GJK Tablets intervene in cartilage homeostasis to protect articular cartilage of mice with knee osteoarthritis

doi:10.12307/2026.661

Abstract

Abstract: BACKGROUND: Previous studies have found that GJK Tablets can improve joint pain and function in patients with knee osteoarthritis, but the specific mechanism remains unclear.
OBJECTIVE: To explore the mechanism underlying the chondroprotective effect of GJK Tablets in a mouse model of knee osteoarthritis.
METHODS: Network pharmacology was used to obtain the common targets of GJK Tablets and cartilage homeostasis, and a protein-protein interaction network was constructed. Key signaling pathways were screened through gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. Experimental mice were randomly divided into a control group, a model group, low-, medium-, and high-dose GJK Tablet groups, and a positive drug group (glucosamine hydrochloride capsules). In the latter five groups, the modified Hulth method was used to establish the mouse model of knee osteoarthritis. In the control group, only the relevant tissues of mice were exposed but the ligaments and menisci were not removed. After modeling, the low, medium-, and high-dose groups were intragastrically administered with 187.5, 375, and 750 mg/kg GJK Tablet suspension, respectively. The positive drug group was intragastrically administered with 187.5 mg/kg glucosamine hydrochloride suspension. The model group and the control group were intragastrically administered with an equal volume of normal saline. The administration was carried out every other day for 8 weeks. After the intragastric administration, the pathological morphology of the knee joint cartilage of the mice was observed by hematoxylin-eosin and safranin O-fast green staining, and the Osteoarthritis Research Society International (OARSI) score for the cartilage was evaluated. The protein expressions of SRY-Box transcription factor 9 (Sox9), type II collagen, and matrix metalloproteinase 13 in the mouse cartilage tissue were detected by immunohistochemical staining.
RESULTS AND CONCLUSION: (1) A total of 140 intersection targets of GJK Tablets and cartilage homeostasis were obtained, and Sox9 was one of the targets. (2) Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the main signaling pathways involved were cancer-related pathways, hypoxia-inducible factor 1, MAPK, PI3K/Akt, nuclear factor κB, transforming growth factor β and other signaling pathways. (3) The results of hematoxylin-eosin staining and safranin O-fast green staining showed that compared with the control group, the cartilage contour in the model group was deformed, the defect was stratified, the number of chondrocytes was significantly reduced, the tidemark was blurred, and the OARSI score was higher. Compared with the model group, the cartilage surface in the low-, medium-, and high-dose GJK Tablet groups was more intact, the cartilage thickness and the number of chondrocytes were increased, the tidemark was clearer, and the OARSI scores were significantly decreased. (4) The immunohistochemical results indicated that compared with the control group, the protein expressions of Sox9 and type II collagen in the model group were significantly decreased, and the expression of matrix metalloproteinase 13 was significantly increased (P < 0.01). Compared with the model group, the expression of matrix metalloproteinase 13 in the low-, medium-, and high-dose GJK Tablet groups was significantly decreased (P < 0.01). The positive expressions of Sox9 and type II collagen in the medium- and high-dose GJK Tablet groups were significantly increased (P < 0.01), and the positive expressions showed an increasing trend from the low-dose group to the high-dose group (P < 0.01). To conclude, GJK Tablets can intervene in cartilage homeostasis by regulating Sox9, playing a chondroprotective role in knee osteoarthritis. Signaling pathways such as hypoxia-inducible factor 1, MAPK, PI3K/Akt, nuclear factor κB, and transforming growth factor β may be the main pathways through which GJK Tablets intervene in cartilage homeostasis.

Key words: GJK Tablets, knee osteoarthritis, cartilage homeostasis, articular cartilage, Sox9

CLC Number:

Li Yijin, Li Jiahao, Zhang Haitao, Huang Yiwei, Chen Jinlun, Zeng Yirong, Feng Wenjun. GJK Tablets intervene in cartilage homeostasis to protect articular cartilage of mice with knee osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(12): 2994-3004.

Figures/Tables 10

2.1 关节康片成分靶点与软骨稳态靶点交集分析通过TCMSP、BATMAN-TCM数据库，设置生物利用度≥30%，类药性≥0.18，去除重复项，共检索到关节康片活性成分321个。使用SwissTargetPrediction数据库对以上有效成分进行靶点预测，剔除可信度为0的靶点，共获得500个潜在靶点。检索GeneCards数据库得到软骨稳态靶点7 407个，靶点relevance score最高为74.27，最低为0.21，取relevance score排名前1 000的靶点作为潜在靶点。OMIM数据库检索到45个靶点，DisGeNET数据库检索到0个靶点。合并以上数据库靶点，去除重复项，最终得到软骨稳态相关靶点1 036个。将关节康片潜在靶点与软骨稳态靶点取交集，共得到“关节康片-软骨稳态”交集靶点140个，见图1。 2.2 交集靶点PPI网络构建将交集靶点导入String平台，设置可信度为≥0.4，同时删除游离节点，构建关节康片-软骨稳态PPI网络，共有139个节点，3 514条边。将该PPI网络数据导入Cytoscape 3.10.3 软件，使用degree值进行排序，发现最高degree值为232，最低为2，其中Sox9的degree值为88，最后将PPI网络进行可视化展示，见图2。 2.3 GO和KEGG富集分析通过DAVID数据库对关节康片-软骨稳态交集靶点进行GO和KEGG富集分析，并筛选P值≤0.01的结果，最终得到GO分析条目673条，KEGG分析条目142条。使用微生信平台，取GO富集前10位条目进行气泡图可视化，见图3。GO分析显示，生物过程主要涉及基因表达的正向调控、RNA聚合酶Ⅱ介导的转录正向调控、凋亡过程的负向调控、细胞群体增殖的正向调控等，细胞组分主要涉及含蛋白质的复合物、细胞外区域、内质网腔、膜筏等，分子功能主要涉及酶结合、核受体活性、转录共激活因子结合、转录共调节因子结合等。取KEGG富集前20位条目进行柱状图可视化，见图4。KEGG分析显示，关节康片干预软骨稳态主要涉及癌症相关通路、缺氧诱导因子1(Hypoxia Inducible Factor-1，HIF-1)、丝裂原活化蛋白激酶(Mitogen- Activated Protein Kinase，MAPK)、磷脂酰肌醇3-激酶/蛋白激酶B(Phosphatidylinositol 3-Kinase/Akt，PI3K/Akt)、核因子κB(Nuclear Factor-κB，NF-κB)、转化生长因子β(Transforming Growth Factor-β，TGF-β)等信号通路。"

References

[1] GELBER AC. Knee Osteoarthritis. Ann Intern Med. 2024;177(9):ITC129-ITC144.
[2] YAO Q, WU X, TAO C, et al. Osteoarthritis: pathogenic signaling pathways and therapeutic targets. Signal Transduct Target Ther. 2023; 8(1):56.
[3] PENG Z, SUN H, BUNPETCH V, et al. The regulation of cartilage extracellular matrix homeostasis in joint cartilage degeneration and regeneration. Biomaterials. 2021;268:120555.
[4] SHAO R, SUO J, ZHANG Z, et al. H3K36 methyltransferase NSD1 protects against osteoarthritis through regulating chondrocyte differentiation and cartilage homeostasis. Cell Death Differ. 2024;31(1):106-118.
[5] SHARMA L. Osteoarthritis of the Knee. N Engl J Med. 2021;384(1):51-59.
[6] GIORGINO R, ALBANO D, FUSCO S, et al. Knee Osteoarthritis: Epidemiology, Pathogenesis, and Mesenchymal Stem Cells: What Else Is New? An Update. Int J Mol Sci. 2023;24(7):6405.
[7] 傅永升, 谭茗月, 王卫国, 等. 中药调控膝骨关节炎相关信号通路的研究进展[J].中国实验方剂学杂志,2023,29(22):231-243.
[8] 陈丹丹, 武晏屹, 姬叔梅, 等. 基于文献分析的治疗膝骨关节炎中药应用规律分析[J].中药药理与临床,2022,38(4):186-191.
[9] 李晓峰, 刘元禄, 关雪峰, 等. 补肾活血方剂治疗膝骨关节炎研究进展[J]. 辽宁中医药大学学报,2023,25(10):142-146.
[10] 杨浩宇, 王世坤, 杨东元, 等.补肾活血中药治疗膝骨性关节炎与骨质疏松症“共病”机制研究进展[J].中草药,2024,55(11):3898-3905.
[11] 唐立明, 李鹏飞, 庞智晖, 等. 樊粤光治疗膝骨性关节炎经验介绍[J].新中医,2017,49(5):146-147.
[12] 梁笃, 樊粤光, 王海彬. 关节康治疗膝骨性关节炎22例临床观察[J]. 新中医,2009,41(6):59-60.
[13] 梁笃, 樊粤光, 王海彬. 关节康结合关节镜技术治疗膝骨性关节炎23例[J]. 江西中医药,2009,40(7):28-30.
[14] 曹厚然, 冯文俊, 陈昭, 等. 关节康片调控白细胞介素6表达促进膝关节损伤修复的机制及相关生物标志物的测定[J]. 广州中医药大学学报,2020,37(11):2210-2218.
[15] 周斌, 樊粤光, 曾意荣. 中药关节康对骨性关节炎软骨TGF-β1、IL-1β表达的影响[J]. 中国中医骨伤科杂志,2009,17(2):11-13.
[16] 梁笃, 樊粤光, 王海彬, 等. 关节康中药血清促进去分化软骨细胞再分化的实验研究[J]. 新中医,2009,41(4):101-103.
[17] FUJII Y, LIU L, YAGASAKI L, et al. Cartilage Homeostasis and Osteoarthritis. Int J Mol Sci. 2022;23(11):6316.
[18] ZHANG X, WU S, ZHU Y, et al. Exploiting Joint-Resident Stem Cells by Exogenous SOX9 for Cartilage Regeneration for Therapy of Osteoarthritis. Front Med (Lausanne). 2021;8:622609.
[19] MUTHU S, KORPERSHOEK JV, NOVAIS EJ, et al. Failure of cartilage regeneration: emerging hypotheses and related therapeutic strategies. Nat Rev Rheumatol. 2023;19(7):403-416.
[20] YEE JL, HUANG CY, YU YC, et al. Potential Mechanisms of Guizhi Fuling Wan in Treating Endometriosis: An Analysis Based on TCMSP and DisGeNET Databases. J Ethnopharmacol. 2024;329:118190.
[21] KONG X, LIU C, ZHANG Z, et al. BATMAN-TCM 2.0: an enhanced integrative database for known and predicted interactions between traditional Chinese medicine ingredients and target proteins. Nucleic Acids Res. 2024;52(D1):D1110-D1120.
[22] PESARE E, VICENTI G, KON E, et al. Italian Orthopaedic and Traumatology Society (SIOT) position statement on the non-surgical management of knee osteoarthritis. J Orthop Traumatol. 2023;24(1):47.
[23] HERRERO-BEAUMONT G,LARGO R.Glucosamine and O-GlcNAcylation:a novel immunometabolic therapeutic target for OA and chronic, low-grade systemic inflammation? Ann Rheum Dis. 2020;79(10):1261-1263.
[24] 程丽丽, 黄传兵, 陈君洁, 等. 重骨颗粒干预膝骨关节炎大鼠细胞焦亡的机制[J].中国骨质疏松杂志,2024,30(9):1317-1322.
[25] Ji X, Du W, Che W, et al. Apigenin Inhibits the Progression of Osteoarthritis by Mediating Macrophage Polarization. Molecules. 2023;28(7):2915.
[26] ZHU S, QU W, HE C. Evaluation and management of knee osteoarthritis.J Evid Based Med. 2024;17(3):675-687.
[27] COURTIES A, KOUKI I, SOLIMAN N, et al. Osteoarthritis year in review 2024: Epidemiology and therapy. Osteoarthritis Cartilage. 2024; 32(11):1397-1404.
[28] YOU B, ZHOU C, YANG Y. MSC-EVs alleviate osteoarthritis by regulating microenvironmental cells in the articular cavity and maintaining cartilage matrix homeostasis. Ageing Res Rev. 2023;85:101864.
[29] LIU X, JIANG S, JIANG T, et al. Bioenergetic-active exosomes for cartilage regeneration and homeostasis maintenance. Sci Adv. 2024; 10(42):eadp7872.
[30] WANG Z, EFFERTH T, HUA X, et al. Medicinal plants and their secondary metabolites in alleviating knee osteoarthritis: A systematic review. Phytomedicine. 2022;105:154347.
[31] ZENG L, ZHOU G, YANG W, et al. Guidelines for the diagnosis and treatment of knee osteoarthritis with integrative medicine based on traditional Chinese medicine. Front Med (Lausanne). 2023;10:1260943.
[32] 李慧, 周祥, 刘伟, 等. 补肾活血法治疗膝关节骨性关节炎机制研究进展[J]. 实用中医药杂志,2024,40(5):1027-1030.
[33] 陈继鑫, 周沁心, 余伟杰, 等. 补肾活血法治疗膝骨关节炎随机对照试验结局指标的现状分析[J]. 中国中药杂志,2024,49(6):1661-1672.
[34] KIM SM, JO SY, PARK HY, et al. Investigation of Drug-Interaction Potential for Arthritis Dietary Supplements:Chondroitin Sulfate, Glucosamine,and Methylsulfonylmethane. Molecules. 2023;28(24):8068.
[35] MENG Z, LIU J, ZHOU N. Efficacy and safety of the combination of glucosamine and chondroitin for knee osteoarthritis: a systematic review and meta-analysis. Arch Orthop Trauma Surg. 2023;143(1):409-421.
[36] RABADE A, VISWANATHA GL, NANDAKUMAR K, et al. Evaluation of efficacy and safety of glucosamine sulfate, chondroitin sulfate, and their combination regimen in the management of knee osteoarthritis: a systematic review and meta-analysis. Inflammopharmacology. 2024;32(3):1759-1775.
[37] ROELOFS AJ, DE BARI C. Osteoarthritis year in review 2023: Biology.Osteoarthritis Cartilage. 2024;32(2):148-158.
[38] ARAI Y, CHA R, NAKAGAWA S, et al. Cartilage Homeostasis under Physioxia. Int J Mol Sci. 2024;25(17):9398.
[39] TAHEEM DK, JELL G, GENTLEMAN E.Hypoxia Inducible Factor-1alpha in Osteochondral Tissue Engineering. Tissue Eng Part B Rev. 2020; 26(2):105-115.
[40] LU R, WANG YG, QU Y, et al. Dihydrocaffeic acid improves IL-1beta-induced inflammation and cartilage degradation via inhibiting NF-kappaB and MAPK signalling pathways. Bone Joint Res. 2023;12(4):259-273.
[41] TIAN B, ZHANG L, ZHENG J, et al. The role of NF-kappaB-SOX9 signalling pathway in osteoarthritis. Heliyon. 2024;10(17):e37191.
[42] KLAMPFLEUTHNER F, LOTZ B, RENKAWITZ T, et al.Stage-Dependent Activity and Pro-Chondrogenic Function of PI3K/AKT during Cartilage Neogenesis from Mesenchymal Stromal Cells. Cells. 2022;11(19):2965.
[43] XU R, WU J, ZHENG L, et al. Undenatured type II collagen and its role in improving osteoarthritis. Ageing Res Rev. 2023;91:102080.
[44] SONG H, PARK KH. Regulation and function of SOX9 during cartilage development and regeneration. Semin Cancer Biol. 2020;67(Pt 1):12-23.
[45] DANALACHE M, UMRATH F, RIESTER R, et al. Proteolysis of the pericellular matrix: Pinpointing the role and involvement of matrix metalloproteinases in early osteoarthritic remodeling. Acta Biomater. 2024;181:297-307.
[46] HU Q, ECKER M. Overview of MMP-13 as a Promising Target for the Treatment of Osteoarthritis. Int J Mol Sci. 2021;22(4):1742.
[47] OUYANG Y, WANG W, TU B, et al. Overexpression of SOX9 alleviates the progression of human osteoarthritis in vitro and in vivo. Drug Des Devel Ther. 2019;13:2833-2842.
[48] SHAO Y, ZHANG H, GUAN H, et al. PDZK1 protects against mechanical overload-induced chondrocyte senescence and osteoarthritis by targeting mitochondrial function. Bone Res. 2024;12(1):41.
[49] FU B, SHEN J, ZOU X, et al. Matrix stiffening promotes chondrocyte senescence and the osteoarthritis development through downregulating HDAC3. Bone Res. 2024;12(1):32.
[50] SUN Y, YOU Y, WU Q, et al. Senescence-targeted MicroRNA/Organoid composite hydrogel repair cartilage defect and prevention joint degeneration via improved chondrocyte homeostasis. Bioact Mater. 2024;39:427-442.
[51] GUAN Z, JIN X, GUAN Z, et al. The gut microbiota metabolite capsiate regulate SLC2A1 expression by targeting HIF-1alpha to inhibit knee osteoarthritis-induced ferroptosis. Aging Cell. 2023;22(6):e13807.
[52] LIU J, JIA S, YANG Y, et al. Exercise induced meteorin-like protects chondrocytes against inflammation and pyroptosis in osteoarthritis by inhibiting PI3K/Akt/NF-kappaB and NLRP3/caspase-1/GSDMD signaling. Biomed Pharmacother. 2023;158:114118.
[53] ZHANG Z, MA J, YI Y, et al. Isoliensinine suppresses chondrocytes pyroptosis against osteoarthritis via the MAPK/NF-kappaB signaling pathway. Int Immunopharmacol. 2024;143(Pt 3):113589.
[54] WANG L, XU H, LI X, et al. Cucurbitacin E reduces IL-1beta-induced inflammation and cartilage degeneration by inhibiting the PI3K/Akt pathway in osteoarthritic chondrocytes. J Transl Med. 2023;21(1):880.
[55] LIU L, ZHAO C, ZHANG H, et al. Asporin regulated by miR-26b-5p mediates chondrocyte senescence and exacerbates osteoarthritis progression via TGF-beta1/Smad2 pathway. Rheumatology (Oxford). 2022;61(6):2631-2643.
[56] PRIMORAC D, MOLNAR V, MATISIC V, et al.Comprehensive Review of Knee Osteoarthritis Pharmacological Treatment and the Latest Professional Societies’ Guidelines. Pharmaceuticals (Basel). 2021; 14(3):205.