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SUMMARY: Senile osteoporosis is mainly caused by reduced osteoblast differentiation and has become the leading cause of fractures in the elderly worldwide. Natural organics are emerging as a potential option for the prevention and treatment of osteoporosis. This study was designed to study the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteoporosis mice. A mouse model of osteoporosis was established by subcutaneous injection of dexamethasone and treated with resveratrol administered by gavage. In vivo and in vitro, we used western blot to detect protein expression, and evaluated osteogenic differentiation of BMSCs by detecting the expression of osteogenic differentiation related proteins, calcium deposition, ALP activity and osteocalcin content. Resveratrol treatment significantly increased the body weight of mice, the level of serum Ca2+, 25(OH)D and osteocalcin, ration of bone weight, bone volume/total volume, trabecular thickness, trabecular number, trabecular spacing and cortical thickness in osteoporosis mice. In BMSCs of osteoporosis mice, resveratrol treatment significantly increased the expression of Runx2, osterix (OSX) and osteocalcin (OCN) protein, the level of calcium deposition, ALP activity and osteocalcin content. In addition, resveratrol treatment also significantly increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT in BMSCs of osteoporosis mice. In vitro, resveratrol increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT, Runx2, OSX and OCN protein, the level of calcium deposition, ALP activity and osteocalcin content in BMSCs in a concentration-dependent manner, while SIRT1 knockdown significantly reversed the effect of resveratrol. Resveratrol can attenuate osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and the mechanism may be related to the regulation of SIRT1/PI3K/AKT pathway.
La osteoporosis senil es causada principalmente por una diferenciación reducida de osteoblastos y se ha convertido en la principal causa de fracturas en las personas mayores en todo el mundo. Los productos orgánicos naturales están surgiendo como una opción potencial para la prevención y el tratamiento de la osteoporosis. Este estudio fue diseñado para estudiar el efecto del resveratrol en la diferenciación osteogénica de las células madre mesenquimales de la médula ósea (BMSC) en ratones con osteoporosis. Se estableció un modelo de osteoporosis en ratones mediante inyección subcutánea de dexametasona y se trató con resveratrol administrado por sonda. In vivo e in vitro, utilizamos Western blot para detectar la expresión de proteínas y evaluamos la diferenciación osteogénica de BMSC detectando la expresión de proteínas relacionadas con la diferenciación osteogénica, la deposición de calcio, la actividad de ALP y el contenido de osteocalcina. El tratamiento con resveratrol aumentó significativamente el peso corporal de los ratones, el nivel sérico de Ca2+, 25(OH)D y osteocalcina, la proporción de peso óseo, el volumen óseo/ volumen total, el espesor trabecular, el número trabecular, el espaciado trabecular y el espesor cortical en ratones con osteoporosis. En BMSC de ratones con osteoporosis, el tratamiento con resveratrol aumentó significativamente la expresión de las proteínas Runx2, osterix (OSX) y osteocalcina (OCN), el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina. Además, el tratamiento con resveratrol también aumentó significativamente la expresión de SIRT1, p-PI3K/PI3K y p-AKT/AKT en BMSC de ratones con osteoporosis. In vitro, el resveratrol aumentó la expresión de las proteínas SIRT1, p-PI3K/PI3K y p- AKT/AKT, Runx2, OSX y OCN, el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina en BMSC de manera dependiente de la concentración, mientras que La caída de SIRT1 revirtió significativamente el efecto del resveratrol. El resveratrol puede atenuar la osteoporosis al promover la diferenciación osteogénica de las células madre mesenquimales de la médula ósea, y el mecanismo puede estar relacionado con la regulación de la vía SIRT1/PI3K/AKT.
Assuntos
Animais , Masculino , Camundongos , Osteoporose/tratamento farmacológico , Resveratrol/administração & dosagem , Osteogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Western Blotting , Modelos Animais de Doenças , Sirtuína 1 , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Resveratrol/farmacologia , Camundongos Endogâmicos C57BLRESUMO
Objective To investigate the effect and mechanism of transplantation of neuregulin1(NRG1)gene-modified bone marrow mesenchymal stem cells(BMSCs)on the repair of hemi-transected spinal cord injury(SCI)in rats.Methods Isolated and cultured rat BMSCs,followed by transfection with the NRG1 gene.The levels of NRG1 in BMSCs lysate and culture supernatant was deected by ELISA method,and the proliferation activity of the BMSCs was detected by cell counting method.Forty-three healthy 8-week-old SD rats were randomly divided into control group(n=10),SCI model group(n=10),BMSCs group(n=10),and NRG1-BMSCs group(n=13).After establishing the spinal cord hemisection model,animals received in-situ transplantation of BMSCs or NRG1-BMSCs.On the 1,7,14,21,and 28 days after transplantation,the hind limb motor function was evaluated using BBB score and inclined plate test;on the 7th day after transplantation,the migration and distribution of transplanted cells was monitored using a fluorescence microscope;on the 28th day after transplantation,the pathological changes of rat spinal cord tissues was examined using HE staining and Nissl staining;cell apoptosis using TUNEL staining,and levels of endoplasmic reticulum stress-related proteins[X-box binding protein 1(XBP1),C/EBP homologous protein(CHOP),activating transcription factor 4(ATF4),ATF6,glucose-regulated protein 78(GRP78)]and apoptosis-related proteins[B-cell lymphoma-2(Bcl-2)and Bcl-2-associated protein X(Bax)]in rat spinal cord tissues using Western blotting.Results BMSCs were successfully isolated,cultured,and transfected with the NRG1 gene.ELISA method results showed that the NRG1 contents in the NRG1-BMSCs lysate and culture supernatant were significantly higher than that of BMSCs in a time-dependent manner(P<0.05).The proliferation activity of NRG1-BMSCs was significantly higher than that of BMSCs(P<0.05).On the 21 and 28 days after transplantation,the BBB score and the slope angle of the inclined plate in NRG1-BMSCs group were higher than those in SCI model group or BMSCs group(P<0.05).However,it did not reverse to the level in control group(P<0.05).On the 28th day after transplantation,compared with the SCI model group and BMSCs group,neuronal pyknosis reduced,the Nissl body density increased,the expression levels of XBP1,CHOP,ATF4,ATF6,GRP78,and Bax,and the rate of TUNEL-positive cells significantly reduced in NRG1-BMSCs group(P<0.05),and the expression level of Bcl-2 significantly increased(P<0.05).Conclusion Transplantation of NRG1 gene-modified BMSCs can alleviate SCI and improve the recovery of motor function in rats.The mechanism may be related to promoting the proliferation activity of BMSCs,inhibiting cell apoptosis,and mitigating endoplasmic reticulum stress.
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Objective:To explore the mechanism of zoledronic acid (ZOL) affects osteogenic differentiation and bone formation through the regulation of sirtuin 3 (SIRT3) / P53 expression.Methods:Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate into osteogenic cells, the expression of SIRT3 in the cells was detected, and the targeting regulation relationship between SIRT3 and P53 was analyzed. The intracellular expressions of SIRT3 and P53 were intervened and ZOL was used to treat the cells. MTT method, Western blot method and kit were used to detect cell viability, osteogenesis-related genes Osteoprotegerin (OPG), runt-related transcription factor 2 (Runx2) expression, alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining, respectively. Ovariectomy (OVX) was used to construct a rat model and explore the effect of ZOL on the progression of osteoporosis (OP) in vivo.Results:ZOL promoted osteogenic differentiation of BMSCs. The expression of SIRT3 was down-regulated in the serum of OP patients (0.78±0.23) compared with that of healthy subjects (1.00±0.26 vs. 0.78±0.23. t=3.85, P<0.001). During the osteogenic differentiation of BMSCs, the expression level of SIRT3 gradually increased with the prolonged induction of osteogenesis. Compared with the p53 protein expression and BMSCs activity in the control group, SIRT3 knockout could increase the expression level of p53 protein (0.59±0.05 vs. 1.01±0.11. t=6.02, P=0.004) but inhibited the activity of BMSCs (100.00±8.41 vs. 51.26±5.59. t=8.36, P=0.001). After ZOL treatment, the inhibitory effect of SIRT3 on cell viability (49.61±5.11 vs. 87.61±7.31. t=7.38, P=0.002) and osteogenesis was relieved, and the level of P53 was inhibited (1.10±0.10 vs. 0.69±0.04. t=6.59, P=0.003). P53 overexpression partially offseted the effects of ZOL on cell viability (84.61±6.52 vs. 66.54±5.47. t=3.68, P=0.021) and osteogenesis. Compared with the sham surgery group, the OVX group showed inhibition of osteogenesis in rats, and ZOL treatment significantly improved osteogenic inhibition. ZOL treatment increased the expression level of SIRT3 protein in bone tissue of OVX rats, but inhibited the expression level of P53. Conclusion:ZOL promoted osteogenic differentiation and bone formation of BMSCs by promoting the ubiquitination and degradation of P53 by SIRT3.
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Objective To observe the effect of rat bone marrow mesenchymal stem cells(BMSCs)on the apoptosis of rat peritoneal mesothelium cells(PMCs)induced by high glucose peritoneal dialysis fluid(PDF),and to explore its possible molecular mechanism.Methods The primary BMSCs and PMCs were extracted and identified.Apoptosis of PMCs was induced by high glucose PDF.Cell supernatant from BMSCs after 24 h of culture was collected as the conditioned medium(BMSCs-CM).PMCs were co-cultured with BMSCs by conditioned media or Transwell chambers.PMCs were randomly divided into the control group,the PDF group and the PDF+BMSCs-CM group.The viability of PMCs was measured by CCK-8 in each group.The depolarization of mitochondrial membrane potential was measured by JC-1 method.TUNEL staining was used to detect cell apoptosis.Western blot assay was used to detect the expression levels of apoptosis related proteins B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),Cleaved cysteine aspartase-3(Cleaved Caspase-3)and pathway related protein serine/threonine protein kinase(Raf),mitogen-activated extracellular signal-regulated kinase(MEK),extracellular-signal regulated protein kinase(ERK)and their phosphorylated proteins in each group.Results Compared with the control group,the proliferative activity and mitochondrial membrane potential of PMCs were decreased in the PDF group,while the apoptosis rate and the ratio of Bax/Bcl-2,Cleaved Caspase-3/Caspase-3,p-Raf/Raf,p-MEK/MEK and p-ERK/ERK were increased(P＜0.05).Compared with the PDF group,the proliferative activity and mitochondrial membrane potential of PMCs were increased in the PDF+BMSCs-CM group,while the apoptosis rate and the ratio of Bax/Bcl-2,Cleaved Caspase-3/Caspase-3,p-Raf/Raf,p-MEK/MEK and p-ERK/ERK were decreased(P＜0.05).Conclusion BMSCs can reduce the apoptosis of PMCs induced by high glucose PDF,and its mechanism maybe related to inhibiting the activation of Raf/MEK/ERK signaling pathway.
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Fluorine is an important element widely present in nature, and moderate intake can prevent dental caries and promote bone development. However, long-term excessive intake can lead to fluorosis, damaging tissues or organs such as teeth, bones, heart muscle, and blood vessels. Bone marrow mesenchymal stem cells (BMSCs) play an important role in the repair process of bone injury due to their excellent multi-directional differentiation potential. Therefore, studying BMSCs is of great value in the treatment of fluorosis caused by fluoride poisoning. This article summarize the progress on the effect of fluoride on BMSCs, providing new ideas for the study of the pathogenesis and clinical treatment of fluorosis.
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Aim To investigate the effect of miR-141-5p/ZNF705A in chronic myeloid leukemia(CML)cell-derived exosome(Exo)on the adhesion of bone marrow mesenchymal stem cells(BMSCs). Methods The morphology and size of Exo in peripheral blood from CML patients and K562 cells were examined by electron microscopy and NTA particle size analysis. The expressions of Exo and BMSCs marker molecules and adhesion proteins in K562 cells were detected by qRT-PCR and Western blot before and after transfection. The adhesion ability of BMSCs was detected by cell adhesion assay, and the cellular activity of BMSCs was examined using CCK-8. miR-141-5p binding to ZNF705A was detected by luciferase assay. Results qRT-PCR results showed that miR-141-5p expression was significantly reduced in both CML patients and K562 cell-derived Exo. qRT-PCR, Western blot and other results showed that BMSCs in CML patients had significantly reduced the expression of adhesion proteins CD44 and CXCL12, and were able to phagocytose K562 cell-derived Exo. Further, K562-derived Exo was found to reduce CD44 and CXCL12 expression and adhesion in Exo-promoted BMSCs compared with CD34+ cells. Meanwhile, the results of dual luciferase reporter assay verified that miR-141-5p targeted binding to ZNF705A. Finally, we found ZNF705A could be targeted by up-regulating miR-141-5p expression in Exo of K562 cells, which in turn inhibited the adhesion of BMSCs. Conclusions K562 cells down-regulate miR-141-5p expression in Exo and inhibit the adhesion function of BMSCs by targeting ZNF705A, thus regulating the bone marrow hematopoietic function in CML patients.
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The senescence of bone marrow mesenchymal stem cells (BM-MSCs) will induce age-related bone tissue degeneration and chronic inflammation, and reduce its application effect for cell therapy. More and more active ingredients of traditional chinese medicine have been proved to intervene BM - MSCs senescence, playing an important role in bone diseases prevention and treatment, and improving the therapeutic effect of BM-MSCs. In this paper, the latest research progress on the molecular mechanism of traditional chinese medicine active ingredients interfering BM-MSCs senescence was summarized, in order to provide new direction and reference basis for senescence intervention research and clinical application improvement of BM-MSCs.
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Objective@#To investigate the osteogenic properties of a methacrylated gelatin (GelMA) / bone marrow mesenchymal stem cells (BMSCs) composite hydrogel applied to the skull defect area of rats and to provide an experimental basis for the development of bone regeneration biomaterials.@*Methods@#This study was approved by the Animal Ethics Committee of Nanjing University. A novel photocurable composite biohydrogel was developed by constructing photoinitiators [lthium phenyl (2,4,6-trimethylbenzoyl) phosphinate, LAP], GelMA, and BMSCs. The surface morphology and elemental composition of the gel were examined using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The compressive strength of the gel was evaluated using an electronic universal testing machine. After in vitro culture for 1, 2, and 5 days, the proliferation of the BMSCs in the hydrogels was assessed using a CCK-8 assay, and their survival and morphology were examined through confocal microscopy. A 5 mm critical bone deficiency model was generated in a rat skull. The group receiving composite hydrogel treatment was referred to as the GelMA/BMSCs group, whereas the untreated group served as the control group. At the 4th and 8th weeks, micro-CT scans were taken to measure the bone defect area and new bone index, while at the 8th week, skull samples from the defect area were subjected to H&E staining, van Gieson staining, and Goldner staining to evaluate the quality of bone regeneration and new bone formation.@*Results@#SEM observed that the solidified GelMA showed a 3D spongy gel network with uniform morphology, the porosity of GelMA was 73.41% and the pore size of GelMA was (28.75 ± 7.13) μm. EDX results showed that C and O were evenly distributed in the network macroporous structure of hydrogel. The hydrogel compression strength was 152 kPa. On the 5th day of GelMA/BMSCs culture, the cellular morphology transitioned from oval to spindle shaped under microscopic observation, accompanied by a significant increase in cell proliferation (159.4%, as determined by the CCK-8 assay). At 4 weeks after surgery, a 3D reconstructed micro-CT image revealed a minimal reduction in bone defect size within the control group and abundant new bone formation in the GelMA/BMSCs group. At 8 weeks after surgery, no significant changes were observed in the control group's bone defect area, with only limited evidence of new bone growth; however, substantial healing of skull defects was evident in the GelMA/BMSCs group. Quantitative analysis at both the 4- and 8-week examinations indicated significant improvements in the new bone volume (BV), new bone volume/total bone volume (BV/TV), bone surface (BS), and bone surface/total bone volume (BS/TV) in the GelMA/BMSCs group compared to those in the control group (P<0.05). Histological staining showed continuous and dense formation of bone tissue within the defects in the GelMA/BMSCs group and only sporadic formation of new bone, primarily consisting of fibrous connective tissue, at the defect edge in the control group.@*Conclusion@#Photocuring hydrogel-based stem cell therapy exhibits favorable biosafety profiles and has potential for clinical application by inducing new bone formation and promoting maturation within rat skull defects.
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OBJECTIVE@#To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ).@*RESULTS@#Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area.@*CONCLUSION@#After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
Assuntos
Feminino , Camundongos , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/farmacologia , PPAR gama/metabolismo , Esteroide 12-alfa-Hidroxilase/metabolismo , Camundongos Endogâmicos C57BL , Diferenciação Celular , Osteogênese , Células-Tronco Mesenquimais , Ácidos e Sais Biliares/farmacologia , Células da Medula Óssea , Células Cultivadas , Compostos AzoRESUMO
ObjectiveTo explore the mechanism of Buyang Huanwutang combined with bone marrow mesenchymal stem cell （BMSC） transplantation in the treatment of spinal cord injury （SCI）. MethodDifferent concentrations （12.5， 25， 50 g·kg-1） of Buyang Huanwutang were administrated to rats by gavage. The spinal cord function of rats was measured by modified Tarlov score， and the most suitable concentration of Buyang Huanwutang was screened out. SD rats were then divided into 6 groups， namely， the sham operation group （gavage of equal amount of normal saline）， the model group （gavage of equal amount of normal saline）， the Buyang Huanwutang group （gavage of 25 g·kg-1 Buyang Huanwutang）， the BMSC transplantation group （tail vein injection of BMSCs 1 mL）， the Buyang Huanwutang+BMSC group （gavage of 25 g·kg-1 Buyang Huanwutang and tail vein injection of BMSCs 1 mL）， the Buyang Huanwutang+BMSC+LY294002 group （gavage of 25 g·kg-1 Buyang Huanwutang and tail vein injection of BMSCs 1 mL and 40 mg·kg-1 LY294002）， with 10 rats in each group. The spinal cord function was measured by the modified Tarlov score， inclined plate test， and latency of cortical somatosensory evoked potential. Immunohistochemistry was used to detect the number of 5-bromo-2-deoxyuracil nucleoside （Brdu）-labeled positive cells in the spinal cord tissue. The protein expression levels of phosphorylated protein kinase B （p-Akt）， glycoprotein 130 （gp130）， and interleukin-6 （IL-6） in spinal cord were detected by Western blot. ResultAs compared with the sham operation group， the Tarlov score and the critical angle of tilt plane in the model group were significantly decreased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 were significantly increased （P<0.05）. As compared with the model group， the Tarlov score and the critical angle of tilt plane in the sham operation group and each treatment group were significantly increased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 were significantly decreased （P<0.05）. As compared with the BMSC group， the Tarlov score and the critical angle of inclined plane in the Buyang Huanwutang+BMSC group increased （P<0.05）， the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 decreased （P<0.05）， and the number of Brdu-labeled positive cells increased 5 weeks after transplantation （P<0.05）. As compared with the Buyang Huanwutang+BMSC group， the Tarlov score and the critical angle of the inclined plane in the Buyang Huanwutang+BMSC+LY294002 group increased （P<0.05）， and the latency of cortical somatosensory evoked potential wave and the protein expression levels of p-Akt， gp130， and IL-6 decreased significantly （P<0.05）. Five weeks after transplantation， the number of Brdu-labeled positive cells increased significantly in the Buyang Huanwutang+BMSC+LY294002 group （P<0.05）. ConclusionBuyang Huanwutang can promote BMSCs migration and restore spinal cord function by inhibiting phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signal.