RESUMEN
【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.
RESUMEN
OBJECTIVE： To establish pre- column derivatization-HPLC fingerprint of polysaccharide from Achyranthes bidentata，and to determine the contents of monosaccharide components ，so as to provide reference for quality evaluation of A. bidentata decoction pieces. METHODS ：Taking 10 batches of A. bidentata decoction pieces from different manufacturers as samples，the polysaccharides were extracted by water extraction and alcohol precipitation ，hydrolyzed by trifluoroacetic acid ，and then derivatized by 1-phenyl-3-methyl-5-pyrazolone for HPLC analysis. The determination was performed on Hanbon Hedera C 18 column with column temperature of 30 ℃ at the flow rate of 1.2 mL/min. The mobile phase consisted of acetonitrile- 0.02 mol/L ammonium acetate solution （gradient elution ）. The detection wavelength was set at 250 nm，and sample size was 20 μL. HPLC fingerprint was established and similarity evaluation was performed for 10 batches of A. bidentata polysaccharide by using TCM Chromatogramic Fingerprint Similarity Evaluation System （2012A edition ）. The common peak was identified by comparing with the reference substance ，and cluster analysis was performed by using SPSS 25.0 software. The contents of identified monosaccharides were determined by HPLC. RESULTS ：The similarity of 10 batches of A. bidentata polysaccharide were all higher than 0.95. Nine common peaks were fixed and a total of 5 common peaks were identified ，namely anhydrous glucose （peak 1）， mannose（peak 2），rhamnose（peak 4），galacturonic acid （peak 5）and arabinose （peak 7）. Results of cluster analysis showed that S1 sample was classified into one category ；S2，S5，S8 and S 9 samples were clustered into one category ；S3，S4，S6，S7 and S10 samples were clustered into one category. Results of content determination showed that the contents of anhydrous glucose in 10 batches of samples ranged from 6.17 to 17.55 mg/g；those of mannose ranged from 3.31 to 7.66 mg/g；those of rhamnose ranged from 38.80 to 73.97 mg/g；those of galacturonic acid ranged from 2.49 to 8.95 mg/g；those of arabinose ranged from 11.30 to 28.58 mg/g. CONCLUSIONS ：Established pre-column derivatization HPLC fingerprints and content determination method can provide reference for quality evaluation of A. bidentata decoction pieces.