Tougu Xiaotong Capsule promotes repair of osteoarthritis cartilage damage in mice by activating the CXCL12/GDF5 pathway

doi:10.12122/j.issn.1673-4254.2025.06.02

Abstract

Abstract:

Objective To explore the mechanism by which Tougu Xiaotong Capsule (TXC) promotes chondrogenic differentiation and cartilage repair in mice with osteoarthritis (OA). Methods Fifty 8-week-old male C57BL mice were randomly divided into normal control group, cartilage damage (induced by subchondral ring-shaped drilling) model group and TXC treatment groups at low, moderate and high doses (184, 368 and 736 mg/kg, respectively). Saline (in normal control and model groups) and TXC were administered after modeling by daily gavage for 6 consecutive weeks. The changes of cartilage damage in the mice were assessed by measuring thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) and using micro-CT, modified safranine O and fast green staining, HE staining, and qPCR. Primary cultures of mouse synovial mesenchymal stem cells (SMSCs) with lentivirus vector transfection for interfering CXCL12, TXC treatment, or both for 24 h were examined for chondrogenic differentiation using immunofluorescence staining, scratch assay, immunocytochemistry, and Western blotting. Results In mouse models with cartilage damage, TXC treatment at the moderate dose significantly alleviated joint pain, promoted cartilage repair, and upregulated the mRNA expression levels of CXCL12, GDF5, collagen II, aggrecan, Comp and Sox9 in the cartilage tissue. In primary mouse SMSCs, CXCL12 knockdown resulted in significant reduction of GDF5 protein expression, migration ability and Sox9 protein expression, and these changes were obviously reversed by TXC treatment. Conclusion TXC promotes chondrogenic differentiation of mouse SMSCs to promote repair of cartilage damage in mice by activating the CXCL12/GDF5 pathway.

Key words: Tougu Xiaotong Capsule, synovial mesenchymal stem cells, cartilage damage, CXCL12/GDF5 pathway

Changlong FU, Lu XU, Ruolan CHEN, Jinghang YANG, Yan LUO, Yanfeng HUANG. Tougu Xiaotong Capsule promotes repair of osteoarthritis cartilage damage in mice by activating the CXCL12/GDF5 pathway[J]. Journal of Southern Medical University, 2025, 45(6): 1122-1130.

Figures/Tables 8

Tab.1 Criteria for Mankin's Scoring of joint cartilage damage

Score	Cartilage structure	Chondrocytes	Matrix staining	Tidemark
0	Normal	Normal	Normal	Complete
1	Irregular surface	Diffuse cellular hyperplasia	Mild weakening	Incomplete
2	Surface irregularity with pannus	Focal cellular hyperplasia	Moderate weakening	-
3	Fissures extending into the transitional zone	Hypocellularity	Severe weakening	-
4	Fissures penetrating the radial zone	-	Uncolored	-
5	Fissures invading the calcified layer	-	-	-
6	Complete structural disruption	-	-	-

Tab.2 Primer sequences for RT-qPCR

Gene	Primer sequence	Primer length
CXCL12	F:5'-GCTCTGCATCAGTGACGGTA-3' R:5'-GCCGTGCAACAATCTGAAGG-3'	143 bp
GDF5	F:5'-CACGCAGTCATTCAGACCCT-3' R:5'-TACACCACGTTGTTGGCAGA-3'	125 bp
Collagen II	F:5'-ACCAGATTGAGAGCATCCGC-3' R:5'-CAGCCCTGGTTGGGATCAAT-3'	127 bp
Aggrecan	F:5'-AGCACTACCTCCGACATAGAC-3' R:5'-TGATGGCAACATTCACCTCTG-3'	127 bp
Comp	F:5'-CATCCTACCGCTGGTTCCTG-3' R:5'-CCGTGTCCAACACCACATTG-3'	108 bp
Sox9	F:5'-CCACGGAACAGACTCACATC-3' R:5'-CCTCTCGCTTCAGATCAACTT-3'	192 bp
GAPDH	F:5'-TGGAAAGCTGTGGCGTGATG-3' R:5'-TACTTGGCAGGTTTCTCCAGG-3'	189 bp

Fig.1 Assessment of the therapeutic efficacy of Tougu Xiaotong Capsule (TXC) in mouse models with joint cartilage damage. A: Mechanical withdrawal threshold (MWT). B: Thermal withdrawal latency (TWL). C: Mankin's Score. D: Micro-CT findings. E, F: Safranine O and fast green staining results (Scale bar=100 μm). *P<0.05 vs NC group; #P<0.05 vs Model group.

Fig.2 Effect of TXC on mRNA levels of CXCL12, GDF5, collagen II, aggrecan, Comp and Sox9 in mouse cartilage tissue. *P<0.05 vs NC group; #P<0.05 vs Model group.

Fig.3 Culture, identification, and CXCL12 lentiviral transfection of mouse synovial mesenchymal stem cells (SMSCs). A: Primary cultures of mouse SMSCs. B: Identification of mouse SMSCs. C: Mouse SMSCs with CXCL12 lentiviral transfection (Scale bar=25 μm).

Fig.4 TXC promotes the expression of GDF5 fluorescence protein (scale bar=100 μm).

Fig.5 TXC promotes expression of GDF5 fluorescence protein in mouse SMSCs (Scale bar=100 μm).

Fig.6 TXC promotes chondrogenic differentiation of mouse SMSCs. A: Immunohistochemical staining for collagen II (scale bar=25 μm). B: Protein expression of CXCL12, GDF5, and Sox9 detected by Western blotting. *P<0.05 vs Control group; #P<0.05 vs sh-CXCL12 group.

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