snail and ZEB1(P<0.05),and promoted migration of HCoEpiCs(P<0.05),with over expression of Smads including Smad2,p-Smad2,Smad3,p-Smad3 and Smad4(P<0.05),which were abolished by a transforming growth factor-β(TGF-β)receptor inhibitor LY364947 and by cBerEvo in a concentration dependent manner.In addition,cBerEvo-inhibited ratios of p-Smad2/Smad2 and p-Smad3/Smad3 were also dose dependent.Conclusion The above results suggest that cBerEvo can regulate the differentiation of colon epithelial cells induced by CCD-18 Co through suppressing activity of TGF-β/Smads signaling pathway.
文摘目的 探讨Wnt/β-catenin信号通路对大鼠腹膜间皮细胞上皮-间充质转化(EMT)进程的影响作用。方法 选择SPF级雄性Wistar大鼠20只,体质量180~200 g。随机分为对照组、模型组。采用5/6肾切除法+高糖腹膜透析液+脂多糖(LPS)法制作腹膜纤维化模型;大鼠于4周末次腹膜透析后,留取腹膜组织,采用苏木精-伊红(HE)染色观察腹膜组织形态学变化并测量腹膜组织厚度;采用实时定量聚合酶链式反应(PCR)、Western blot和免疫组织化学等方法检测腹膜组织中E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、Collagen-Ⅰ、β-catenin、Wnt-1和淋巴增强因子1(LEF1)的蛋白和mRNA表达。结果 与对照组相比,模型组大鼠腹膜组织明显增厚。模型组大鼠腹膜组织中E-cadherin、α-SMA、Collagen-Ⅰ、β-catenin、Wnt-1和LEF1蛋白与对照组比较明显升高(0.21±0.13 vs 1.33±0.41,0.27±0.20 vs 1.22±0.06,0.26±0.08 vs 1.08±0.13,0.21±0.11 vs 1.11±0.46,0.24±0.17 vs 1.99±0.36,0.37±0.15 vs 1.49±0.46。均P <0.01);mRNA表达水平明显升高(1.00±0.00 vs 3.07±0.85,1.00±0.00 vs 9.36±1.25,1.00±0.00 vs 13.5±0.31,1.00±0.00 vs 7.89±0.63,1.00±0.00 vs 18.2±1.79,1.00±0.00 vs 11.8±0.55。均P <0.01)。结论 Wnt/β-catenin信号通路显著影响EMT和腹膜纤维化的发展进程。
