Background:The casein kinase 2-interacting protein-1(CKIP-1)is important in the development of osteoblasts and cardiomyocytes.However,the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells(MSCs)remain un...Background:The casein kinase 2-interacting protein-1(CKIP-1)is important in the development of osteoblasts and cardiomyocytes.However,the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells(MSCs)remain unclear.This study aimed to determine whether CKIP-1 affects osteogenic differentiation in MSCs and explore the relationship of CKIP-1 and inflammation.Methods:Bone marrow MSCs of CKIP-1 wild type(WT)and knockout(KO)mice were cultivated in vitro.Cell phenotype was analyzed by flow cytometry,colony formation was detected to study the proliferative ability.Osteogenic and adipogenic induction were performed.The osteogenic ability was explored by alizarin red staining,alkaline phosphatase(ALP)staining and ALP activity detection.Quantitative real-time polymerase chain reaction(qRT-PCR)was carried out to determine the mRNA expression levels of osteoblast marker genes.The adipogenic ability was detected by oil red O staining.Content of the bone was analyzed to observe the differences of bone imaging parameters including trabecular bone volume/tissue volume(BV/TV),bone surface area fraction/trabecular BV,trabecular number(Tb.N),and trabecular spacing(Tb.sp).Interleukin(IL)-1b was injected on WT mice of 2 months old and 18 months old,respectively.Difference in CKIP-1 expression was detected by RT-PCR and western blot.The relationship between CKIP-1 and inflammation was explored by RT-PCR and western blot.Results:ALP assays,alizarin red staining,and qRT-PCR showed that MSCs derived from CKIP-1 KO mice exhibited a stronger capability for osteogenesis.Micro-computed tomography detection showed that among 18-month-old mice,CKIP-1 KO mice presented significantly higher bone mass compared withWTmice(P=0.02).No significant difference was observed in 2-month-old mice.In vivo data showed that expression of CKIP-1 was higher in the bone marrow of aging mice than in young mice(4.3-fold increase at themRNA level,P=0.04).Finally,the expression levels of CKIP-1 in bone marrow(3.2-fold increase at themRNA level,P=0.03)and cultured MSCs were up-regulated on chronic inflammatory stimulation by IL-1b.Conclusions:CKIP-1 is responsible for negative regulation of MSC osteogenesis with age-dependent effects.Increasing levels of inflammation with aging may be the primary factor responsible for higher expression levels of CKIP-1 but may not necessarily affect MSC aging.展开更多
OBJECTIVE:To investigate the mechanism of Dan Ze mixture(丹泽合剂,DZM)in the treatment of lipotoxic cardiomyopathy.METHODS:Ultra-performance liquid chromatography tandem mass spectrometry was employed to characterize ...OBJECTIVE:To investigate the mechanism of Dan Ze mixture(丹泽合剂,DZM)in the treatment of lipotoxic cardiomyopathy.METHODS:Ultra-performance liquid chromatography tandem mass spectrometry was employed to characterize the serum migration constituents of DZM.A lipotoxic cardiomyopathy rat model was established through high-fat diet and intervened by different doses of DZM.The cardiac function was assessed using echocardiography,and hematoxylin and eosin,oil red O,and Masson staining were conducted to evaluate morphological changes,lipid accumulation,and fibrosis in myocardial tissue.Serum myocardial enzyme activity,lipid levels,and lipid content of myocardial tissue were measured,while fluorescent staining and colorimetry were used to assess oxidation levels in myocardial tissue.Mitochondrial membrane potential was detected by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanineiodide(JC-1).Transmission electron microscopy was employed to observe ultrastructure and mitochondrial structure changes in myocardial tissue.Fluorescence double staining and colocalization were utilized to observe the binding of autophagosomes and mitochondria,while immunohistochemical staining was used to detect the expression of mitophagy-related proteins.Terminal deoxynucleoitidyl transferase mediated nick end labeling staining was employed for the identification of apoptosis in myocardial tissue,while quantitative real-time reverse transcriptase polymerase chain reaction(q RT-PCR)and Western blot were utilized for the detection of apoptosis,B-cell lymphoma-2 adenovirus E1B 19 k Da-interacting protein 3(BNIP3)/mitophagy signaling pathway-related genes and proteins.In palmitic acid-induced Rat H9C2 cardiomyocytes(H9c2)cells,various cellular parameters including cell viability,lactate dehydrogenase release,apoptosis rate,oxidative stress level,mitochondrial structure and function,and mitophagy level were assessed after the treatment of DZM drug-containing serum for a duration of 24 h.The cellular expressions of BNIP3/mitophagy signaling pathway relevant genes and proteins were further evaluated using q RT-PCR and Western blot techniques.RESULTS:A total of 295 prototypes(e.g.,phenolic acids,quinones,terpenoids)were identified in serum of rats after oral administration of DZM.In vivo,DZM therapy has been shown to effectively enhance cardiac function,mitigate high-fat diet-induced myocardial structural damage and lipid accumulation.Furthermore,DZM has demonstrated the ability to reduce lipid levels,attenuate cell apoptosis,combat oxidative stress,enhance mitochondrial structure and function,and activate the BNIP3/mitophagy signaling pathway.Furthermore,the silencing of BNIP3 has been shown to exacerbate palmitic acid-induced damages in H9c2 cells,while inhibiting the BNIP3/mitophagy signaling pathway can mitigate the inhibitory effects of DZM on palmitic acidinduced apoptosis,lipid deposition and oxidative stress.CONCLUSION:This study presents preliminary evidence for the therapeutic efficacy of DZM on lipotoxic cardiomyopathy through the activating BNIP3/mitophagy signaling pathway.展开更多
基金Supported by the grants from the National Key Research and Development Project(No.2017YFB1304300)Conversion Fund of PLA General Hospital(No.2017tm-018)the Clinical Research Support Fund of PLA General Hospital(No.2017fc-tsys-2013).
文摘Background:The casein kinase 2-interacting protein-1(CKIP-1)is important in the development of osteoblasts and cardiomyocytes.However,the effects of CKIP-1 on osteoblast precursor mesenchymal stem cells(MSCs)remain unclear.This study aimed to determine whether CKIP-1 affects osteogenic differentiation in MSCs and explore the relationship of CKIP-1 and inflammation.Methods:Bone marrow MSCs of CKIP-1 wild type(WT)and knockout(KO)mice were cultivated in vitro.Cell phenotype was analyzed by flow cytometry,colony formation was detected to study the proliferative ability.Osteogenic and adipogenic induction were performed.The osteogenic ability was explored by alizarin red staining,alkaline phosphatase(ALP)staining and ALP activity detection.Quantitative real-time polymerase chain reaction(qRT-PCR)was carried out to determine the mRNA expression levels of osteoblast marker genes.The adipogenic ability was detected by oil red O staining.Content of the bone was analyzed to observe the differences of bone imaging parameters including trabecular bone volume/tissue volume(BV/TV),bone surface area fraction/trabecular BV,trabecular number(Tb.N),and trabecular spacing(Tb.sp).Interleukin(IL)-1b was injected on WT mice of 2 months old and 18 months old,respectively.Difference in CKIP-1 expression was detected by RT-PCR and western blot.The relationship between CKIP-1 and inflammation was explored by RT-PCR and western blot.Results:ALP assays,alizarin red staining,and qRT-PCR showed that MSCs derived from CKIP-1 KO mice exhibited a stronger capability for osteogenesis.Micro-computed tomography detection showed that among 18-month-old mice,CKIP-1 KO mice presented significantly higher bone mass compared withWTmice(P=0.02).No significant difference was observed in 2-month-old mice.In vivo data showed that expression of CKIP-1 was higher in the bone marrow of aging mice than in young mice(4.3-fold increase at themRNA level,P=0.04).Finally,the expression levels of CKIP-1 in bone marrow(3.2-fold increase at themRNA level,P=0.03)and cultured MSCs were up-regulated on chronic inflammatory stimulation by IL-1b.Conclusions:CKIP-1 is responsible for negative regulation of MSC osteogenesis with age-dependent effects.Increasing levels of inflammation with aging may be the primary factor responsible for higher expression levels of CKIP-1 but may not necessarily affect MSC aging.
基金Scientific Research Project of Hebei Province Administration of Traditional Chinese Medicine:to Explore the Protective Effect and Mechanism of Zexie Decoction on Lipotoxic Cardiomyopathy based on the p-mitogen-activated protein kinases/Peroxisome proliferator-activated receptorγcoactivator 1-alpha(p MAPK/PGC-1α)Signaling Pathway(No.2022096)Medical Science Research Project of Hebei Province:the Effect of 23-acetyl Alismol-B on Mitochondrial Function in Palmitic Acid-induced H9c2 Cells Was Investigated based on the Ca2+-Cyclic Adenosine Monophosphate(c AMP)-Response Element Binding Protein/c AMP Response Element(CREB/CRE)-PGC-1αSignaling Pathway(No.20221490)+1 种基金Hebei province natural science fund project:Study on the Mechanism of Danshen Zexie Decoction in Activating Nuclear Factor Erythroid 2-related Factor 2 Signaling Pathway to Trigger 0mi/Htr A2,Restoring Autophagic Flux and Enhancing Metabolism-Related Fatty Liver Disease(No.H2023423064)Hebei graduate student innovation ability funding training project:to Investigate the Protective Effects and Underlying Mechanisms of Zexie Decoction on Lipotoxic Cardiomyopathy,with A Focus on the PGC-1a Signaling Pathway(No.CXZZBS2022096)。
文摘OBJECTIVE:To investigate the mechanism of Dan Ze mixture(丹泽合剂,DZM)in the treatment of lipotoxic cardiomyopathy.METHODS:Ultra-performance liquid chromatography tandem mass spectrometry was employed to characterize the serum migration constituents of DZM.A lipotoxic cardiomyopathy rat model was established through high-fat diet and intervened by different doses of DZM.The cardiac function was assessed using echocardiography,and hematoxylin and eosin,oil red O,and Masson staining were conducted to evaluate morphological changes,lipid accumulation,and fibrosis in myocardial tissue.Serum myocardial enzyme activity,lipid levels,and lipid content of myocardial tissue were measured,while fluorescent staining and colorimetry were used to assess oxidation levels in myocardial tissue.Mitochondrial membrane potential was detected by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanineiodide(JC-1).Transmission electron microscopy was employed to observe ultrastructure and mitochondrial structure changes in myocardial tissue.Fluorescence double staining and colocalization were utilized to observe the binding of autophagosomes and mitochondria,while immunohistochemical staining was used to detect the expression of mitophagy-related proteins.Terminal deoxynucleoitidyl transferase mediated nick end labeling staining was employed for the identification of apoptosis in myocardial tissue,while quantitative real-time reverse transcriptase polymerase chain reaction(q RT-PCR)and Western blot were utilized for the detection of apoptosis,B-cell lymphoma-2 adenovirus E1B 19 k Da-interacting protein 3(BNIP3)/mitophagy signaling pathway-related genes and proteins.In palmitic acid-induced Rat H9C2 cardiomyocytes(H9c2)cells,various cellular parameters including cell viability,lactate dehydrogenase release,apoptosis rate,oxidative stress level,mitochondrial structure and function,and mitophagy level were assessed after the treatment of DZM drug-containing serum for a duration of 24 h.The cellular expressions of BNIP3/mitophagy signaling pathway relevant genes and proteins were further evaluated using q RT-PCR and Western blot techniques.RESULTS:A total of 295 prototypes(e.g.,phenolic acids,quinones,terpenoids)were identified in serum of rats after oral administration of DZM.In vivo,DZM therapy has been shown to effectively enhance cardiac function,mitigate high-fat diet-induced myocardial structural damage and lipid accumulation.Furthermore,DZM has demonstrated the ability to reduce lipid levels,attenuate cell apoptosis,combat oxidative stress,enhance mitochondrial structure and function,and activate the BNIP3/mitophagy signaling pathway.Furthermore,the silencing of BNIP3 has been shown to exacerbate palmitic acid-induced damages in H9c2 cells,while inhibiting the BNIP3/mitophagy signaling pathway can mitigate the inhibitory effects of DZM on palmitic acidinduced apoptosis,lipid deposition and oxidative stress.CONCLUSION:This study presents preliminary evidence for the therapeutic efficacy of DZM on lipotoxic cardiomyopathy through the activating BNIP3/mitophagy signaling pathway.
