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\begin{document}

\title{Contribution of Inwardly Rectifying Potassium Channel 4.1 in Orofacial
Neuropathic Pain: Regulation of Pannexin 3 via the Reactive Oxygen
Species-Activated P38 MAPK Signal Pathway}



\author[1]{Yuheng Feng}%
\author[1]{Renjie Tang}%
\author[1]{Yanyan Zhang}%
\author[1]{Jiu Lin}%
\author[1]{Yajing Liu}%
\author[1]{Yike Li}%
\author[1]{Chunjie Li}%
\author[2]{Cheng Zhou}%
\author[1]{Fei Liu}%
\author[1]{Jie-Fei Shen}%
\affil[1]{Sichuan University West China Hospital of Stomatology}%
\affil[2]{Translational Neuroscience Center, West China Hospital of Sichuan University Tianfu Life Science Park}%


\vspace{-1em}



  
  \date{June 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The involvement of inwardly rectifying potassium channel 4.1 (Kir4.1) in
neuropathic pain has been established. However, there is limited
understanding of the downstream mechanism through which Kir4.1
contributes to orofacial neuropathic pain. The objective of this study
was to examine the regulation of Kir4.1 on the expression of pannexin 3
(Panx3) in the trigeminal ganglion (TG) and the underlying mechanism in
the context of orofacial neuropathic pain caused by chronic constriction
injury of the infraorbital nerve (CCI-ION). The study observed a
significant increase in Panx3 expression in the TG of mice with CCI-ION.
Inhibition of Panx3 in the TG of CCI-ION mice resulted in alleviation of
orofacial mechanical allodynia. Furthermore, conditional knockdown (CKD)
of Kir4.1 in the TG of both male and female mice led to mechanical
allodynia and up-regulation of Panx3 expression. Conversely,
overexpression of Kir4.1 decreased Panx3 levels in the TG and relieved
mechanical allodynia in CCI-ION mice. In addition, silencing Kir4.1 in
satellite glial cells (SGCs) decreased Panx3 expression and increased
the phosphorylation of P38 MAPK. Moreover, silencing Kir4.1 in SGCs
increased the levels of reactive oxygen species (ROS). The elevated
phosphorylation of P38 MAPK resulting from Kir4.1 silencing was
inhibited by using a superoxide scavenger known as the tempol. Silencing
Panx3 in the TG in vivo attenuated the mechanical allodynia caused by
Kir4.1 CKD. In conclusion, these findings suggest that the reduction of
Kir4.1 promotes the expression of Panx3 by activating the ROS-P38 MAPK
signaling pathway, thus contributing to the development of orofacial
neuropathic pain.%
\end{abstract}%



\sloppy


\textbf{Title Page}

\textbf{Informative title:}

\textbf{Contribution of Inwardly Rectifying Potassium Channel 4.1 in
Orofacial Neuropathic Pain: Regulation of Pannexin 3 via the Reactive
Oxygen Species-Activated P38 MAPK Signal Pathway}

\textbf{Running title:}

\textbf{Kir4.1 regulate Panx3 through the ROS-P38 MAPK}

Yu-Heng Feng\textsuperscript{1,2}, Ren-Jie Tang\textsuperscript{1},
Yan-Yan Zhang\textsuperscript{1,2}, Jiu Lin\textsuperscript{1,2},
Ya-Jing Liu\textsuperscript{1,2}, Yi-Ke Li\textsuperscript{1,2},
Chun-Jie Li\textsuperscript{1,2}, Cheng Zhou\textsuperscript{3}, Fei
Liu\textsuperscript{1,2*}, Jie-Fei Shen\textsuperscript{1,2*}

\section*{Institutional affiliations:}

{\label{institutional-affiliations}}

1. State Key Laboratory of Oral Diseases \& National Center for
Stomatology \& National Clinical Research Center for Oral Disease, West
China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan,
China

2. Department of Prosthodontics, West China Hospital of Stomatology,
Sichuan University, Chengdu, China

3. Laboratory of Anesthesia and Critical Care Medicine, Translational
Neuroscience Center, West China Hospital of Sichuan University, Chengdu,
China

\subsection*{*These authors are the co-corresponding
authors:}

{\label{these-authors-are-the-co-corresponding-authors}}

\textbf{Prof. Jie-Fei Shen,}

State Key Laboratory of Oral Diseases

National Center for Stomatology

National Clinical Research Center for Oral Diseases

West China Hospital of Stomatology

Sichuan University,

No. 14, Section 3, Renmin Road South, Chengdu 610041, Sichuan, China.

E-mail address: shenjiefei@scu.edu.cn

\textbf{Dr. Fei Liu,}

State Key Laboratory of Oral Diseases

National Center for Stomatology

National Clinical Research Center for Oral Diseases

West China Hospital of Stomatology

Sichuan University,

No. 14, Section 3, Renmin Road South, Chengdu 610041, Sichuan, China.

E-mail address: liufei.hxkq@scu.edu.cn

\textbf{Abstract}

The involvement of inwardly rectifying potassium channel 4.1 (Kir4.1) in
neuropathic pain has been established. However, there is limited
understanding of the downstream mechanism through which Kir4.1
contributes to orofacial neuropathic pain. The objective of this study
was to examine the regulation of Kir4.1 on the expression of pannexin 3
(Panx3) in the trigeminal ganglion (TG) and the underlying mechanism in
the context of orofacial neuropathic pain caused by chronic constriction
injury of the infraorbital nerve (CCI-ION). The study observed a
significant increase in Panx3 expression in the TG of mice with CCI-ION.
Inhibition of Panx3 in the TG of CCI-ION mice resulted in alleviation of
orofacial mechanical allodynia. Furthermore, conditional knockdown (CKD)
of Kir4.1 in the TG of both male and female mice led to mechanical
allodynia and up-regulation of Panx3 expression. Conversely,
overexpression of Kir4.1 decreased Panx3 levels in the TG and relieved
mechanical allodynia in CCI-ION mice. In addition, silencing Kir4.1 in
satellite glial cells (SGCs) decreased Panx3 expression and increased
the phosphorylation of P38 MAPK. Moreover, silencing Kir4.1 in SGCs
increased the levels of reactive oxygen species (ROS). The elevated
phosphorylation of P38 MAPK resulting from Kir4.1 silencing was
inhibited by using a superoxide scavenger known as the tempol. Silencing
Panx3 in the TG in vivo attenuated the mechanical allodynia caused by
Kir4.1 CKD. In conclusion, these findings suggest that the reduction of
Kir4.1 promotes the expression of Panx3 by activating the ROS-P38 MAPK
signaling pathway, thus contributing to the development of orofacial
neuropathic pain.

\textbf{Keywords:} inwardly rectifying potassium channel 4.1; pannexin
3; reactive oxygen species; mitogen-activated protein kinases;
trigeminal nerve injury; orofacial neuropathic pain.



\textbf{Introduction}

Orofacial neuropathic pain, characterized by allodynia and hyperalgesia,
is a debilitating condition often caused by trigeminal nerve injury.
Unfortunately, the pathomechanism of this condition remains unclear, and
effective treatments are lacking (Liu et al., 2023). The development and
persistence of orofacial neuropathic pain are attributed, in part, to
peripheral sensitization of the trigeminal nervous system (Liu et al.,
2022). In our previous research, we discovered a significant role of
inwardly rectifying potassium channel 4.1 (Kir4.1) expressed
specifically in trigeminal ganglion (TG) satellite glial cells (SGCs) in
peripheral sensitization and the subsequent induction of orofacial
neuropathic pain resulting from inferior alveolar nerve injury (Li et
al., 2023a). However, the downstream signaling pathway through which
Kir4.1 regulates the development of orofacial neuropathic pain remains
poorly understood.

Following sciatic nerve injury, the levels of reactive oxygen species
(ROS) increase in both the dorsal root ganglion (DRG) and sciatic nerve.
This increase in ROS has been identified as a contributing factor to the
development of neuropathic pain (Kallenborn-Gerhardt et al., 2012; Xu et
al., 2018; Uslusoy et al., 2020). Cellular studies have shown that
elevated ROS production can activate the mitogen-activated protein
kinases (MAPK) signal pathway, specifically the ERKs, JNKs, and p38
MAPKs (Son et al., 2011; Jaenen et al., 2021). Activation of the MAPK
signal pathway is well-established to play a crucial role in both
neuropathic and inflammatory pain (Liu et al., 2019; Zhang et al., 2019;
Gui et al., 2020). Interestingly, it has been observed that increasing
the intracellular potassium concentration in auditory cells can mitigate
ROS generation and reduce the side effects of cisplatin (Kim \& Park,
2013). This suggests that the activation of the ATP-sensitive potassium
(K\textsubscript{ATP}) channel, a specific subtype of inwardly
rectifying potassium channel (Kir), can regulate ROS production and
potentially prevent ultraviolet-induced skin aging (Cao et al., 2007;
Fellows et al., 2023). Likewise, Kir4.1 is known to play a significant
role in the regulation of extracellular and intracellular potassium
levels in both normal and pathological cellular states (Nwaobi et al.,
2016). However, the precise role of Kir4.1 in the TG in the development
of orofacial neuropathic pain through the ROS-MAPK signal pathway
remains unclear.

Pannexins, a group of membrane channel proteins consisting of three
subtypes (Panx1, Panx2, and Panx3), play a role in facilitating
electrical communication between cells as gap junctions and act as
conduits for ATP (Bruzzone et al., 2003; Sohl et al., 2005; O'Donnell \&
Penuela, 2021). These proteins have been implicated in the development
of chronic pain, as ATP is known to be involved in this process (Karatas
et al., 2013; Li et al., 2021; Belonogova et al., 2023). For instance,
the up-regulation of Panx3 in intervertebral disk disorder has been
linked to the promotion of chronic back pain (Belonogova et al., 2023).
Notably, previous studies have suggested that the function of the
pannexin family can be influenced by ROS (Xu et al., 2018). Based on
these findings and our previous research, we postulate that Kir4.1 in
the SGCs may play a role in orofacial neuropathic pain by modulating
Panx3 through the ROS-MAPK signal pathway following trigeminal nerve
injury.

In this study, an orofacial neuropathic pain model was established
through chronic constriction injury of the infraorbital nerve (CCI-ION).
The findings demonstrated that both CCI-ION and \emph{Kcnj10}conditional
knockdown (CKD) led to an increase in Panx3 levels in the TG of mice,
resulting in orofacial mechanical allodynia. However, this effect was
reversed when Kir4.1 was overexpressed in the TG. Furthermore,
\emph{Kcnj10} CKD was found to activate P38 MAPK, but this activation
was inhibited by the administration of a ROS scavenger. Importantly, the
inhibition of Panx3 in the TG effectively alleviated orofacial allodynia
caused by CCI-ION. Overall, this study provides novel insights into the
pathomechanism of orofacial neuropathic pain induced by trigeminal nerve
injury by establishing a connection between Kir4.1 and Panx3 through the
ROS-P38 MAPK signal pathway. These findings not only enhance our
understanding of orofacial neuropathic pain but also propose potential
therapeutic targets for future exploration.

\textbf{Materials and Methods}

\textbf{Animals}

C57BL/6 wild-type (WT) mice aged 6 to 8 weeks and
GFAP-CreER\textsuperscript{T2+/-};
\emph{Kcnj10\textsuperscript{f/f}}mice were used in our study. The
C57BL/6 mice were procured from the Laboratory Animal Center of Sichuan
University. The GFAP-CreER\textsuperscript{T2+/-};
\emph{Kcnj10\textsuperscript{f/f}}mice, weighing between 20 and 35 g,
were obtained through the breeding of mice carrying \emph{Kcnj10-floxed}
alleles (\emph{Kcnj10\textsuperscript{f/f}} mice) with transgenic mice
expressing -CreER\textsuperscript{T2} recombinase under the
glia-specific glial fibrillary acidic protein (GFAP) promoter
(GFAP-CreER\textsuperscript{T2+/-} mice). The mice were housed in groups
of five per cage under standard conditions, including a constant
temperature and a 12-hour light-dark cycle. They were provided with
ample water and food to ensure their well-being. All experimental
procedures were conducted following the guidelines of the International
Association for the Study of Pain and were approved by the Ethics
Committee of the West China Hospital of Stomatology, Sichuan University
(WCHSIRB-D-2023-001). We took measures to minimize the number of animals
used and any potential discomfort or pain experienced by the animals.

\textbf{Chronic constriction injury of the infraorbital nerve}

CCI-ION procedure was conducted on the ION following previously
established methods(Lin \emph{et al.} , 2022a). Before the surgery, mice
were anesthetized using Isoflurane, and a 2 mm incision was made in the
mucosa of the right palate to explore the ION. Subsequently, two pieces
of chromic gut 4-0 suture were loosely tied around the ION to induce
constriction of the blood flow in the perineural vasculature. The 7--0
absorbable sutures were used to close the incision. The same surgical
procedure was performed without nerve ligation for the sham operation
group.

\textbf{Conditional knockdown of \emph{Kcnj10}}

GFAP-CreER\textsuperscript{T2+/-}; \emph{Kcnj10\textsuperscript{f/f}}
mice were employed in our study to achieve CKD of Kir4.1 since mice with
complete Kir4.1 gene deficiency exhibit early mortality. To generate
GFAP-CreER\textsuperscript{T2+/-};\emph{Kcnj10\textsuperscript{f/f}}
mice, the\emph{Kcnj10\textsuperscript{f/f}} and
CreER\textsuperscript{T2+/-}mouse lines were crossed. The \emph{Kcnj10}
contains two loxP sites flanking its entire open reading frame within
intronic regions, and abnormal expression of Kir4.1 has been observed in
the\emph{Kcnj10\textsuperscript{f/f}} mouse line (McLellan \emph{et al.}
, 2017). The glia-specific GFAP promoter was utilized to drive the
expression of CreER\textsuperscript{T2-} recombinase in the
GFAP-CreER\textsuperscript{T2+} mouse line, where in the mutant
ligand-binding region of the estrogen receptor is fused with the Cre
enzyme (Hirrlinger \emph{et al.} , 2006). In
GFAP-CreER\textsuperscript{T2+/-};
\emph{Kcnj10\textsuperscript{f/f}}mice, CreER\textsuperscript{T2-}
recombinase is expressed in glial cells but remains inactive and
sequestered in the cytoplasm (Weinstock \emph{et al.} , 2019).
Tamoxifen, a synthetic steroid ligand, can bind to
CreER\textsuperscript{T2-} recombinase (Hirrlinger \emph{et al.} ,
2006). Upon tamoxifen administration, CreER\textsuperscript{T2-}
recombinase is released from the heat shock 90 anchor protein and
translocates to the nucleus. It recognizes the loxP sites and excises
the floxed\emph{Kcnj10} gene (Szabo \emph{et al.} , 2009). Genotyping of
the mice was performed by PCR using tail genomic DNA to confirm the
presence of the \emph{Cre} gene (Leneuve \emph{et al.} , 2001). The PCR
primers used for \emph{Cre} gene detection were as follows: forward:
5'-GGTCGATGCAACGAGTGATGAGG and reverse: 5'-GCTAAGTGCCTTCTCTACACCTGCG. To
induce Cre-mediated deletion of \emph{Kcnj10} , tamoxifen (\#6342;
Tocris Bioscience, Bristol, UK) dissolved in corn oil (Sigma-Aldrich,
St. Louis, MO, USA) was administered intraperitoneally to
GFAP-CreER\textsuperscript{T2+/-};
\emph{Kcnj10\textsuperscript{f/f}}mice for seven consecutive days.
Simultaneous intraperitoneal injections of the vehicle (corn oil) were
administered to the control mice. The allocation of mice to the
tamoxifen and vehicle groups in the GFAP-CreER\textsuperscript{T2+/-};
\emph{Kcnj10\textsuperscript{f/f}}strain was done randomly. Tamoxifen
exhibits robust CreER\textsuperscript{T2} induction approximately three
weeks after injections, and its effects persist for an extended duration
(Kumar \emph{et al.} , 2022).

\textbf{Intro-ganglionic injection}

In our study, recombinant adeno-associated virus (AAV) was used to
facilitate gene transfer and overexpress Kir4.1 in the TG. A
cre-dependent Kir4.1-overexpressing AAV (AAV-Kir4.1,
hEF1a-DIO-kcnj10-2A-eGFP-WPRE-pA; [?]1E + 13.V.G./mL; cat\# S0270-8;
Shanghai Taitool Bioscience Co., Ltd., Shanghai, China) and an AAV
carrying the Cre enzyme (GFAP-iCRE-WPRE-pA; [?]2E + 12.V.G./mL; cat\#
S0611-8; Shanghai Taitool Bioscience Co., Ltd.) were injected into the
TG of wild-type C57BL/6J mice. For the control group, a control AAV
(AAV-control, hEF1aDIO-eGFP-WPRE-pA; [?]1E + 13.V.G./mL; cat\# S0270-8;
Shanghai Taitool Bioscience Co., Ltd.) and the AAV containing the Cre
enzyme were both administered. CCI-ION surgery was performed on both
groups three weeks after the AAV injection.

In vivo transfection reagents (EntransterTM-in vivo, Engreen Biosystem
Co, Ltd.) and a mixture of 21 nucleotide-long siRNAs targeting Panx3
(5'-GGCCAGUACAAGGUGAAAUTT -3' and 5'-AUUUCACCUUGUACUGGCCTT -3') were
employed to decrease the expression of Panx3 in the TG. For each mouse,
2 \selectlanguage{greek}μ\selectlanguage{english}L of transfection reagents were mixed with 4 \selectlanguage{greek}μ\selectlanguage{english}L of siRNA (20 \selectlanguage{greek}µ\selectlanguage{english}M) and
incubated at room temperature for 15 minutes for in vivo transfection.

The AAV and the mixture of transfection reagents with Panx3 siRNA were
injected into the right TG using a stereotaxic apparatus. The mice were
placed under anesthesia and mounted on the apparatus, and the anterior
fontanelle was used as the reference point for the X, Y, and Z axes. The
position of the skull surface was marked by adjusting the stereotaxic
device to coordinates of 4.00 mm (X-axis) and 1.75 mm (Y-axis). A
micro-syringe (Hamilton, Shanghai, China) was inserted vertically from
this position into the TG, reaching a depth of 6.00 mm (Z-axis) below
the surface of the skull. Subsequently, the medications were slowly
delivered over 1 minute, and the needles were gently removed. The
accuracy of the injection was confirmed upon brain removal.

\textbf{Assessment of orofacial mechanical allodynia}

Von Frey filaments (NJKEWBIO, China) were used to assess changes in
sensitivity to mechanical stimulation of the face. This method has been
utilized in our previous experiments ( Li\emph{et al.} , 2021; Lin
\emph{et al.} , 2022b; Zhang \emph{et al.} , 2022a;). A suitable
metallic mesh cage, which allowed the mice to move freely without
constraints, was used to evaluate the mechanical head-withdrawal
threshold (HWT) in both WT and \emph{Kcnj10} CKD (Zhang \emph{et al.} ,
2020) mice. Before baseline testing, the right whisker pad of the mice
was shaved, and a firm tip with a diameter of 1 mm was applied until the
animals responded by evading. Von Frey filaments of varying gauge were
perpendicularly applied to the whisker pad area based on the behavioral
reactions observed upon stimulation and a positive reaction was
considered when the mice abruptly withdrew their heads or flinched after
removing the filaments. The sequence of positive and negative responses
was recorded, and Dixon's up-down method was employed to determine the
50\% HWT. To maintain accuracy in evaluating behavioral responses, all
observations were carried out by a researcher who was blind to the
experimental conditions.

\textbf{Preparation of satellite glial cells}

C57BL/6 mice (1-2 weeks) were sterilized with 75\% alcohol and the skin,
skull, and brain were removed to expose the TG. The TG was obtained with
a micro dissecting instrument to make sure that blood vessels around the
TG were utterly removed under a stereomicroscope. 4 TGs were collected
in 60 mm Petri dishes with DMEM complete~medium, digested with cell
dissociation buffer for 5 minutes under 37\selectlanguage{ngerman}°C and terminated with a
complete medium. After that, cells were seeded into 60mm plates
containing DMEM primary~medium (4mL per well). All operations were
performed on ice. The preparation of the solutions is listed in Table 1.

\textbf{Table 1. Liquid components for cells}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& Composition & Content & Manufacturer/catalog number\tabularnewline
\midrule
\endhead
DMEM complete~medium & DMEM & 89\% & Gibco/11965084\tabularnewline
& Fetal bovine serum & 10\% & Gibco/10099-141\tabularnewline
& Penicillin-Streptomycin Solution & 1\% & Gibco/15140122\tabularnewline
DMEM primary~medium & DMEM & 83\% & Gibco/11965084\tabularnewline
& Fetal bovine serum & 15\% & Gibco/10099-141\tabularnewline
& Penicillin-Streptomycin Solution & 2\% & Gibco/15140122\tabularnewline
Digestive fluid & DMEM & 50\% & Gibco/11965084\tabularnewline
& Collagenase & 1mg/L & Gibco/17101015\tabularnewline
& Trypsin & 50\% & Gibco/25200072\tabularnewline
\bottomrule
\end{longtable}

\textbf{siRNA transfection and drug administration on SGCs}

Cell passaging was performed 48h before transfection to make the density
of SGCs reach 60-80\%. Cells in 6-well plates were transfected with
150pmol siRNA oligonucleotides (GenePharma) using 6ul GP-transfect-MATE
reagent (GenePharma). We used 21 nucleotide-long siRNAs for knock-down
of Kir4.1 (Kir4.1 siRNA) (5'-GGA GCA CAU UGC UGA CAA ATT-3' and 5'-UUU
GUC AGC AAU GUG CUC CTT-3'). As a control group, the negative control
siRNA (NC siRNA) (5'-UUC UCC GAA CGU GUC ACG UTT-3 and 5'-ACG UGA CAC
GUU CGG AGA ATT-3') was used. The cells were collected after 48h of
transfection for PCR and 72h for WB.

Selective inhibitors were used to pretreat SGCs to explore the effect of
MAPKs pathway in SGCs. The drugs used were U0126 (ERK 1/2 inhibitor;
10mM; Apexbio), SB203580 (p38-MAPK inhibitor; 10mM; Apexbio) and
SP600125 (JNK1/2/3 inhibitor; 10mM; Apexbio). All drugs were diluted to
the required quantities with normal saline containing 5%0 DMSO. The
control vehicle used 5%0 DMSO. Selective inhibitors were added into SGC
cultures for 30min before transfection to yield the following groups: NC
siRNA (75 pM) + DMSO (5 \selectlanguage{greek}μ\selectlanguage{english}M), Kir4.1 siRNA (75 pM), Kir4.1 siRNA (75 pM)
+ U0126 (5 \selectlanguage{greek}μ\selectlanguage{english}M), Kir4.1 siRNA (75 pM) + SB203580 (5 \selectlanguage{greek}μ\selectlanguage{english}M), Kir4.1 siRNA (75
pM) + SP600125 (5 \selectlanguage{greek}μ\selectlanguage{english}M). The medicine doses listed above were based on
earlier research (Zhang \emph{et al.} , 2020).

\textbf{Measurement of superoxide anion}

To assess the superoxide content, we employed the Reactive Oxygen
Species Assay Kit (Beyonc\selectlanguage{ngerman}é, S0033S). DCFH-DA was diluted with serum-free
culture medium at a ratio of 1:1000 to achieve a concentration of 10
\selectlanguage{greek}µ\selectlanguage{english}mol/L and then was loaded into the cells 24 hours after transfection
with Kir4.1 siRNA in SGCs. 1 ml DCFH-DA solution was added to each well
of a 12-well plate. The plate was then incubated for 20 minutes at 37
\selectlanguage{ngerman}°C. Another group was treated with tempol (a superoxide scavenger) for
30 minutes. The cells were washed with medium to remove the residual
DCFH-DA. The samples was conducted using laser confocal microscop and
also collected for fluorescence enzyme marker detection.

\textbf{Quantitative reverse transcription-polymerase chain reaction.}

We use quantitative reverse transcription-polymerase chain reaction
(RT-qPCR) to analyze the mRNA level of Kir4.1 and Panx3. Total RNA was
extracted using RNAiso Plus. Concentration and purity of the RNA were
determined by measuring the absorbance at 260 and 280 nm using the
NanoDrop One Microvolume UV--Vis Spectrophotometer (\#840-317400,
ThermoFisher Scientific, Waltham, MA, USA). PrimeScript\selectlanguage{english} RT reagent Kit
with gDNA Eraser (Takara Bio Inc., Kusatsu, Japan) was used for the
removal of genomic DNA and reverse transcription reaction. To detect
mRNA expression, eraser cDNA was diluted and added to a real-time
fluorescence quantitative PCR machine. The primers are showed in Table
2. After the dissolution curves were plotted, the
2\selectlanguage{english}\textsuperscript{-ΔΔCt} method was used to assess the final data.

\textbf{Table 2. Main characteristics of the genes and sequences of the
real-time quantitative polymerase chain reaction primers.}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Geen name/NCBI Gene ID & Location & Primer sequences
(5'-3')\tabularnewline
\midrule
\endhead
Panx3/208098 & NC\_000075.7 & (forward) GGAACGCCCTCTTACTCC (reverse)
AGCAGCCCTGTCTTGATG\tabularnewline
Kcnj10/16513 & NC\_000067.7 & (forward) GGAACGCCCTCTTACTCC (reverse)
AGCAGCCCTGTCTTGATG\tabularnewline
GAPDH/2597 & NC\_000072.7 & (forward) GGAACGCCCTCTTACTCC (reverse)
AGCAGCCCTGTCTTGATG\tabularnewline
\selectlanguage{greek}β-\selectlanguage{english}actin/11461 & NC\_000071.7 & (forward) GGAACGCCCTCTTACTCC (reverse)
AGCAGCCCTGTCTTGATG\tabularnewline
\bottomrule
\end{longtable}

\textbf{Western blotting}

The samples were homogenized using RIPA lysis buffer (Thermo Fisher
Scientific) supplemented with 1 Halt Protease and Phosphatase Inhibitor
Cocktail (Thermo Fisher Scientific). The protein concentration was
tested using the BCA Protein Assay Kit (Thermo Fisher Scientific). A
total protein concentration of 200 mg was loaded onto NuPAGE 4--12\%
Bis-Tris gels (Thermo Fisher Scientific). Subsequently, the proteins
were transferred to nitrocellulose membranes. The membranes were
incubated for 2 hours with 5\% BSA in Tris-buffered saline plus Tween
(TBST) and then incubated with primary antibodies overnight at 4\selectlanguage{ngerman}°C,
secondary antibodies at room temperature (RT) for 1 hour. The signals
were detected using an autoradiograph and enhanced chemiluminescence
system (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were
imaged using an image analysis system (LAS-3000, Fujifilm). The
antibodies used for Western blot analysis are listed in Table 3.

\textbf{Table 3. Antibodies used in WB.}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Antibody name & Manufacturer/catalog number/RRID/host species/clonality
& Concentration\tabularnewline
\midrule
\endhead
Anti-Panx3 & Invitrogen/433270/AB\_2532206/Rabbit/Polyclonal &
1:125\tabularnewline
Anti-Kir4.1 & Proteintech/12503-1-AP/AB\_2234144/Rabbit/Unconjugated &
1:10000\tabularnewline
Anti-P38 & Proteintech/14064-1-AP/AB\_2878007/Rabbit/Unconjugated &
1:1000\tabularnewline
Anti-p-P38 & ZENBIO/310091/Rabbit/Polyclonal & 1:500\tabularnewline
Anti-ERK & ZENBIO/343830/Rabbit/Polyclonal & 1:500\tabularnewline
Anti-p-ERK & ZENBIO/310065/Rabbit/Polyclonal & 1:500\tabularnewline
Anti-JNK & ZENBIO/R22866/Rabbit/Unconjugated & 1:500\tabularnewline
Anti-p-JNK & ZENBIO/381100/Rabbit/Polyclonal & 1:500\tabularnewline
Anti-GAPDH & Abcam/Ab9485/AB\_307275/rabbit/ polyclonal &
1:1000\tabularnewline
Anti-\selectlanguage{greek}α-\selectlanguage{english}tublin & Proteintech/11224-1-AP/AB\_2210206/Rabbit/Unconjugated &
1:10000\tabularnewline
\bottomrule
\end{longtable}

\textbf{Immunohistochemistry}

To identify the expression of Panx3, NeuN (a specific marker of
neurons), and glutamine synthetase (GS, a specific marker of SGCs),
fluorescent immunohistochemistry was performed on isolated TG, following
the previously described methods (Li, Liu, et al., 2021; Zhang et al.,
2019). The TGs were removed and incubated in 4\% paraformaldehyde for 4
hours, then were transferred to a 30\% sucrose solution for a night and
incubated for 24 hours at 4\selectlanguage{ngerman}°C. Afterwards the samples were cut into
10-\selectlanguage{greek}µ\selectlanguage{english}m sections using a cryostat (CM1950, Leica Microsystems, Wetzlar,
Germany), embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo,
Japan), and treated with a 0.25\% Triton X-100 solution (Solarbio Life
Science, Beijing, China) for 15 minutes. To block nonspecific binding,
the sections were then incubated with normal goat serum (10\%, Solarbio
Life Science) at RT for 30 minutes. Next, the sections were incubated
overnight at 4\selectlanguage{ngerman}°C with primary antibodies specific to Panx3, NeuN, and GS
and secondary antibodies for 1 hour at RT. Finally, the nuclei were
stained with DAPI (1:1000; Beyotime Biotechnology, Shanghai, China) for
5 minutes at RT. Confocal laser scanning microscopy (FV3000; Olympus,
Tokyo, Japan) was used for imaging the stained sections. The antibodies
are listed in Table 4.

\textbf{Table 4. Antibodies used in fluorescent immunohistochemistry}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Antibody name & Manufacturer/catalog number/RRID/host species/clonality
& Concentration\tabularnewline
\midrule
\endhead
Anti-Panx3 & Invitrogen/433270/RRID: AB\_2532206/Rabbit/Polyclonal &
1:100\tabularnewline
anti-GS & Abcam/Ab64613/RRID: AB\_1140869/mouse/monoclonal &
1:500\tabularnewline
anti-NeuN & Abcam/Ab17345/RRID: AB\_776808/mouse/polyclonal &
1:500\tabularnewline
anti-rabbit Alexa Fluor 488 & Proteintech/SA00013-2/RRID:
AB\_2797132/goat/polyclonal secondary & 1:500\tabularnewline
anti-rabbit Alexa Fluor 594 & Abcam/Ab150080/RRID:
AB\_2650602/goat/polyclonal secondary & 1:400\tabularnewline
anti-mouse Alexa Fluor 488 & Proteintech/sms1AF488-1/RRID:
AB\_2827579/goat/polyclonal secondary & 1:500\tabularnewline
anti-mouse Alexa Fluor 594 & Proteintech/SA00013-3/RRID:
AB\_2797133/goat/polyclonal secondary & 1:500\tabularnewline
\bottomrule
\end{longtable}

\textbf{Statistical analysis.}

All data were analyzed using SPSS software (IBM, Chicago) and presented
as mean \selectlanguage{ngerman}± standard deviation (SD). Repeated-measure analysis of variance
(ANOVA) followed by the Bonferroni test was used to analyze the time
tendency of behavioral data. One-way ANOVA followed by the least
significance of difference (LSD) test was used to assess the differences
between each time points within groups and between each group within the
same time point. A two-tailed Student's \emph{t} -test was used to
determine statistical differences between the two groups at each time
point. Differences were considered statistically significant at
\emph{P}\textless{} 0.05.



\textbf{Results}

\textbf{CCI-ION induced orofacial mechanical allodynia and up-regulation
of Panx3 in the TG}

To confirm the successful establishment of the orofacial neuropathic
pain model using CCI-ION, mechanical allodynia in the ipsilateral
Whisker pad of mice was evaluated using the von Frey filament
experiment. The results revealed that mice in the CCI-ION group
exhibited significantly lower HWT from day 1 to at least day 14 after
surgery compared to the sham group, indicating the successful induction
of orofacial neuropathic pain through the CCI-ION model (Fig. 1B).
Administration of Panx3 siRNA via intra-ganglionic injection in the
CCI-ION mice resulted in a significant increase in HWT compared to the
CCI-ION group treated with negative control siRNA (Fig. 1C). On day 7
and 14 post-surgery, both RT-qPCR and Western blot analysis demonstrated
a significant upregulation of Panx3 mRNA and protein expression in the
TG of mice from the CCI-ION group compared to the sham group (Fig.
1D-E). Additionally, immunofluorescence staining showed co-localization
of Panx3 with NeuN (a specific neuronal marker) and GS (a specific
satellite glial cell marker) in the TG of CCI-ION mice (Fig. 1F-G),
suggesting the expression of Panx3 in both neuronal cells and satellite
glial cells in the TG. The evidence above supported the regulatory role
of Panx3 in orofacial neuropathic pain following trigeminal nerve
injury.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig. 1. Effects of CCI-ION on the orofacial mechanical allodynia and
Panx3 expression in the TG. (A) Procedure and timing for treatments and
tests in different groups. (B) CCI-ION decreased the HWT from day 1 to
day 14 (n = 6 per group; repeated-measure ANOVA followed by Bonferroni
test, ** \emph{P} \textless{} .01, *** \emph{P}\textless{} .001, vs. BL;
two-tailed Student's \emph{t} -test,\textsuperscript{\#\#} \emph{P}
\textless{} .01,\textsuperscript{\#\#\#} \emph{P} \textless{} .001 vs.
SHAM). (C) Transfection of Panx3 in the TG of CCI-ION mice increased the
HWT 1 day after injection (n = 6 per group; repeated-measure ANOVA
followed by Bonferroni test; *** \emph{P} \textless{} .001 vs. BL;
One-way ANOVA followed by the LSD test, \textsuperscript{\#\#\#}
\emph{P}\textless{} .001 vs. CCI-NC siRNA). (D) Quantification of Panx3
mRNA levels in the TG on days 7 and 14 (n = 4 per group; One-way ANOVA
followed by the LSD test; ** \emph{P} \textless{} .01 vs. SHAM). (E)
Representative WB band images and dot plot of WB-based quantification of
Panx3 protein expression in the TG (n = 5 per group; One-way ANOVA
followed by the LSD test; * \emph{P} \textless{} .05 vs. SHAM). (F-G)
Fluorescent immunohistochemical staining of Panx3 in the TG of mice.
Co-localization of Panx3 and NeuN (blue: DAPI, green: Panx3, red: NeuN)
indicated by arrows. Co-localization of Panx3 and GS (blue: DAPI, green:
GS, red: Panx3) indicated by empty arrows (n = 3; scale bars: 20 \selectlanguage{greek}μ\selectlanguage{english}m).

\textbf{The expression of Panx3 in the TG is regulated by Kir4.1}

In our previous study, we discovered that trigeminal nerve injury leads
to the down-regulation of Kir4.1 in the TG and that Kir4.1 knockdown in
the TG is associated with orofacial mechanical allodynia (Li et al.,
2023b). To further investigate the potential involvement of Kir4.1 in
the regulation of Panx3 and its impact on orofacial neuropathic pain, we
utilized male and female mice with conditional knockdown of Kir4.1
(\emph{Kcnj10} CKD) in the TG (Fig. 2A). The HWT in both male and female
mice with \emph{Kcnj10} CKD was significantly lower compared to control
mice (Fig. 2B), indicating the occurrence of orofacial mechanical
allodynia. Furthermore, we observed a decrease in the expression of
Kir4.1 mRNA and protein, as well as an increase in the expression of
Panx3 mRNA and protein, in both female and male mice in the\emph{Kcnj10}
CKD group compared to the control group (Fig. 2C-G). Additionally, there
were no significant differences in Kir4.1 mRNA and protein expression
between female and male mice (Fig. 2C-G). Therefore, it was proved that
the decreased expression of Kir4.1 in the TG is involved in orofacial
mechanical allodynia through the down-regulation of Panx3 .

To further observe the potential regulatory role of Kir4.1 in orofacial
mechanical allodynia through Panx3, we performed AAV-Kir4.1 transfection
in the TG of both sham and CCI-ION mice (Fig. 2H). In the CCI-ION mice,
we observed a significantly higher HWT in the AAV-Kir4.1 group compared
to the AAV-control group (Fig. 2I), indicating that Kir4.1
overexpression in the TG alleviated orofacial allodynia induced by
CCI-ION. However, the overexpression of Kir4.1 did not affect the HWT in
the sham group (Fig. 2I). Moreover, the expression of Kir4.1 mRNA and
protein was increased in both the sham and CCI-ION groups transfected
with AAV-Kir4.1 compared to the corresponding control groups (Fig. 2J,
L, M). Conversely, we observed a decrease in the expression of Panx3
mRNA and protein in the sham and CCI-ION groups transfected with
AAV-Kir4.1 compared to the control groups, indicating that the
overexpression of Kir4.1 inhibited the expression of Panx3 (Fig. 2K, L,
N). These findings suggested that Kir4.1 modulates orofacial neuropathic
pain by regulating the expression of Panx3.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig. 2. The effects of Kir4.1 knockdown and overexpression on the HWT
and expression of Panx3 in the TG of mice. (A) The timeline of the
treatments and tests in this experiment. (B) \emph{Kcnj10} CKD group in
male and female mice decreased the HWT compared to the control groups (n
= 6 per group; two-tailed Student's \emph{t} -test, ***
\emph{P}\textless{} .001, vs. Control). (C-D) Quantification of
Kir4.1and Panx3 mRNA levels in the TG (n = 4 per group; two-tailed
Student's\emph{t} -test, ** \emph{P} \textless{} .01, vs. Control). (E)
Representative WB band images showing Kir4.1 and Panx3 protein
expression in the TG. (F-G) Dot plot of WB-based quantification of
Kir4.1 and Panx3 protein expression in the TG (n = 4 per group;
two-tailed Student's \emph{t} -test; * \emph{P} \textless{} .05,
**\emph{P} \textless{} .01, vs. Control). (H) The timeline of the
treatments and tests in this experiment. (I) HWT was elevated in the
AAV-Kir4.1 group compared with the AAV-control group (n = 5 per group;
One-way ANOVA followed by the LSD test, *** \emph{P} \textless{} .001,
vs. AAV-control-SHAM, \textsuperscript{\#\#} \emph{P} \textless{} .01
vs. AAV-Kir4.1-CCI). (J-K) Quantification of Kir4.1 and Panx3 mRNA
levels in the TG (n = 4 per group; One-way ANOVA followed by the LSD
test; ** \emph{P} \textless{} .01 vs.
AAV-control-SHAM,\textsuperscript{\#\#\#} \emph{P} \textless{} .001 vs.
AAV-control-CCI). (L) Representative WB band images showing Kir4.1 and
Panx3 protein expression in the TG of the AAV-control-SHAM group and the
AAV-Kir4.1 group. (M-N) Dot plot of WB-based quantification of Kir4.1
and Panx3 protein expression in the TG (Kir4.1, n = 6 per group; Panx3,
n = 4 per group; One-way ANOVA followed by the LSD test, *
\emph{P}\textless{} .05, ** \emph{P} \textless{} .01 vs.
AAV-control-SHAM,\textsuperscript{\#} \emph{P} \textless{} .05 vs.
AAV-control-CCI).

\textbf{Inhibition of P38 MPAK reversed the up-regulation of Panx3 in
the SGCs caused by Kir4.1 silence}

Considering the findings from previous studies, we aimed to investigate
the involvement of the MAPK signaling pathway in elucidating the
mechanisms by which Kir4.1 modulates Panx3 expression in cultured
satellite glial cells (SGCs) in vitro. RT-qPCR analysis confirmed a
decrease in Kir4.1 mRNA levels in SGCs transfected with Kir4.1 siRNA
compared to the negative control siRNA (vehicle) group (Fig. 3A).
Notably, there were no significant changes in Kir4.1 mRNA levels between
the Kir4.1 siRNA group and the groups treated with inhibitors of P38
MAPKs, ERK1/2, or JNKs, suggesting that the inhibition of the MAPK
signaling pathway did not affect Kir4.1 expression (Fig. 3A). We also
observed an upregulation of Panx3 mRNA in SGCs transfected with Kir4.1
siRNA compared to the vehicle group (Fig. 3B). Furthermore, the
Kir4.1-silenced SGCs treated with SB203580 (a P38 MAPK blocker) showed a
decreased expression of Panx3 mRNA compared to the Kir4.1 siRNA group.
However, there were no significant changes in the expression of Panx3
mRNA in the Kir4.1-silenced SGCs treated with U0126 (an ERK1/2 blocker)
or SP600125 (a JNK blocker) compared to the Kir4.1 siRNA group (Fig.
3B). The aforementioned findings suggest that the regulation of Kir4.1
on Panx3 expression involves the P38 MAPK pathway, while the ERK1/2 and
JNK pathways are not involved. Consistent with the RT-qPCR results, the
Western blot analysis also revealed a significantly lower level of
Kir4.1 protein and a higher level of Panx3 protein in SGCs transfected
with Kir4.1 siRNA compared to the control group (Fig. 3C-E). Treatment
of Kir4.1 silenced SGCs with SB203580 resulted in a decrease in Panx3
levels compared to the Kir4.1 group (Fig. 3E). Interestingly, there was
a notable increase in phosphorated P38 MAPK levels in the Kir4.1
silenced SGCs compared to the control group (Fig. 3F). Furthermore,
treatment of Kir4.1 silenced SGCs with SB203580 led to a reduction in
phosphorated P38 MAPK levels compared to the Kir4.1 siRNA group (Fig.
3F). However, there were no significant changes observed in phosphorated
ERK1/2 or JNK levels between the control and Kir4.1 siRNA groups (Fig.
G-J). Overall, the results above indicated that the downregulation of
Kir4.1 promotes Panx3 expression by activating the P38 MAPK pathway,
while the ERK1/2 and JNK pathways remain unaffected.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Fig. 3. Panx3 was affected by Kir4.1 through the P38 MAPK pathway. (A-B)
Quantification of Kir4.1 and Panx3 mRNA levels in the SGCs. (n = 4 per
group; One-way ANOVA followed by the LSD test; ***
\emph{P}\textless{} .001, ** \emph{P} \textless{} .01 * \emph{P}
\textless{} .05 vs Vehicle, \textsuperscript{\#\#} \emph{P}
\textless{} .01 vs Kir4.1 siRNA). (C) Representative WB band images
showing Kir4.1, Panx3, P38, and p-P38 protein expression in the SGCs of
different groups. The Kir4.1 siRNA group increased the phosphorylation
level of the P38 pathway and the expression of Panx3 compared to the
Vehicle. The level of P38 pathway phosphorylation decreased after using
SB203580, and the protein expression of Panx3 returned to normal levels.
(D-F) Dot plot of WB-based quantification of Kir4.1, Panx3 and p-P38/P38
protein expression in the SGCs (Kir4.1, Panx3: n = 5 per group;
p-P38/P38: n =4 per group; One-way ANOVA followed by the LSD test, *
\emph{P}\textless{} .05, ** \emph{P} \textless{} .01 vs.
Vehicle,\textsuperscript{\#} \emph{P} \textless{} .05,
\textsuperscript{\#\#}\emph{P} \textless{} .01 vs Kir4.1 siRNA
+SB2003580 group). (G) Representative WB band images showing ERK1/2,
p-ERK1/2, JNK, and p-JNK protein expression in the SGCs of different
groups. (H-I) Dot plot of WB-based quantification of p-ERK1/2/ERK1/2 and
p-JNK/JNK protein expression in the SGCs (n = 4 per group; two-tailed
Student's\emph{t} -test).

\textbf{Inhibition of superoxide dismutase inhibits P38 MAPK
phosphorylation induced by Kir4.1 silence}

Based on a previous study indicating the crucial role of ROS in the
elevated phosphorylation levels of P38 MAPK (Son et al., 2011), we
investigated the involvement of ROS in the regulation of Kir4.1 on the
activation of the P38 MAPK pathway. Initially, the immunofluorescence
test for superoxide revealed an increased immunofluorescence intensity
of ROS in SGCs transfected with Kir4.1 siRNA (Fig. 4A). Importantly, the
immunofluorescence intensity of ROS in Kir4.1 siRNA SGCs treated with
tempol showed a significant decrease compared to the Kir4.1 siRNA group
(Fig. 4A). Similarly, the ROS assay demonstrated a substantially higher
fluorescence intensity of ROS in the Kir4.1 siRNA group compared to the
control group. Conversely, the application of tempol resulted in a
significant reduction in fluorescence intensity (Fig. 4B). The Western
blot results showed a significant decrease in the level of
phosphorylated P38 MAPK in Kir4.1 silenced SGCs treated with tempol (a
superoxide scavenger) compared to the Kir4.1 siRNA group (Fig. 4C and
D). These findings implied that the downregulation of Kir4.1 promotes
the phosphorylation of P38 MAPK pathway by elevating the level of ROS.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Fig. 4. Kir4.1 silence increased the phosphorylation of P38 MAPK by
elevating the intracellular ROS concentration. (A) Fluorescence
photographs of ROS in the SGCs treated with or without siRNA and tempol.
(B) ROS measurement represented by fluorescence intensity after 24h
transfection of SGCs treated as indicated (n = 6 per group; One-way
ANOVA followed by the LSD test; *** \emph{P} \textless{} .001 vs.
Vehicle group, \textsuperscript{\#\#\#} \emph{P} \textless{} .001 vs.
Kir4.1 siRNA + tempol group. (C) Representative WB band images showing
P38 MAPK and p-P38 MAPK protein expression in the SGCs of different
groups. SGCs transfected by Kir4.1 siRNA increased the phosphorylation
level of the P38 MAPK pathway, and the effect can be blocked by using
tempol. (D) Dot plot of WB-based quantification of p-P38/P38 protein
expression in the SGCs (n =4 per group; One-way ANOVA followed by the
LSD test, ** \emph{P} \textless{} .01 vs. Vehicle
group,\textsuperscript{\#\#} \emph{P} \textless{} .01 vs. Kir4.1 siRNA +
tempol group).

\textbf{Inhibition of Panx3 in the TG relieves orofacial mechanical
allodynia induced by Kir4.1 CKD}

In order to further investigate the contribution of Panx3 in the
development of orofacial mechanical allodynia induced by Kir4.1 CKD, we
conducted Panx3 siRNA transfection in mice with Kir4.1 CKD (Fig. 5A).
The results from both RT-qPCR and Western blot analysis revealed a
significant inhibition of Kir4.1 expression in the trigeminal ganglion
(TG) of mice with Kir4.1 CKD. Furthermore, transfection of Panx3 siRNA
resulted in a significant inhibition of Panx3 expression, without
affecting the expression of Kir4.1 mRNA and protein levels (Fig. 5B-F).
Importantly, the von Frey filament assay demonstrated a notable increase
in the withdrawal threshold to mechanical stimuli (HWT) one day after in
vivo transfection of Panx3 siRNA within the TG (Fig. 5G). These findings
provided novel evidence that the decrease in Kir4.1 expression in the TG
contributes to orofacial mechanical allodynia through the upregulation
of Panx3.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Fig. 5. Inhibition of Panx3 in the TG attenuated CKD Kir4.1-evoked
mechanical allodynia. (A) The timeline of the treatments and tests in
this experiment. (B) Quantification of Kir4.1 and Panx3 mRNA levels in
the TG on day 1 after Panx3 siRNA transfection in Kir4.1 CKD mice (n = 4
per group; One-way ANOVA followed by the LSD test; *
\emph{P}\textless{} .05, *** \emph{P} \textless{} .001 vs.
Control;\textsuperscript{\#} \emph{P} \textless{} .05 vs. \emph{Kcnj10}
CKD + Panx3 siRNA). (D) Representative Western blot (WB) band images
showing Kir4.1 and Panx3 protein expression in the TG on 1 day after
Panx3 siRNA transfection in Kir4.1 knockdown mice. (E) Dot plot of
WB-based quantification of Kir4.1 and Panx3 protein expression in the TG
(Kir4.1: n = 4 per group; Panx3: n = 6 per group; two-tailed
Student's\emph{t} -test; * \emph{P} \textless{} .05 vs. \emph{Kcnj10}
CKD). (G) Transfection of Panx3 in TG of kir4.1 knockout mice increased
the HWT 1 day after injection (n = 6 per group; repeated-measure ANOVA
followed by Bonferroni test; *** \emph{P} \textless{} .001 vs.
BL-\emph{Kcnj10}CKD+Panx3 siRNA, \textsuperscript{\#\#\#} \emph{P}
\textless{} .001 vs.\emph{Kcnj10} CKD).

\textbf{Discussion}

In recent years, there has been a growing interest in the role of Kir4.1
in diseases such as anxiety-like behavior, diabetic retinopathy,
multiple sclerosis, and dystrophin (Curry \emph{et al.} , 2023; Fang
\emph{et al.} , 2023; Fujimoto \emph{et al.} , 2023; Kapell \emph{et
al.} , 2023; Zheng \emph{et al.} , 2023). There is also a lot of
research indicating that Kir4.1 plays a role in pain development (Vit
\emph{et al.} , 2008; Takeda \emph{et al.} , 2011; Hanani \& Spray,
2020). And our previous work has shown that Kir4.1 contributes to the
development of orofacial neuropathic pain induced by inferior alveolar
nerve injury (Li \emph{et al.} , 2023b). But the underlying mechanism by
which Kir4.1 regulates orofacial neuropathic pain remains unclear. This
study was the first to focus on the effects and mechanisms of Kir4.1 on
Panx3 expression, which contribute to the development of orofacial
neuropathic pain induced by trigeminal nerve injury. The results of the
present study showed that (1) increased Panx3 within the TG was firstly
identified as a mediator to promote orofacial neuropathic pain after
trigeminal nerve injury; (2) Decreased Kir4.1 caused orofacial
neuropathic pain by modulating the Panx3 expression after trigeminal
nerve injury, while overexpression of Kir4.1 alleviated orofacial
neuropathic pain by inhibiting the upregulation of Panx3 induced by
trigeminal nerve injury; (3) Decreased Kir4.1 promoted Panx3 expression
by activating P38 MAPK pathway through increasing ROS. The above
findings demonstrate that Kir4.1 contributes to orofacial neuropathic
pain induced by trigeminal nerve injury through the ROS-P38 MAPK-Panx3
signaling pathway.

Kir4.1 are preferentially expressed by glial cells, including SGCs and
astrocytes, where they regulate membrane potential and serve as channels
for K\textsuperscript{+} uptake from the extracellular space, and play
an essential role in the development of neuropathic pain (Hanani \&
Spray, 2020; Ou \emph{et al.} , 2023). Our previous study has shown that
trigeminal nerve injury causes a reduction of Kir4.1 in the TG of mice
(Li \emph{et al.} , 2023b). Besides, silencing the expression of Kir4.1
in the SGCs of the rat TG results in pain-like behavior in the absence
of nerve injury (Vit \emph{et al.} , 2008). Similarly, the results of
the present study also demonstrated that Kir4.1 CKD in the TG of male
and female mice induced orofacial mechanical allodynia. Moreover,
overexpression of Kir4.1 in the TG alleviated the orofacial neuropathic
pain induced by CCI-ION. These findings support the conclusion that
trigeminal nerve injury-induced reduction of Kir4.1 in the TG promotes
orofacial neuropathic pain after trigeminal nerve injury.

It is well-known that Kir4.1 serves as a downstream target of some pain
modulators, such as metabotropic glutamate receptor 5 (mGluR5),
N-methyl-d-aspartate receptor (NMDAR), and methyl-CpG-binding protein 2
(MeCP2) to play a critical role in neuropathic pain by affecting
K\textsuperscript{+} homeostasis (Vit\emph{et al.} , 2008; Zhang
\emph{et al.} , 2022b; Li \emph{et al.} , 2023b; Ou \emph{et al.} ,
2023). However, the downstream mechanisms of the change in the
intracellular K\textsuperscript{+} homeostasis in the glial cells
regulated by Kir4.1 under neuropathic pain remain unclear. In the
present study, Panx3, a subtype channel protein of the pannexin family,
was first proved to be a novel downstream target regulated by Kir4.1 in
the TG, which contributed to the development of orofacial neuropathic
pain induced by trigeminal nerve injury. Pannexins represent a family of
membrane conduits for releasing ATP into the extracellular space and
consist of three members, Panx1, Panx2, and Panx3 (Bao \emph{et al.} ,
2004; Penuela \emph{et al.} , 2014; Li \emph{et al.} , 2015), which are
involved in many pathologies such as epilepsy, ischemic stroke, cancer,
and Parkinson's disease, as well as in neuropathic pain (Penuela
\emph{et al.} , 2014; Laird \& Penuela, 2021; Crocetti \emph{et al.} ,
2023). The findings of this study indicated that silencing the
expression of Panx3 in the TG alleviated the orofacial mechanical
allodynia induced by CCI-ION. Moreover, CCI-ION caused a significant
increase in the expression of Panx3 in the TG, which was located both in
the SG Cs and neurons. Interestingly, Kir4.1 CKD also significantly
promoted the expression of Panx3 in the TG of male and female mice with
orofacial mechanical allodynia. Furthermore, overexpression of Kir4.1 in
the TG inhibited the upregulation of Panx3 caused by CCI-ION. The in
vitro study showed that the expression of Panx3 was increased in the
SGCs transfected with Kir4.1 siRNA. Therefore, these findings suggest
that Kir4.1 in the SGCs mediates orofacial neuropathic pain by
regulating the expression of Panx3.

Next, the underlying mechanism by which Kir4.1 regulates Panx3 was
further investigated in the cultured SGCs in vitro. A previous study has
reported that the pannexin family can be regulated by ROS (Xu \emph{et
al.} , 2018). Moreover, ROS is also one of the key factors in regulating
pain (Kim \emph{et al.} , 2010). In the meantime, the ROS can promote
the activation of the MAPK signal pathway in endothelial cells, renal
cortex, and outer medulla (Babilonia \emph{et al.} , 2006; Milovanova
\emph{et al.} , 2008; Son \emph{et al.} , 2011). Furthermore, ROS
generation is prevented by increased intracellular K\textsuperscript{+}
concentration in the auditory cells (Kim \& Park, 2013). Notably,
activation of the K\textsubscript{ATP}~channels, a subtype of Kir
channels, can regulate ROS production (Cao \emph{et al.} , 2007; Fellows
\emph{et al.} , 2023). Therefore, we further investigated whether the
ROS-MAPK signal pathway was involved in the effect of Kir4.1 on the
expression of Panx3 in the SGCs.

MAPKs are highly conserved and ubiquitous serine-threonine protein
kinases with three conventional types: P38 MAPKs, JNKs, and ERKs
(Widmann \emph{et al.} , 1999; Pearson \emph{et al.} , 2001). This study
showed that the phosphorylation level of P38 MAPK was significantly
increased in the SGCs with Kir4.1 silence, while the phosphorated JNKs
and ERK1/2 levels were not affected by silencing Kir4.1. In addition,
the increase in the expression of Panx3 was reversed in the Kir4.1
silent SGCs treated with P38 MAPK blocker. However, the blockers of JNKs
and ERK1/2 did not affect the expression of Panx3 in the Kir4.1 silent
SGCs. These findings suggest that Kir4.1 regulates Panx3 through the P38
MAPK pathway. However, the underlying mechanism by which Kir4.1
activates the P38 MAPK pathway is unclear.

The ROS~plays a vital role in various diseases, including neuropathic
pain (Pieczenik \& Neustadt, 2007).~The superoxide anion
O\textsuperscript{2-} as one of the ROS (Afonso \emph{et al.} , 2007),
is critically important in developing pain (Salvemini \emph{et al.} ,
2011). Moreover, superoxide is reported to activate the P38 MAPK pathway
(Babilonia \emph{et al.} , 2006). Therefore, we envisioned whether
superoxide plays a role in activating the P38 MAPK signal pathway
regulated by Kir4.1. The results of the present study demonstrated that
the level of superoxide in the Kir4.1 silent SGCs was increased compared
with the vehicle group. Meanwhile, the application of superoxide
scavenger tempol inhibited the upregulated-phosphorylation of P38 MAPK
caused by Kir4.1 siRNA. These results display that the downregulation of
Kir4.1 induces activation of the P38 MAPK signal pathway through
superoxide in the SGCs.

Increasing evidence has demonstrated that extracellular ATP is crucial
for the cross-talk between SGCs and neurons through activating P2X and
P2Y receptors expressed on the SGCs and neurons, further promoting the
development of pain in the peripheral nervous system (Bele \& Fabbretti,
2015; Zhang \& Li, 2019; Hanani \& Spray, 2020; Yang \emph{et al.} ,
2023). Pannexins are a family of glycoproteins releasing ATP to the
extracellular space (Harcha \emph{et al.} , 2021; Rusiecka \emph{et al.}
, 2022). For example, Panx3 activates the purinergic receptor
P2X\textsubscript{7} by releasing ATP (Xiao \emph{et al.} , 2023). In
the present study, Panx3 was increased in the TG after trigeminal nerve
injury. Therefore, the increasing Panx3 induced by the reduction of
Kir4.1 in the SGCs further results in the increase of extracellular ATP
between SGCs and neurons, which might activate P2X and P2Y receptors
expressed on the SGCs and neurons to promote the orofacial neuropathic
pain. Further study is needed to prove this deduction.

However, there was a limitation to this study. The immunofluorescence
staining showed that Panx3 was expressed not only in the SGCs but also
in neurons. Therefore, the role of Panx3 expressed in the neurons in the
effect of intra-ganglion injection of Panx3 siRNA on the orofacial
neuropathic pain induced by CCI-ION can not be excluded. The function of
Panx3 in neurons under the condition of trigeminal nerve injury needs to
be further explored.

In conclusion, this study first reported that Panx3 was increased in the
TG after trigeminal nerve injury. And inhibition of Panx3 alleviated
orofacial neuropathic pain induced by CCI-ION. Moreover, Kir4.1
contributed to the development of orofacial mechanical allodynia by
regulating Panx3 expression in the SGCs in the TG. Furthermore,
silencing Kir4.1 activated the P38 MPAK signal pathway by affecting the
expression of superoxide. These results demonstrate that the reduction
of Kir4.1 induced by trigeminal nerve injury promotes orofacial
neuropathic pain by regulating the expression of Panx3 by activating the
ROS-P38 MPAK signal pathway. Our study brings a novel insight into the
underlying mechanism of orofacial neuropathic pain and indicates that
Panx3 is a potential therapeutic target of neuropathic pain.

\textbf{Acknowledgments}

This study was funded by the National Natural Science Foundation of
China (Grant No. 81870800, 82071149, 82271014), the Department of
Science and Technology of Sichuan Province (Grant No. 2021JDRC0166,
2020YJ0224), and China Postdoctoral Science Foundation (Grant Number:
2020M683329). We thank Ms. Xin Guan and Mr. Fan-Zi Wu for their
technical assistance.

\textbf{Author contributions}

Yu-Heng Feng: Conceptualization; data curation; formal analysis;
investigation; visualization; methodology; writing---original draft.
Ren-Jie Tang: Data curation. Yan-Yan Zhang: Supervision; methodology;
writing---review and editing. Jiu Lin: Conceptualization; data curation.
Ya-Jing Liu: Data curation. Yi-Ke Li: Methodology. Chun-Jie Li:
Supervision. Cheng Zhou: Methodology. Fei Liu: Conceptualization; data
curation; formal analysis; investigation; methodology; supervision;
validation; visualization; writing---review and editing. Jie-Fei Shen:
Conceptualization; data curation; formal analysis; funding acquisition;
investigation; methodology; project administration; resources;
supervision; validation; visualization; writing---review and editing.

\textbf{Conflict of interest}

The authors declare no conflicts of interest.

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