Mast Cell-Associated Serotonin in Acupoint Contributes to Acupuncture Analgesia in Arthritis Rats by Mediating ATP Release

¹ Shanghai Key Laboratory for Acupuncture Mechanism and Acupoint Function, Department of Aeronautics and Astronautics, Fudan University, 200433 Shanghai, China

² School of Acupuncture-Moxibustion and Tuina, Shanghai University of Traditional Chinese Medicine, 201203 Shanghai, China

³ Shanghai Research Center for Acupuncture and Meridians, 200433 Shanghai, China

^*Correspondence: lnwang@shutcm.edu.cn (Li-Na Wang); dizhang@fudan.edu.cn (Di Zhang)
Academic Editor: Graham Pawelec

Front. Biosci. (Landmark Ed) 2023, 28(1), 1; https://doi.org/10.31083/j.fbl2801001

Submitted: 3 September 2022 | Revised: 10 November 2022 | Accepted: 28 November 2022 | Published: 9 January 2023

This is an open access article under the CC BY 4.0 license.

Abstract

Background: The activation of subcutaneous mast cells (MCs) helps to trigger the analgesic effect induced by acupuncture (AP), a traditional oriental therapy, that has been gradually accepted worldwide. This work aimed to reveal whether the serotonin (5-hydroxytryptamine, 5-HT) released from MCs plays an important role in this process, which has a controversial effect in the mechanism of pain. Methods: In vivo tests, a 20-min session of AP was applied at Zusanli acupuncture point (acupoint) of acute ankle arthritis rats. Pain thresholds of the injured hindpaw were assessed to reflect the pain state, and the targeting substances in the interstitial space of the treated acupoint were sampled by microdialysis. In vitro experiments, exogenous 5-HT (exo-5-HT) was introduced to mediate adenosine triphosphate (ATP) release from cultured MCs. Results: Needling promoted 5-HT accumulation at the Zusanli acupoint, which was prevented by sodium cromolyn. AP’s analgesic effect was suppressed by the inhibition of 5-HT receptors at the acupoint, especially 5-HT ${}_{\text{1A}}$ subtype. In vitro tests, mechanical perturbation mimicking needling stimulation induced MCs to release 5-HT. 1 $\mu{}$ M and 10 $\mu{}$ M of exo-5-HT facilitated ATP release, which was restrained by blocking of 5-HT ${}_{1}$ receptors rather than 5-HT ${}_{3}$ receptors. As 5-HT, ATP and adenosine were also transiently accumulated in the treated acupoint during needling. Promoting ATP hydrolysis or activation adenosine A1 receptors duplicated AP analgesic effect. Finally, the inhibition of ATP receptors by suramin or pyridoxal phosphate-6-azo tetrasodium salt hydrate (PPADS) prevented AP analgesic effect. Conclusions: Our results suggest that MC-associated 5-HT release at acupoints contributes to AP analgesia, and the mediation of ATP secretion through 5-HT ${}_{\text{1A}}$ receptors might be the underlying mechanism at play. ATP could facilitate adenosine production or the propagation of needling signals.

Keywords

acupuncture analgesia

mast cells

5-HT

5-HT_1A receptor

ATP

Funding

81574076/National Natural Science Foundation of China

82174488/National Natural Science Foundation of China

81590953/National Natural Science Foundation of China

81574053/National Natural Science Foundation of China

2021LK098/Budget Research Project of Shanghai Education Commission

21DZ2271800/Shanghai Key Laboratory for Acupuncture Mechanism and Acupoint Function

22ZR1461500/Shanghai Natural Science Foundation

Figures

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Fig. 1.

AP mobilized 5-HT release from MCs in acupoint, which contributed to AP-analgesic effect (n = 4–10, mean $\pm{}$ SE). (A) Time-course of inters. 5-HT concentration in response to needling on AA model rats in the absence and presence of CRO (0.02 g/mL, 20 $\mu{}$ L) pre-injected in ST 36 (n = 4). Inters. 5-HT was collected by microdialysis technique and determined by ELISA. ### p $<$ 0.001 vs. baseline (time = 0 min); ** and ***, p $<$ 0.01 and 0.001 vs. +CRO, respectively. Two-way ANOVA and LSD. test were used. (B) Changes of inters. 5-HT amount in response to AP in the absence and presence of CRO (n = 4). They were calculated based on the area under the curve in (A). ## and ###, p $<$ 0.01 and 0.001 vs. baseline (time = -30–0 min); *, ** and ***, p $<$ 0.05, 0.01 and 0.001 vs. +CRO, respectively. (C,D) Effects of AP treatment on CFA-induced tactile allodynia (PWT) and thermal hyperalgesia (PWL) of injured-side hindpaws, respectively. ### p $<$ 0.001 vs. baseline (Day 0). $\bullet$ $\bullet$ $\bullet$ p $<$ 0.001 vs. Day 2, before AP. Two-way ANOVA and LSD. test were used to perform within-group comparisons. (E,F) Effects of pre-co-injection of Methiothepin (Meth.) and Granisetron (Gran.) in ST 36 on AP-relieved PWT and PWL, respectively. Data illustrate the final behavioral tests. ** and ***, p $<$ 0.01 and 0.001, respectively. One-way ANOVA and LSD. test were used to perform between-group comparisons. (G) AP/Mechanical stimulation induced MCs to degranulate. Allows point to degranulated MCs. (i) Paraffin sections prepared from ST36 of AA model group (left) and AP group (right). MCs were stained with toluidine blue and were marked in purple. (ii) Connective tissue slices isolated from ST 36 of normal rat before (left) and after (right) hypotonic shock. (iii) HMC-1 cells before (left) and after (right) hypotonic shock. In (ii) and (iii), the tissue slice and HMC-1 cells were incubated in 200 mOSm/kg H2O-hypotonic solution for 25 min and 15 min, respectively. The plasma membrane of degranulated MCs became rough and the granules scattered around the cells. Scale bar: 50 $\mu{}$ m. Mech. stim.: Mechanical stimulation. (H) Release of tryptase, ATP and 5-HT from HMC-1 cells in response to 50% hypotonic shock (n = 7–10). Unpaired t-test was performed for two groups’ comparison. *** p $<$ 0.001. In (E–G), numbers above each column denote sample size (n).

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