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Citation: Yao, D.; Wang, Y.; Chen, Y.;
Chen, G. The Analgesia Effect of
Aucubin on CFA-Induced
Inflammatory Pain by Inhibiting
Glial Cells Activation-Mediated
Inflammatory Response via
Activating Mitophagy.
Pharmaceuticals 2023,16, 1545.
https://doi.org/10.3390/
ph16111545
Academic Editor: Abdeslam
Chagraoui
Received: 18 September 2023
Revised: 10 October 2023
Accepted: 30 October 2023
Published: 1 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceuticals
Article
The Analgesia Effect of Aucubin on CFA-Induced Inflammatory
Pain by Inhibiting Glial Cells Activation-Mediated
Inflammatory Response via Activating Mitophagy
Dandan Yao 1,2 , Yongjie Wang 3, Yeru Chen 1and Gang Chen 1,*
1Department of Anesthesiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University,
Hangzhou 310058, China
2Department of Anesthesiology, School of Medicine, Shaoxing University, Shaoxing 312000, China
3Key Laboratory of Elemene Class Anti-Cancer Chinese Medicines, Engineering Laboratory of Development
and Application of Traditional Chinese Medicines, Collaborative Innovation Center of Traditional Chinese
Medicines of Zhejiang Province, Hangzhou Normal University, Hangzhou 311121, China
*Correspondence: chengang120@zju.edu.cn
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: aucubin; inflammation; pain; PINK1; mitophagy
1. Introduction
Inflammatory pain is characterized by persistent nociceptive hypersensitivity, which
includes both allodynia and hypersensitivity in the injured site and adjacent tissues, leading
to an unpleasant sensory and emotional experience [
1
]. It affects at least 25% of the general
population and results in a significant economic load on patients and healthcare systems
Pharmaceuticals 2023,16, 1545. https://doi.org/10.3390/ph16111545 https://www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2023,16, 1545 2 of 16
worldwide [
2
]. Currently, the efficacy of most analgesics (including opioids, non-steroidal
anti-inflammatory drugs, and anticonvulsants) in addressing chronic pain is limited, and
can even lead to severe side effects [
3
]. Therefore, it is imperative to urgently investigate the
specific molecular mechanisms that underlie the generation and persistence of chronic pain
with diverse etiologies. Recently, increased attention has been devoted to the development
of novel therapeutics for pain management, focusing on traditional medicinal herbs and
dietary supplements within the realm of drug discovery.
In recent years, researchers have found that glial cells, especially microglia and as-
trocytes, are non-neuronal cells in the central nervous system (CNS) that provide support
and maintain the overall function of neurons, playing a crucial role in the development
and modulation of pain [
4
,
5
]. Microglia were activated during the initial disease phase,
whereas astrocyte activation occurred in the subsequent sustaining phase. In response to
injury or inflammation, glial cells undergo activation and release pro-inflammatory cy-
tokines, including interleukin-1
β
(IL-1
β
), interleukin-6 (IL-6), and tumor necrosis factor-
α
(TNF-
α
). Pro-inflammatory cytokines serve as signaling molecules of the immune response
that promote neuroinflammation. Activated glial cells release proinflammatory cytokines,
amplifying pain transmission by activating and sensitizing neurons, and, reciprocally,
activated neurons communicate with glial cells, fostering persistent inflammation and
prolonged pain sensitization [6].
Mitophagy is a selective autophagy process that specifically eliminates impaired mi-
tochondria from within cells, thus contributing to maintaining cellular homeostasis [
7
].
Multiple mitochondrial quality control pathways have been identified to mediate the
degradation of misfolded mitochondrial proteins, mitochondrial fission/fusion, and the
phagocytosis and degradation of damaged mitochondria (mitophagy) [
8
]. Dysfunctional
mitophagy or defective mitochondrial biogenesis leads to an overall less efficient mitochon-
drial pool with damaged ATP production and enhanced mitochondrial ROS generation,
potentially affecting sensory processing [
9
]. PTEN-induced protein kinase 1 (PINK1) is a
neuroprotective protein that induces depolarization of the inner mitochondrial membrane
as a response to mitochondrial damage, resulting in the accumulation of PINK1 on the
outer mitochondrial membrane [
10
]. Studies have demonstrated that PINK1 stabilizes at
the mitochondrial outer membrane upon mitochondrial damage and activates the Parkin
ubiquitin ligase via phosphorylating Parkin and ubiquitin [
11
,
12
]. Parkin promotes the
recruitment of the ubiquitin-binding adaptor SQSTM1/p62 (p62) and recruits ubiquiti-
nated cargo to autophagosomes by binding to LC3, thereby mediating mitophagy [
13
].
Autophagy also plays a crucial role in maintaining a balanced inflammatory response due
to its regulatory function [
14
]. Preclinical and clinical evidence suggests that modulation
of mitochondrial function holds promise for alleviating or eliminating pain, providing
possible advantages across a range of rheumatic diseases. [15].
Aucubin (Figure 1), an iridoid glucoside, is found in various plant species that ex-
hibit potent antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective ef-
fects [
16
]. Recent studies have demonstrated that aucubin effectively inhibits the activation
of microglia and astrocytes, resulting in a significant inhibition of pro-inflammatory cy-
tokines [
17
–
20
]. Furthermore, aucubin has been observed to elevate autophagy levels
and inhibit apoptosis, thereby conferring neuronal protection [
21
,
22
]. Consequently, we
formulated the hypothesis that aucubin could mitigate inflammatory pain by modulating
the inflammatory response and promoting mitophagy. To validate this hypothesis, an in-
flammatory pain model was established in mice through intraplantar injection of complete
Freund’s adjuvant (CFA), and subsequently, the role of aucubin on inflammatory pain
was investigated.
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Figure 1. Chemical structure of aucubin.
2. Results
2.1. Effect of Aucubin on CFA-Induced Inflammatory Pain in Mice
We established an inflammatory pain model by injecting CFA into the left hindpaw
of mice. Subsequently, we evaluated the effects of CFA-induced pain by detecting me-
chanical allodynia and thermal hypersensitivity. Behavioral tests revealed that the PWT
and PWL in mice injected with CFA were significantly reduced compared to the control
group (Figure 2A, B), indicating the successful induction of inflammatory pain. Remark-
ably, aucubin treatment significantly increased both PWT and PWL in CFA-injected mice
(Figure 2A,B), suggesting its efficacy in alleviating mechanical allodynia and thermal hy-
persensitivity pain induced by CFA.
Chronic pain often co-occurs with psychiatric disorders, including anxiety, in clinical
seings [23]. Thus, we employed the EPM and OFT to assess the anxiolytic effects of
aucubin. CFA-treated mice exhibited a significant decrease in the number of entries into
the open arms of the EPM, as well as a reduction in the time spent in the open arms, com-
pared to the control mice (Figure 2C–E). Similarly, in the OFT, CFA-injected mice dis-
played reduced distance moved in the central area and a decreased time spent in the cen-
tral area compared to the controls (Figure 2G–I). These results collectively indicate the
presence of anxiety-like behavior in CFA-treated mice. In contrast, aucubin-treated mice
demonstrated a significant increase in the time spent in the open arm and the number of
open arm entries in the EPM (Figure 2C–E). Additionally, aucubin also increased the time
spent in the center area and the distance moved in the central area of the OFT compared
to the control group (Figure 2G–I). Crucially, the total distance traveled by the mice re-
ceiving either OFT or CFA did not show any discernible variations between the groups
(Figure 2F,J), indicating that neither aucubin nor CFA affected the miceʹs locomotor activ-
ity. Taken together, our results indicate that aucubin has the potential to alleviate pain and
anxiety-like behavior in CFA-injected mice.
Figure 1. Chemical structure of aucubin.
2. Results
2.1. Effect of Aucubin on CFA-Induced Inflammatory Pain in Mice
We established an inflammatory pain model by injecting CFA into the left hindpaw
of mice. Subsequently, we evaluated the effects of CFA-induced pain by detecting me-
chanical allodynia and thermal hypersensitivity. Behavioral tests revealed that the PWT
and PWL in mice injected with CFA were significantly reduced compared to the control
group (
Figure 2A,B
), indicating the successful induction of inflammatory pain. Remark-
ably, aucubin treatment significantly increased both PWT and PWL in CFA-injected mice
(Figure 2A,B), suggesting its efficacy in alleviating mechanical allodynia and thermal
hypersensitivity pain induced by CFA.
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 4 of 16
Figure 2. Effect of aucubin on CFA-induced inflammatory pain in mice. (A, B) Aucubin aenuates
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) Sum-
marized 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.
2.2. Effects of Aucubin on Inflammatory Responses in Mice Induced by CFA
To assess the effect of aucubin on inflammatory responses triggered by CFA, we uti-
lized immunofluorescence and Western blot to examine its effect on the spinal glial cells
and proinflammatory cytokines in mice. Specifically, we employed Ionized Calcium-Bind-
ing Adapter Molecule 1 (Iba-1) and Glial Fibrillary Acidic Protein (GFAP) as specific mark-
ers for astrocytes and microglia, respectively. As shown in Figure 3A–D, our results indi-
cated that the intensity of Iba-1 and GFAP in the spinal dorsal horn of mice in the CFA
group was dramatically increased compared to the control group, whereas treatment with
aucubin (10 mg/kg) resulted in a noteworthy reduction of GFAP and Iba-1 expression.
Furthermore, previous study has demonstrated that activated astrocytes and micro-
glia in the spinal cord produce and release a considerable amount of proinflammatory
cytokines, which may directly sensitize nociceptive sensory neurons, thereby leading to
pain [24]. Consistent with this, in the CFA group, the levels of IL-1β, IL-6, and TNF-α
exhibited a marked elevation in comparison to the control group (Figure 3E–H). Con-
versely, aucubin treatment led to a notable decrease in the expression of IL-1β, IL-6, and
TNF-α in CFA-injected mice (Figure 3E–H).
These results indicate that aucubin effectively alleviates CFA-induced inflammatory
responses in the spinal cord.
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. Repre-
sentative 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.
Pharmaceuticals 2023,16, 1545 4 of 16
Chronic pain often co-occurs with psychiatric disorders, including anxiety, in clinical
settings [
23
]. Thus, we employed the EPM and OFT to assess the anxiolytic effects of
aucubin. CFA-treated mice exhibited a significant decrease in the number of entries into the
open arms of the EPM, as well as a reduction in the time spent in the open arms, compared
to the control mice (Figure 2C–E). Similarly, in the OFT, CFA-injected mice displayed
reduced distance moved in the central area and a decreased time spent in the central area
compared to the controls (Figure 2G–I). These results collectively indicate the presence of
anxiety-like behavior in CFA-treated mice. In contrast, aucubin-treated mice demonstrated
a significant increase in the time spent in the open arm and the number of open arm entries
in the EPM (Figure 2C–E). Additionally, aucubin also increased the time spent in the center
area and the distance moved in the central area of the OFT compared to the control group
(Figure 2G–I). Crucially, the total distance traveled by the mice receiving either OFT or
CFA did not show any discernible variations between the groups (Figure 2F,J), indicating
that neither aucubin nor CFA affected the mice’s locomotor activity. Taken together, our
results indicate that aucubin has the potential to alleviate pain and anxiety-like behavior in
CFA-injected mice.
2.2. Effects of Aucubin on Inflammatory Responses in Mice Induced by CFA
To assess the effect of aucubin on inflammatory responses triggered by CFA, we
utilized immunofluorescence and Western blot to examine its effect on the spinal glial
cells and proinflammatory cytokines in mice. Specifically, we employed Ionized Calcium-
Binding Adapter Molecule 1 (Iba-1) and Glial Fibrillary Acidic Protein (GFAP) as specific
markers for astrocytes and microglia, respectively. As shown in Figure 3A–D, our results
indicated that the intensity of Iba-1 and GFAP in the spinal dorsal horn of mice in the CFA
group was dramatically increased compared to the control group, whereas treatment with
aucubin (10 mg/kg) resulted in a noteworthy reduction of GFAP and Iba-1 expression.
Furthermore, previous study has demonstrated that activated astrocytes and microglia
in the spinal cord produce and release a considerable amount of proinflammatory cytokines,
which may directly sensitize nociceptive sensory neurons, thereby leading to pain [
24
].
Consistent with this, in the CFA group, the levels of IL-1
β
, IL-6, and TNF-
α
exhibited a
marked elevation in comparison to the control group (Figure 3E–H). Conversely, aucubin
treatment led to a notable decrease in the expression of IL-1
β
, IL-6, and TNF-
α
in CFA-
injected mice (Figure 3E–H).
These results indicate that aucubin effectively alleviates CFA-induced inflammatory
responses in the spinal cord.
2.3. RNA-Seq Analysis in the Spinal Cord of CFA-Injected Mice Treated with Aucubin
To further investigate the potential mechanism by which aucubin alleviates inflam-
matory pain induced by CFA, we employed RNA sequencing to identify differentially
expressed genes (DEGs) in CFA-injected mice with and without aucubin treatment. By
analyzing the q-values, we identified the top 100 DEGs that significantly distinguished
the two groups, as indicated by the heatmap (Figure 4A). The volcano plot displayed
22 upregulated DEGs and 15 downregulated DEGs in the spinal cord of CFA-injected mice
after 3 days of aucubin treatment (Figure 4B), with upregulated genes shown in red and
downregulated genes in blue. Moreover, the Gene Ontology (GO) enrichment analysis
of DEGs demonstrated that the DEGs within the CFA + Aucubin group were primarily
associated with mitochondrion, mitochondrial inner membrane, cytochrome C oxidase
activity, oxidative phosphorylation, electron transport chain, and proton transmembrane
transport when compared to CFA + Veh group (Figure 4C). The Kyoto Encyclopedia of
Genes and Genomes (KEGG) pathway enrichment analysis indicated that the DEGs were
significantly enriched in oxidative phosphorylation, thermogenesis, ribosome function,
myocardial contraction, PPAR signaling pathway, and mitophagy (Figure 4D). Collec-
tively, these results suggest that aucubin may mitigate CFA-induced inflammatory pain by
modulating mitochondrial function.
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Figure 3. Effect of aucubin on CFA-induced glial cell activation and proinflammatory cytokine pro-
duction in the spinal cord. (A,B) Representative immunofluorescence staining for microglia and as-
trocytes 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.
2.3. RNA-Seq Analysis in the Spinal Cord of CFA-Injected Mice Treated with Aucubin
To further investigate the potential mechanism by which aucubin alleviates inflam-
matory pain induced by CFA, we employed RNA sequencing to identify differentially
expressed genes (DEGs) in CFA-injected mice with and without aucubin treatment. By
analyzing the q-values, we identified the top 100 DEGs that significantly distinguished the
two groups, as indicated by the heatmap (Figure 4A). The volcano plot displayed 22 up-
regulated DEGs and 15 downregulated DEGs in the spinal cord of CFA-injected mice after
3 days of aucubin treatment (Figure 4B), with upregulated genes shown in red and down-
regulated genes in blue. Moreover, the Gene Ontology (GO) enrichment analysis of DEGs
demonstrated that the DEGs within the CFA + Aucubin group were primarily associated
with mitochondrion, mitochondrial inner membrane, cytochrome C oxidase activity,
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
µ
m and 20
µ
m (magnification). (
E
–
H
) Representative bands of Western
blot and quantitative analysis of the relative expression of TNF-
α
, IL-1
β
, and IL-6 (n= 3). Data are
presented as mean ±SEM. * p< 0.05, ** p< 0.01, and *** p< 0.001.
Pharmaceuticals 2023,16, 1545 6 of 16
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 6 of 16
oxidative phosphorylation, electron transport chain, and proton transmembrane transport
when compared to CFA + Veh group (Figure 4C). The Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway enrichment analysis indicated that the DEGs were signifi-
cantly enriched in oxidative phosphorylation, thermogenesis, ribosome function, myocar-
dial contraction, PPAR signaling pathway, and mitophagy (Figure 4D). Collectively, these
results suggest that aucubin may mitigate CFA-induced inflammatory pain by modulat-
ing mitochondrial function.
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 down-
regulated 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.
2.4. Effects of Aucubin on Mitophagy in CFA-Injected Mice
The PINK1 gene is closely associated with mitochondrial function and integrity,
playing a vital role in activating mitophagy, a process linked to intracellular mitochon-
drial homeostasis and cell survival [25]. To explore the impact of aucubin on the PINK1
pathway, we examined spinal cord tissue from a mouse model of CFA-induced inflam-
matory pain using Western bloing and immunofluorescence. First, we assessed the ex-
pression levels of PINK1 and Parkin proteins of mice (Figure 5A). The results showed that
the expression of PINK1 and Parkin was elevated after CFA injection compared to the
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.
2.4. Effects of Aucubin on Mitophagy in CFA-Injected Mice
The PINK1 gene is closely associated with mitochondrial function and integrity, play-
ing a vital role in activating mitophagy, a process linked to intracellular mitochondrial
homeostasis and cell survival [
25
]. To explore the impact of aucubin on the PINK1 pathway,
we examined spinal cord tissue from a mouse model of CFA-induced inflammatory pain
using Western blotting and immunofluorescence. First, we assessed the expression levels
of PINK1 and Parkin proteins of mice (Figure 5A). The results showed that the expression
of PINK1 and Parkin was elevated after CFA injection compared to the control group
(Figure 5B,C). Conversely, treatment with aucubin resulted in a decrease in PINK1 and
Parkin expression compared to the CFA group (Figure 5B,C).
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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.
2.5. CCCP Significantly Alleviated CFA-Induced Inflammatory Pain in Mice by Activating
Mitophagy
To further investigate the correlation between mitophagy and inflammatory pain, we
employed CCCP as a pharmacological inducer of mitophagy. Mitophagy plays a vital role
in the timely elimination of damaged mitochondria, but excessive autophagy activation
may also lead to mitochondrial damage. Acting as a proton-selective ionophore, CCCP
induces PINK1 accumulation, thus triggering mitophagy [27]. Previous study has showed
that CCCP administration results in a dose-dependent increase in PINK1 and LC3B ex-
pression, effectively alleviating pain in a neuropathic pain model [28]. As illustrated in
Figure 6A,B, there was a significant reduction in PWT and PWL in mice following CFA
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.
We then evaluated the expression of LC3B and p62 proteins through Western blotting.
LC3B is a mammalian autophagy protein that localizes to the membrane of cytoplasmic
autophagosome, while p62 serves as a well-known autophagic substrate degraded by
LC3B and ubiquitinated substrates [
13
]. The accumulation of p62 indicates autophagic
impairment, suggesting that autophagic flux is inhibited. Notably, a recent study has
confirmed that autophagic degradation is blocked in osteoarthritis [
26
]. Consistent with this,
CFA-injected mice exhibited increased expression of LC3B and p62 proteins (
Figure 5D–E
),
indicating impaired autophagy in the spinal cord. Remarkably, aucubin treatment resulted
in an increased LC3B expression and a decreased p62 expression compared to CFA-treated
Pharmaceuticals 2023,16, 1545 8 of 16
mice (Figure 5D–E), indicating that aucubin could ameliorate the impaired autophagic flow
induced by CFA.
To further explore the cellular localization of PINK1, we conducted double immunoflu-
orescence using three cell markers: NeuN, GFAP, and Iba-1. As shown in Figure 5F, the
findings demonstrated that PINK1 exhibited co-localization with neurons, but not with
microglia and astrocytes, in the spinal cord. Collectively, these results suggest that PINK1 is
predominantly expressed in neuronal cells and that mitophagy in neuronal cells is involved
in the pain process.
2.5. CCCP Significantly Alleviated CFA-Induced Inflammatory Pain in Mice by
Activating Mitophagy
To further investigate the correlation between mitophagy and inflammatory pain,
we employed CCCP as a pharmacological inducer of mitophagy. Mitophagy plays a
vital role in the timely elimination of damaged mitochondria, but excessive autophagy
activation may also lead to mitochondrial damage. Acting as a proton-selective ionophore,
CCCP induces PINK1 accumulation, thus triggering mitophagy [
27
]. Previous study has
showed that CCCP administration results in a dose-dependent increase in PINK1 and LC3B
expression, effectively alleviating pain in a neuropathic pain model [
28
]. As illustrated in
Figure 6A,B, there was a significant reduction in PWT and PWL in mice following CFA
injection compared with baseline values on day 0. However, after CCCP treatment, we
observed an upward trend in PWT and PWL, strongly suggesting that CCCP effectively
reverses CFA-induced mechanical pain and thermal hypersensitivity.
Subsequently, we performed Western blotting to assess the protein expression of
PINK1, Parkin, LC3B, and p62 in the spinal cord (Figure 6C). The results demonstrated that
CCCP treatment led to a tendency of increased PINK1 expression and decreased Parkin
expression compared with the CFA group (Figure 6D–E), indicating the effective activation
of mitophagy by CCCP. Moreover, in the CCCP-treated groups, there was an increase in
LC3B expression and a decrease in p62 levels compared to the CFA group (Figure 6F,G).
These findings indicate that CCCP effectively restored the impaired autophagic flow in the
spinal cord of mice subjected to CFA-induced inflammatory pain.
Pharmaceuticals 2023,16, 1545 9 of 16
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 9 of 16
injection compared with baseline values on day 0. However, after CCCP treatment, we
observed an upward trend in PWT and PWL, strongly suggesting that CCCP effectively
reverses CFA-induced mechanical pain and thermal hypersensitivity.
Subsequently, we performed Western bloing to assess the protein expression of
PINK1, Parkin, LC3B, and p62 in the spinal cord (Figure 6C). The results demonstrated
that CCCP treatment led to a tendency of increased PINK1 expression and decreased Par-
kin expression compared with the CFA group (Figure 6D–E), indicating the effective acti-
vation of mitophagy by CCCP. Moreover, in the CCCP-treated groups, there was an in-
crease in LC3B expression and a decrease in p62 levels compared to the CFA group (Figure
6F,G). These findings indicate that CCCP effectively restored the impaired autophagic
flow in the spinal cord of mice subjected to CFA-induced inflammatory pain.
Figure 6. CCCP significantly alleviated CFA-induced inflammatory pain in mice by activating mi-
tophagy. (A,B) CCCP aenuates 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.
3. Discussion
The present study endeavors to elucidate the effects and underlying mechanisms of
aucubin on inflammatory pain induced by CFA in mice. Our findings demonstrate that
aucubin effectively reduces CFA-induced inflammatory pain and anxiety-like behavior.
Furthermore, subsequent experiments revealed that aucubin exerts inhibitory effects on
the activation of spinal astrocytes and microglia in CFA-injected mice, concomitantly re-
ducing the expression of pro-inflammatory cytokines. Interestingly, aucubin also exhibits
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.
3. Discussion
The present study endeavors to elucidate the effects and underlying mechanisms of
aucubin on inflammatory pain induced by CFA in mice. Our findings demonstrate that
aucubin effectively reduces CFA-induced inflammatory pain and anxiety-like behavior.
Furthermore, subsequent experiments revealed that aucubin exerts inhibitory effects on the
activation of spinal astrocytes and microglia in CFA-injected mice, concomitantly reducing
the expression of pro-inflammatory cytokines. Interestingly, aucubin also exhibits the ability
to reverse the impaired autophagic flux induced by CFA injection and activate mitophagy.
Taken together, our research results indicate that aucubin exhibits potential as a thera-
peutic avenue for ameliorating pain and neuroinflammation. These results offer a critical
theoretical foundation for the continued advancement of relevant treatment approaches.
Mounting evidence highlights the pivotal role of neuroinflammation, characterized by
glial cell activation and pro-inflammatory cytokine expression, in the establishment and per-
sistence of central sensitization and pain [
29
]. Notably, microglia, as resident immune cells
in the CNS, and astrocytes, with their star-shaped morphology and role in neuronal regula-
tion, are key players in this process. Upon activation, microglia release pro-inflammatory
cytokines that further stimulate astrocytes, initiating a cascade of inflammatory processes.
Conversely, activated astrocytes can produce additional pro-inflammatory cytokines, re-
sulting in the activation of glial cells and neurons, ultimately establishing a neuro-glial
positive feedback loop, resulting in the sustained release of pain mediators [30].
Pharmaceuticals 2023,16, 1545 10 of 16
Autophagy serves as a lysosomal degradation process responsible for clearing dam-
aged proteins and organelles to maintain cellular homeostasis. It holds a crucial function in
neuronal protection, as defects in autophagy or mitophagy are often linked to neuronal
loss and cognitive decline in aging or CNS neurodegenerative disorders [
31
]. Mitophagy
specifically governs the turnover of mitochondria, eliminating damaged ones, thus being
essential in regulating mitochondrial quality and maintaining mitochondrial homeostasis.
The outcome of mitophagy, whether beneficial or harmful to cell survival, depends on
the level of mitophagy activation [
32
]. A pivotal pathway mediating mitophagy is the
PINK1/Parkin pathway. Normally, PINK1 levels remain low under steady-state conditions,
but upon mitochondrial damage, the PINK1 pathway becomes activated, causing the accu-
mulation of PINK1 on the outer mitochondrial membrane and the recruitment of Parkin to
damaged mitochondria [33].
A previous study has shown that models of osteoarthritis exhibit an augmentation in
the expression of PINK1 and Parkin. [
34
]. Furthermore, studies have indicated that PINK1
expression is selectively induced in spinal dorsal horn neurons during neuropathic pain,
leading to abnormal mitochondrial flux [
35
,
36
]. Consistent with these findings, our study
demonstrates an upregulation of PINK1 and Parkin expression in CFA-injected mice, which
was subsequently reversed by aucubin treatment. The potential mechanism underlying
this effect might be attributed to aucubin promoting smooth mitophagy flow, resulting
in the degradation of PINK1 anchored to the damaged mitochondrial outer membrane,
consequently reducing the number of damaged mitochondria.
LC3B serves as a reliable marker of autophagic activity, and its upregulation can
suggest enhanced autophagic flux as well as defective clearance of autophagosomes [
37
].
Additionally, we investigated p62, one of the autophagy-specific substrates that binds to
LC3 and facilitates the recognition of damaged mitochondria by autophagosomes, aiding
in their delivery to lysosomes for degradation [
13
]. Indeed, increased expression of p62
has been observed under autophagy impairment [
38
]. Previous study has shown that
CFA administration led to elevated level of LC3B and p62 accumulation in the spinal cord,
indicating blocked autophagic flux [
39
]. Similarly, our research demonstrates that in the
CFA group, LC3B levels are elevated and accompanied by significant p62 accumulation.
However, aucubin treatment significantly enhances LC3B levels and reduces P62 content,
further activating mitophagy and restoring the blocked autophagic flux.
The relationship between autophagy and pain has been extensively investigated. Stud-
ies have indicated that impaired autophagy in glial cells in neuropathic pain models can
lead to the secretion of pro-inflammatory cytokines, thereby exacerbating mechanical allo-
dynia and thermal hyperalgesia [
40
,
41
]. Conversely, rapamycin has shown the ability to
alleviate allodynia, hyperalgesia, and glial cell activation by inducing autophagy. Further-
more, increasing autophagy levels in neuronal cells have also been found to suppress the
neuroinflammatory response and alleviate pain [
42
,
43
]. In our research, we demonstrated
that the activation of mitophagy by CCCP effectively mitigated CFA-induced inflamma-
tory pain. Similar studies have indicated that CCCP-induced activation of mitophagy
also reduces pain sensitivity in neuropathic pain model [
28
]. This evidence suggests that
mitophagy holds significant potential for future molecular pain research.
4. Materials and Methods
4.1. Chemicals and Reagents
Complete Freund’s adjuvant (CFA) was acquired from Sigma (St. Louis, MO, USA).
Aucubin (HY-N0664) and carbonyl cyanide m-chlorophenylhydrazone (CCCP, HY-100941)
were obtained from MedChemExpress (St. Louis, MO, USA). RIPA buffer (Catalog No.:
HY-K1001) and PMSF (CAS No.: 329-98-6) were procured from MedChemExpress. Pri-
mary antibodies against the following proteins were obtained from ABclonal Technology
(Wuhan, China): PINK1, TNF-
α
, IL-1
β
, IL-6, and
β
-actin (designated as A7131, A11534,
A16288, A2447, and AC026, respectively). Additionally, primary antibodies against Iba-1
(#17198), GFAP (#3670), SQSTM1/p62 (#5114), and LC3B (#2775) were obtained from Cell
Pharmaceuticals 2023,16, 1545 11 of 16
Signaling Technology Company (Danvers, MA, USA). Antibodies against Iba-1 (ab283319)
and NeuN (ab104224) were purchased from Abcam (Cambridge, MA, USA), while PINK1
(sc-517353) and Parkin (sc-32282) antibodies were obtained from Santa Cruz Biotechnol-
ogy (Santa Cruz, CA, USA). Antibodies against Iba-1 (ab283319) and NeuN (ab104224)
were purchased from Abcam (Cambridge, MA, USA), while PINK1 (sc-517353) and Parkin
(
sc-32282
) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Fluorescently labeled secondary antibodies (ab150105 and ab150064) were procured from
Abcam (Cambridge, MA, USA), and HRP-conjugated secondary antibodies (HA1001 and
HA1006) were obtained from HuaBio (Hangzhou, China). Enhanced chemiluminescent
solution (ECL) was obtained from Absin Biotechnology (Shanghai, China). The reagents of
the TransZol Up Plus RNA Kit (ER501) used in the study were acquired from Transgene
Biotechnology (Beijing, China).
4.2. Animals
Male C57BL/6 mice, aged 8–10 weeks and weighing 20–30 g, were obtained from the
Zhejiang University Laboratory Animal Center. The mice were kept in standard laboratory
conditions with free access to food and water. Prior to the behavioral tests, the mice were
given about one week to acclimate to the laboratory environment. All necessary measures
were taken to minimize the usage of animals and to ensure their ethical treatment and
welfare during the study. Special attention was given to reducing any potential suffering
experienced by the animals.
4.3. Experimental Protocol and Treatment Schedule
Inflammatory pain was induced by subcutaneously injecting 10
µ
L of CFA into the
plantar surface of the left hindpaw of the mice, whereas the control group was given an
equal volume of sterile saline in the hindpaw.
Mice were allocated randomly into four groups to examine the anti-nociceptive effect
of aucubin on CFA-induced inflammatory pain: Con + Veh, Con + Aucubin, CFA + Veh
and CFA + Aucubin. After 3 days following the saline/CFA injection, mice received
an intraperitoneal (i.p.) administration of 10 mg/kg aucubin or normal saline for three
consecutive days, as illustrated in Figure 7A.
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 12 of 16
Figure 7. (A,B) Schematic representation of the experimental protocols.
Throughout the experiment, the behavioral tests were conducted at a fixed time dur-
ing the testing days, and the animals were allowed a 30 min habituation period in the
testing room before the behavioral tests. Following the completion of the behavioral tests,
the animals were sacrificed, and the L4-5 spinal cord segments were harvested for further
processing and analysis.
4.4. Mechanical Allodynia
Mechanical allodynia was evaluated using the von Frey filaments following the up-
down method as described by Chaplan [44]. Mice were placed on an elevated mesh plat-
form and granted a 30 min habituation period before commencement of the test. Mechan-
ical stimuli were applied to the middle of the plantar surface of each paw by calibrated
von Frey filaments. The withdrawal thresholds were computed and documented as the
paw withdrawal threshold (PWT).
4.5. Thermal Hyperalgesia
Thermal hyperalgesia was evaluated by the Hargreaves test [45]. Mice were placed
individually on a raised box positioned on a glass plate, and a radiant heat source was
positioned beneath the hind paw. The heat stimulus was applied to the plantar surface of
the hind paw, and an automated timer was employed to measure the paw withdrawal
latency (PWL). The test was repeated three times, with a 5 min interval between each rep-
etition, and subsequently, the average PWL was calculated. To prevent tissue damage, a
20 s cut-off time was implemented, after which the heat stimulus was automatically ter-
minated.
4.6. Elevated Plus Maze Test
The elevated plus maze (EPM) test was performed as previously established protocol
[46]. The maze was composed of two open arms and two closed arms, elevated above the
floor. Mice were individually placed at the center of the maze, oriented toward one of the
open arms, and their behavior was recorded for a 5 min period. Prior to testing the next
mouse, the maze was cleaned with 75% ethanol. Movements of the mice were tracked and
quantified by ANY-maze 6.32 software (Stoelting, Wood Dale, IL 60191, USA).
Figure 7. (A,B) Schematic representation of the experimental protocols.
Mice were assigned randomly to four groups to further validate whether the anal-
gesia effect of aucubin was mediated by activating mitophagy: CFA, CFA + Aucubin,
Pharmaceuticals 2023,16, 1545 12 of 16
CFA + CCCP
and CFA + CCCP + Aucubin. It has been previously shown that CCCP
induces the activation of PINK1 and promotes Parkin Ser65 phosphorylation, thereby trig-
gering mitophagy. After the establishment of inflammatory pain induced by CFA, aucubin
(10 mg/kg) or CCCP (5 mg/kg) was administrated i.p. from day 4 to day 6, as indicated in
Figure 7B.
Throughout the experiment, the behavioral tests were conducted at a fixed time during
the testing days, and the animals were allowed a 30 min habituation period in the testing
room before the behavioral tests. Following the completion of the behavioral tests, the
animals were sacrificed, and the L4-5 spinal cord segments were harvested for further
processing and analysis.
4.4. Mechanical Allodynia
Mechanical allodynia was evaluated using the von Frey filaments following the up-
down method as described by Chaplan [
44
]. Mice were placed on an elevated mesh
platform and granted a 30 min habituation period before commencement of the test. Me-
chanical stimuli were applied to the middle of the plantar surface of each paw by calibrated
von Frey filaments. The withdrawal thresholds were computed and documented as the
paw withdrawal threshold (PWT).
4.5. Thermal Hyperalgesia
Thermal hyperalgesia was evaluated by the Hargreaves test [
45
]. Mice were placed
individually on a raised box positioned on a glass plate, and a radiant heat source was
positioned beneath the hind paw. The heat stimulus was applied to the plantar surface of the
hind paw, and an automated timer was employed to measure the paw withdrawal latency
(PWL). The test was repeated three times, with a 5 min interval between each repetition,
and subsequently, the average PWL was calculated. To prevent tissue damage, a 20 s cut-off
time was implemented, after which the heat stimulus was automatically terminated.
4.6. Elevated Plus Maze Test
The elevated plus maze (EPM) test was performed as previously established proto-
col [
46
]. The maze was composed of two open arms and two closed arms, elevated above
the floor. Mice were individually placed at the center of the maze, oriented toward one of
the open arms, and their behavior was recorded for a 5 min period. Prior to testing the next
mouse, the maze was cleaned with 75% ethanol. Movements of the mice were tracked and
quantified by ANY-maze 6.32 software (Stoelting, Wood Dale, IL 60191, USA).
4.7. Open Field Test
The open field test (OFT) was performed following previously described methods [
47
].
Prior to the experiments, mice were habituated to the testing condition and light level for
30 min. Mice were placed individually in a square arena (dimensions: 45
×
45
×
45 cm
3
)
and allowed to explore freely for a duration of 15 min. After each test, the open field arena
was cleansed using 75% ethanol. During the test, locomotor and exploratory behaviors of
the mice were recorded through ANY-maze software.
4.8. Immunofluorescence Staining
Mice were anesthetized by intraperitoneal administration of sodium pentobarbital
(50 mg/kg), followed by transcardial perfusion using phosphate-buffered saline (PBS)
and subsequently 4% paraformaldehyde (PFA). Tissue sections were prepared from the
spinal cord L4-5, fixed in PFA overnight at 4
◦
C, and subsequently permeabilized with
30% sucrose until saturation. Samples were freeze-mounted in OCT compound and
sliced into
30-µm
sections at
−
20
◦
C using a freezing microtome (NX50, Thermo Sci-
entific,
Waltham, MA, USA
). For immunofluorescence staining, the sections were blocked
in a solution of 5% normal donkey serum in 0.3% Triton-X in PBS at room temperature for
1 h. Subsequent to blocking, the sections were incubated at 4
◦
C overnight with primary
Pharmaceuticals 2023,16, 1545 13 of 16
antibodies against PINK1 (1:100), Iba-1 (1:100), Iba-1 (1:100), GFAP (1:100) and NeuN (1:100).
Subsequently, the sections were subjected to appropriate fluorescently labeled secondary
antibodies at 1:200 for 1h at room temperature. Nuclei were counterstained with DAPI.
Images were captured using a fluorescence microscope (VS120, Olympus,
Tokyo, Japan
)
and analyzed with Image J 2 software (NIH, Bethesda, Rockville, MD, USA). Initially, we
imported the fluorescence images, followed by separating the RGB channels and converting
them into 8-bit grayscale images. Subsequently, we delineated the spinal dorsal horn
region within the images, and we consistently applied a uniform threshold to standardize
the process for quantifying the mean gray value. Notably, a minimum of three mice
were included in each experimental group, with three randomly selected slices from each
individual mouse were analyzed. Finally, average values were computed for each group to
facilitate subsequent data analysis.
4.9. Western Blot Analysis
Under deep anesthesia, animals were sacrificed in order to harvest the L4-5 spinal cord
segments. Tissue samples were collected and homogenized with RIPA buffer containing
PMSF. The tissue samples were thoroughly mashed, and the protein extracts were obtained
through centrifugation at 12,000
×
gfor 15 min at 4
◦
C. The supernatants containing the
protein extracts were collected. Protein concentrations were determined by the BCA assay,
in accordance with the manufacturer’s instructions. Equivalent protein quantities from
each sample were loaded and subjected to separation by SDS-PAGE gel electrophoresis.
The proteins were then transferred onto PVDF membranes. To block non-specific binding
sites, the membranes were blocked with 5% non-fat milk in PBST at room temperature
for 1h to prevent nonspecific binding. Following this, the membranes were incubated
overnight at 4
◦
C with appropriate primary antibodies against TNF-
α
(1:1000), IL-1
β
(1:1000), IL-6 (1:1000), PINK1 (1:250), Parkin (1:500), SQSTM1/p62 (1:1000), LC3B (1:1000),
and
β
-actin (1:3000). The membranes were washed with PBST, followed by an incubation
with appropriate secondary antibodies (goat anti-rabbit/mouse HRP) at a dilution of 1:5000
for 1h at room temperature. Protein bands were acquired using the ECL luminescence
Reagent, and the images were captured by ChemiDoc Touch Imaging System (Bio-Rad,
Hercules, CA, USA). The quantification of protein band intensities was performed using
the Image J software.
4.10. RNA Sequencing
Three days after the CFA injection, mice were administered aucubin (10 mg/kg) for
three consecutive days. Subsequently, the L4-5 spinal cord tissue was extracted. Three
replicate samples were collected from each experimental group. RNA extraction was
performed using the TransZol Up Plus RNA Kit reagents, followed by mRNA-seq analysis
and data processing conducted by LC-Bio Technology Co. (Zhejiang, China).
4.11. Statistical Analyses
Statistical analyses were performed using GraphPad Prism 9.0 software (GraphPad,
San Diego, CA, USA). All the data are expressed as mean
±
SEM. For comparisons involving
multiple groups, one-way or two-way analysis of variance (ANOVA) was performed,
followed by appropriate post hoc tests to ascertain statistical significance. p< 0.05 was
considered statistically significant.
5. Conclusions
In summary, our data demonstrate that mitochondrial dysfunction in mice with
CFA-induced inflammatory pain. Aucubin exhibits potential as a candidate for allevi-
ating inflammatory pain, likely attributed to its capacity to enhance autophagy, restore
autophagic flux, and inhibit glial cell activation and pro-inflammatory cytokine expression.
Consequently, our research sheds light on the crucial role of mitochondrial function in pain
modulation, offering novel therapeutic avenues for pain management.
Pharmaceuticals 2023,16, 1545 14 of 16
Author Contributions:
Conceptualization, G.C.; Investigation, D.Y., Y.W. and Y.C.; Writing—Original
Draft Preparation, D.Y.; Writing—Review and Editing, G.C., Y.W. and Y.C. All authors have read and
agreed to the published version of the manuscript.
Funding:
This research was supported by the National Natural Science Foundation of China
(
No. 82171176
and No. 82001424), and the Zhejiang Provincial Department of Medicine and Health
Science and Technology (No. YH42021010).
Institutional Review Board Statement:
All experimental procedures involving animals were con-
ducted in accordance with the guidelines approved by the Institutional Animal Care and Use
Committee at Zhejiang University (protocol number: ZJU20230241), and approval date 26 June 2023.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
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