BACKGROUND The transforming growth factor β(TGFβ) signaling pathway plays a crucial role in the development of liver fibrosis by activating TGFβ type Ⅱ receptor(TGFβR2), followed by the recruitment of TGFβR1 fin...BACKGROUND The transforming growth factor β(TGFβ) signaling pathway plays a crucial role in the development of liver fibrosis by activating TGFβ type Ⅱ receptor(TGFβR2), followed by the recruitment of TGFβR1 finally triggering downstream signaling pathway.AIM To find drugs targeting TGFβR2 that inhibit TGFβR1/TGFβR2 complex formation, theoretically inhibit TGFβ signaling pathway, and thereby ameliorate liver fibrosis.METHODS Food and Drug Administration-approved drugs were screened for binding affinity with TGFβR2 by virtual molecular docking. We identified 6 candidates and further explored their potential by Cell Counting Kit-8(CCK-8) cell cytotoxic experiment to validate toxicity and titrated the best cellular working concentrations. Next, we further demonstrated the detailed molecular working mechanisms using mutagenesis analysis. Finally, we used a mouse model to investigate its potential anti-liver fibrosis effect.RESULTS We identified 6 drug candidates. Among these 6 drugs, dihydroergotamine(DHE) shows great ability in reducing fibrotic gene expressions such as collagen, p-SMAD3, and α-SMA in TGFβ induced cellular model of liver fibrosis in LX-2 cells. Furthermore, we demonstrated that DHE binds to TGFβR2. Moreover, mutation of Leu27, Phe30, Thr51, Ser52, Ile53, and Glu55 of TGFβR2 disrupted the binding of TGFβR2 with DHE. In addition, DHE significantly improved liver fibrosis, as evidenced by Masson’s trichrome staining of liver sections. This is further supported by the width and the velocity of the portal vein, and serum markers of liver function. In line with those observations, DHE also decreased macrophages infiltration and extracellular matrix deposition in the liver.CONCLUSION DHE alleviates liver fibrosis by binding to TGFβR2 thereby suppressing TGFβ signaling pathway. We show here that as far as drug repurposing, DHE has great potential to treat liver fibrosis.展开更多
The transforming growth factor-β(TGF-β)family controls embryogenesis,stem cell differentiation,and tissue homeostasis.However,how post-translation modifications contribute to fine-tuning of TGF-βfamily signaling re...The transforming growth factor-β(TGF-β)family controls embryogenesis,stem cell differentiation,and tissue homeostasis.However,how post-translation modifications contribute to fine-tuning of TGF-βfamily signaling responses is not well understood.Inhibitory(I)-Smads can antagonize TGF-β/Smad signaling by recruiting Smurf E3 ubiquitin ligases to target the active TGF-βreceptor for proteasomal degradation.A proteomic interaction screen identified Vpr binding protein(VprBP)as novel binding partner of Smad7.Mis-expression studies revealed that VprBP negatively controls Smad2 phosphorylation,Smad2–Smad4 interaction,as well as TGF-βtarget gene expression.VprBP was found to promote Smad7–Smurf1–TβRI complex formation and induce proteasomal degradation of TGF-βtype I receptor(TβRI).Moreover,VprBP appears to stabilize Smurf1 by suppressing Smurf1 poly-ubiquitination.In multiple adult and mouse embryonic stem cells,depletion of VprBP promotes TGF-βor Activin-induced responses.In the mouse embryo VprBP expression negatively correlates with mesoderm marker expression,and VprBP attenuated mesoderm induction during zebrafish embryogenesis.Our findings thereby uncover a novel regulatory mechanism by which Smurf1 controls the TGF-βand Activin cascade and identify VprBP as a critical determinant of embryonic mesoderm induction.展开更多
Objective Chronic renal failure(CRF)is a worldwide public health burden.Niaoduqing granules(NDQ)is widely used for CRF treatment in China.However,the underlying mechanism of NDQ is not fully studied.This study is aime...Objective Chronic renal failure(CRF)is a worldwide public health burden.Niaoduqing granules(NDQ)is widely used for CRF treatment in China.However,the underlying mechanism of NDQ is not fully studied.This study is aimed to investigate whether NDQ ameliorate CRF by inhibiting transforming growth factor-β1(TGF-β1)-induced EMT in human renal tubular epithelial HK-2 cells.Methods 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide assay and colony formation assay were used to investigate the cytotoxicity of NDQ in HK-2 cells.Morphological changes of HK-2 cells after TGF-β1 or/and NDQ treatment were observed under a microscope.Wound-healing,migration and invasion assays were performed to determine the cell movement,migratory and invasive abilities,respectively.Western blot analysis was carried out to examine the protein levels of TGF-βreceptor I(TβRI)and epithelial-mesenchymal transition(EMT)-associated factors.Fluorescence confocal microscopy was applied to observe the organization of filamentous actin.Results NDQ suppressed TβRI expression dose-dependently.NDQ inhibited TGF-β1-stimulated EMT in HK-2 cells,supported by the evidences that NDQ prevented morphology change,attenuated cell migration and invasion,downregulated EMT factors and reorganized filamentous actin distribution in TGF-β1-stimulated HK-2 cells.Conclusions NDQ attenuates chronic renal failure which may be associated with inhibition of TβRI expression and EMT process.展开更多
基金Supported by the Special Research Project for Capital Health Development,No.2022-2-2174the Beijing Municipal Science and Technology Commission,No.Z191100007619037.
文摘BACKGROUND The transforming growth factor β(TGFβ) signaling pathway plays a crucial role in the development of liver fibrosis by activating TGFβ type Ⅱ receptor(TGFβR2), followed by the recruitment of TGFβR1 finally triggering downstream signaling pathway.AIM To find drugs targeting TGFβR2 that inhibit TGFβR1/TGFβR2 complex formation, theoretically inhibit TGFβ signaling pathway, and thereby ameliorate liver fibrosis.METHODS Food and Drug Administration-approved drugs were screened for binding affinity with TGFβR2 by virtual molecular docking. We identified 6 candidates and further explored their potential by Cell Counting Kit-8(CCK-8) cell cytotoxic experiment to validate toxicity and titrated the best cellular working concentrations. Next, we further demonstrated the detailed molecular working mechanisms using mutagenesis analysis. Finally, we used a mouse model to investigate its potential anti-liver fibrosis effect.RESULTS We identified 6 drug candidates. Among these 6 drugs, dihydroergotamine(DHE) shows great ability in reducing fibrotic gene expressions such as collagen, p-SMAD3, and α-SMA in TGFβ induced cellular model of liver fibrosis in LX-2 cells. Furthermore, we demonstrated that DHE binds to TGFβR2. Moreover, mutation of Leu27, Phe30, Thr51, Ser52, Ile53, and Glu55 of TGFβR2 disrupted the binding of TGFβR2 with DHE. In addition, DHE significantly improved liver fibrosis, as evidenced by Masson’s trichrome staining of liver sections. This is further supported by the width and the velocity of the portal vein, and serum markers of liver function. In line with those observations, DHE also decreased macrophages infiltration and extracellular matrix deposition in the liver.CONCLUSION DHE alleviates liver fibrosis by binding to TGFβR2 thereby suppressing TGFβ signaling pathway. We show here that as far as drug repurposing, DHE has great potential to treat liver fibrosis.
基金This research was supported by Cancer Genomics Centre Netherlands and a grant from the National Natural Science Foundation of China(31471315).
文摘The transforming growth factor-β(TGF-β)family controls embryogenesis,stem cell differentiation,and tissue homeostasis.However,how post-translation modifications contribute to fine-tuning of TGF-βfamily signaling responses is not well understood.Inhibitory(I)-Smads can antagonize TGF-β/Smad signaling by recruiting Smurf E3 ubiquitin ligases to target the active TGF-βreceptor for proteasomal degradation.A proteomic interaction screen identified Vpr binding protein(VprBP)as novel binding partner of Smad7.Mis-expression studies revealed that VprBP negatively controls Smad2 phosphorylation,Smad2–Smad4 interaction,as well as TGF-βtarget gene expression.VprBP was found to promote Smad7–Smurf1–TβRI complex formation and induce proteasomal degradation of TGF-βtype I receptor(TβRI).Moreover,VprBP appears to stabilize Smurf1 by suppressing Smurf1 poly-ubiquitination.In multiple adult and mouse embryonic stem cells,depletion of VprBP promotes TGF-βor Activin-induced responses.In the mouse embryo VprBP expression negatively correlates with mesoderm marker expression,and VprBP attenuated mesoderm induction during zebrafish embryogenesis.Our findings thereby uncover a novel regulatory mechanism by which Smurf1 controls the TGF-βand Activin cascade and identify VprBP as a critical determinant of embryonic mesoderm induction.
基金This work was supported by the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme(GDHVPS2018)the National Hong Kong Scholars program(XJ2016059)+2 种基金the Natural Science Foundation of Guangdong Province(2020A1515011239)the Guangzhou Science and Technology Project(202102021241)the Administration of Traditional Chinese Medicine of Guangdong Province(20211253)and the Guangzhou Consun Pharmaceuticals Co.,Ltd..These funding bodies played no role in the design of the study,collection,analysis and interpretation of data,and in writing the manuscript.
文摘Objective Chronic renal failure(CRF)is a worldwide public health burden.Niaoduqing granules(NDQ)is widely used for CRF treatment in China.However,the underlying mechanism of NDQ is not fully studied.This study is aimed to investigate whether NDQ ameliorate CRF by inhibiting transforming growth factor-β1(TGF-β1)-induced EMT in human renal tubular epithelial HK-2 cells.Methods 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide assay and colony formation assay were used to investigate the cytotoxicity of NDQ in HK-2 cells.Morphological changes of HK-2 cells after TGF-β1 or/and NDQ treatment were observed under a microscope.Wound-healing,migration and invasion assays were performed to determine the cell movement,migratory and invasive abilities,respectively.Western blot analysis was carried out to examine the protein levels of TGF-βreceptor I(TβRI)and epithelial-mesenchymal transition(EMT)-associated factors.Fluorescence confocal microscopy was applied to observe the organization of filamentous actin.Results NDQ suppressed TβRI expression dose-dependently.NDQ inhibited TGF-β1-stimulated EMT in HK-2 cells,supported by the evidences that NDQ prevented morphology change,attenuated cell migration and invasion,downregulated EMT factors and reorganized filamentous actin distribution in TGF-β1-stimulated HK-2 cells.Conclusions NDQ attenuates chronic renal failure which may be associated with inhibition of TβRI expression and EMT process.
