Bushen Tiaogan Prescription containing serum in the treatment of osteoarthritis by promoting  chondrocyte autophagy in rats

doi:10.12307/2022.617

Abstract

Abstract: BACKGROUND: Previous studies have found that Bushen Tiaogan Prescription has obvious clinical efficacy in the treatment of osteoarthritis, but there is a lack of research on its mechanism.
OBJECTIVE: To study the effect of Bushen Tiaogan Prescription containing serum on the autophagy of rat chondrocytes, and to explore its internal mechanism.
METHODS: The third-generation rat chondrocytes subcultured were randomly divided into four groups: dimethyl sulfoxide (DMSO), 10 μg/L interleukin-1β (IL-1β), 10 μg/L IL-1β+10% Bushen Tiaogan Prescription medicated serum (BSTG), and 5 μmol/L rapamycin groups, and chondrocytes in each group were treated correspondingly for 24 hours. Dansylcadaverine staining was used to detect the expression of chondrocyte autophagosomes. Real-time fluorescence quantitative PCR was used to detect the expression of autophagy-related genes ATG5 and ATG7 mRNAs, and the expression levels of Beclin1, LC3A/B, phosphorylated protein kinase B (p-AKT), and phosphorylated mammalian target of rapamycin (p-mTOR) proteins were analyzed by western blot assay.
RESULTS AND CONCLUSION: Compared with the DMSO group, the number of autophagosomes was significantly reduced in the IL-1β group. Compared with the DMSO group, the number of autophagosomes was significantly increased in the rapamycin group. Compared with the IL-1β group, the number autophagosomes was significantly increased in the 10 μg/L IL-1β+10% BSTG group. Compared with the DMSO group, the mRNA expressions of chondrocyte autophagy-related genes ATG5, ATG7 and the protein expressions of Beclin1 and LC3A/B-II/LC3A/B-I protein expression were significantly decreased in the IL-1β
group (P < 0.05, P < 0.01, P < 0.01, P < 0.05), while the protein expressions of p-AKT and p-mTOR were significantly increased (P < 0.05, P < 0.05). Compared with the DMSO group, the mRNA expressions of ATG5 and ATG7 and the protein expressions of Beclin1 and LC3A/B-II/LC3A/B-I were significantly increased in the rapamycin group (P < 0.01, P < 0.05, P < 0.05, P < 0.05), while the protein expressions of p-AKT and p-mTOR were significantly decreased (P < 0.01, P < 0.01). Compared with the IL-1β group, the mRNA expressions of ATG5 and ATG7 the protein expressions of Beclin1 and LC3A/B-II/LC3A/B-I were significantly increased in the 10 μg/L IL-1β+10% BSTG group (P < 0.01, P < 0.01, P < 0.05, P < 0.01), while the protein expressions of p-AKT and p-mTOR were significantly decreased (P < 0.05, P < 0.05). To conclude, Bushen Tiaogan Prescription containing serum increases the autophagy ability of chondrocytes by inhibiting the AKT/mTOR signaling pathway in chondrocytes and alleviating the degeneration of articular cartilage, and thereby plays a role in the treatment of osteoarthritis.

Key words: autophagy, osteoarthritis, Bushen Tiaogan Prescription, articular cartilage, AKT/mTOR

CLC Number:

Fan Shuai, Wu Chunfei, Liang Zujian, Xu Zhaohui, Xie Pingjin, Zhu Genfu. Bushen Tiaogan Prescription containing serum in the treatment of osteoarthritis by promoting chondrocyte autophagy in rats[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(20): 3178-3183.

Figures/Tables 4

References

[1] ACKERMAN IN, BOHENSKY MA, STEIGER RD, et al. Substantial rise in the lifetime risk of primary total knee replacement surgery for osteoarthritis from 2003 to 2013: an international, population-level analysis. Osteoarthritis Cartilage. 2016;25(4):455-461.
[2] HARRELL CR, MARKOVIC BS, FELLABAUM C, et al. Mesenchymal stem cell-based therapy of osteoarthritis: Current knowledge and future perspectives. Biomed Pharmacother. 2019;109:2318-2326.
[3] JEON OH, KIM C, LABERGE RM, et al. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat Med. 2017;23(6):775-781.
[4] VINA ER, KWOH CK. Epidemiology of osteoarthritis: literature update. Curr Opin Rheumatol. 2018;30(2):160-167.
[5] LOESER RF. The Role of Aging in the Development of Osteoarthritis. Trans Am Clin Climatol Assoc. 2017;128:44-54.
[6] O’NEILL TW, MCCABE PS, MCBETH J. Update on the epidemiology, risk factors and disease outcomes of osteoarthritis. Best Pract Res Clin Rheumatol. 2018;32(2):312-326.
[7] CUTOLO M, BERENBAUM F, HOCHBERG M, et al. Commentary on recent therapeutic guidelines for osteoarthritis. Semin Arthrit Rheum. 2015;44:611-617.
[8] NAKAMURA S, YOSHIMORI T. New insights into autophagosome-lysosome fusion. J Cell Sci. 2017;130:1209-1216.
[9] YU L, CHEN Y, TOOZE SA. Autophagy pathway: cellular and molecular mechanisms. Autophagy. 2018;14(2):207-215.
[10] TAKAYAMA K, KAWAKAMI Y, KOBAYASHI M, et al. Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis. Arthrit Res Ther. 2014;16(6):482-482.
[11] CHENG NT, GUO A, CUI YP. Intra-articular injection of Torin 1 reduces degeneration of articular cartilage in a rabbit osteoarthritis model. Bone Joint Res. 2016;5(6):218-224.
[12] 梁桂洪,黄和涛,潘建科,等.龙鳖胶囊含药血清对YAP抑制剂诱导人软骨细胞凋亡的保护作用及机制研究[J].中国药房,2021, 32(12):1442-1448.
[13] 吴春飞, 易俊, 梁桂洪, 等. 补肾调肝方治疗肝郁肾虚膝骨关节炎的临床研究[J]. 中医临床研究,2017,9(36):74-76.
[14] 范帅, 林杰彬, 吴春飞, 等. 补肾调肝方含药血清对白细胞介素1β诱导软骨细胞凋亡的影响[J]. 中国组织工程研究,2021,25(35): 5594-5598.
[15] 策力木格, 张小峰, 苏都娜, 等.血清药物化学在中药领域的应用现状[J].中国药房,2017,28(7):978-981.
[16] 李若飞, 于春萍, 兰丁璇, 等.中药血清药理学实验方法与相关问题探讨[J].全科口腔医学电子杂志,2019,6(31):125-136.
[17] LI B, YANG J, LU Z, et al. A study on the mechanism of rapamycin mediating the sensitivity of pancreatic cancer cells to cisplatin through PI3K/AKT/mTOR signaling pathway. J BUON. 2019;24(2):739-745.
[18] WILKINSON DJ, HABGOOD A, LAMB HK, et al. Matriptase induction of metalloproteinase-dependent aggrecanolysis in vitro and in vivo: promotion of osteoarthritic cartilage damage by multiple mechanisms promote cartilage damage in osteoarthritis. Arthritis Rheumatol. 2017; 69(8):1601-1611.
[19] HE Y, LI Z, ALEXANDER PG, et al. Pathogenesis of Osteoarthritis: Risk Factors, Regulatory Pathways in Chondrocytes, and Experimental Models. Biology. 2020;9(8):194.
[20] ZHANG G, CAO J, YANG E, et al. Curcumin improves age-related and surgically induced osteoarthritis by promoting autophagy in mice. Biosci Rep. 2018;38(4):BSR20171691.
[21] DUAN R, XIE H, LIU ZZ. The Role of Autophagy in Osteoarthritis. Front Cell Dev Biol. 2020;8:1437.
[22] LAPLANTE M, SABATINI DM. mTOR signaling in growth control and disease. Cell. 2012;149(2):274-293.
[23] VASHEGHANI F, ZHANG Y, LI YH, et al. PPARγ deficiency results in severe, accelerated osteoarthritis associated with aberrant mTOR signalling in the articular cartilage. Ann Rheum Dis. 2015;74(3):569-578.
[24] DON AS, TSANG CK, KAZDOBA TM, et al. Targeting mTOR as a novel therapeutic strategy for traumatic CNS injuries. Drug Discov Today. 2012;17(15-16):861-868.
[25] KIM YC, GUAN KL. mTOR: a pharmacologic target for autophagy regulation. J Clin Invest. 2015;125(1):25-32.
[26] ZHANG Y, VASHEGHANI F, LI YH, et al. Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis. Ann Rheum Dis. 2014;74(7):1432-1440.
[27] NEW J, THOMAS SM. Autophagy-dependent secretion: mechanism, factors secreted, and disease implications. Autophagy. 2019;15(10): 1682-1693.
[28] ZHANG Y, VASHEGHANI F, LI Y, et al. Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis. Ann Rheum Dis. 2015;74(7):1432-1440.
[29] QIN N, WEI L, LI W, et al. Local intra-articular injection of resveratrol delays cartilage degeneration in C57BL/6 mice by inducing autophagy via AMPK/mTOR pathway. J Pharmacol Sci. 2017;134(3):166-174.
[30] XUE JF, SHI ZM, ZOU J, et al. Inhibition of PI3K/AKT/mTOR signaling pathway promotes autophagy of articular chondrocytes and attenuates inflammatory response in rats with osteoarthritis. Biomed Pharmacother. 2017;89:1252-1261.
[31] MUSUMECI G, CASTROGIOVANNI P, TROVATO FM, et al. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis. Int J Mol Sci. 2015;16(9):20560-20575.
[32] YU Y, ZHAO J. Modulated Autophagy by MicroRNAs in Osteoarthritis Chondrocytes. Biomed Res Int. 2019;2019:1484152.
[33] MATSUZAKI T, ALVAREZ-GARCIA O, MOKUDA S, et al. FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and osteoarthritis. Sci Transl Med. 2018;10(428): eaan0746.
[34] TANG Q, ZHENG G, FENG Z, et al. Trehalose ameliorates oxidative stress-mediated mitochondrial dysfunction and ER stress via selective autophagy stimulation and autophagic flux restoration in osteoarthritis development. Cell Death Dis. 2017;8(10):e3081.
[35] LUO P, GAO F, NIU D, et al. The Role of Autophagy in Chondrocyte Metabolism and Osteoarthritis: A Comprehensive Research Review. Biomed Res Int. 2019;2019:5171602.
[36] WANG C, YAO Z, ZHANG Y, et al. Metformin Mitigates Cartilage Degradation by Activating AMPK/SIRT1-Mediated Autophagy in a Mouse Osteoarthritis Model. Front Pharmacol. 2020;11:1114.
[37] TIAN F, WANG J, ZHANG Z, et al. LncRNA SNHG7/miR-34a-5p/SYVN1 axis plays a vital role in proliferation, apoptosis and autophagy in osteoarthritis. Biol Res. 2020;53(1):9.
[38] ZHONG G, LONG H, MA S, et al. miRNA-335-5p relieves chondrocyte inflammation by activating autophagy in osteoarthritis. Life Sci. 2019; 226:164-172.
[39] CAI C, MIN S, YAN B, et al. MiR-27a promotes the autophagy and apoptosis of IL-1beta treated-articular chondrocytes in osteoarthritis through PI3K/AKT/mTOR signaling. Aging (Albany NY). 2019;11(16):6371-6384.
[40] LIN Z, MIAO J, ZHANG T, et al. JUNB-FBXO21-ERK axis promotes cartilage degeneration in osteoarthritis by inhibiting autophagy. Aging Cell. 2021;20(2):e13306.