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Objective@#To investigate the therapeutic and immunomodulatory effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on adjuvant arthritis(AA) rats.@*Methods@#Twenty-four male Sprague-Dawley(SD) rats were established with AA by complete Freund's adjuvant method.They were randomly divided into model group and hUC-MSCs group (2×106 cells/mL, 5×106 cells/mL, tail vein injection), and the Yisaipu group (2.8mg/kg, subcutaneous injection), 6 rats in each group.Another 6 male SD rats were used as the control group.After the model was established, the body weight and paw volume were recorded weekly, the whole body score and the arthritis index score were calculated, and the joint swelling number was calculated.The animals were sacrificed after d35, the weight of thymus and spleen were weighed, and the corresponding index was calculated, the histopathological changes of the ankle joint were observed by HE staining.The percentages of CD4+ CD44+ T cell and CD4+ CD62L+ T cell were detected by flow cytometry.The levels of TNF-α, IL-1β in the serum of AA rats were detected by ELISA.@*Results@#hUC-MSCs relieved paw volume, the whole body score and arthritis index score, and the joint swelling number in AA rats (F=20.573, 89.092, 14.161, 10.914, all P<0.01). hUC-MSCs reduced thymus index[(0.120±0.032), (0.120±0.031)] and spleen index[(0.250±0.070), (0.240±0.018)] (F=6.339, 4.105, all P<0.01), improved structural damage of ankle joint.hUC-MSCs could regulate the percentage of T cell subsets(CD4+ CD62L+ )[(7.0±1.4)%, (7.9±2.2)%], (CD4+ CD44+ )[(15.0±3.6)%, (12.0±1.9)%] in spleen (F=6.331, 12.719, all P<0.01), and down-regulate the levels of TNF-α [(172.0±13.0)ng/L, (150.0±12.0)ng/L] and IL-1β [(75.0±36.0)ng/L, (74.0±20.0)ng/L] in serum (F=8.221, 3.581, all P<0.05) of AA rats by tail vein injection.@*Conclusion@#hUC-MSCs can promote the treatment of AA rats by regulating the function of T cells.
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To investigate the therapeutic and immunomodulatory effects of human umbilical cord-derived mesenchymal stem cells ( hUC-MSCs) on adjuvant arthritis (AA) rats.Methods Twenty -four male Sprague-Dawley( SD) rats were established with AA by complete Freund＇s adjuvant method.They were randomly divided into model group and hUC -MSCs group (2×106 cells／mL,5×106 cells／mL,tail vein injection),and the Yisaipu group (2.8mg／kg,subcutaneous injection ),6 rats in each group.Another 6 male SD rats were used as the control group.After the model was established ,the body weight and paw volume were recorded weekly ,the whole body score and the arthritis index score were calculated ,and the joint swelling number was calculated.The animals were sacrificed after d35,the weight of thymus and spleen were weighed ,and the corresponding index was calculated ,the histopathological changes of the ankle joint were observed by HE staining .The percentages of CD4 +CD44 +T cell and CD4 +CD62L+T cell were detected by flow cytometry.The levels of TNF-α,IL-1βin the serum of AA rats were detected by ELISA.Results hUC-MSCs relieved paw volume ,the whole body score and arthritis index score ,and the joint swelling number in AA rats (F＝20.573,89.092,14.161,10.914,all P＜0.01).hUC-MSCs reduced thymus index[(0.120 ±0.032),(0.120 ±0.031)] and spleen index[(0.250 ±0.070),(0.240 ±0.018)] ( F＝6.339,4.105,all P＜0.01),improved structural damage of ankle joint.hUC-MSCs could regulate the percentage of T cell subsets(CD4 +CD62L+)[(7.0 ±1.4)%,(7.9 ±2.2)%],( CD4 +CD44 +) [(15.0 ±3.6)%,(12.0 ± 1.9)%] in spleen (F＝6.331,12.719,all P＜0.01),and down -regulate the levels of TNF -α[(172.0 ± 13.0)ng／L,(150.0 ±12.0)ng／L] and IL-1β[(75.0 ±36.0)ng／L,(74.0 ±20.0)ng／L] in serum (F＝8.221, 3.581,all P＜0.05) of AA rats by tail vein injection.Conclusion hUC-MSCs can promote the treatment of AA rats by regulating the function of T cells.
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BACKGROUND: miRNA plays a critical regulatory role in the development and plasticity of spinal cord, and pathological changes after spinal cord injury. OBJECTIVE: To study the effect of miR-136-5p on the A20 expression in mouse astrocytes stimulated by interleukin-17 (IL-17). METHODS: C57BL/6 mouse astrocytes were cultured in vitro, identified by immunofluorescence staining, and then stimulated by 100 μg/L IL-17 for 0, 3, 6, 12 and 24 hours, respectively. The relative mRNA expression levels of IL-6 and tumor necrosis factor-α were detected by RT-PCR to determine the optimal stimulation time of IL-17. The mouse astrocytes were respectively stimulated by 10, 20, 50, 100 and 200 μg/L IL-7 for 6 hours, and similarly, the relative mRNA expression levels of IL-6 and tumor necrosis factor-α were detected to determine the optimal concentration of IL-17. At 6 hours after IL-17 (50 μg/L) stimulation, the mRNA expression levels of miR-136-5p and A20 in mouse astrocytes were detected by RT- PCR, and the protein expression level of A20 was detected by western blot assay. In addition, the lentiviral expression vector (miR-136-5p-inhibition) was constructed and transfected into the mouse astrocytes that were also stimulated by IL-7 to detect the expression levels of miR-136-5p, A20 mRNA and A20 protein. RESULTS AND CONCLUSION: Compared with the blank control group, the expression level of miR-136-5p in the miR-136-5p-inhibition group was significantly decreased after 6-hour IL-17 stimulation (P 0.05). To conclude, miR-136-5p makes certain effect on the expression of A20 protein in astrocytes after IL-17 stimulation.
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Objective To investigate PSP on bone microstructures,Ca,P,OPG and RANKL of osteoporotic rat model.Methods Thirty female rats randomly divided into five groups:Sham,OVX,H-,M-,L-PSP.Sham and OVX were irrigated stomachsaline;PSP solution was gavaged to other groups.After 8-week,bone microstructures of tibial metaphyseal,Ca,P,OPG and RANKL were measured.Results Body weight,Ca,P,RANKL,Tb.Sp of OVX were significantly increased compared to Sham,OPG,BV/TV,Tb.Th,Tb.N decreased.Body weight of H-,M-PSP,Ca and Tb.Sp of PSP,P and RANKL in H-PSP were decreased compared to OVX,OPG in H-,M-PSP,BV/TV,Tb.Th,Tb.N of PSP group increased.The differences were statistically significant (P ＜ 0.05).Conclusion PSP prevents osteoporosis by improving the microstructure of trabecular bone,reducing bone turnover,increasing OPG and reducing RANKL expression.
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BACKGROUND:Our previous studies have found that polygonatum sibiricum polysaccharide (PSP) promotes osteogenic differentiation of bone marrow mesenchymal stem cel s (BMSCs) by Wnt/β-catenin signaling pathway, but the molecular mechanism is unclear.OBJECTIVE:To investigate the effect of PSP promoting the osteogenic differentiation via Wnt signaling pathways in BMSCs after LRP5 silencing. METHODS:LRP5 interference vectors were constructed and then transfected into C57BL/6 mouse BMSCs cultured in vitro. The transfection efficiency of cel s was calculated under fluorescence inverted microscope and the expression of LRP5 protein was detected by western blot assay. The osteogenic potential of BMSCs after LRP5-siRNA transfection was analyzed by alkaline phosphatase staining, alizarin red staining and western blot assay. Effect of PSP on the osteogenic differentiation of LIRP5-silenced mouse BMSCs was detected by real-time PCR and dual luciferase assay. RESULTS AND CONCLUSION:Compared with the control group, the mineralization ability, the mRNA expressions of Runx2 and Osterix, and the protein expression of LRP5 were significantly decreased in the LRP5-siRNA group (P<0.05). PSP could promote LRP5-siRNA transfected mouse BMSCs differentiating into osteoblasts and significantly upregulated the expressions ofβ-catenin and Osterixin, and also induced the high expression of luciferase reporter gene (TOPFlash) containing wild type TCF binding sites (P<0.05). To conclude, LRP5 plays an important role in the process of mouse BMSCs differentiating into osteoblasts. PSP can promote the osteogenic differentiation of mouse BMSCs by activating the Wnt/β-catenin signaling pathway independent on LRP5.
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BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNCs) hold the potential of differentiating into osteoclasts. Polygonatum sibiricum polysaccharide (PSP) may inhibit the differentiation of BM-MNCs into osteoclasts and it is expected to become a new drug for the treatment of osteoporosis. OBJECTIVE: To investigate the effect of PSP on the differentiation of mouse BM-MNCs into osteoclasts induced by receptor activator of nuclear factor kappa-B ligand (RANKL) and bone resorption in vivo. METHODS: Mouse bone marrow-derived macrophages cultured in vitro, the effect of macrophage colony stimulating factor and PSP (5, 10, 20, 40, 80,160, 320, 640, 1280, 2560 mg/L) on the proliferation of mouse BM-MNCs was detected by cell counting kit-8 assay to determine the PSP concentration range; the mouse BMMs were cultured and induced in DMEM medium containing macrophage colony stimulating factor, RANKL and 5, 10, 20, 40, 80,160, 320, 640 mg/L PSP, respectively; those cultured without PSP served as control group. The morphological changes of cells were observed under an inverted microscope.; the number of osteoclasts was detected by tartrate-resistant acid phosphatase staining; the mRNA expression levels of osteoclast-related genes including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 were evaluated by quantitative real-time PCR. A mouse model of calvarial osteolysis induced by lipopolysaccharide was established to receive PSP intervention, and then micro CT scanning, three-dimensional reconstruction and relevants software were used for quantitative analysis of bone volume/volume percentage, trabecular number, trabecular bone spacing and thickness. The number of osteoclasts was identified by tartrate-resistant acid phosphatase staining and quantitative analysis of bone resorption area was conducted. RESULTS AND CONCLUSION: Compared with the control group, the concentration of PSP below 640 mg/L showed no significant effect on the proliferation of BMMs (P > 0.05). Different concentrations of PSP (40-640 mg/L) significantly reduced the number of osteoclasts, osteoclast differentiation and maturation, and the mRNA expression levels of tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 TRAP, MMP-9, CtsK and NFATc1 (P < 0.05). Compared with lipopolysaccharide, PSP could effectively alleviate the lipopolysaccharide-induced calvarial osteolysis, and the bone volume/volume percentage, trabecular number, and trabecular bone spacing were significantly decreased (P < 0.05); additionally, the number of osteoclasts and the area of bone resorption were decreased significantly (P < 0.01). To conclude, PSP can inhibit the differentiation and maturation of mouse BMMs to osteoclasts and alleviate lipopolysaccharide-induced calvarial osteolysis.