Peripheral ERK modulates acupuncture-induced brain neural activity and its functional connectivity

Abstract

Acupuncture has been widely used as a therapeutic intervention, and the brain network plays a crucial role in its neural mechanism. This study aimed to investigate the acupuncture mechanism from peripheral to central by identifying how the peripheral molecular signals induced by acupuncture affect the brain neural responses and its functional connectivity. We confirmed that peripheral ERK activation by acupuncture plays a role in initiating acupuncture-induced peripheral proteomic changes in mice. The brain neural activities in the neocortex, hippocampus, thalamus, hypothalamus, periaqueductal grey, and nucleus of the solitary tract (Sol) were significantly changed after acupuncture, and these were altered by peripheral MEK/MAPK inhibition. The arcuate nucleus and lateral hypothalamus were the most affected by acupuncture and peripheral MEK/MAPK inhibition. The hypothalamic area was the most contributing brain region in contrast task PLS analysis. Acupuncture provoked extensive changes in brain functional connectivity, and the posterior hypothalamus showed the highest betweenness centrality after acupuncture. After brain hub identification, the Sol and cingulate cortex were selected as hub regions that reflect both degree and betweenness centrality after acupuncture. These results suggest that acupuncture activates brain functional connectivity and that peripheral ERK induced by acupuncture plays a role in initiating brain neural activation and its functional connectivity.

Figure 1

The role of peripheral ERK in the acupuncture-induced thermal pain threshold changes and the acupuncture-induced peripheral molecular signals. (A,B) Peripheral administration of U0126, an ERK inhibitor, before acupuncture treatment significantly blocked the effect of acupuncture in increasing the thermal pain threshold (each n = 5). (C,D) The activated p-ERK after acupuncture treatment was inhibited by U0126; however, it was not affected by CPZ, a TRPV1 inhibitor and DPCPX, an A1R inhibitor. The activated p-HSP27 were inhibited by U0126, CPZ, and DPCPX (each n = 3). The western blot image represent the cropped blots. Full length blots are presented in Supplementary Figure S2. CON control, CON + U U0126 administration, ACU acupuncture treatment, ACU + U U0126 administration followed by acupuncture treatment, ACU + C CPZ administration followed by acupuncture treatment, ACU + D DPCPX administration followed by acupuncture treatment, N-ACU acupuncture treatment at non-acupoint on the hips. *P < 0.05, ***P < 0.001 compared to the CON group, ^#P < 0.05, ^###P < 0.001 compared to the ACU group, ^$P < 0.05, ^$$P < 0.01 compared to the ACU + U group. One-way ANOVA was followed by the Newman–Keuls post-hoc test. Data are expressed as the mean ± SEM.

Figure 2

Schematic diagram of experimental schedule for functional connectivity study. Acupuncture was performed at bilateral GB34 (Yangneungcheon, Yanglingquan) for 10 min and inhibitor was administrated 15 min before acupuncture treatment. The brains were removed 90 min after acupuncture treatment for identifying the brain neural responses and the brain functional connectivity. The c-Fos positive cells were quantified in each of the 34 brain regions, and a set of inter-regional correlations were computed. Finally, functional networks were generated. Abbreviations for each brain region are shown in Table 1.

Figure 3

Expression of c-Fos after acupuncture treatment expressed in each brain region. (A,B) The number of c-Fos-positive cells changed after acupuncture or acupuncture with U0126 administration. (C) Increased percentage of c-Fos-positive cells after acupuncture treatment compared to the control group. (D) Decreased percentage of c-Fos-positive cells after U0126 administration followed by acupuncture treatment compared to the acupuncture treatment only. (E) The overlap between brain regions ranked above the 80th percentile for acupuncture increased, and U0126 decreased-c-Fos activation. CON control, ACU acupuncture treatment, ACU + U U0126 administration followed by acupuncture treatment. Abbreviations for each brain region are shown in Table 1. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the CON group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared to the ACU group. One-way ANOVA was followed by the Newman–Keuls post-hoc test. Data are expressed as the mean ± SEM. The number of animals in each group is shown in Table 1.

Figure 4

Contrast task PLS analysis of brain c-Fos expression in CON vs. ACU and ACU vs. ACU + U. (A,B) Two pairs of ACU vs. CON and ACU vs. ACU + U were compared in the Contrast task PLS analysis of c-Fos expression. The contrast (left) reflects the experimental condition in CON vs. ACU (A) and ACU vs. ACU + U (B). Salience scores (right) identified that the brain regions which differed in c-Fos expression between the experimental conditions were intensely similar between the ACU vs. CON and ACU vs. ACU + U. In both comparisons, c-Fos expression in multiple brain regions contributed to the contrast, and the hypothalamus contributed the most strongly to this contrast. CON control, ACU acupuncture treatment, ACU + U U0126 administration followed by acupuncture treatment. Abbreviations for each brain region are shown in Table 1.

Figure 5

Matrices of inter-regional correlations and network construction for brain c-Fos expression within each group. (A) Colour-coded matrices showing inter-regional correlations for c-Fos activation between the 34 brain regions. (B) The threshold square of inter-regional correlations for c-Fos activation. Acupuncture produces a high correlation between the brain regions of the cortex, and these correlations were mostly altered by U0126 administration. Red colour indicates high correlation and blue indicates low-correlation (scale, right). (C) A circular layout grouped by major brain subdivision to show the connectivity between the brain regions. Nodes are connected by the edges of super-threshold inter-regional correlations. CON control; ACU acupuncture treatment, ACU + U U0126 administration followed by acupuncture treatment. Abbreviations for each brain region are shown in Table 1.

Figure 6

Network clustering and hub-identification. (A) A force atlas format to show the degree and betweenness centrality. The number of edges was represented as degree, and the number of shortest paths of all possible pairs of nodes represented betweenness centrality. (B) Brain regions were ranked in descending order for degree and betweenness centrality of the CON, ACU, and ACU + U groups. Brain regions ranked above the 80th percentile for degree and betweenness centrality were indicated by a red-coloured box. (C) Venn diagram shows the overlap between brain regions ranked above the 80th percentile for degree and betweenness centrality in the ACU and ACU + U groups. Two putative hub regions (Sol and Cg2 in the ACU group; S1 and PV in the ACU + U group) were identified as hub brain regions in each group. CON control, ACU acupuncture treatment, ACU + U U0126 administration followed by acupuncture treatment. Abbreviations for each brain region are shown in Table 1.

Figure 7

Schematic diagram of local molecular signaling and brain function after acupuncture treatment. The peripheral mechanism (right) and central mechanism (left) after acupuncture treatment are illustrated. Acupuncture-induced ERK1/2 activation acts as a triggering molecule that induces multiple signaling transduction around the acupoint. These local signals are received by the peripheral nerve ending and then transmitted to the afferent sensory nervous tract. The brain regions of Sol and Cg are mainly involved in acupuncture-mediated brain functional connectivity, and these brain regions are functionally connected with variable brain regions of SC, MC, HIP, Insul, HyTH, SN, and RMg. These central signals might activate the descending inhibitory pathway that provokes the acupuncture effect. The solid line is the mechanism of the findings in this study, and the dotted line is the potential prediction mechanism. Cg cingulate cortex, Insul insular cortex, HIP hippocampus, HYTH hypothalamus, MC motor cortex, PAG periaqueductal grey, RMg raphe magnus nucleus, SC somatosensory cortex, SN substantia nigra, Sol nucleus of solitary tract, ST striatum, TH thalamus.