Therapeutic Effects of Fire Needling Acupuncture on Pain Relief and Cartilage Protection in MIA-Induced Knee Osteoarthritis Rats: The Role of Macrophage Polarization in Synovium and Angiogenesis in Subchondral Bone

Abstract

Purpose: Knee osteoarthritis (KOA) is a prevalent degenerative disease impacting bone and joint health. Clinical studies indicate that fire needling acupuncture can alleviate joint stiffness, pain, and dysfunction in KOA. Previous research demonstrated its efficacy in reducing pain, mitigating cartilage damage, and regulating macrophage polarization, but its effects on subchondral bone remain unclear. This study aimed to evaluate the therapeutic effects of fire needling acupuncture on subchondral bone in KOA.

Methods: Sprague-Dawley rats were divided into three groups: control (CON), monosodium iodoacetate (MIA), and fire needling acupuncture (FNA) (n=6 per group). A KOA model was established using 0.3 mg/50 µL MIA, followed by acupuncture at acupoints SP10, ST34, ST35, EX-LE5, and ST36 twice weekly. Evaluations included body weight, joint diameter, weight distribution, and mechanical withdrawal threshold. Micro-CT imaging assessed tibial plateau bone mass changes. Histological evaluations used HE, Safranin O/Fast Green, and toluidine blue staining. Immunohistochemistry examined COL2, MMP9, and MMP13 expression, while macrophage polarization was analyzed using immunofluorescence for F4/80, iNOS, and Arg-1. TRAP staining assessed osteoclast activity, and immunofluorescence for CD31/Emcn and VEGF evaluated angiogenesis in subchondral bone.

Results: Fire needling acupuncture significantly improved weight distribution and mechanical withdrawal thresholds, reduced synovial inflammation and abnormal changes in subchondral bone, and preserved cartilage integrity in MIA-induced KOA rats. Notably, F4/80 and iNOS expression levels decreased, while Arg-1 expression increased after treatment. Additionally, TRAP, CD31/Emcn, and VEGF expression in subchondral bone decreased following fire needling acupuncture.

Conclusion: Fire needling acupuncture mitigates pain behavior, synovial inflammation, cartilage degradation, and abnormal changes in subchondral bone in MIA-induced KOA rats. The therapeutic mechanism may involve modulation of synovial macrophage polarization and subchondral bone angiogenesis. Further research is warranted to elucidate the precise molecular pathways and the interaction between macrophage polarization and angiogenesis.

Keywords: cartilage degradation; fire needling acupuncture; knee osteoarthritis; macrophages polarization; subchondral bone angiogenesis.

Figure 1

(A) Flowchart of animal experiment. (B) Body weight analysis. (C) Joint diameter analysis. ***P<0.001, ****P<0.0001, vs CON group. (D) Weight bearing analysis. *P<0.05, **P<0.01, ***P<0.001, vs MIA group. (E) Paw withdrawal threshold analysis. *P<0.05, **P<0.01, ***P<0.001, vs MIA group. Data are expressed as the mean ± SEM, n = 6 for each group.

Figure 2

(A) Macroscopic observation of knee joints. (B) Micro-CT comparison of the knee joint in MIA-induced KOA rats. (C) Hematoxylin and eosin (HE) staining, Safranin O/Fast Green (Saf-O) staining, and Toluidine blue staining of the knee joint cartilage of the rats. Scale bars = 200μm or 40μm. (D–J) Micro-CT analysis of bone volume (BV), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular pattern factor (Tb.Pf), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineral density (BMD) of the tibial plateau of KOA rats. (K) Quantification of OARSI scores. Data are expressed as the mean ± SEM, n = 3 for each group. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

(A) Images of Immunohistochemistry staining of COL2, MMP9 and MMP13 of rat knee joints. Scale bars = 200μm or 100μm. (B–D) Quantification of IHC staining of the number of positive cells of COL2, MMP9 and MMP13. Data are expressed as the mean ± SEM, n = 3 for each group. *P<0.05, ***P<0.001.

Figure 4

(A) Images of HE staining and Saf-O staining of the synovium of rat knee joint. Scale bars = 100μm or 40μm. (B–D) Images of Immunofluorescence staining of F4/80, iNOS and Arg-1 of the synovium of rat knee joint. Scale bars = 40μm or 20μm. (E) Quantification of synovial inflammation scores. (F–H) Quantification of the number of positive cells of F4/80, iNOS and Arg-1. Data are expressed as the mean ± SEM, n = 3 for each group. *P<0.05, **P<0.01.

Figure 5

(A) Micro-CT images and Saf-O staining images. The green rectangles represent subchondral bone cysts. (B) Saf-O staining images of subchondral bone of KOA. Scale bars = 200μm or 40μm. (C) TRAP staining images of subchondral bone of KOA. Scale bars = 400μm or 100μm. (D) Quantification of TRAP⁺. Data are expressed as the mean ± SEM, n = 3 for each group. **P<0.01, ***P<0.001.

Figure 6

(A) Images of Immunofluorescence staining and fluorescence intensity of CD31/Emcn of rat knee joints. Scale bars = 200μm or 40μm. (A and B) Images of Immunofluorescence staining of VEGF of rat knee joints. Scale bars = 200μm or 40μm. (C) Quantification of CD31^hi/Emcn^hi. (D) Quantification of VEGF. Data are expressed as the mean ± SEM, n = 3 for each group. **P<0.01, ***P<0.001.

Figure 7

Schematic diagram illustrating how fire needling acupuncture regulates macrophage polarization, reduces cartilage damage, and inhibits abnormal angiogenesis in the subchondral bone.