牛膝总皂苷调控Nampt/NAD/Sirt1轴诱导骨髓间充质干细胞向髓核样细胞分化
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摘要
背景:研究表明,骨髓间充质干细胞可向髓核样细胞分化,但分化机制尚不明确。目的:利用牛膝总皂苷诱导大鼠骨髓间充质干细胞向髓核样细胞分化,并探讨Nampt/NAD/Sirt1轴在其中的作用。方法:取第3代SD大鼠骨髓间充质干细胞,随机分成4组:(1)骨髓间充质干细胞组;(2)骨髓间充质干细胞+牛膝总皂苷组;(3)骨髓间充质干细胞+牛膝总皂苷+烟酰胺单核苷酸组(烟酰胺单核苷酸为促NAD合成的外源性小分子物质);(4)骨髓间充质干细胞+牛膝总皂苷+FK866组(FK866为烟酰胺磷酸核糖转移酶抑制剂)。连续诱导培养14d,阿尔新蓝染色法检测细胞内糖胺多糖含量,RT-PCR检测髓核样细胞相关基因COL2、Aggrecan、KRT19和PAX1 mRNA表达,Western blot检测COL2的蛋白表达,Sirt1测定试剂盒检测Sirt1活性,NAD~+/NADH试剂盒检测NAD+含量。结果与结论:(1)与骨髓间充质干细胞组相比,骨髓间充质干细胞+牛膝总皂苷组能够使糖胺多糖、髓核样细胞相关基因表达、COL2蛋白表达、Sirt1活性、NAD~+含量明显增加(P <0.05);(2)与骨髓间充质干细胞+牛膝总皂苷组相比,骨髓间充质干细胞+牛膝总皂苷+烟酰胺单核苷酸组糖胺多糖、髓核样细胞相关基因表达、COL2蛋白表达、Sirt1活性和NAD~+含量增加(P<0.05);相反,骨髓间充质干细胞+牛膝总皂苷+FK866组上述指标均降低(P <0.05);(3)结果表明,牛膝总皂苷可诱导大鼠骨髓间充质干细胞向髓核样细胞分化,Nampt/NAD/Sirt1轴在其中发挥促进作用。
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.
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.
引文
[1]周校黎.FGF-2单独以及与TGF-beta联合对人间充质干细胞向髓核样细胞分化的影响[D].杭州:浙江大学,2016.
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[6]刘洋,王静成,冯新民,等.正常与退变椎间盘来源髓核间充质干细胞的生物学性能比较[J].中华实验外科杂志,2018,35(6):1111-1114.
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[8]Martin BI,Deyo RA,Mirza SK,et al.Expenditures and health status among adults with back and neck problems.JAMA.2008;299(6):656-664.
[9]张钰,刘志礼,黄山虎.线粒体与椎间盘退变[J].中国老年学杂志,2016,36(17):4393-4396.
[10]Gelalis ID,Papadopoulos DV,Giannoulis DK,et al.Spinal motion preservation surgery:indications and applications.Eur J Orthop Surg Traumatol.2018;28(3):335-342.
[11]Donk RD,Verbeek ALM,Verhagen WIM,et al.What's the best surgical treatment for patients with cervical radiculopathy due to single-level degenerative disease?A randomized controlled trial.PLoS One.2017;12(8):e0183603.
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[13]Richardson SM,Walker RV,Parker S,et al.Intervertebral disc cell-mediated mesenchymal stem cell differentiation.Stem Cells.2006;24(3):707-716.
[14]Fernandes M,Valente SG,Sabongi RG,et al.Bone marrow-derived mesenchymal stem cells versus adiposederived mesenchymal stem cells for peripheral nerve regeneration.Neural Regen Res.2018;13(1):100-104.
[15]Sevivas N,Teixeira FG,Portugal R,et al.Mesenchymal Stem Cell Secretome:A Potential Tool for the Prevention of Muscle Degenerative Changes Associated With Chronic Rotator Cuff Tears.Am J Sports Med.2017;45(1):179-188.
[16]Maerz T,Herkowitz H,Baker K.Molecular and genetic advances in the regeneration of the intervertebral disc.Surg Neurol Int.2013;4(Suppl 2):S94-S105.
[17]Li Y,He J,He X,et al.Nampt expression increases during osteogenic differentiation of multi-and omnipotent progenitors.Biochem Biophys Res Commun.2013;434(1):117-123.
[18]Hassan B,Baroukh B,Llorens A,et al.NAMPT expression in osteoblasts controls osteoclast recruitment in alveolar bone remodeling.J Cell Physiol.2018;233(9):7402-7414.
[19]Yoon J,Lee KJ,Oh GS,et al.Regulation of Nampt expression by transcriptional coactivator NCOA6 in pancreaticβ-cells.Biochem Biophys Res Commun.2017;487(3):600-606.
[20]李金亭,胡正海.牛膝类药材的生物学与化学成分的研究进展[J].中草药,2006,37(6):952-956.
[21]张小鸿.牛膝总皂苷保护软骨细胞作用及其机制研究[D].泉州:华侨大学,2014.
[22]孙奋勇,潘秋辉,洪岸.牛膝促进成骨细胞增殖的作用与机理研究[J].中药材,2004,27(4):264-266.
[23]李摇娟,毕志明,肖雅洁,等.怀牛膝的三萜皂苷成分研究[J].中国药学杂志,2007,42(3):178-180.
[24]岳宗进,刘汝银,于露,等.miR146通过靶向Notch 1促进骨髓问充质干细胞向髓核样细胞分化[J].西安交通大学学报(医学版),2016,37(5):663-668.
[25]Sakai D,Andersson GB.Stem cell therapy for intervertebral disc regeneration:obstacles and solutions.Nat Rev Rheumatol.2015;11(4):243-256.
[26]Imai S.The NAD World:a new systemic regulatory network for metabolism and aging--Sirt1,systemic NAD biosynthesis,and their importance.Cell Biochem Biophys.2009;53(2):65-74.
[27]王荥,王延洲,刘禄,等.Deltex-1对骨髓间充质干细胞增殖及其向平滑肌细胞分化的影响[J].第三军医大学学报,2018,40(2):109-114.
[28]Revollo JR,Grimm AA,Imai S.The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells.J Biol Chem.2004;279(49):50754-20763.
[29]段琳琳,李红梅.来源于大肠杆菌的NMN转移酶的克隆表达与酶学性质研究[J].工业微生物,2016,46(2):30-35.
[30]Wang P,Li WL,Liu JM,et al.NAMPT and NAMPT-controlled NAD Metabolism in Vascular Repair.J Cardiovasc Pharmacol.2016;67(6):474-481.
[2]Li Y,Samartzis D,Campbell DD,et al.Two subtypes of intervertebral disc degeneration distinguished by large-scale population-based study.Spine J.2016;16(9):1079-1089.
[3]Khan AN,Jacobsen HE,Khan J,et al.Inflammatory biomarkers of low back pain and disc degeneration:a review.Ann N Y Acad Sci.2017;1410(1):68-84.
[4]魏强强,殷嫦嫦,殷明,等.GDF-5联合地塞米松诱导大鼠BMSCs向类髓核样细胞分化[J].基础医学与临床,2014,34(8):1037-1043.
[5]梁航,邓享誉,邵增务,等.椎间盘内源性干细胞在椎间盘组织修复再生中的研究进展[J].中国修复重建外科杂志,2017,31(10):1267-1272.
[6]刘洋,王静成,冯新民,等.正常与退变椎间盘来源髓核间充质干细胞的生物学性能比较[J].中华实验外科杂志,2018,35(6):1111-1114.
[7]Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999;284(5411):143-147.
[8]Martin BI,Deyo RA,Mirza SK,et al.Expenditures and health status among adults with back and neck problems.JAMA.2008;299(6):656-664.
[9]张钰,刘志礼,黄山虎.线粒体与椎间盘退变[J].中国老年学杂志,2016,36(17):4393-4396.
[10]Gelalis ID,Papadopoulos DV,Giannoulis DK,et al.Spinal motion preservation surgery:indications and applications.Eur J Orthop Surg Traumatol.2018;28(3):335-342.
[11]Donk RD,Verbeek ALM,Verhagen WIM,et al.What's the best surgical treatment for patients with cervical radiculopathy due to single-level degenerative disease?A randomized controlled trial.PLoS One.2017;12(8):e0183603.
[12]Liang L,Li X,Li D,et al.The characteristics of stem cells in human degenerative intervertebral disc.Medicine(Baltimore).2017;96(25):e7178.
[13]Richardson SM,Walker RV,Parker S,et al.Intervertebral disc cell-mediated mesenchymal stem cell differentiation.Stem Cells.2006;24(3):707-716.
[14]Fernandes M,Valente SG,Sabongi RG,et al.Bone marrow-derived mesenchymal stem cells versus adiposederived mesenchymal stem cells for peripheral nerve regeneration.Neural Regen Res.2018;13(1):100-104.
[15]Sevivas N,Teixeira FG,Portugal R,et al.Mesenchymal Stem Cell Secretome:A Potential Tool for the Prevention of Muscle Degenerative Changes Associated With Chronic Rotator Cuff Tears.Am J Sports Med.2017;45(1):179-188.
[16]Maerz T,Herkowitz H,Baker K.Molecular and genetic advances in the regeneration of the intervertebral disc.Surg Neurol Int.2013;4(Suppl 2):S94-S105.
[17]Li Y,He J,He X,et al.Nampt expression increases during osteogenic differentiation of multi-and omnipotent progenitors.Biochem Biophys Res Commun.2013;434(1):117-123.
[18]Hassan B,Baroukh B,Llorens A,et al.NAMPT expression in osteoblasts controls osteoclast recruitment in alveolar bone remodeling.J Cell Physiol.2018;233(9):7402-7414.
[19]Yoon J,Lee KJ,Oh GS,et al.Regulation of Nampt expression by transcriptional coactivator NCOA6 in pancreaticβ-cells.Biochem Biophys Res Commun.2017;487(3):600-606.
[20]李金亭,胡正海.牛膝类药材的生物学与化学成分的研究进展[J].中草药,2006,37(6):952-956.
[21]张小鸿.牛膝总皂苷保护软骨细胞作用及其机制研究[D].泉州:华侨大学,2014.
[22]孙奋勇,潘秋辉,洪岸.牛膝促进成骨细胞增殖的作用与机理研究[J].中药材,2004,27(4):264-266.
[23]李摇娟,毕志明,肖雅洁,等.怀牛膝的三萜皂苷成分研究[J].中国药学杂志,2007,42(3):178-180.
[24]岳宗进,刘汝银,于露,等.miR146通过靶向Notch 1促进骨髓问充质干细胞向髓核样细胞分化[J].西安交通大学学报(医学版),2016,37(5):663-668.
[25]Sakai D,Andersson GB.Stem cell therapy for intervertebral disc regeneration:obstacles and solutions.Nat Rev Rheumatol.2015;11(4):243-256.
[26]Imai S.The NAD World:a new systemic regulatory network for metabolism and aging--Sirt1,systemic NAD biosynthesis,and their importance.Cell Biochem Biophys.2009;53(2):65-74.
[27]王荥,王延洲,刘禄,等.Deltex-1对骨髓间充质干细胞增殖及其向平滑肌细胞分化的影响[J].第三军医大学学报,2018,40(2):109-114.
[28]Revollo JR,Grimm AA,Imai S.The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells.J Biol Chem.2004;279(49):50754-20763.
[29]段琳琳,李红梅.来源于大肠杆菌的NMN转移酶的克隆表达与酶学性质研究[J].工业微生物,2016,46(2):30-35.
[30]Wang P,Li WL,Liu JM,et al.NAMPT and NAMPT-controlled NAD Metabolism in Vascular Repair.J Cardiovasc Pharmacol.2016;67(6):474-481.