Proteomic analysis on cartilage differentiation of rabbit bone marrow mesenchymal stem cells induced by Achyranthes bidentata alcohol extract

doi:10.12307/2023.641

Abstract

Abstract: BACKGROUND: Preliminary studies have found that Achyranthes bidentata alcohol extract can induce the directional chondrocyte differentiation of bone marrow mesenchymal stem cells, but the specific protein targets and network mechanisms are unknown.
OBJECTIVE: To observe the proteomic analysis of cartilage differentiation of rabbit bone marrow mesenchymal stem cells induced by Achyranthes bidentata alcohol extract and the construction of protein interaction network.
METHODS: Bone marrow mesenchymal stem cells of New Zealand white rabbits were isolated and cultured by density gradient centrifugation combined with bone marrow adherent method. Passage 3 cells were randomly divided into five groups: blank group, control group, low-dose Achyranthes bidentata alcohol extract group, medium-dose Achyranthes bidentata alcohol extract group, and high-dose Achyranthes bidentata alcohol extract group. After continuous induction and culture for 21 days, qRT-PCR was used to detect the expression of type II collagen mRNA and toluidine blue staining was used to identify the formation of chondrocytes. The differentially expressed proteins of different groups were identified by absolute quantitative isotope labeling (iTRAQ) combined with two-way liquid chromatography tandem mass spectrometry, and the differentially expressed proteins were analyzed by GO analysis, KEGG analysis and protein interaction network analysis.
RESULTS AND CONCLUSION: (1) The qRT-PCR results showed that the expression of type II collagen mRNA in the high-dose Achyranthes bidentata alcohol extract group was significantly higher than that in other groups (P < 0.05), and toluidine blue staining displayed positive reaction in the high-dose Achyranthes bidentata alcohol extract group. A total of 1 354 differential protein spots were identified by proteomic analysis, including 633 up-regulated proteins and 721 down-regulated proteins. (2) According to the results of qRT-PCR, the differentially expressed proteins of blank group, high-dose Achyranthes bidentata alcohol extract group and control group were analyzed. Go analysis showed that these differentially expressed proteins were involved in metabolism, cell differentiation, cell cycle and apoptosis, inflammatory response, immune regulation, oxidative stress, phosphorylation, ubiquitination, cancer and so on. KEGG analysis obtained 10 typical signal pathways most related to osteoarthritis: interleukin-17 signaling pathway, Toll like receptor signaling pathway, Wnt signaling pathway, PI3K-Akt signaling pathway, mTOR signaling pathway, JAK-STAT signaling pathway, NF-kappa B signaling pathway, MAPK signaling pathway, AMPK signaling pathway, and HIF-1 signaling pathway. According to the protein difference between groups, Cytoscape 3.6.0 software was used to successfully construct protein interaction network diagram. (3) These results suggest that Achyranthes bidentata alcohol extract can induce chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells by regulating oxidation, cell cycle and apoptosis, changes in cell structure, cell differentiation, metabolism and inflammatory injury. It has multi-target and multi-center network regulation, and its specific mechanism needs to be further studied.

Key words: Achyranthes bidentata alcohol extract, proteomics, bone marrow mesenchymal stem cell, cartilage differentiation, iTRAQ technology

CLC Number:

Ma Dujun, Zhu Houjun, Liu Leshi, Peng Liping, Zhao Jing, Liao Zhouwei. Proteomic analysis on cartilage differentiation of rabbit bone marrow mesenchymal stem cells induced by Achyranthes bidentata alcohol extract[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(24): 3795-3802.

Figures/Tables 12

References

[1] 高子茏,李婷,吕政,等.补骨脂素联合转化生长因子β1诱导骨髓间充质干细胞向软骨细胞的分化[J].中国组织工程研究,2022,26(30):4884-4888.
[2] 田能,孔祥英,王荣田,等.不同引经药对股骨头坏死模型兔骨髓干细胞归巢的影响[J].中国中药杂志,2012,37(11):1624-1628.
[3] 黄勇,黄秀深,胡一梅,等.“左归丸”配伍规律对骨髓源成体干细胞定向分化的调控作用[J].成都中医药大学学报,2010,33(1):48-52.
[4] 马笃军,彭力平,王立新,等.牛膝醇提物诱导兔骨髓间充质干细胞软骨定向分化的实验研究[J].中国中医骨伤科杂志,2017,25(2):6-11.
[5] 马笃军,彭力平,王立新,等.实验兔骨髓间充质干细胞的分离、培养及鉴定[J].基因组学与应用生物学,2017,36(1):99-104.
[6] AKGUN I, UNLU MC, ERDAL OA, et al. Matrix-induced autologous mesenchymal stem cell implantation versus matrix-induced autologous chondrocyte implantation in the treatment of chondral defects of the knee: a 2-year randomized study. Arch Orthop Trauma Surg. 2015;135(2):251-263.
[7] 李强强,谢亚东,杨国清,等.骨髓间充质干细胞成骨分化的研究进展[J].医学综述,2022,28(3):434-438.
[8] 高松,孙珍珍.近20年中医药治疗骨关节炎临床用药规律分析[J].西南医科大学学报,2021,44(1):73-77.
[9] 黄艳峰,谢新宇,林晴,等.基于计算机模拟探讨牛膝治疗骨关节炎软骨退变机制[J].中国实验方剂学杂志,2021,27(15):148-155.
[10] 张巍,张慧,张振凌.牛膝、酒牛膝饮片色度测定方法的建立及检测[J].中医药导报,2022,28(1):59-62.
[11] LI Z, MA D, PENG L, et al. Compatibility of Achyranthes bidentata components in reducing inflammatory response through Arachidonic acid pathway for treatment of Osteoarthritis. Bioengineered. 2022;13(1):1746-1757.
[12] 肖伟,马笃军,彭力平,等.牛膝醇提物对兔骨关节炎软骨细胞体外增殖及糖胺聚糖的干预作用[J].中国医药导报,2017,14(5):20-24.
[13] 马笃军,彭力平,蒋顺琬,等.牛膝醇提物干预BMSC-Exos对 OA模型兔局部骨组织超微结构及炎症小体的影响[J].中医药导报,2022,28(1):12-18.
[14] 刘璐,刘传慧,段智霞.膝关节炎患者血清RANKL、IL-17水平与疾病严重程度的关系[J].中国实用医刊,2021,48(19):16-19.
[15] 孙桂芳,张雪锋,茅瑜,等.温针灸治疗轻中度膝骨关节炎疗效观察及其对TLR4/NF-κB信号通路的影响[J].上海针灸杂志,2021,40(12):1452-1457.
[16] 刘晶,林巧璇,卢莉铭,等.基于Wnt3a/β-catenin信号通路探讨针刀对膝骨关节炎兔股直肌纤维化的影响[J].中华中医药杂志,2022,37(1):136-140.
[17] 邓欢,吕艺蓁,刘宣,等.PI3K/AKT信号通路调控骨关节疾病软骨细胞自噬及损伤的机制[J].西安交通大学学报(医学版),2022,43(2):309-314.
[18] 曹舒兴,周楠,王进,等.姜黄素通过Akt-mTOR信号通路调控细胞自噬对骨关节炎大鼠的保护作用研究[J].河北医药,2021,43(24):3685-3689.
[19] 徐斌,周明旺,李盛华,等.JAK/STAT信号通路在骨关节炎致病机制及治疗靶点的研究进展[J].中国疼痛医学杂志,2022,28(2):125-129.
[20] 郑晓慧,董博,袁普卫,等.NF-κB信号通路在骨性关节炎软骨破坏中的研究进展[J].中国疼痛医学杂志,2021,27(7):540-544.
[21] 冯帅华,吴官保,文哲,等.超微肿痛贴对兔膝骨关节炎模型的软骨中JNK/p38 MAPK信号通路的影响[J].湖南中医药大学学报,2022,42(1):31-36.
[22] 马笃军,华树良,彭力平,等.牛膝醇提物对实验兔膝骨关节炎模型AMPK/Wnt/MAPK信号通路串话的影响[J].中医药导报,2021,27(7):16-21,26.
[23] 徐高丽,张建兴,周健,等.静压力下缺氧诱导因子-1α信号通路对髁突软骨细胞增殖与凋亡的调控作用[J].中华老年病研究电子杂志,2021,8(3):24-29.