The Role of p38 Mitogen-Activated Protein Kinase-Mediated F-Actin in the Acupuncture-Induced Mitigation of Inflammatory Pain in Arthritic Rats

Abstract

The analgesic efficacy of acupuncture has been widely recognized. However, the mechanism by which manual acupuncture-generated mechanical stimuli translate into biological signals remains unclear. This study employed a CFA-induced inflammatory pain rat model. Acupuncture intervention was then performed following standardized procedures. Enzyme-linked immunosorbent assay (ELISA) assessed inflammatory cytokines levels, while immunofluorescence and qRT-PCR screened the level of p38 and F-actin expression in the ST36 acupoint area of rats. Results indicated increased inflammatory factors, including IL-1β and TNFα, with reduced paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) in CFA rats compared to unmodeled rats. After acupuncture intervention, the heightened expression level of F-actin and p38 mRNA and the phosphorylation of p38 in the acupoint area was observed alongside decreased inflammatory factors in diseased ankle joints. The application of lifting and thrusting manipulations further enhanced the effect of acupuncture, in which the molecular expression level of muscle and connective tissue increased most significantly, indicating that these two tissues play a major role in the transformation of acupuncture stimulation. Moreover, antagonizing p38 expression hindered acupuncture efficacy, supporting the hypothesis that p38 MAPK-mediated F-actin transduces mechanical signals generated by acupuncture and related manipulation into biological signals.

Keywords: acupoint area; acupuncture; inflammatory pain; p38 MAPK pathway.

Figure 1

The details of modeling and acupuncture treatment. (A) Swollen left hindfoot metatarsophalangeal joint one day after modeling. (B) Location of the acupuncture point ST36 used in this study. (C) The self-made acupuncture needle cannula used to control the amplitude of the lifting and thrusting manipulations.

Figure 2

Effects of acupuncture on nociceptive behaviors in the CFA-induced rat arthritic model. (A) PWMT and (B) PWTL were evaluated 24 h before CFA injection (baseline), 1 day after CFA injection (injection), and 1, 3, and 7 days after acupuncture treatment. PWMT, the paw withdrawal mechanical threshold; PWTL, the paw withdrawal thermal latency. * p < 0.05 and ** p < 0.001 compared with the control group; # p < 0.05 compared with the CFA group; § p < 0.05 compared with the LTM group. N = 8 in each group. PWMT, paw withdrawal mechanical threshold; PWTL, paw withdrawal thermal latency; CFA, complete Freund’s adjuvant; MA, minimum acupuncture; LTM, lifting and thrusting manipulations.

Figure 3

(A) Interleukin-1 beta (IL-1β) and (B) tumor necrosis factor-alpha (TNF-α) levels in the inflamed ankle joint of CFA-induced rat arthritic model after a seven-day intervention. Data are shown as mean ± SD. ** p < 0.001 compared with the control group; ## p < 0.001 compared with the CFA group; & p < 0.05 compared with the MA group; § p < 0.05 and §§ p < 0.001 compared with the LTM group. N = 8 in each group. CFA, complete Freund’s adjuvant; MA, minimum acupuncture; LTM, lifting and thrusting manipulations.

Figure 4

Real-time PCR analysis of p38 mRNA expression in the ST36 acupoint area of the CFA-induced rat arthritic model after a seven-day intervention. Relative quantification was performed using the comparative Ct method (2^–ΔΔCt). Results are expressed as arbitrary units (control = 1) and presented as mean ± SD. ## p < 0.001 compared with the CFA group; && p < 0.001 compared with the MA group; §§ p < 0.001 compared with the LTM group. N = 8 in each group. CFA, complete Freund’s adjuvant; MA, minimum acupuncture; LTM, lifting and thrusting manipulations.

Figure 5

F-actin expression and p-p38 level in fibroblasts of the ST36 acupoint area after a seven-day intervention. (A,B) Fluorescence intensity of F-actin filaments and p-p38 in fibroblasts from skin, connective, and muscle tissues. # p < 0.05 and ## p < 0.001 compared with the CFA group; && p < 0.001 compared with the MA group; §§ p < 0.001 compared with the LTM group. N = 12 in each group. (C,D) The linear correlation between F-actin expression and p-p38 level in the MA and LTM groups. (E,F) Immunofluorescence staining for (E) F-actin and (F) p-p38 (red), Vimentin (a fibroblast marker) (green), and DAPI (blue) in fibroblasts of the acupoint area with different interventions. The orange and yellow zones (indicated by arrows) denote the colocalization of all three elements within the loose subcutaneous connective tissue and intermuscular connective tissue in both the MA and LTM groups. Therefore, fibroblasts expressing F-actin or p-p38 positively are predominantly distributed in these regions. Scale bar, 40×: 100 μm. p-p38, phosphorylated p38 MAPK; CFA, complete Freund’s adjuvant; MA, minimum acupuncture; LTM, lifting and thrusting manipulations.