| Evidence Sentence: |
First, a genome-wide microarray of the isolated skin layer at the GB34-equivalent acupoint of C57BL/6 mice 1 hour after acupuncture found that a total of 236 genes had changed and that extracellular signal-regulated kinase (ERK) activation was the most prominent bio-candidate. |
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Second, in mouse pain models using formalin and complete Freund adjuvant, we found that acupuncture attenuated the nociceptive behavior and the mechanical allodynia; these effects were blocked when ERK cascade was interrupted by the mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) inhibitor U0126 (.8 mug/muL). |
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Based on these results, we suggest that ERK phosphorylation following acupuncture needling is a biochemical hallmark initiating the effect of acupuncture including analgesia. |
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This article presents the novel evidence of the local molecular signaling in acupuncture analgesia by demonstrating that ERK activation in the skin layer contributes to the analgesic effect of acupuncture in a mouse pain model. |
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Acupuncture-Induced Changes in the Proteins of the MAPK Pathway: Remarkable Increase of Phospho-ERK After Acupuncture Needling |
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To find a specific biomarker, we focused on the MAPK pathway, as suggested by the results of the previous experiment, and we analyzed the activity of the MAPK family, ERK, p38, and JNK after acupuncture needling. |
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Five minutes after acupuncture needling, the activation of phospho-ERK was significantly higher than in the CON group (P < .05) and then it gradually decreased for 30 minutes (Fig 4A). |
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Five minutes after acupuncture needling, the phosphorylation of p38 was also elevated compared with the CON group(P <.05),but the elevation was not as high as that of phospho-ERK (Fig 4B). |
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To investigate the location of ERK activation in the skin layer, a histologic analysis with immunohistochemistry and immunofluorescence was performed. |
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Five minutes after acupuncture needling, the activation of ERK was detected in the epidermis and the dermis, and these signals were diminished after U0126 (MEK/MAPK inhibitor) administration (Fig 5). |
| Evidence Sentence: |
To investigate the type of cell undergoing ERK activation, the tissue was costained for phospho-ERK, a fibroblast marker, and a keratinocyte marker, and then visualized by immunofluorescence (Fig 6). |
| Evidence Sentence: |
Five minutes after acupuncture needling, activation of ERK was detected in the keratinocytes of the epidermis (colocalized with CK7) and in the fibroblast of the dermis (colocalized with 5B5), suggesting that acupuncture phosphorylates ERK in both the keratinocytes and fibroblast at the acupuncture point. |
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To examine whether acupuncture-induced ERK activation is involved in the analgesic effects of acupuncture, we used the MEK/MAPK inhibitor U0126 in the formalin-induced and CFA-induced mouse pain model. |
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To verify that the intradermal injection of U0126 worked successfully in the skin layer, the level of ERK phosphorylation was determined by Western blotting. |
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Five minutes after acupuncture needling, phospho-ERK activation was elevated and U0126 (.4 and .8 mug/muL) inhibited the acupuncture-induced ERK activation in a dose-dependent manner (Supplementary Fig S3A). |
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As a result, U0126 inhibited acupuncture-induced ERK activation only on the left leg and did not affect the other side (Supplementary Fig S3B), indicating that U0126 successfully inhibited ERK activation locally. |
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Finally, we investigated whether local ERK activation caused by acupuncture needling was linked to analgesia using the formalin-induced and CFA-induced pain models. |
| Evidence Sentence: |
From Peripheral to Central: The Role of ERK Signaling Pathway in Acupuncture Analgesia |
