Objective To explore the mechanism of ubiquitin specific peptidase 21 (USP21) increasing the stability of forkhead box protein M1 (FOXM1) and promoting M2 polarization of macrophages in endometriosis (EM). Methods Eutopic endometrial stromal cells (EESC) collected from patients and normal endometrial stromal cells (NESC) from routine health examiners were cultured in vitro, and the expression levels of USP21 and FOXM1 were detected using RT-qPCR and Western blot. EESCs were co-cultured with macrophages. M1 polarization markers of interleukin 6 (IL-6) and CXC chemokine ligand 10 (CXCL10) and M2 polarization markers of CD206 and fibronectin 1 (FN1) were tested using RT-qPCR. M2 marker CD206 was further detected by flow cytometry. IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, and transforming growth factor-beta (TGF-β) levels in cell supernatant were detected by ELISA. Co-immunoprecipitation was used to assess the interaction between USP21 and FOXM1, and the ubiquitination level of FOXM1. FOXM1 protein stability was detected through cycloheximide (CHX) assay. Results USP21 and FOXM1 expression levels in the EESC group were significantly increased compared with those in the NESC group; compared with the NESC + M0 group, the EESC + M0 group showed no significant difference in the expression of M1 polarization markers (IL-6 and CXCL10), but increased expression of M2 polarization markers (CD206 and FN1), along with notably increased number of M2 macrophages; there was no significant difference in IL-6 and TNF-α levels, but increased levels of IL-10 and TGF-β in the cell supernatant. The above findings indicated that the deubiquitinase USP21 was highly expressed in EM, promoting M2 polarization of macrophages. Knocking down USP21 or FOXM1 can inhibit M2 polarization of EM macrophages. USP21 interacted with FOXM1 in EESC, leading to a decrease in FOXM1 ubiquitination level and an increase in FOXM1 protein stability. Overexpression of FOXM1 reversed the inhibitory effect of knocking down USP21 on M2 polarization of EM macrophages. Conclusion The deubiquitinase USP21 interacts with FOXM1 to increase the stability of FOXM1 and promote M2 polarization of EM macrophages.
Humans ; Forkhead Box Protein M1/genetics* ; Female ; Macrophages/cytology* ; Endometriosis/genetics* ; Ubiquitin Thiolesterase/genetics* ; Cells, Cultured ; Endometrium/metabolism* ; Ubiquitination ; Adult ; Interleukin-10/metabolism* ; Interleukin-6/metabolism* ; Protein Stability ; Stromal Cells/metabolism*

BackgroundCorneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear.

MethodsADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens.

Results:ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40-200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = -82.742, P < 0.01; collagen II: t = -72.818, P < 0.01; collagen III: t = -104.452, P < 0.01; collagen IV: t = -133.426, P < 0.01, and collagen V: t = -294.019, P < 0.01; and fibronectin: t = -92.491, P < 0.01, respectively).

Conclusion:The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

Adipose Tissue ; cytology ; Animals ; Cell Proliferation ; physiology ; Cell Survival ; physiology ; Cells, Cultured ; Exosomes ; metabolism ; Extracellular Matrix ; metabolism ; Fibroblasts ; cytology ; metabolism ; Matrix Metalloproteinases ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rabbits

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.
Acute Lung Injury/chemically induced/*therapy ; Angiopoietin-1/*genetics ; Animals ; Bronchoalveolar Lavage Fluid ; Cytokines/metabolism ; Endotoxins ; Genetic Therapy ; Interleukin-10/metabolism ; Interleukin-6/metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung/metabolism ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/metabolism ; Neutrophils/metabolism ; Rats ; Transforming Growth Factor beta1/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Umbilical Cord/*cytology

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.
Epithelial Cells/*physiology ; Epithelium/metabolism ; Hepatic Stellate Cells/*physiology ; Humans ; Liver/*cytology/injuries/*physiology ; Liver Cirrhosis/etiology/prevention & control ; Liver Regeneration/*physiology ; Mesenchymal Stromal Cells/physiology ; Myofibroblasts/physiology

OBJECTIVETo explore the role of CXCR4/STAT3 in mesenchymal stromal cell (MSC)-mediated drug resistance of AML cells.

METHODSAML cell lines U937 and KG1a and primary AML cells were co-cultured with MSC from bone marrow of healthy donors. The AML cell lines cultured alone were used as control. Apoptosis induced by mitoxantrone was measured by flow cytometry. Expression of CXCR4 and STAT3 protein were detected by Western blot. After incubated with STAT3 inhibitor Cucurbitacin I or CXCR4 antagonist AMD3100, the apoptosis of AML cells induced by mitoxantrone was evaluated.

RESULTSApoptosis of AML cells (U937 and KG1a) and primary AML cells induced by mitoxantrone significantly decreased in cocultured group than that of control group [U937 cells: (20.08±1.53)% vs (45.33 ± 1.03)% , P=0.004; KG1a cells: (25.60 ± 1.82)% vs (40.33 ± 3.29)% , P=0.020]. Expression of phosphorylated STAT3 and CXCR4 protein in AML cells were upregulated in cocultured group. After addition of Cucurbitacin I into the co-culture system, the apoptosis rate of primary AML cells significantly increased. Similar results of the apoptosis rates were also detected when the inhibitor of CXCR4 AMD3100 was added to overcome the stromal cell-mediated drug resistance. Besides, the expression of p-STAT3 in AML cells after incubated with AMD3100 decreased significantly.

CONCLUSIONSAML cells cocultured with MSC leads to the up-regulation of phosphorylated STAT3 and CXCR4 proteins, which resulted in AML cells resistance to chemotherapeutic drugs. Therefore targeting STAT3 or CXCR4 could be a new therapeutic strategy of AML.

Acute Disease ; Apoptosis ; Coculture Techniques ; Drug Resistance, Neoplasm ; Flow Cytometry ; Gene Expression Regulation, Leukemic ; Heterocyclic Compounds ; Humans ; Leukemia ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Receptors, CXCR4 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; U937 Cells ; Up-Regulation

OBJECTIVETo investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts.

METHODS(1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post scratch hour (PSH) 0 (immediately after scratch), 12, 24, 48, and 72, the migration distance of cells was observed and measured with inverted phase contrast microscope. (6) Human fibroblasts were grouped and treated as in (5), with 3 battles in each group, and apoptosis rate of cells was detected by flow cytometer. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, one-way analysis of variance, LSD test, and t test.

RESULTS(1) On culture day 3, most hAMSCs were in large form, and spindle-shaped with much prominences like fibroblasts or in flat polygonal shape. hAMSCs of the third passage were spindle-shaped. The expression of vimentin of hAMSCs of the third passage was strongly positive, and the expressions of surface markers CD90, CD73, and CD105 of the cells were positive, while the expression of CD45 of the cells was negative. (2) The content of IGF-Ⅰ, VEGF, EGF, and bFGF in hAMSCs-CS were respectively (11.7±1.0), (316±68), (6.1±0.4), and (1.49±0.05) pg/mL. (3) At culture hour 12-72, the proliferation activity of human fibroblasts in each hAMSCs-CS group was significantly higher than that in blank control group (with P values below 0.01), and the proliferation activity of human fibroblasts in 50% hAMSCs-CS group was the highest. (4) The width of scratch in two groups was nearly the same at PSH 0. The migration distance of cells in 50% hAMSCs-CS group was significantly longer than that in blank control group at PSH 12-72 (with P values below 0.01). (5) The apoptosis rate of human fibroblasts in blank control group was (16.2±2.4)%, which was significantly higher than that in 50% hAMSCs-CS group [(7.4±3.6)%, t=6.710, P<0.01].

CONCLUSIONShAMSCs-CS can promote proliferation and migration of human fibroblasts and inhibit the apoptosis of human fibroblasts.

Amnion ; cytology ; Apoptosis ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; Enzyme-Linked Immunosorbent Assay ; Epidermal Growth Factor ; metabolism ; Female ; Fibroblast Growth Factor 2 ; metabolism ; Fibroblasts ; cytology ; drug effects ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; metabolism ; Male ; Mesenchymal Stromal Cells ; chemistry ; Pregnancy ; Vascular Endothelial Growth Factor A ; metabolism

Objective To compare the effecacy of human mesenchymal stromal cell (hMSC) with human mononuclear cell (hMNC) in treating rat cerebral infarct.Methods The SD rat models of cerebral infarct were established by distal middle cerebral artery occlusion (dMCAO). Rats were divided into four groups: sham,ischemia vehicle,MSC,and MNC transplantation groups. For the transplantation group,1×10hMSCs or hMNCs were intravascularly transplanted into the tail vein 1 hour after the ischemia onset. The ischemia vehicle group received dMCAO surgery and intravascular saline injection 1,3,5,and 7 days after the ischemia onset,and then behavioral tests were performed. At 48 h after the ischemia onset,the abundance of Iba- 1,the symbol of activated microglia,was evaluated in the peri-ischemia striatum area; meanwhile,the neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in ipsilateral peri-ischemia striatum area were also measured. Results The relative infarct volume in ischemia vehicle group,hMSC group,and hMNC transplantation group were (37.85±4.40)%,(33.41±3.82)%,and (30.23±3.63)%,respectively. The infarct volumes of MSC group (t=2.100,P=0.034) and MNC group (t=2.109,P=0.0009) were significantly smaller than that of ischemia vehicle group,and that of MNC group was significantly smaller than that of MSC group (t=1.743,P=0.043). One day after transplantation,the score of ischemia vehicle group in limb placing test was (4.32±0.71)%,which was significantly lower than that in sham group (9.73±0.36)% (t=2.178,P=8.61×10). The scores of MSC and MNC group,which were (5.09±0.62)% (t=2.1009,P=0.024) and (5.90±0.68)% (t=2.1008,P=0.0001),respectively,were significantly higher than that of ischemia vehicle group; also,the score of MNC group was significantly higher than that of MSC group(t=2.1009,P=0.0165). The contralateral forelimb scores of MSC and MNC groups in beam walking test were (5.56±0.86)% (t=2.120,P=0.020) and (5.13±0.95)% (t=2.131,P=0.003),were both significantly lower than that of ischemia vehicle group [(6.47±0.61)%]. Three days after the transplantation,the limb placing test score of MNC group [(6.91±1.10)%] was significantly higher than that of ischemia vehicle group (5.80±0.82)% (t=2.110,P=0.027). The score of MSC group [(6.30±0.77)%] showed no statistic difference with that of ischemia vehicle group(t=2.101,P=0.199).The contralateral forelimb scores of MNC group in beam walking test [(4.34±0.58)%] was significantly lower than that of ischemia vehicle group [(5.31±0.65)%] (t=2.100,P=0.006) and MSC group [(4.92±0.53)%] (t=2.100,P=0.041); there was no statistic difference between MSC group and ischemia vehicle group (t=2.109,P=0.139). The relative abundance of Iba- 1 in sham,ischemia vehicle,MSC,and MNC groups was 1.00+0.00,1.72±0.21,1.23±0.08,and 1.48±0.06,respectively. The Iba-1 relative abundance of ischemia vehicle group was significantly higher than that of sham group (t=2.262,P=2.9×10). The Iba-1 relative abundances of both MSC (t=2.178,P=3.91×10)and MNC (t=2.200,P=0.007)groups were significantly lower than that of ischemia vehicle group. It was also significantly lower in MNC group than in MSC group also (t=2.120,P=7.09×10). Three days after transplantation,the BDNF and GDNF levels of MSC group,which were (531.127±73.176)pg/mg (t=2.109,P=0.003)and(127.780±16.733)pg/mg(t=2.100,P=2.76×10),respectively,were significantly higher than those of ischemia vehicle group,which were (401.988±89.006)pg/mg and (86.278±14.832) pg/mg,respectively. The BDNF and GDNF levels of MNC group,which were (627.429±65.646)pg/mg (t=2.144,P=0.017) and (153.117±20.443)pg/mg (t=2.109,P=0.010),respectively,were all significantly higher than that of MSC group. At day 7,the BDNF and GDNF levels of MSC group,which were (504.776±83.282)pg/mg (t=2.101,P=0.005) and (81.641±11.019)pg/mg (t=2.100,P=0.002),respectively,were significantly higher than those of ischemia vehicle group,which were (389.257±70.440)pg/mg and (64.322±9.855) pg/mg,respectively. The BDNF and GDNF levels of MNC group,which were (589.068±63.323)pg/mg (t=2.100,P=0.027) and (102.161±19.932)pg/mg (t=2.144,P=0.017),respectively,were all significantly higher than that of MSC group. Conclusions Both hMSC and hMNC are beneficial to the ischemia-damaged brain when they are intravascularly transplanted within 1 h after the onset of ischemia. The anti-inflammation ability and secretion of neurotrophic factors are the underlying mechanisms of the therapeutic effects. MNC is more effective than MSC in reducing infarct area and improving behaviors,which might be explained by the fact that MNC induces more GDNF and BDNF in brain than MSC.
Animals ; Bone Marrow ; Brain Ischemia ; therapy ; Brain-Derived Neurotrophic Factor ; metabolism ; Disease Models, Animal ; Fetus ; Glial Cell Line-Derived Neurotrophic Factor ; metabolism ; Humans ; Infarction, Middle Cerebral Artery ; therapy ; Leukocytes, Mononuclear ; cytology ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells.

METHODSThe co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b.

RESULTSUC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells.

CONCLUSIONUC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.

Cell Differentiation ; Cell Line, Tumor ; Humans ; Interleukin-6 ; metabolism ; Leukemia, Promyelocytic, Acute ; pathology ; Mesenchymal Stromal Cells ; metabolism ; Tretinoin ; pharmacology ; Umbilical Cord ; cytology

BACKGROUNDThe functional improvement following bone marrow stromal cells (BMSCs) transplantation after stroke is directly related to the number of engrafted cells and neurogenesis in the injured brain. Here, we tried to evaluate whether 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), a free radical scavenger, might influence BMSCs migration to ischemic brain, which could promote neurogenesis and thereby enhance treatment effects after stroke.

METHODSRat transient middle cerebral artery occlusion (MCAO) model was established. Two separate MCAO groups were administered with either MCI-186 or phosphate-buffered saline (PBS) solution to evaluate the expression of stromal cell-derived factor-1 (SDF-1) in ischemic brain, and compared to that in sham group (n = 5/ group/time point[at 1, 3, and 7 days after operation]). The content of chemokine receptor-4 (CXCR4, a main receptor of SDF-1) at 7 days after operation was also observed on cultured BMSCs. Another four MCAO groups were intravenously administered with either PBS, MCI-186, BMSCs (2 × 106), or a combination of MCI-186 and BMSCs (n = 10/group). 5-bromo-2-deoxyuridine (BrdU) and Nestin double-immunofluorescence staining was performed to identify the engrafted BMSCs and neuronal differentiation. Adhesive-removal test and foot-fault evaluation were used to test the neurological outcome.

RESULTSMCI-186 upregulated the expression of SDF-1 in ischemic brain and CXCR4 content in BMSCs was enhanced after hypoxic stimulation. When MCAO rats were treated with either MCI-186, BMSCs, or a combination of MCI-186 and BMSCs, the neurologic function was obviously recovered as compared to PBS control group (P < 0.01 or 0.05, respectively). Combination therapy represented a further restoration, increased the number of BMSCs and Nestin+ cells in ischemic brain as compared with BMSCs monotherapy (P < 0.01). The number of engrafted-BMSCs was correlated with the density of neuronal cells in ischemic brain (r = 0.72 , P < 0.01) and the improvement of foot-fault (r = 0.70, P < 0.01).

CONCLUSIONMCI-186 might promote BMSCs migration to the ischemic brain, amplify the neurogenesis, and improve the effects of cell therapy.

Animals ; Antipyrine ; analogs & derivatives ; therapeutic use ; Bone Marrow Cells ; cytology ; physiology ; Brain Ischemia ; drug therapy ; metabolism ; therapy ; Chemokine CXCL12 ; metabolism ; Disease Models, Animal ; Infarction, Middle Cerebral Artery ; drug therapy ; metabolism ; therapy ; Male ; Mesenchymal Stromal Cells ; physiology ; Neurogenesis ; physiology ; Rats ; Rats, Sprague-Dawley ; Stroke ; drug therapy ; metabolism ; therapy

OBJECTIVETo observe the effect of Jianpi Bushen Qingchang Huashi Recipe (JBQHR) on proliferation and migration of bone marrow mesenchymal stem cells (BMSCs).

METHODSBMSCs were isolated and cultured in vitro with adherence screening method to prepare cell suspension. No drug intervention was given to BMSCs in the vehicle control group. JBQHR at 0.39, 0.78, 1.56 µg/mL was added in BMSCs of low, mid, and high dose JBQHR groups for co-incubation. Its effect on the proliferation of BMSCs was detected by CCK-8. BMSCs migration and chemotactic ability was detected using Transwell method. Each dose JBQHR combined ERK kinase inhibitor U0126 was set up as control. The phosphorylation of extracellular regulated protein kinase (ERK) and CAMP responsive element-binding protein (CREB) were detected by Western blot.

RESULTSCompared with the vehicle control group, the proliferation of BMSCs and BMSCs migration number could be promoted in the 3 JBQHR groups (P < 0.05). Besides, the proliferation of BMSCs was better in mid and high dose JBQHR groups than in the low dose JBQHR group (P < 0.05). Compared with the vehicle control group, the phosphorylation of ERK and CREB could be elevated in the 3 JBQHR groups (P < 0.05), and could be inhibited by U0126 (P < 0.01). Compared with the low dose JBQHR group, the phosphorylation of ERK increased in mid and high dose JBQHR groups with statistical difference (P < 0.05).

CONCLUSIONJBQHR could promote the proliferation and migration of BMSCs, and its mechanism might be related to ERK/CREB signaling pathway

Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; MAP Kinase Signaling System ; Mesenchymal Stromal Cells ; cytology ; drug effects