The Analgesia Effect of Aucubin on CFA-Induced Inflammatory Pain by Inhibiting Glial Cells Activation-Mediated Inflammatory Response via Activating Mitophagy

Abstract

Background: Inflammatory pain, characterized by sustained nociceptive hypersensitivity, represents one of the most prevalent conditions in both daily life and clinical settings. Aucubin, a natural plant iridoid glycoside, possesses potent biological effects, encompassing anti-inflammatory, antioxidant, and neuroprotective properties. However, its impact on inflammatory pain remains unclear. The aim of this study is to investigate the therapeutic effects and underlying mechanism of aucubin in addressing inflammatory pain induced by complete Freund's adjuvant (CFA).

Methods: The CFA-induced inflammatory pain model was employed to assess whether aucubin exerts analgesic effects and its potential mechanisms. Behavioral tests evaluated mechanical and thermal hyperalgesia as well as anxiety-like behaviors in mice. The activation of spinal glial cells and the expression of pro-inflammatory cytokines were examined to evaluate neuroinflammation. Additionally, RNA sequencing was utilized for the identification of differentially expressed genes (DEGs). Molecular biology experiments were conducted to determine the levels of the PINK1 gene and autophagy-related genes, along with PINK1 distribution in neural cells. Furthermore, mitophagy induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) was employed to examine the roles of PINK1 and mitophagy in pain processing.

Results: Aucubin significantly ameliorated pain and anxiety-like behaviors induced by CFA in mice and reduced spinal inflammation. RNA sequencing indicated PINK1 as a pivotal gene, and aucubin treatment led to a significant downregulation of PINK1 expression. Further GO and KEGG analyses suggested the involvement of mitochondrial function in the therapeutic regulation of aucubin. Western blotting revealed that aucubin markedly decreased PINK1, Parkin, and p62 levels while increasing LC3B expression. Immunofluorescence showed the predominant co-localization of PINK1 with neuronal cells. Moreover, CCCP-induced mitophagy alleviated mechanical and thermal hyperalgesia caused by CFA and reversed CFA-induced mitochondrial dysfunction.

Conclusions: In summary, our data suggest that aucubin effectively alleviates CFA-induced inflammatory pain, potentially through triggering the PINK1 pathway, promoting mitophagy, and suppressing inflammation. These results provide a novel theoretical foundation for addressing the treatment of inflammatory pain.

Keywords: PINK1; aucubin; inflammation; mitophagy; pain.

Figure 1

Chemical structure of aucubin.

Figure 2

Effect of aucubin on CFA-induced inflammatory pain in mice. (A, B) Aucubin attenuates CFA-induced mechanical allodynia and thermal hyperalgesia in mice. Results are expressed as mean ± SEM (n = 8). *** p < 0.001, and **** p < 0.0001, CFA + Veh vs. CFA + Aucubin. Representative trajectories of locomotor activity in the EPM (C) and OFT (G). (D–F) Summarized data showed the time spent in the open arms, open arm entries, and total distance traveled in the EPM. (H–J) Summarized data showed the time spent in the central area, distance moved in the central area, and total distance moved in the OFT. Data are expressed as mean ± SEM (n = 8). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 3

Effect of aucubin on CFA-induced glial cell activation and proinflammatory cytokine production in the spinal cord. (A,B) Representative immunofluorescence staining for microglia and astrocytes in the spinal dorsal cord of mice. (C,D) Mean immunofluorescence intensity of Iba-1 and GFAP (n = 3). Scale bars = 200 μm and 20 μm (magnification). (E–H) Representative bands of Western blot and quantitative analysis of the relative expression of TNF-α, IL-1β, and IL-6 (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

RNA−Seq analysis in the spinal cord of CFA−injected mice treated with aucubin. (A) Heatmap analysis of DEGs in CFA + Veh and CFA + Aucubin groups. The x−axis represents different groups, while the y−axis represents the DEGs. Upregulated genes are depicted in red, and downregulated genes are represented in blue. The arrow points to PINK1 gene. (B) Volcano plot of DEGs. Red indicates upregulated DEGs, blue represents downregulated DEGs, and grey represents non-significant DEGs. (C) GO enrichment analysis. (D) KEGG pathway analysis. The size of the circles corresponds to the number of DEGs, and the color gradient from red to blue indicates decreasing significance. The arrow points to mitophagy pathway.

Figure 5

Effects of aucubin on mitophagy in CFA-injected mice. (A–E) Representative bands of Western blot and quantitative analysis of the relative expression of PINK1, Parkin, LC3B, and p62. Data are expressed as the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.01. (F) Double immunofluorescence staining of Neun (green), Iba-1 (green), GFAP (green) with PINK1 (red) after CFA injection for 6 days. Scale bar = 50 μm.

Figure 6

CCCP significantly alleviated CFA-induced inflammatory pain in mice by activating mitophagy. (A,B) CCCP attenuates mechanical allodynia and thermal hypersensitivity induced by CFA in mice (n = 9). (C–G) Representative bands of Western blot and quantitative analysis of the relative expression of PINK 1, Parkin, LC3B and p62 (n = 3). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.001.

Figure 7

(A,B) Schematic representation of the experimental protocols.