Résumé
【Objective】 To investigate the effect and mechanism of achyranthes bidentata polysaccharides （ABPS） on the differentiation of bone mesenchymal stem cells （BMSCs） into nucleus pulposus-like cells. 【Methods】 Five 4-week-old SD rats were sacrificed and their BMSCs were isolated and purified by repeated adherent method. The third-generation BMSCs were treated with different concentrations of ABPS for 48 h. The cell viability was detected by MTT method. The highest concentration of ABPS was selected for subsequent experiments. According to different treatment methods， cells were divided into control group （conventional culture）， ABPS group （200 mg/L of ABPS）， Notch1 group （transfected with Notch1）， Notch1 negative control group （transfected with Notch1 negative control）， and ABPS+Notch1 group （200 mg/L of ABPS was added after transfection with Notch1）. After 14 days of induction， the cell survival rate was measured by MTT method. CollagenⅡ was detected by immunohistochemical staining. The mRNA and protein expression levels of collagenⅡ， aggrecan， Notch1 and Hes1 were detected by qPCR and Western Blotting respectively. The glucosaminoglycan （GAG） was detected by alcian blue method on the 1st， 4th， 7th， 10th and 14th day of cell culture. 【Results】 After BMSCs were treated with 400 mg/L of ABPS for 48 h， the cell survival rate was significantly lower than that in the control group （P<0.05）. When the concentration was ≤200 mg/L， ABPS had no significant toxic effect on the growth of BMSCs. After 14 days of induction， compared with the control group， A value， GAG content in the medium， the expressions of collagen II and aggrecan mRNA and protein in the ABPS group were increased， and the expressions of Notch1 and Hes1 mRNA and protein were decreased （P<0.05）. The relative cell viability， GAG content in the medium， the expressions of collagen II and aggrecan mRNA and protein in the Notch1 group were decreased， and the expressions of Notch1 and Hes1 mRNA and protein were increased （P<0.05）. ABPS+Notch1 could partially reverse the inhibitory effect of Notch1 overexpression on the differentiation of BMSCs into nucleus pulposus-like cells. 【Conclusion】 ABPS can promote the differentiation of BMSCs into nucleus pulposus-like cells， and the mechanism may be related to the inhibition of Notch1 pathway.
Résumé
BACKGROUND: Studies have shown that bone marrow mesenchymal stem cells (BMSCs) can differentiate into nucleus pulposus-like cells, but the mechanism of differentiation is not clear. OBJECTIVE: To induce bone BMSCs into nucleus pulposus-like cells using Achyranthes bidentata Bl. saponins (ABS) and to explore the role of Nampt/NAD/Sirt1 axis in the differentiation of BMSCs. METHODS: BMSCs were collected from Sprague-Dawley rats. Cells at passage 3 were divided into four groups: BMSCs group, BMSCs+ABS group, BMSCs+ABS+nicotinamide mononucleotide (an exogenous small molecule substance promoting NAD synthesis) group, BMSCs+ABS+FK866 (nicotinamide phosphoribosyltransferase inhibitor) group, in which the cells were induced for 14 days. Alcian blue staining was used to show the changes of glycosaminoglycan in the cells. RT-PCR was used to detect the mRNA expression of COL2, Aggrecan, KRT19, Pax1. The protein expression level of COL2 was detected by western blot. The activity of Sirt1 was detected by Sirt1 assay kit and the content of NAD+ was measured by NAD+/NADH kit. RESULTS AND CONCLUSION: (1) Compared with the BMSCs group, BMSCs+ABS group showed significant increases in the expression levels of glycosaminoglycan, Aggrecan, KRT19, and Pax1 (P < 0.05), and the protein expression levels of COL2, activity of Sirt1, and content of NAD+ were also significantly increased (all P < 0.05). (2) Compared with the BMSCs+ABS group, the above-mentioned indicators were significantly increased in the BMSCs+ABS+nicotinamide mononucleotide (P < 0.05); on the contrary, these indicators were all decreased significantly in the BMSCs+ABS+FK866 group (P < 0.05). To conclude, ABS could induce the differentiation of rat BMSCs into nucleus pulposus-like cells, in which the Nampt/NAD/Sirt1 axis might play a promotion role.
Résumé
BACKGROUND: Studies have shown that bone marrow mesenchymal stem cells (BMSCs) can differentiate into nucleus pulposus-like cells, but the mechanism of differentiation is not clear. OBJECTIVE: To induce bone BMSCs into nucleus pulposus-like cells using Achyranthes bidentata Bl. saponins (ABS) and to explore the role of Nampt/NAD/Sirt1 axis in the differentiation of BMSCs. METHODS: BMSCs were collected from Sprague-Dawley rats. Cells at passage 3 were divided into four groups: BMSCs group, BMSCs+ABS group, BMSCs+ABS+nicotinamide mononucleotide (an exogenous small molecule substance promoting NAD synthesis) group, BMSCs+ABS+FK866 (nicotinamide phosphoribosyltransferase inhibitor) group, in which the cells were induced for 14 days. Alcian blue staining was used to show the changes of glycosaminoglycan in the cells. RT-PCR was used to detect the mRNA expression of COL2, Aggrecan, KRT19, Pax1. The protein expression level of COL2 was detected by western blot. The activity of Sirt1 was detected by Sirt1 assay kit and the content of NAD+ was measured by NAD+/NADH kit. RESULTS AND CONCLUSION: (1) Compared with the BMSCs group, BMSCs+ABS group showed significant increases in the expression levels of glycosaminoglycan, Aggrecan, KRT19, and Pax1 (P < 0.05), and the protein expression levels of COL2, activity of Sirt1, and content of NAD+ were also significantly increased (all P < 0.05). (2) Compared with the BMSCs+ABS group, the above-mentioned indicators were significantly increased in the BMSCs+ABS+nicotinamide mononucleotide (P < 0.05); on the contrary, these indicators were all decreased significantly in the BMSCs+ABS+FK866 group (P < 0.05). To conclude, ABS could induce the differentiation of rat BMSCs into nucleus pulposus-like cells, in which the Nampt/NAD/Sirt1 axis might play a promotion role.
Résumé
Objective To explore effect of 5-HT,VEGF and mineral content in osteoporosis rat by Morinda officinalis.Methods 90 SPF male SD rats were selected, 15 rats were randomly selected as the normal control group, the rest were to establish the model of osteoporosis.When the model was established successfully according to the different treatment methods were divided into control group, model group, positive medicine group, low, middle and high does group.Bone mineral density, 5-HT, VEGF and the levels of the mineral were compared after 4 weeks treatment.Results Compared with the normal control group, BMD of model control group and low dose group was lower, Compared with the positive drug group, BMD of middle dose and high dose was higher ( P<0.05 ).Compared with model group,5-HT level of positive medicine group, high dose group and middle dose group was higher, compared with positive drug group, 5-HTP of middle dose group and high dose group was higher ( P <0.05 ).Compared with model group, positive drug group, VEGF levels of middle dose group and high dose group were higher(P <0.05), compared with positive drug group, VEGF of middle dose group and high dose group were higher(P<0.05), compared with model control group, positive drug group, serum P content of middle dose group was higher, compared with positive drug, serum P content of high dose group and high dose group was less.Conclusion Morindae Officinalis can increase 5-HT, VEGF in a rat model of osteoporosis, improve the level of serum P, has the guiding sense to the clinical.