Mast cell degranulation mediates analgesic effects of acupuncture at different stimulation durations in arthritic rats

Abstract

Background: To understand the effect and mechanism of different acupuncture durations on analgesic effects.

Methods: An adjuvant-induced arthritis rat model evaluated the therapeutic effects of acupuncture at the Zusanli acupoint (ST36). A robotic arm provided stable lifting-thrusting stimulation for different durations (2, 5, 10, 20, and 30 min). Mechanical and thermal pain thresholds were measured before and after treatment to assess the analgesic effects. Additionally, tissue sections from ST36 were stained and analyzed to assess mast cell degranulation rates, and in vitro stretching experiments were performed at different stretching times (0-60 min) to measure the release of histamine, 5-hydroxytryptamine, and leukotrienes.

Results: Within the 30 min acupuncture duration range set in this study, the pain threshold recovery rate in rats exhibited a steady upward trend as the acupuncture duration gradually increased, reaching its optimal level between 20 and 30 min. The mast cell degranulation rate exhibited a similar time-related trend, reaching 80-90 % at 20-30 min. Correlation analysis revealed a significant positive correlation between the pain threshold recovery rate and mast cell degranulation rate. In the cell-stretching experiment, the release of biochemical mediators was time-dependent.

Conclusions: In the adjuvant-induced arthritis rat model, the analgesic effect observed at 20-30 min was superior to that observed at shorter durations, which was associated with the mast cell degranulation rate and release of biochemical mediators in the tissue.

Keywords: Acupuncture duration; Adjuvant-induced rat model; Analgesia; Mast cell; Pain threshold.

Fig. 1

General overview of the experimental process. (A) Schematic diagram of the experimental procedure in rats. Before modeling, the rats needed to adapt for 2 d. On day 0, complete Freund's adjuvant was injected into the left ankle joint of the rats to induce acute inflammation. Two days later, acupuncture treatment (AP treatment) was performed on the left ST36. Pain thresholds were measured before complete Freund's adjuvant injection (L₀=$\frac{1}{2}$[adaption #1 + adaptation #2]), before AP (L₁) and after AP (L₂). Tissue preparation included tissue extraction from the acupoint area, embedding, sectioning, and toluidine blue staining. (B) After peritoneal mast cells were extracted from the mouse abdomen, they were cultured in cell culture plates for 28 d and inoculated into Flexcell culture plates. Cell supernatants were extracted after stretching and subsequently measured using specific Enzyme-Linked Immunosorbent Assay (ELISA) kits.

Fig. 2

Overview of mechanical and thermal pain threshold test results for rats, coupled with the corresponding pain threshold recovery ratio (PTRR) (n = 6 per group). (A and B) The mechanical and thermal pain thresholds of rats across eight distinct groups. The figure arranges them in sequence from left to right: the blank group, the needle-retaining group, and groups subjected to robotic-assisted acupuncture for 2, 5, 10, 20, and 30 min, followed by the manual stimulation group, with six rats in each group. The pain thresholds of each rat were evaluated at four different time points: Adaptation #1, Adaptation #2, modeling, and post-acupuncture treatment. A notable asterisk (*) signifies significant deviations from the baseline adaptation pain threshold (L₀), whereas a hash (#) marks significant differences between post-acupuncture (L₂) and post-modeling (L₁) data. (C and D) The PTRR for mechanical and thermal pain thresholds in the same eight groups. The groups are arranged identically from left to right. The statistical significance of differences in comparison with the blank group is clearly indicated. The levels of significance are denoted as follows: **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and NS (not significant).

Fig. 3

Mast cell (MC) degranulation post-acupuncture treatment (n = 6 per group). (A) Histological sections from the skin and muscle tissues at the ST36 acupoint, with a focus on two specific groups: the 2 min stimulus and 20 min stimulus groups. MCs within these sections are stained and distinctly marked with triangles for clarity. Red triangles signify activated and degranulated cells, whereas blue triangles denote cells that have not undergone degranulation. (B) MC degranulation rate of different groups. P values of comparisons between the blank and other groups were calculated after analysis of variance analysis. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, NS (not significant).

Fig. 4

Correlation between mast cell (MC) degranulation rate and pain threshold recovery ratio (PTRR) under the influence of acupuncture duration (n = 6 per group). (A) Thermal PTRR and MC degranulation rate. (B) Mechanical PTRR and MC degranulation rate.

Fig. 5

Substances released by mast cells after cell stretching (n = 6 per group). (A) Release of 5-hydroxytryptamine (5-HT) over time. (B) Release of histamine over time. (C) Release of leukotrienes over time. NS indicates statistically not significant (P > 0.05).

Fig. 6

Mechanism of pain relief induced by mast cell (MC) activation through acupuncture. Acupuncture directly induces MC degranulation, resulting in the release of 5-hydroxytryptamine, histamine, and other analgesic substances. These substances stimulate further MC degranulation and also activate nerve endings, causing axonal reflexes that release additional mediators, which, in turn, induce MC degranulation. Additionally, acupuncture can trigger electrical potential changes in nerve cells, leading to analgesic effects.