Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia

Abstract

Background: Acupuncture has been proven effective for various types of pain, and peripheral molecular signals around acupuncture-treated areas have been suggested to contribute to the analgesic effects of acupuncture. However, the underlying mechanism from these peripheral molecular signals to central ones remains unclear. The purpose of this study was to investigate whether peripheral Rho-associated protein kinase (ROCK) activation induced by acupuncture treatment mediates acupuncture analgesia, and also to investigate the relationship between ROCK activation and extracellular signal-regulated kinase (ERK), which has previously been proven to mediate acupuncture analgesia and other related molecular changes during acupuncture.

Methods: Acupuncture was treated at the bilateral GB34 acupoints of C57BL/6 mice, after which changes in ROCK activation and the location of its expression in the skin were analyzed. To verify the role of ROCK in acupuncture analgesia, we administrated ROCK inhibitor Y-27632 (0.3 μg/ul) into the skin before acupuncture treatment with formalin and complete Freund adjuvant (CFA) induced pain models, then the nociceptive responses were analyzed.

Results: Acupuncture treatment produced ROCK2 activation in the skin after 30 and 60 min, and the histological analyses revealed that ROCK2 was activated in the fibroblast of the dermis. The acupuncture-induced ROCK2 expression was significantly attenuated by the ERK inhibitor, whereas phospho-ERK expression was not inhibited by ROCK inhibitor. In both the formalin- and CFA-induced mouse pain models, acupuncture analgesia was blocked by ROCK inhibitor administration.

Conclusion: Acupuncture treatment-induced ROCK2 expression is a downstream effector of phospho-ERK in the skin and plays a crucial role in acupuncture analgesia.

Keywords: Acupuncture; Analgesia; Molecular mechanism; Peripheral; Rho associated protein kinase.

Fig. 1

Experimental schedule of this study. CFA, complete Freund adjuvant.

Fig. 2

ROCK1, ROCK2, and p-ERM expression in the skin after acupuncture treatment. Expression levels of (A) ROCK1, (B) ROCK2, and (C) p-ERM in the skin were determined at 5, 10, 30, and 60 min after acupuncture treatment via western blotting analyses. ROCK2 activation increased significantly at 30 and 60 min after acupuncture treatment compared to that in the control group (B). Activation of p-ERM was increased significantly 5 min after acupuncture treatment (C). Here, *p < 0.05 compared to the CON group followed by the Student's t-test (each n = 3–5) and error bars indicate the SEM. CON, treated with no acupuncture; ROCK, Rho-associated protein kinase; p-ERM, phosphorylated ezrin-radixin-moesin.

Fig. 3

ROCK2 expression in the skin after acupuncture treatment, and inhibition of acupuncture-induced ROCK2, p-ERM, and p-ERK activation by U0126 (i.e., ERK inhibitor) and Y-27632 (i.e., ROCK inhibitor). (A-B) ROCK2 was activated in the skin, especially in the fibroblasts of the dermis 30 min after acupuncture treatment. 5B5, fibroblast marker. Blue: counterstain with 40,6-diamidino-2-phenylindole (DAPI), Green: expression of ROCK2, Red: expression of 5B5, a fibroblast marker. (C-D) Acupuncture induced ROCK2 and p-ERM activation was attenuated by U0126 administration, injected at the GB34 acupuncture point intradermally. (E) Acupuncture-induced ROCK2 activation in the skin was significantly attenuated by 0.3 μg/ul of Y-27632. (F) Acupuncture induced p-ERK expression was attenuated by U0126, but not by Y-27632 administration. Acupuncture induced p-ERM expression was attenuated by U0126 and Y-27632 administration. Scale bar: 100 µm (A) and 20 µm (B). ^∗∗p < 0.01 compared to the CON group; ^$$p < 0.01 compared to the ACU group followed by the Student's t-test (each n = 3). Error bars indicate the SEM. ACU, treated with acupuncture; ACU+U, treated with U0126 and acupuncture; ACU+Y, treated with Y-27632 and acupuncture; CON, treated with no acupuncture; p-ERK, phosphorylated extracellular signal-regulated kinase; p-ERM, phosphorylated ezrin-radixin-moesin; ROCK, Rho-associated protein kinase.

Fig. 4

The analgesic effects of acupuncture treatment were reversed by Y-27632 administration in mouse pain models induced by formalin and complete Freund's adjuvant (CFA). (A-B) The duration of the formalin-evoked nociceptive response, such as flinching, licking, and biting of the right hind paw, was analyzed during 10-30 min after the formalin injection (A) and during each 5-minute block (B). (C-D) The frequency of positive responses was determined using a von Frey filament exerting 0.6 g of force (C: ipsilateral, D: contralateral) every 10 min after acupuncture treatment in a CFA-induced mouse pain model. *p < 0.05 and ^⁎⁎⁎p < 0.001 compared to the CON group; ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 compared to the FOR or CFA group; ^$$p < 0.01, ^$$$p < 0.001 compared to the ACU group. One-way ANOVA was followed by the Newman–Keuls test (A) and 2-way ANOVA was followed by the Bonferroni test (B-D) (A-B: each n = 8, C-D: each n = 7). Error bars indicate SEM. ACU, injected with formalin or CFA, and treated with acupuncture; ACU+Y, injected with formalin or CFA, administered Y-27632, and treated with acupuncture; CFA, injected with CFA; CFA+Y, injected with CFA and administered Y-27632. CON, treated with control procedures; FOR, injected with formalin; FOR+Y, injected with formalin and administered Y-27632.

Fig. 5

Hypothesis of signaling transduction mechanism of acupuncture analgesia from peripheral molecular responses to central analgesic pathway. Acupuncture was performed at the GB34 acupuncture point. In the local molecular changes, ERK was activated as a trigger molecule that activates ERM and ROCK proteins and the activated ROCK was strongly involved with acupuncture analgesia. Localized molecular signaling might be connected to the descending inhibitory pathway of supra-spinal tract, resulting in overall pain relief. Displacement, a linear movement of the needle; ERK, extracellular signal–regulated kinase; ERM, ezrin-radixin-moesin; Rev, revolution; ROCK, rho-associated protein kinase.