OBJECTIVE: To prepare clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with high expression of hematopoietic supporting factors and evaluate their stem cell characteristics. METHODS: Fetal umbilical cord tissues were collected from healthy postpartum women during full-term cesarean section. Wharton's jelly was mechanically separated and hUC-MSCs were obtained by explant culture method and enzyme digestion method in an animal serum-free culture system with addition of human platelet lysate. The phenotypic characteristics of hUC-MSCs obtained by two methods were detected by flow cytometry. The differences in proliferation ability between the two groups of hUC-MSCs were identified through CCK-8 assay and colony forming unit-fibroblast (CFU-F) assay. The differences in multilineage differentiation potential between the two groups of hUC-MSCs were identified through induction of adipogenic, osteogenic, and chondrogenic differentiation. The mRNA expression levels of hematopoietic supporting factors such as SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in the two groups of hUC-MSCs were identified by real-time fluorescence quantiative PCR(RT-qPCR). RESULTS: The results of flow cytometry showed that hUC-MSCs obtained by the two methods both expressed high levels of CD73, CD90 and CD105, while lowly expressed CD31, CD45 and HLA-DR. The results of CCK-8 and CFU-F assay showed that the proliferation ability of hUC-MSCs obtained by explant culture method was better than those obtained by enzyme digestion method. The results of the triple lineage differentiation experiment showed that there was no significant difference in multilineage differentiation potential between the two grous of hUC-MSCs. The results of RT-qPCR showed that the mRNA expression levels of hematopoietic supporting factors SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in hUC-MSCs obtained by explant cultrue method were higher than those obtained by enzyme digestion method. CONCLUSION Clinical-grade hUC-MSCs with high expression levels of hematopoietic supporting factors were successfully cultured in an animal serum-free culture system.
Humans ; Mesenchymal Stem Cells/metabolism* ; Umbilical Cord/cytology* ; Cell Differentiation ; Female ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Angiopoietin-1/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Stem Cell Factor/metabolism* ; Flow Cytometry ; Pregnancy

Objective:To investigate the effects and underlying mechanism of ionizing radiation on the adipogenic of mesenchymal stem cells(MSCs).Methods:Mouse MSCs were cultured in vitro and treated with 2 Gy and 6 Gy radiation with 60Co,and the radiation dose rate was 0.98 Gy/min.Bulk RNA-seq was performed on control and irradiated MSCs.The changes of adipogenic differentiation and oxidative stress pathways of MSC were revealed by bioinformatics analysis.Oil Red O staining was used to detect the adipogenic differentiation ability of MSCs in vitro,and real-time fluorescence quantitative PCR(qPCR)was used to detect the expression differences of key regulatory factors Cebpa,Lpl and Pparg after radiation treatment.At the same time,qPCR and Western blot were used to detect the effect of inhibition of Nrf2,a key factor of antioxidant stress pathway,on the expression of key regulatory factors of adipogenesis.Moreover,the species conservation of the irradiation response of human bone marrow MSCs and mouse MSC was determined by qPCR.Results:Bulk RNA-seq suggested that ionizing radiation promotes adipogenic differentiation of MSCs and up-regulation of oxidative stress-related genes and pathways.The results of Oil Red O staining and qPCR showed that ionizing radiation promoted the adipogenesis of MSCs,with high expression of Cebpa,Lpl and Pparg,as well as oxidative stress-related gene Nrf2.Nrf2 pathway inhibitors could further enhance the adipogenesis of MSCs in bone marrow after radiation.Notably,the similar regulation of oxidative pathways and enhanced adipogenesis post irradiation were observed in human bone marrow MSCs.In addition,irradiation exposure led to up-regulated mRNA expression of interleukin-6 and down-regulated mRNA expression of colony stimulating factor 2 in human bone marrow MSCs.Conclusion:Ionizing radiation promotes adipogenesis of MSCs in mice,and oxidative stress pathway participates in this effect,blocking Nrf2 further promotes the adipogenesis of MSCs.Additionally,irradiation activates oxidative pathways and promotes adipogenic differentiation of human bone marrow MSCs.

Objective:To establish an in vitro cell model simulating acute graft-versus-host disease(aGVHD)bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells(MSCs).Methods:The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model,respectively.Bone marrow transplantation(BMT)mouse model(n=20)was established by being injected with bone marrow cells(1 × 10'per mouse)from donor mice within 4-6 hours after receiving a lethal dose(8.0 Gy,72.76 cGy/min)of y ray general irradiation.A mouse model of aGVHD(n=20)was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells(1 × 107 per mouse)and spleen lymphocytes(2 × 106 per mouse).The blood was removed from the eyeballs and the mouse serum was aspirated on the 7th day after modeling.Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2％,5％and 10％BMT mouse serum and aGVHD mouse serum in the medium,respectively.The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining.The expression levels of related proteins PPARy and CEBPα were detected by Western blot.The expression differences of key adipogenic transcription factors including PPARy,CEBPα,FABP4 and LPL were determined by real-time quantitative PCR(RT-qPCR).Results:An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established.Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10％compared with BMT serum.Western blot experiments showed that adipogenesis-related proteins PPARy and CEBPα expressed in MSCs were down-regulated.Further RT-qPCR assay showed that the production of PPARy,CEBPα,FABP4 and LPL,the key transcription factors for adipogenic differentiation of MSC,were significantly reduced.Conclusion:The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.

Objective:To investigate the correlation between serum Chemerin and nerve growth associated protein (GAP) -43 levels and hyperhomocysteinemia (HHcy) in acute ischemic stroke.Methods:The clinical data of 149 patients with acute ischemic stroke admitted to the Sun Simiao Hospital of Beijing University of Traditional Chinese Medicine from March 2021 to May 2023 were retrospectively analyzed, and the patients were divided into observation group (97 cases) and control group (52 cases) according to whether they were combined with HHcy. Observation group patients were divided into mild group ( n=29), moderate group ( n=38) and severe group ( n=30) according to the severity of the disease. The serum Chemerin and GAP-43 levels of the observation group and the control group were compared, and the serum Chemerin and GAP-43 levels of the observation group with different severity of disease were compared, and the correlation between the serum Chemerin and GAP-43 levels and the severity of acute ischemic stroke patients with HHcy was analyzed. The influencing factors of poor prognosis in patients with acute ischemic stroke complicated with HHcy were analyzed, and receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of serum Chemerin and GAP-43 for poor prognosis in patients with acute ischemic stroke complicated with HHcy. Results:The levels of Chemerin and GAP-43 in the observation group were higher than those in the control group (all P<0.05). Serum Chemerin and GAP-43 levels in the severe group were higher than those in the mild group and the moderate group (all P<0.05), and serum Chemerin and GAP-43 levels in the moderate group were higher than those in the mild group (all P<0.05). Pearson correlation analysis showed that serum Chemerin and GAP-3 levels were positively correlated with the severity of acute ischemic stroke patients with HHcy ( r=0.458, 0.497, all P<0.05). Among 97 patients with acute ischemic stroke complicated with HHcy, 19 had poor prognosis and 78 had good prognosis. In the poor prognosis group, the proportion of diabetes, the severity of the disease and the levels of blood glucose, C-reactive protein (CRP), vascular endothelial growth factor (VEGF) and malondialdehyde (MDA) were higher than those in the good prognosis group (all P<0.05), and the superoxide dismutase (SOD) was lower than those in the good prognosis group ( P<0.05). Logistic regression analysis showed that the severity of the disease, CRP, Chemerin and GAP-43 were the influential factors for the poor prognosis of acute ischemic stroke patients with HHcy (all P<0.05). ROC curve results showed that the area under the curve (AUC) of serum Chemerin and GAP-43 levels and their combination in predicting poor prognosis of acute ischemic stroke patients with HHcy were 0.799, 0.804 and 0.907, respectively, and the AUC of the combination was larger ( P<0.05). Conclusions:Serum Chemerin and GAP-43 levels are closely related to the severity and prognosis of acute ischemic stroke patients with HHcy, and their combined value is higher in predicting poor prognosis.

Objective:To investigate the correlation between serum Chemerin and nerve growth associated protein (GAP) -43 levels and hyperhomocysteinemia (HHcy) in acute ischemic stroke.Methods:The clinical data of 149 patients with acute ischemic stroke admitted to the Sun Simiao Hospital of Beijing University of Traditional Chinese Medicine from March 2021 to May 2023 were retrospectively analyzed, and the patients were divided into observation group (97 cases) and control group (52 cases) according to whether they were combined with HHcy. Observation group patients were divided into mild group ( n=29), moderate group ( n=38) and severe group ( n=30) according to the severity of the disease. The serum Chemerin and GAP-43 levels of the observation group and the control group were compared, and the serum Chemerin and GAP-43 levels of the observation group with different severity of disease were compared, and the correlation between the serum Chemerin and GAP-43 levels and the severity of acute ischemic stroke patients with HHcy was analyzed. The influencing factors of poor prognosis in patients with acute ischemic stroke complicated with HHcy were analyzed, and receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of serum Chemerin and GAP-43 for poor prognosis in patients with acute ischemic stroke complicated with HHcy. Results:The levels of Chemerin and GAP-43 in the observation group were higher than those in the control group (all P<0.05). Serum Chemerin and GAP-43 levels in the severe group were higher than those in the mild group and the moderate group (all P<0.05), and serum Chemerin and GAP-43 levels in the moderate group were higher than those in the mild group (all P<0.05). Pearson correlation analysis showed that serum Chemerin and GAP-3 levels were positively correlated with the severity of acute ischemic stroke patients with HHcy ( r=0.458, 0.497, all P<0.05). Among 97 patients with acute ischemic stroke complicated with HHcy, 19 had poor prognosis and 78 had good prognosis. In the poor prognosis group, the proportion of diabetes, the severity of the disease and the levels of blood glucose, C-reactive protein (CRP), vascular endothelial growth factor (VEGF) and malondialdehyde (MDA) were higher than those in the good prognosis group (all P<0.05), and the superoxide dismutase (SOD) was lower than those in the good prognosis group ( P<0.05). Logistic regression analysis showed that the severity of the disease, CRP, Chemerin and GAP-43 were the influential factors for the poor prognosis of acute ischemic stroke patients with HHcy (all P<0.05). ROC curve results showed that the area under the curve (AUC) of serum Chemerin and GAP-43 levels and their combination in predicting poor prognosis of acute ischemic stroke patients with HHcy were 0.799, 0.804 and 0.907, respectively, and the AUC of the combination was larger ( P<0.05). Conclusions:Serum Chemerin and GAP-43 levels are closely related to the severity and prognosis of acute ischemic stroke patients with HHcy, and their combined value is higher in predicting poor prognosis.

ObjectiveTo investigate the effect of Astragalin （AST） on apoptosis of cerebral cortex neurons in APP/PS1 transgenic mice. MethodsEighteen six-month-old male APP/PS1 transgenic mice were randomly divided into APP/PS1 group， APP/PS1+ 40 mg/kg AST group and APP/PS1+ 20 mg/kg Donepezil （DNP） group， with six mice in each group. At the same time， six male C57BL/6 mice were selected as the normal control group. After intraperitoneal injection of AST once a day and continuous administration for one month， we used Tunel staining to detect the apoptosis of neurons in the cerebral cortex of APP/PS1 mice； immunofluorescent staining to examine the expression of apoptosis-related proteins Bax， Bcl-2， Caspase9 and Cleaved-Caspase3 in the cerebral cortex neurons of APP/PS1 mice； Western blot method to evaluate the changes of the expression of Bax， Bcl-2， Caspase9 and Caspase3. ResultsTunel staining showed that 40 mg/kg AST and 20 mg/kg DNP both reduced the apoptosis of neurons in the cerebral cortex of APP/PS1 mice， AST with more significant inhibition effect. Immunofluorescent staining revealed that 40 mg/kg AST and 20 mg/kg DNP both inhibited the expression of Bax， Caspase9， and Cleaved-Caspase3， and icreased the expression of Bcl-2 in the cerebral cortex neurons of APP/PS1 mice. Western blot results further confirmed that 40 mg/kg AST and 20 mg/kg DNP both down-regulated the expression of Bax （P < 0.05， P < 0.05）， Caspase9 （P < 0.005， P < 0.05） and Caspase3 （P < 0.0001， P < 0.0001） ， and up-regulated the expresstion of Bcl-2 （P < 0.05， P < 0.05） in the cerebral cortex neurons of APP/PS1 mice. ConclusionsAST can inhibit the apoptosis of cerebral cortex neurons in APP/PS1 mice.

OBJECTIVE: To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. METHODS: Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). RESULTS: ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). CONCLUSION MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.
Female ; Rats ; Animals ; Tumor Necrosis Factor-alpha ; Osteogenesis ; Rats, Sprague-Dawley ; Core Binding Factor Alpha 1 Subunit ; Melatonin/pharmacology* ; Interleukin-6/genetics* ; Lipopolysaccharides/pharmacology* ; Coloring Agents ; Inflammation

Objective To investigate the influence of volatile oil from Acori graminei Rhizoma (VOA) on expressions of glial fibrillary acidic protein (GFAP), c-Jun N-terminal protein kainse (JNK) and tumour necrosis factor-α (TNF-α) in the spinal cord dorsal horn of imflammatory pain rats. Methods Totally 36 male SD rats were randomly divided into control group (control), sham-operated group (sham), complete Freund' s adjuvant group (CFA), 5 g/(kg·d) low dose VOA+CFA group (VOA-L+CFA), 10 g/(kg·d) medium dose VOA + CFA group (VOA-M+CFA) and 20 g/(kg·d) high dose VOA + CFA group (VOA-H+CFA). All animals were sacrificed immediately after continuous gavage administration for 22 days. The expressions of GFAP, JNK and TNF-α in the spinal cord dorsal horn of rats in each group were detected by immunofluorescence and Western blotting methods. Results The present results showed that the positive expressions of GFAP, JNK and TNF-α in the spinal cord dorsal horn of rats increased significantly in the CFA group, when compared to the control and sham groups (P < 0. 01). The expressions of GFAP, JNK and TNF-α in the spinal cord dorsal horn of rats with VOA treatment reduced in the dose-dependent manner, when compared to the CFA group, the positive expressions of GFAP, JNK and TNF-α reduced significantly in the dorsal horn of the spinal cord of the VOA-H+CFA group (P<0. 05, P<0. 01). Conclusion VOA reduces the expressions of GFAP, JNK and TNF-α in the spinal cord dorsal horn of rats of CFA-induced inflammatory pain.

ObjectiveTo explore the effect of astragalin （AST） on neurons and Aβ plaques in the cortex of APP/PS1 transgenic mice. MethodsTwenty-four 8-month-old male APP/PS1 transgenic mice were randomly divided into APP/PS1 group， 10 mg/kg AST （APP/PS1+AST 10） group， and 20 mg/kg AST （APP/PS1+AST 20） group， 40 mg/kg AST （APP/PS1+AST 40） group， with 6 mice in each group. Six C57BL/6 male mice of the same age served as the control group （WT group）. AST drugs were continuously injected intraperitoneally for one month. Then Immunofluorescent staining was used to observe the deposition of Aβ plaques in the cortex. Nissl staining was used to observe the number and morphological changes of neurons in the cortex， and immunofluorescent multiple staining methods were used to observe the co-expression of LC3B， p62 and NeuN in the cortex. Then the expressions of NeuN， LC3B， and p62 protein were detected by Western blot method. ResultsImmunofluorescent staining results showed 20 mg/kg and 40 mg/kg AST reduced Aβ plaques deposition in the cortex of APP/PS1 mice （P < 0.000 1； P < 0.000 1）. Western blot analysis showed both 20 and 40 mg/kg AST increased the expression of NeuN protein in the cortex of APP/PS1 mice （P = 0.012 1； P < 0.000 1）. Immunofluorescent multiplex staining showed co-expression of LC3B， p62， and NeuN in the cortex of APP/PS1 mice. Western blot analysis showed AST increased the expression of LC3B （P = 0.007， P < 0.000 1） and decreased the expression of p62 （P < 0.000 1， P < 0.000 1） in the cortex of APP/PS1 mice. ConclusionsAST reduces neuronal damage and Aβ plaques deposition in the cortex of APP/PS1 mice by activating autophagy.