Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence

ZHANG Tianchi; LI Muzhe; NIU Yuanyuan; GUO Yang; WANG Lining; TIAN Linkun; WANG Yuheng; WANG Quanquan; MA Yong

doi:10.14148/j.issn.1672-0482.2024.0249

Volume 40 Issue 3

Mar. 2024

Turn off MathJax

Article Contents

Article Navigation > Journal of Nanjing University of traditional Chinese Medicine > 2024 > 40(3): 249-260

ZHANG Tianchi, LI Muzhe, NIU Yuanyuan, GUO Yang, WANG Lining, TIAN Linkun, WANG Yuheng, WANG Quanquan, MA Yong. Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence[J]. Journal of Nanjing University of traditional Chinese Medicine, 2024, 40(3): 249-260. doi: 10.14148/j.issn.1672-0482.2024.0249

Citation:

ZHANG Tianchi, LI Muzhe, NIU Yuanyuan, GUO Yang, WANG Lining, TIAN Linkun, WANG Yuheng, WANG Quanquan, MA Yong. Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence[J]. Journal of Nanjing University of traditional Chinese Medicine, 2024, 40(3): 249-260. doi: 10.14148/j.issn.1672-0482.2024.0249

Citation:

ZHANG Tianchi, LI Muzhe, NIU Yuanyuan, GUO Yang, WANG Lining, TIAN Linkun, WANG Yuheng, WANG Quanquan, MA Yong. Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence[J]. Journal of Nanjing University of traditional Chinese Medicine, 2024, 40(3): 249-260. doi: 10.14148/j.issn.1672-0482.2024.0249

PDF( 6706 KB)

Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence

doi: 10.14148/j.issn.1672-0482.2024.0249

ZHANG Tianchi^{1, 2
,},
LI Muzhe^{1, 2},
NIU Yuanyuan¹,
GUO Yang^{1, 2, 3},
WANG Lining^{1, 3},
TIAN Linkun¹,
WANG Yuheng¹,
WANG Quanquan¹,
MA Yong^{1, 2, 3
,
,}

1.
New Technology Laboratory of Bone Injury Repair and Reconstruction, The First School of Clinical Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China
3.
Wuxi TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Jiangsu Clinical Medical Innovation Center for TCM Degenerative Osteoarthritis, Wuxi 214071, China

Received Date: 2023-10-31
Available Online: 2024-03-15

Abstract

Abstract

OBJECTIVE To study the intervention effects of Wenshen Tongluo Zhitong Recipe(WTZR) on macrophage senescence and senile osteoporosis. METHODS The senescent macrophage model was established using hydrogen peroxide (H₂O₂) and subsequently divided into four groups: control, model, low-dose drug-treated serum, and high-dose drug-treated serum.β-galactosidase staining, Western blot and qPCR were employed to evaluate the mRNA expression of senescence markers p21 and p53. ROS staining and JC-1 staining were applied to assess mitochondrial function in macrophages. The mRNA levels of Interleukin(IL)-6, IL-10, CD206, and inducible nitric oxide synthase (iNOS) were determined by qPCR analysis. Immunofluorescence was used to evaluate arginase(ARG1) and iNOS protein expressions for assessing the impact of drug-containing serum on macrophage polarization. qPCR analysis was conducted to measure osteocalcin(OCN), collagen type Ⅰ alpha 1(Col1a1), runt-related transcription factor 2(Runx2) mRNA levels as osteoblast-related markers; ALP staining along with alizarin red staining were performed to evaluate the effect of macrophage conditioned medium treated with drug-containing serum on bone marrow mesenchymal stromal cell(BMSC)osteogenic differentiation. C57BL/6J mice were randomly allocated into four groups: control group, model group, WTZR low-dose group, and WTZR high-dose group. The senile osteoporosis (SOP) mouse model was established by D-galactose. Micro-CT scanning analyzed femur microstructure while HE staining detected pathological changes in femur bone tissue samples collected from each experimental condition. Furthermore, Western blot was used to detect the senescence-related molecules p21 and p53 and the osteogenesis-related markers OCN and Runx2, qPCR analysis measured tibial expression levels of senescence-related molecules (p21, p53) as well as macrophage polarisation-related molecules (IL-6, iNOS, CD206, and IL-10) to assess the effect of this compound on a mouse model simulating SOP. RESULTS Following intervention with serum containing WTZR, there was a significant decrease in the number of senescent positive cells compared to the model group. Additionally, there was a notable decrease in p21 and p53 mRNA and proteins expression (P < 0.05, P < 0.01, P < 0.001). Furthermore, drug-containing WTZR effectively inhibited ROS production induced by H₂O₂ and mitigated mitochondrial membrane potential reduction in macrophages (P < 0.05, P < 0.001). Treatment with drug-containing WTZR resulted in down-regulated mRNA expression of M1-related gene iNOS (P < 0.05) while up-regulating mRNA expression of M2-related genes CD163 and CD206 (P < 0.05). The drug-containing WTZR significantly reduced fluorescence intensity for iNOS (P < 0.01) while increasing ARG1 (P < 0.05) fluorescence intensity. Moreover, conditioned medium from macrophages treated with drug-containing serum increased ALP positive cell count (P < 0.01, P < 0.001), alizarin red positive area (P < 0.05), as well as Col1a1, Runx2 and OCN mRNA expression levels (P < 0.05, P < 0.01). The Tb.N, BMD, and BV/TV were significantly higher in the WTZR group compared to the model group (P < 0.05, P < 0.01, P < 0.001); meanwhile, Tb.Sp was notably lower than that observed in the model group (P < 0.05, P < 0.01); bone trabeculae were significantly improved, increased in number and widened. Additionally, the compound could significantly inhibit the D-galactose induced up-regulation of tibial senescence-related genes and proteins p21 and p53(P < 0.05, P < 0.001, P < 0.0001), promote the expression of osteogenesis-related markers OCN and Runx2 protein(P < 0.01, P < 0.0001), promote the down-regulation of M1 related genes IL-6 and iNOS (P < 0.05), and promote the expression of M2 related genes IL-10 and CD206 (P < 0.05, P < 0.01). CONCLUSION Wenshen Tongluo Zhitong Recipe may play an anti-osteoporosis effect by inhibiting macrophage senescence and promoting the osteogenic differentiation of BMSC.
- senile osteoporosis,
- Wenshen Tongluo Zhitong Recipe,
- macrophage,
- bone marrow mesenchymal stromal cells,
- senescence

FullText(HTML)

References(26)

References

[1]	COMPSTON J E, MCCLUNG M R, LESLIE W D. Osteoporosis[J]. Lancet, 2019, 393(10169): 364-376. doi: 10.1016/S0140-6736(18)32112-3
[2]	BECKER L, NGUYEN L, GILL J, et al. Age-dependent shift in macrophage polarisation causes inflammation-mediated degeneration of enteric nervous system[J]. Gut, 2018, 67(5): 827-836. doi: 10.1136/gutjnl-2016-312940
[3]	郑苏阳, 马勇, 郭杨, 等. "温肾通络止痛方" 治疗原发性骨质疏松症的疗效观察及处方优化[J]. 辽宁中医杂志, 2016, 43(10): 2098-2100. https://www.cnki.com.cn/Article/CJFDTOTAL-LNZY201610026.htm ZHENG S Y, MA Y, GUO Y, et al. Treating primary osteoporosis with prescription of warming kidney and removing obstruction in channels to relieve pain and formula optimization[J]. Liaoning J Tradit Chin Med, 2016, 43(10): 2098-2100. https://www.cnki.com.cn/Article/CJFDTOTAL-LNZY201610026.htm
[4]	马勇, 汪志芳, 王培民. 自拟温肾通络止痛方治疗骨质疏松症45例临床研究[J]. 中医药学报, 2010, 38(1): 103-104. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYXB201001046.htm MA Y, WANG Z F, WANG P M. Clinical study on 45 cases of osteoporosis treated by self-made recipe of warming kidney, dredging collaterals and relieving pain[J]. Acta Chin Med Pharmacol, 2010, 38(1): 103-104. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYXB201001046.htm
[5]	韩秋革, 郭杨, 马勇, 等. 温肾通络止痛方调控巨噬细胞极化对破骨细胞分化的影响[J]. 时珍国医国药, 2023, 34(6): 1294-1297. https://www.cnki.com.cn/Article/CJFDTOTAL-SZGY202306004.htm HAN Q G, GUO Y, MA Y, et al. Effect of Wenshen Tongluo Zhitong recipe on osteoclast differentiation by regulating macrophage polarization[J]. Lishizhen Med Mater Med Res, 2023, 34(6): 1294-1297. https://www.cnki.com.cn/Article/CJFDTOTAL-SZGY202306004.htm
[6]	程斌, 方驰华, 范应方, 等. 骨髓间充质干细胞提取方法的改良[J]. 中国组织工程研究与临床康复, 2008, 12(47): 9293-9296. https://www.cnki.com.cn/Article/CJFDTOTAL-XDKF200847025.htm CHENG B, FANG C H, FAN Y F, et al. Improvement on the method of extracting bone marrow mesenchymal stem cells[J]. J Clin Rehabil Tissue Eng Res, 2008, 12(47): 9293-9296. https://www.cnki.com.cn/Article/CJFDTOTAL-XDKF200847025.htm
[7]	王永炫, 李梅, 章振林, 等. 《原发性骨质疏松症诊疗指南(2022)》要点解读[J]. 协和医学杂志, 2023, 14(6): 1203-1207. https://www.cnki.com.cn/Article/CJFDTOTAL-XHYX202306012.htm WANG Y X, LI M, ZHANG Z L, et al. Interpretation on the guidelines for the diagnosis and treatment of primary osteoporosis (2022)[J]. Med J Peking Union Med Coll Hosp, 2023, 14(6): 1203-1207. https://www.cnki.com.cn/Article/CJFDTOTAL-XHYX202306012.htm
[8]	黄帝内经素问[M]. 周鸿飞, 范涛, 点校. 郑州: 河南科学技术出版社, 2017. The yellow emperor's inner classic: Basic questions[M]. ZHOU H F, FAN T, punctuated and collated. Zhengzhou: Henan Science and Technology Press, 2017.
[9]	王辉, 郝宝娟, 王磊, 等. 淫羊藿苷的生物学功能及其在动物生产中的应用研究[J]. 现代畜牧兽医, 2023(6): 66-71. https://www.cnki.com.cn/Article/CJFDTOTAL-LNXM202306016.htm WANG H, HAO B J, WANG L, et al. Biological function of icariin and its application in animal production[J]. Mod J Anim Husb Vet Med, 2023(6): 66-71. https://www.cnki.com.cn/Article/CJFDTOTAL-LNXM202306016.htm
[10]	张仲景. 伤寒论[M]. 钱超尘, 郝万山, 整理. 北京: 人民卫生出版社, 2005. ZHANG Z J. Treatise on cold damage[M]. QIAN C, HAO W S, sorted. Beijing: People's Medical Publishing House, 2005.
[11]	李东垣. 脾胃论[M]. 文魁, 丁国华, 整理. 北京: 人民卫生出版社, 2005. LI D Y. Treatise on the spleen and stomach[M]. WEN K, DING G H, sorted. Beijing: People's Medical Publishing House, 2005.
[12]	马丽, 李作孝. 白芍总苷的免疫调节功能及其临床应用[J]. 中国实验方剂学杂志, 2010, 16(17): 244-246. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX201017072.htm MA L, LI Z X. Immunomodulatory effects and clinical application of total glucosides of Paeon[J]. Chin J Exp Tradit Med Formulae, 2010, 16(17): 244-246. https://www.cnki.com.cn/Article/CJFDTOTAL-ZSFX201017072.htm
[13]	樊景坡. 苍耳子、细辛、枸杞子、白术对小鼠组织自由基代谢的影响[J]. 中医药信息, 1994, 11(2): 48. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYXN402.023.htm FAN J P. Effects of fructus xanthii, herba asari, fructus lycii and atractylodis macrocephalae on free radical metabolism in mice[J]. Inf Tradit Chin Med, 1994, 11(2): 48. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYXN402.023.htm
[14]	ACOSTA-RODRIGUEZ E V, NAPOLITANI G, LANZAVECCHIA A, et al. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells[J]. Nat Immunol, 2007, 8(9): 942-949. doi: 10.1038/ni1496
[15]	LOPEZ-OTIN C, BLASCO M A, PARTRIDGE L, et al. The hallmarks of aging[J]. Cell, 2013, 153(6): 1194-1217. doi: 10.1016/j.cell.2013.05.039
[16]	HUNG Y T, TIKHONOVA M A, DING S J, et al. Effects of chronic treatment with diosgenin on bone loss in a D-galactose-induced aging rat model[J]. Chin J Physiol, 2014, 57(3): 121-127. doi: 10.4077/CJP.2014.BAC199
[17]	HORWOOD N J. Macrophage Polarization and Bone Formation: A review[J]. Clin Rev Allergy Immunol, 2016, 51(1): 79-86. doi: 10.1007/s12016-015-8519-2
[18]	PAWELEC G. Age and immunity: What is "immunosenescence"?[J]. Exp Gerontol, 2018, 105: 4-9. doi: 10.1016/j.exger.2017.10.024
[19]	ADELMAN E R, HUANG H T, ROISMAN A, et al. Aging human hematopoietic stem cells manifest profound epigenetic reprogramming of enhancers that may predispose to leukemia[J]. Cancer Discov, 2019, 9(8): 1080-1101. doi: 10.1158/2159-8290.CD-18-1474
[20]	SHI C Y, LI Q, ZHANG X Y. Platycodin D protects human fibroblast cells from premature senescence induced by H₂O₂ through improving mitochondrial biogenesis[J]. Pharmacology, 2020, 105(9/10): 598-608.
[21]	PAJARINEN J, LIN T, GIBON E, et al. Mesenchymal stem cell-macrophage crosstalk and bone healing[J]. Biomaterials, 2019, 196: 80-89. doi: 10.1016/j.biomaterials.2017.12.025
[22]	SONG X, XUE Y W, FAN S Y, et al. Lipopolysaccharide-activated macrophages regulate the osteogenic differentiation of bone marrow mesenchymal stem cells through exosomes[J]. PeerJ, 2022, 10: e13442. doi: 10.7717/peerj.13442
[23]	WU T L, ZHOU X, YE C H, et al. M1 macrophage-derived exosomes moderate the differentiation of bone marrow mesenchymal stem cells[J]. Biocell, 2022, 46(2): 495-503. doi: 10.32604/biocell.2022.015214
[24]	ASSOIAN R K, FLEURDELYS B E, STEVENSON H C, et al. Expression and secretion of type beta transforming growth factor by activated human macrophages[J]. Proc Natl Acad Sci USA, 1987, 84(17): 6020-6024. doi: 10.1073/pnas.84.17.6020
[25]	CHAMPAGNE C M, TAKEBE J, OFFENBACHER S, et al. Macrophage cell lines produce osteoinductive signals that include bone morphogenetic protein-2[J]. Bone, 2002, 30(1): 26-31. doi: 10.1016/S8756-3282(01)00638-X
[26]	LI C J, XIAO Y, SUN Y C, et al. Senescent immune cells release grancalcin to promote skeletal aging[J]. Cell Metab, 2021, 33(10): 1957-1973. doi: 10.1016/j.cmet.2021.08.009

Relative Articles

Supplements(0)

Cited By

Proportional views

Proportional views

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Figures(8) / Tables(1)

Get Citation

PDF

XML

Article Metrics

Article views (95) PDF downloads(17)

Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence

doi: 10.14148/j.issn.1672-0482.2024.0249

Abstract

References

Proportional views

Catalog

通讯作者: 陈斌, bchen63@163.com

Article Metrics

Proportional views

Related

Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence

doi: 10.14148/j.issn.1672-0482.2024.0249

Abstract

References

Proportional views

Catalog

通讯作者: 陈斌, bchen63@163.com

Article Metrics

Proportional views

Related

Export File

Citation

Format

Content