TRPV1 Responses in the Cerebellum Lobules VI, VII, VIII Using Electroacupuncture Treatment for Chronic Pain and Depression Comorbidity in a Murine Model

Abstract

Depression is a prominent complex psychiatric disorder, usually complicated through expression of comorbid conditions, with chronic pain being among the most prevalent. This comorbidity is consistently associated with a poor prognosis and has been shown to negatively impact patient outcomes. With a global rise in this condition presenting itself, the importance of discovering long-term, effective, and affordable treatments is crucial. Electroacupuncture has demonstrated renowned success in its use for the treatment of pain and is a widely recognized therapy in clinical practice for the treatment of various psychosomatic disorders, most notably depression. Our study aimed to investigate the effects and mechanisms of Acid-Saline (AS) inducing states of chronic pain and depression comorbidity in the cerebellum, using the ST36 acupoint as the therapeutic intervention. Furthermore, the role of TRPV1 was relatedly explored through the use of TRPV1^-/- mice (KO). The results indicated significant differences in the four behavioral tests used to characterize pain and depression states in mice. The AS and AS + SHAM group showed significant differences when compared to the Control and AS + EA groups in the von Frey and Hargreaves's tests, as well as the Open-Field and Forced Swimming tests. This evidence was further substantiated in the protein levels observed in immunoblotting, with significant differences between the AS and AS + SHAM groups when compared to the AS + EA and AS + KO groups being identified. In addition, immunofluorescence visibly served to corroborate the quantitative outcomes. Conclusively these findings suggest that AS-induced chronic pain and depression comorbidity elicits changes in the cerebellum lobules VI, VII, VIII, which are ameliorated through the use of EA at ST36 via its action on TRPV1 and related molecular pathways. The action of TRPV1 is not singular in CPDC, which would suggest other potential targets such as acid-sensing ion channel subtype 3 (ASIC3) or voltage-gated sodium channels (Navs) that could be explored in future studies.

Keywords: ST36; TRPV1; cerebellum; chronic pain; depression; electroacupuncture.

Figure 1

Comparative graph of paw withdrawal threshold and latency of the Acid-Saline (AS)-induced chronic pain subjects after electroacupuncture (EA) treatment and transient receptor vanilloid member 1 (TRPV1) gene deletion (KO). Control, AS, AS + EA, AS + SHAM, and AS + KO were tested according to (A) mechanical von Frey and (B) thermal Hargreaves’ nociceptive sensitivities. * p < 0.05 means when compared with the baseline of control group. # p < 0.05 means when compared with the AS and AS + SHAM groups. (C,D) Open Field testing and (E,F) Forced Swimming testing to depict depressive-like behavior. * p < 0.05 means when compared with the baseline of control group n = 6 for five groups.

Figure 2

The expression levels of nociceptive receptors and associated molecules in cerebellum lobule VI. The immunoblotting images depict five lanes of protein in the following order: Control, AS, AS + EA, AS + SHAM, and AS + KO groups. There are significant decreases in protein expression in the AS and AS + SHAM groups of (A) TRPV1, (B) pmTOR, (E) pPI3K, (F) NMDAR1, (G) pPKCε, (H) pAkt, (I) TrkB, (J) pNFκB, (K) GABAAα1, (L) pPKAIIα, (M) pCREB, and (N) pERK levels, which were significantly attenuated in the AS + EA and AS + KO groups, depicting no difference when compared to the Control group. Conversely, the protein expressions of the AS and AS + SHAM groups were significantly increased in (C) Nav1.7 and (D) Nav1.8 when compared to the Control group. Correspondingly, the AS + EA and AS + KO groups were augmented and showed no significant difference in comparison to the Control group. Accordingly, the protein density of the AS + KO group revealed a predicted decrease in (A) TRPV1 (* p < 0.05).

Figure 3

The expression levels of nociceptive receptors and associated molecules in the cerebellum lobule VII. The immunoblotting images depict five lanes of protein in the following order: Control, AS, AS + EA, AS + SHAM, and AS + KO groups. There are significant decreases in protein expression in the AS and AS + SHAM groups of (A) TRPV1, (B) pmTOR, (E) pPI3K, (F) NMDAR1, (G) pPKCε, (H) pAkt, (I) TrkB and (J) pNFκB (K) GABAAα1, (L) pPKAIIα, (M) pCREB, and (N) pERK levels, which were significantly attenuated in the AS + EA and AS + KO groups, depicting no difference when compared to the Control group. Interestingly, the protein expression levels of (C) Nav1.7 and (D) Nav1.8 displayed states of no significant variances across all 5 groups. Accordingly, the protein density of the AS + KO group revealed a predicted decrease in (A) TRPV1 (* p < 0.05).

Figure 4

The expression levels of nociceptive receptors and associated molecules in the cerebellum lobule VIII. The immunoblotting images depict five lanes of protein in the following order: Control, AS, AS + EA, AS + SHAM, and AS + KO groups. There are significant decreases in protein expression in the AS and AS + SHAM groups of (A) TRPV1, (B) pmTOR, (E) pPI3K, (F) NMDAR1, (G) pPKCε, (H) pAkt, (I) TrkB, (J) pNFκB, (K) GABAAα1, (L) pPKAIIα, (M) pCREB, and (N) pERK levels, which were significantly augmented in the AS + EA and AS + KO groups. Both the AS + EA and AS + KO groups displayed no difference when compared to the Control group, depicting an observable improvement of CPDC tendencies. However, the protein expression levels of (C) Nav1.7 and (D) Nav1.8 displayed states of significant increases in the AS and AS + SHAM groups when compared to the Control group. Furthermore, this increase was significantly ameliorated in the AS + EA and AS + KO groups, which retained states of non-significance when similarly compared to the Control group. Accordingly, the protein density of the AS + KO group revealed a predicted decrease in (A) TRPV1 (* p < 0.05).

Figure 5

Immunofluorescence staining of TRPV1 and pNFkB protein expression in the cerebellum lobules VIa and VIb. There are 5 subject groups: Control, AS, AS + EA, AS + SHAM, and AS + KO. (A) The efficacy of EA treatment involves significant increases in TRPV1 and pNFkB densities in the cerebellum lobule VIa. (B) Conversely, no significant variance in pNFkB density was observed in cerebellum lobule VIb among the 5 groups, although TRPV1 maintained an analogous trend of decreased expression in the AS and AS + SHAM group, which was absent in the AS + KO group and increased in the AS + EA group when compared to Control. Scale bar is 50 μm.

Figure 6

Immunofluorescence staining of TRPV1 and pNFkB protein expression in the cerebellum lobules VII and VIII. There are 5 subject groups: Control, AS, AS + EA, AS + SHAM, and AS + KO. The efficacy of EA treatment involves significant increases in TRPV1 and pNFkB densities in the cerebellum lobules (A) VII and (B) VIII. Scale bar is 50 μm.

Figure 7

Illustration of CPDC pathways attenuated by EA in the cerebellum. We found that EA at 1 mA, 2 Hz/20 min, and TRPV1 gene deletion can increase the expressions of TRPV1, Nav1.7, Nav1.8, GABAAα1, NMDAR1, and TrkB receptors in the AS-induced CPDC model, as observed in the AS + EA and AS + KO groups. Furthermore, the relevant increases in related responses cause effective increases in the signaling of pPI3K, pAkt, pERK, pmTOR, pPKCε, pPKAIIα, pNFκB, and pCREB under conditions of CPDC, and these serve to present a basic neuromodulatory pathway of CPDC, TRPV1 signaling, and the molecular function of EA.