Electroacupuncture Regulates Pain Transition by Inhibiting the mGluR5-PKCε Signaling Pathway in the Dorsal Root Ganglia

Abstract

Background: Acute pain can transition to chronic pain, presenting a major clinical challenge. Electroacupuncture (EA) can partly prevent the transition from acute to chronic pain. However, little is known about the mechanisms underlying the effect of EA. This study investigated the effect of EA on pain transition and the activation of metabotropic glutamate receptor 5 (mGluR5)-protein kinase C epsilon (PKCε) signaling pathway in the dorsal root ganglia (DRG).

Methods: The hyperalgesic priming model was established by the sequential intraplantar injection of carrageenan (1%, 100 μL) and prostaglandin E2 (PGE2) into the left hind paw of rats. EA treatment (2/100 Hz, 30 min, once/day) was applied at bilateral Zusanli (ST36) and Kunlun (BL60) acupoints in rats. Von Frey filaments were used to investigate the mechanical withdrawal threshold (MWT) at different time points. The protein expression levels of mGluR5 and PKCε in the ipsilateral L4-L6 DRGs of rats were detected by Western blot. Some pharmacological experiments were performed to evaluate the relationship between mGluR5, PKCε and the MWT. It was also used to test the effects of EA on the expression levels of mGluR5 and PKCε and changes in the MWT.

Results: Sequential injection of carrageenan and PGE2 significantly decreased the MWT of rats and up-regulated the expression level of mGluR5 and PKCε in the ipsilateral L4-L6 DRGs. EA can reverse the hyperalgesic priming induced by sequential injection of carrageenan/PGE and down-regulate the protein expression of mGluR5 and PKCε. Glutamate injection instead of PGE2 can mimic the hyperalgesic priming model. Pharmacological blocking of mGluR5 with specific antagonist MTEP can prevent the hyperalgesic priming and inhibit the activation of PKCε in DRGs. Furthermore, EA also produced analgesic effect on the hyperalgesic priming rats induced by carrageenan/mGluR5 injection and inhibited the high expression of PKCε. Sham EA produced none analgesic and regulatory effect.

Conclusion: EA can regulate pain transition and it may relate with its inhibitory effect on the activation of mGluR5-PKCε signaling pathway in the DRGs.

Keywords: DRGs; PKCε; electroacupuncture; hyperalgesic priming; mGluR5; pain transition.

Figure 1

(A) Schematic of the experimental timeline. (B) The hyperalgesic priming model was established by sequential intraplantar injection of carrageenan/PGE2 into the left hind paw. The MWT were measured before injection, 4, 24, 48, 72 h, 7 d after the 1^st injection and 1, 4, 24, 48 h after the 2^nd injection in all groups. Data are presented as mean ± SEM, n=6; ^##P<0.01 vs N group at the corresponding time point; ^ΔΔP<0.01 vs sham HP group at the corresponding time point.

Figure 2

(A–F) Western blot shows the expression of mGluR5 and PKCε in the L4-6 DRGs extracts from hyperalgesic priming rats at 4 h (A, B), 24 h (C, D), 48 h (E, F) after the 2^nd injection. Data are presented as the mean ± SEM, n = 6; ^##P < 0.01 vs N group; ^ΔΔP < 0.01, ^ΔP < 0.05 vs sham HP group.

Figure 3

(A) The procedure of EA stimulation experiment. (B) Schematic picture of the locations of the acupoints ST36 and BL60 in rat. (C) The analgesic effect of EA stimulation on the hyperalgesic priming rats. (D) Different time points of EA stimulation exerted comparable effects on MWT. (E, F) Western blotting images and relative protein level of mGluR5 and PKCε in rat L4-6 DRG from different groups. Date are mean ± SEM; n = 6; ^ΔΔP<0.01 vs sham HP group; **P<0.01, *P<0.05 vs HP group; ^##P<0.01 vs EAI group; ^&&P<0.01, vs EAII group.

Figure 4

(A) Glutamate was injected into the left hind paw at 7 d after carrageenan injection. And MWT were measured before injection, 4, 72 h, 7 d after the 1^st injection and 1, 4, 24, 48 h after the 2^nd injection in all groups. (B) Glutamate increased the expression of PKCε protein in the L4-6 DRG by Western blot. (C) Pre-injection of MTEP inhibited the reduction of MWT induced by hyperalgesia (D, E) MTEP (mGluR5 inhibitor) decreased the expression of mGluR5 and PKCε protein in the L4-6 DRG. Data are presented as mean ± SEM, n=6; ^##P<0.01 vs N group; ^ΔΔP<0.01 vs sham HP group; *P<0.05, **P < 0.01 vs HP group.

Figure 5

(A) EA stimulation can increase the reduction of MWT induced by hyperalgesic priming caused by glutamate at different time points. (B) EA decreased the expression of mGluR5 protein in the L4-6 DRGs. (C) EA decreased the expression of PKCε protein in the L4-6 DRGs. Data are presented as mean ± SEM, n=6; ^ΔΔP<0.01 vs sham HP group; **P<0.01, *P<0.05 vs Glu group.