Resveratrol Alleviates Osteoporosis by Promoting Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells via SITR1/PI3K/AKT Pathway / El Resveratrol Alivia la Osteoporosis al Promover la Diferenciación Osteogénica de las Células Madre Mesenquimales de la Médula Ósea a Través de la Vía SITR1/PI3K/AKT

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.

Impact of lithocholic acid on the osteogenic and adipogenic differentiation balance of bone marrow mesenchymal stem cells / 中国修复重建外科杂志

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.

Effect and mechanism of transplantation bone marrow stromal stem cells transfected NRG1 gene on repair of hemi-transected spinal cord injury in rats / 解放军医学杂志

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.

Zoledronic acid affects osteogenic differentiation and bone formation by regulating the expression of SIRT3/P53 / 中华内分泌外科杂志

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.

Study on the effect and mechanism of bone marrow mesenchymal stem cells on apoptosis of peritoneal mesothelial cells / 天津医药

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.

Research progress on the effect of fluoride on bone marrow mesenchymal stem cells / 中华地方病学杂志

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.

Reconstruction of rat calvarial defects utilizing an ultraviolet-cured hydrogel loaded with bone marrow mesenchymal stem cells / 口腔疾病防治

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.

Effect of miR-141-5p/ZNF705A on adhesion of bone marrow mesenchymal stem cells in chronic myeloid leukemia cell-derived exosomes / 中国药理学通报

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.

Research progress on molecular mechanism of traditional chinese medicine active ingredients interfering bone marrow mesenchymal stem cells senescence / 中国临床药理学与治疗学

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.

Effect of Wnt/β-catenin signaling pathway in neural differentiation of human bone marrow mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process.@*METHODS@#The identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot.@*RESULTS@#When compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A ( P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D ( P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased ( P<0.05), while NSE, MAP-2, and GFAP genes significantly increased ( P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E ( P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E ( P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E ( P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E ( P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E ( P<0.05).@*CONCLUSION@#Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.

Effect of Human Bone Marrow Mesenchymal Stem Cells with Ectopic High OCT4 Expression on T Lymphocyte Function / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of human bone marrow mesenchymal stem cells (MSCs) with ectopic high OCT4 expression on T-cell proliferation, activation and secretion in vitro.@*METHODS@#Peripheral blood mononuclear cells were isolated from healthy children. Anti-CD3 and anti-CD28 monoclonal antibodies were used to activate T lymphocytes, which were stimulated by interleukin (IL)-2 for one week in vitro. Then MSCs with ectopic high OCT4 expression (MSC-OCT4) were co-cultured with activated T lymphocytes. After one week of co-culture, the supernatant was collected and the levels of Th1/Th2 cytokines [IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ] were determined by flow cytometry. The lymphocytes after one week of co-culture were collected and counted by Countstar software. After the proportions of activated/inactivated T cell subsets were determined by flow cytometry, the absolute lymphocyte counts were calculated and expressed as mean ± standard deviation.@*RESULTS@#Compared with control T cell alone culture group, the proliferation of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells were significantly inhibited in MSC group and MSC-OCT4 group. Compared with MSC, MSC-OCT4 could inhibit CD3+CD8+ T cell proliferation better (P =0.049), and mainly inhibited early T cell activation. Compared with control T cell alone culture group, the levels of IL-2 and INF-γ were significantly down-regulated both in MSC group and MSC-OCT4 group.After co-culture with T cells for one week, the level of IL-6 significantly increased in MSC group and MSC-OCT4 group compared with that before co-culture. Compared with control MSC group, MSC-OCT4 group had higher viable cell numbers after 1 week of co-culture (P =0.019), and could resist the inhibition of proliferation by higher concentration of mitomycin C.@*CONCLUSION@#Both MSC and MSC-OCT4 can inhibit the proliferation and activation of IL-2-stimulated T cells in vitro. After overexpression of OCT4, MSC has better proliferation ability in vitro and can inhibit the proliferation of CD3+CD8+ T cells more effectively, which may have a better and more lasting immunosuppressive ability to regulate the balance of Th1/Th2.

The research progress on the impact of bone marrow mesenchymal stem cell-derived extracellular vesicles on traumatic brain injury / 中国神经精神疾病杂志

Traumatic brain injury(TBI)is a multifaceted disease with a complex pathogenesis for which there are currently no effective therapeutic interventions.Research has shown that bone marrow mesenchymal stem cell-derived extracellular vesicles(BMSC-EVs)may play a therapeutic role in TBI.They attenuate neuroinflammatory responses at the site of the lesion and promote neurovascular regeneration.However,the exact mechanisms underlying their actions are not fully understood.This article aims to review the current state of research on the therapeutic mechanisms of BMSC-EVs in TBI.It also aims to discuss possible future research directions and potential clinical applications of BMSC-EVs.

BMSCs inhibit inflammation in mice with LPS-induced acute lung injury by regulating TLR4/MyD88/NF-κB signaling pathway / 安徽医科大学学报

Objective To investigate the effect of bone marrow mesenchymal stem cells(BMSCs)on the inflamma-tory response of lipopolysaccharide(LPS)induced acute lung injury(ALI)in mice.Methods 32 SPF KM mice,aged 4 weeks were randomly divided into four groups,control group,LPS group,dexamethasone treatment group(LPS+DEX)and BMSCs treatment group(LPS+BMSCs).The latter three groups were injected with LPS by tra-cheal puncture to establish mouse ALI model 24 h after modeling,BMSCs isolated from the femur of mice were in-jected into the caudal vein,and DEX were injected into caudal vein at the same time in LPS+DEX group for 3 consecutive days.On the 4th day after cell transplantation or 24 h after DEX injection,the survival quantity of mice was recorded,lung function was detected,and the wet/dry weight ratio(W/D)of lung was measured.Then in-flammatory cells in bronchoalveolar lavage fluid(BALF),lung pathological changes and serum inflammatory cyto-kines were collected.Green fluorescent protein(GFP)staining was used to observe the homing of BMSCs in lung tissues.The mRNA and protein expression of TLR4,MyD88 and NF-κB in lung tissues were detected by RT-PCR and Western blot assay respectively.Results Compared with the control group,LPS model group showed de-creased lung function,significantly increase in the W/D weight ratio of lung,inflammatory cytokines in serum and inflammatory cells in BALF,and severe damage in lung tissue.Compared with LPS group,LPS+DEX group and LPS+BMSCs group showed improved lung function,reduced lung tissue damage,significantly decrease in the W/D weight ratio of lung,inflammatory cytokines in serum and inflammatory cells in BALF.And the expression of TLR4-MyD88-NF-κB signaling pathway-related genes and proteins decreased,the survival quantity increased.Conclusion Homologous BMSCs transplantation can effectively treat LPS-induced acute lung injury,and the mechanism may be related to the regulation of TLR4-MyD88-NF-κB signaling pathway and the reduction of inflam-matory response.These findings provide the experimental basis for BMSCs homologous transplantation for ALI.

bFGF and IGF-1 promotes differentiation of bone mesenchymal stem cells into cardiomyocyte-like cells and its related mechanisms / 中国药理学通报

Aim To elucidate the biological effects of insulin-like growth factor-1 ( IGF-1 ) and basic fibro-blast growth factor (bFGF) alone or in combination on the differentiation of bone mesenchymal stem cells (BMSCs) into cardiomyocytes (CMs), and to explore the mechanism in the differentiation process of BMSCs into CMs induced by IGF-1 or bFGF. Methods After four weeks of BMSCs induced by induction differentiation medium ( with or without LY294002) containing IGF-1 and/or bFGF, the expression levels of proteins associated with the cardiomyogenic phenotype in BMSCs were detected via immunocytochemistry, immuno-fluorescence staining, and Western blot. Meanwhile, the expression levels of pathway related proteins were detected by Western blot. The cell ultrastructure was observed by transmission electron microscopy (TEM) . The expression levels of myocardial specific genes were measured via RT-qPCR. Results Compared with the control group, the expression levels of myocardial specific proteins and genes significantly increased in the IGF-1, bFGF and combination groups and were the highest in the coinduction group. The TEM of the conduction group showed parallel myofilaments, mitochondria, endoplasmic reticulum and so on. The additon of LY294002 decreased the expression levels of myocardial specific proteins, genes and phosphorylation Akt. Conclusions The effect of IGF-1 combined with bFGF on the differentiation of BMSCs into CMs is markedly better than that induced by IGF-1 or bFGF a-lone, and the differentiation process may depend on the PI3K/Akt signaling pathway.

Chinese Medicine Regulates Signaling Pathways Related to Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells： A Review / 中国实验方剂学杂志

Bone marrow mesenchymal stem cells （BMSCs） are derived from stem cells isolated from bone marrow and have the potential for multidirectional differentiation and self-renewal. Under certain conditions， BMSCs can be induced to differentiate into osteoblast （OB）， chondrocyte， adipocyte， fibroblast， etc. BMSCs play an important role in maintaining the stability of bone structure and balancing bone metabolism. Promoting the proliferation of BMSCs and inducing their differentiation into OB of great significance for the clinical prevention and treatment of osteoporosis， bone defects， fracture healing， and other diseases. Because the proliferation and osteogenic differentiation of BMSCs are complex processes controlled by multiple genes and regulated by multiple signal transduction pathways， traditional Chinese medicine （TCM） happens to have the advantages of multi-bioactive component， multi-target， and multi-pathway synergism， which can affect the proliferation and differentiation of BMSCs through multiple channels and induce the proliferation of BMSCs. The transcription and expression of genes related to osteogenesis can be enhanced to promote the differentiation of BMSCs into OB， so as to achieve the purpose of preventing and treating osteoporosis， bone defects， and other bone diseases. Based on the literature on the intervention of TCM monomers and compounds in the proliferation and osteogenic differentiation of BMSCs， this study reviewed TCM monomers and compounds in promoting the proliferation and osteogenic differentiation of BMSCs by regulating secreted glycoprotein （Wnt）， neurogenic locus notch homolog protein （Notch）， mitogen-activated protein kinase （MAPK）， phosphatidylinositol-3 kinase （PI3K） /protein kinase B （Akt）， bone morphogenetic protein （BMP）/Smad， Janus kinase （JAK）/signal transducer and activator of transcription protein （STAT）， osteoprotegerin （OPG）/receptor activator of nuclear factor-kappa B （RANK）/RANK ligand （RANKL）， and other signaling pathways to provide new ideas for the research and clinical application of Chinese medicine in the prevention and treatment of orthopedic diseases.

Effects of Cyclic Strain on Neural Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells / 医用生物力学

Objective To study the mechanical effects of cyclic strain on neural differentiation of rat bone marrow mesenchymal stem cells (rBMSCs). Methods The rBMSCs were subjected to cyclic strain for 24 hours andthen cultured for 5 days. The expression of neural markers and the phosphorylation of relative signaling pathway proteins were evaluated. The stress distribution on cell surface was analyzed by finite element method. The differentially expressed genes induced by strain were identified by RNA sequencing analysis. Results The 0. 5 Hz strain with 5% magnitude could significantly induce higher expression of neural markers and elevated phosphorylation level of extracellular-signal-regulated kinase (ERK), protein kinase B (AKT) and mammalian target of rapamycin ( mTOR). KEGG pathway analysis showed that the focal adhesion and ECM-receptor interaction were significantly enriched under cyclic strain. Conclusions Cyclic strain could change the interaction of cells with the extracellular matrix ( ECM) and enhance the AKT/ mTOR and ERK pathway, finally promote rBMSC neural differentiation. Knowledge about the impact of mechanical stimulation on BMSC neural differentiation is expected to improve the efficiency of stem cell differentiation, shed light on device design for tissue engineering, and promote clinical application of mesenchymal stem cells in neural issue repair and regeneration.

Effects of bone marrow mesenchymal stem cell transplantation on bone metabolism and RANKL/OPG/TRAF6 signaling pathway in osteoporotic rats / 中华内分泌外科杂志

Objective:To investigate the effect of bone marrow derived mesenchymal stem cells (BMSC) transplantation on bone metabolism and its mechanism in ovariectomized osteoporosis rats.Methods:Forty clean SD female rats aged 7 weeks were divided into 4 groups according to the random number table method: sham operation group, model group, the transplantation group, positive control group, in addition to control the rest of the group were performed bilateral oophorectomy build osteoporosis rats model, after 2 months of model establishment, rats in transplantation group were injected with 80 μl/kg PBS solution containing bone marrow mesenchymal stem cells through tail vein, rats in sham operation group and model group were injected with the same amount of PBS solution through tail vein, and rats in positive control group were given Xianlinggubao (0.5 g/100 g) by gavage every day. Serum and femur were collected 14 days after treatment. Hematoxylin and eosin staining (HE) was used to observe the histopathological changes of femur. Micro-CT was used to measure bone mineral density and bone parameters. The expression levels of osteocalcin, osteoprotegerin, alkaline phosphatase and insulin-like growth factor 1 were detected by enzyme-linked immunosorbent assay (ELISA) kit. The serum levels of calcium, phosphorus and magnesium were measured by spectrophotometer. The protein expressions of RANKL, OPG, TRAF6 and NF-KB1 in femur of each group were detected by Western blot.Results:Compared with the sham operation group, the bone mineral density (BMD) of the model group was decreased by (0.28±0.01) g/cm 3, bone volume fraction (BMD) was decreased by (0.28±0.01) g/cm 3. BV/TV) decreased by (19.73±2.02) %, trabecular thickness (Tb.Th) decreased by (0.082±0.008) mm, trabecular number (Tb.N) decreased by (1.60±0.17) mm -1 and trabecular separation/spacing (Tb.Sp) increased (0.273±0.024) mm, osteoprotegerin (489.49±55.29) ng/L, alkaline phosphatase (229.13±15.05) U/L, insulin-like growth factor-1 (236.64±14.32) μg/L, and osteocalcin were decreased (1.866±0.109) μg/L, calcium (11.98±1.09) mg/dl, phosphorus (6.85±0.68) mg/dl, and magnesium decreased (0.62±0.04) mg/dl) , the relative expression level of RANKL increased (1.05±0.09) , the relative expression level of OPG decreased (0.58±0.08) , the relative expression level of RANKL increased (0.74±0.10) , and the relative expression level of NF-kB1 increased (1.01±0.11) ( P<0.05) ; bone mineral density, bone mineral density, bone mineral density BMD (0.38±0.04 g/cm 3, BV/TV (26.73±2.74) %, Tb.Th (0.094±0.006) mm, Tb.N (2.67±0.09) mm-1 and Tb.Sp were decreased (0.241±0.026) mm) , osteoprotegerin (720.09±67.41) ng/L, alkaline phosphatase (269.48±14.15) U/L, insulin-like growth factor 1 (IGF-1) decreased (335.95±24.13) μg/L, and osteocalcin increased (1.392±0.153) μg/L, calcium (7.12±0.53) mg/dl, phosphorus (4.54±0.32) mg/dl, magnesium (0.87±0.08) mg/dl. RANKL relative expression level increased (0.59±0.05) , OPG relative expression level decreased (0.97±0.10) , RANKL relative expression level increased (0.45±0.06) , NF-kB1 relative expression level increased (0.72±0.06) ( P<0.05) ;bone mineral density, bone mineral density, bone mineral density BMD (0.36±0.05) g/cm 3, BV/TV (28.72±3.20) %, Tb.Th (0.096±0.011) mm, Tb.N (2.85±0.24) mm -1 Tb.Sp was basically unchanged (0.241±0.027) mm, osteoprotegerin was decreased (716.78±36.90) ng/L, alkaline phosphatase was basically unchanged (270.65±18.59) U/L, and insulin-like growth factor 1 was decreased (336.94±17.50) μg/L, osteocalcin (1.377±0.101) μg/L, calcium (7.13±0.80) mg/dl, phosphorus (4.58±0.71) mg/dl, and magnesium (0.89±0.04) remained unchanged mg/dl, the relative expression level of RANKL increased (0.55±0.08) , the relative expression level of OPG decreased (0.98±0.13) , the relative expression level of RANKL was basically unchanged (0.40±0.05) , and the relative expression level of NF-kB1 increased (0.65±0.09) ( P<0.05) . Conclusion:Bone marrow mesenchymal stem cell transplantation can improve osteoporosis in ovariectomized rats by regulating bone metabolism and serum levels of calcium, phosphorus and magnesium, which may be related to RANKL/OPG/TRAF6 pathway.

Mechanism of Buyang Huanwutang Combined with Bone Marrow Mesenchymal Stem Cell Transplantation in Treatment of Spinal Cord Injury Based on PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

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.

Bone marrow mesenchymal stem cell-derived exosomal miR-30e-5p ameliorates high-glucose induced renal proximal tubular cell pyroptosis / 西安交通大学学报(医学版)

【Objective】 To study the effects of miR-30e-5p from bone marrow mesenchymal stem cell-derived exosomes(BMSC-exos) on high glucose (HG)-induced HK-2 cell pyroptosis and explore an alternative strategy to manage diabetic kidney disease (DKD). 【Methods】 BMSC-exos were isolated and internalized into HK-2 cells treated with HG to measure viability and cytotoxicity. The secretion of IL-1β and IL-18 was measured by ELISA. Pyroptosis was assessed by flow cytometry. The levels of miR-30e-5p, IL-1β, and IL-18 were measured. The expression of pyroptosis-associated cytokine proteins was determined. 【Results】 BMSC-exos decreased LDH, IL-1β, and IL-18 secretion and inhibited the expression of the pyroptosis-related factors (IL-1β, caspase-1, GSDMD-N, and NLRP3) in HG-induced HK-2 cells. Moreover, miR-30e-5p depletion in BMSC-exos promoted HK-2 cell pyroptosis. 【Conclusion】 BMSC-derived exosomal miR-30e-5p inhibits caspase-1-mediated pyroptosis in HG-induced HK-2 cells, which might provide a new strategy for treating DKD.

Experimental study of exosome derived from bone marrow mesenchymal stem cells in treating pancreatic cancer through regulation of tumor-associated macrophages polarization / 中华肝胆外科杂志

Objective:To investigate the therapeutic effect of exosomes divided from bone marrow mesenchymal stem cell (BMSC) on pancreatic cancer in vivo through regulation of tumor-associated macrophages (TAM) polarization.Methods:Ten male C57BL/6 mice weighing approximately 20 g, ages 4 weeks, were used for BMSC exosomes extraction and PKH26 labelling. Thirty female SPF BALB/c-nu/nu nude mice weighing approximately (18.56±0.85) g, ages 4-6 weeks, were adopted for pancreatic carcinoma models. The models were randomly divided into 3 groups with 10 in each: control group (injected with PBS through tail vein), portal vein treatment group (injected with exosomes in PBS through portal vein), tail vein treatment group (injected with exosomes in PBS through tail vein). After the models were executive 8 weeks later, the percentage of PKH26 positive-exosomes in pancreas tissue was quantified by flow cytometry technique. The volume of the primary pancreatic tumor, the tumor volume of inhibitory rate, the number of metastatic nodule, and the ascitic fluid were assess. Also, the weight of liver and the tumor weight were evaluated. The expression of M1 and M2 macrophage-activate biomarkers and the content of pancreatic cancer marker B7-H4 tumor carbohydrate antigen 199 in peripheral blood was detected. Moreover, the expression of Survivin and matrix metalloproteinase-9 (MMP-9) in pancreatic cancer tissue cells was also detected.Results:There was no significant difference for the tumor volume of inhibitory rate between portal vein treatment group (72.4±21.6)% and tail vein treatment group (70.1±20.7)% ( t=0.24, P=0.811). Compared with control group, the volume of the primary pancreatic tumor, the weight of liver, the tumor weight, the number and rate of liver metastatic nodule, the ascitic fluid, and the number of other metastatic nodule were totally lower in the 2 treatment groups with significant difference (all P<0.05). Compared with the control group, iNOS and CD68 in portal vein treatment group and tail vein treatment group were increased, while Arginase, CD206, B7-H4, tumor carbohydrate antigen 199, Survivin and matrix metalloprotein-9 were decreased, with statistically significant differences (all P<0.05). Conclusion:Exosomes derived from BMSC can inhibit the polarization of TAM to the M2 phenotype and induce their polarization to the M1 phenotype, thereby suppressing the proliferation, invasion and migration of pancreatic cancer.