Animals

The animal study was approved by the Ethics Committee of West China Hospital of Stomatology Sichuan University (Number: WCHSIRB-D-2022-021) and complies with the ARRIVE 2.0 guidelines. A total of 273 wild-type male C57BL/6 mice and 89 female C57BL/6 mice (6-8 weeks, 20-25g) were used for this study. Every single animal was defined as an experimental unit. All animals were kept in a specific pathogen-free experimental animal center (West China Hospital Experimental Animal Center) on a 12:12 light-dark cycle at 22 ± 1 °C. Food and water were available at liberty. In all assays, the animals were randomly allocated to the experimental groups using a random number table. The experimenters were blinded for the treatment groups during the analysis of the data in each assay.

Cell culture and transfection

Bilateral TG was dissociated from connective tissue and minced. Then the tissue was enzymatically digested with 8 U/ml papain and incubated at 37°C for 20 minutes. An equal volume of complete medium was added to stop digestion and gently blow the digested tissue fluid several times with a sterile Pasteur pipette to mechanically dissociate neurons. Cell precipitation was obtained by centrifugation and resuspended in the neurobasal medium (Thermo Fischer, USA) containing 2% B27 (Invitrogen, USA) supplements and 1% GlutaMAX (Invitrogen, USA), and then plated in a twelve-well plate coated by poly-D-lysine (Invitrogen, USA) in 37℃ in humidified incubators with 5% CO2 and 95% air. The LV-Anxa10-203 (MOI = 5) and DHX30 siRNA or negative control siRNA (10 nM) were transfected into TG neurons (TGNs) on the second day. The Anxa10-203 oe+siDHX30 was the experimental group, and the Anxa10-203 oe+siNC (negative control siRNA) served as the control group. The LV-Anxa10-203 was purchased from Sangon (Shanghai, China). The siRNA and siNC were synthesized by GenePharma (Shanghai, China) and were transfected with HieffTrans in vitro siRNA/miRNA Transfection Reagent (Yeasen, China) following the manufacturer’s protocol. The sequences of siRNA are listed in Table 1.

Table 1 The sequences of siRNA/shRNA in this studyChronic constriction injury of the infraorbital nerve (CCI-ION) surgery

Mice were anesthetized with isoflurane. For the CCI-ION group, two chronic gut ligatures (5-0) were placed loosely around the infraorbital nerve (ION) [26]. For the Sham group, the same surgical procedures were operated without ligation of the ION. The incision was sutured with 5-0 silk. The mice were placed on a heat recovery pad until they awoke from the anesthesia. Animals were monitored at least once daily after surgery.

TG microinjection

The TG microinjection of mice was performed according to our previous study [27]. Briefly, after anesthesia, the siRNA Anxa10-203 (0.5 ug/μl), LV-Anxa10-203 (MOI = 5) or AAV2/9-CMV-GFP-shDHX30 (2E+9 vg/μl) were injected into the TG (coordinates: AP + 1 mm, ML + 4 mm, and DV -6.5 mm relative to bregma) under the guidance of a stereotaxic apparatus, and the maximum injection volume was 2 μl. A negative control siRNA or AAV2/9-CMV-GFP-shNC was used as control. The AAVs were synthesized by Xuanzun Bioscience (Chongqing, China). The siRNA was synthesized by GenePharma (Shanghai, China) and was transfected with EntransterTM-in vivo (Engreen Biosystem, China) following the manufacturer’s protocol.

Behavioral tests

The Von Frey test was used to measure the 50% head withdrawal threshold (HWT) in response to mechanical stimuli according to our previous studies [27]. In brief, a series of Von Frey filaments were applied to the whisker pad area providing a force ranging from 0.04 g to 4 g. A positive response was recognized as sharp head withdrawal. The test was performed blinded.

Reverse transcription and quantitative PCR

RNAiso regent (Takara, Japan) was used to obtain total RNA. cDNA was synthesized using PrimeScript™ RT reagent Kit (Takara, Japan). The RT-qPCR was performed by QuantStudio 7 Flex System (Applied Biosystems, USA). The primer sequences used are listed in Table 2. The relative expression of the target gene was normalized to GAPDH and calculated with the 2-ΔΔCT method.

Table 2 The primer sequences used in this studySubcellular cell fraction

The cytoplasmic and nuclear fractions of the TG were collected using a Nuclear and Cytoplasmic Extraction Reagents Kit (Beyotime, China) following the manufacturer’s protocol. RNA from cytoplasmic and nuclear fractions was extracted with RNAiso regent and analyzed by RT-qPCR. U6 and Gapdh were defined as nuclear and cytoplasmic control respectively.

Immunofluorescence (IF) staining

The frozen sections were washed and permeabilized. The slices were blocked with 10% goat serum and then incubated with primary antibody overnight at 4℃. After washing with PBS, the sections were then incubated with a secondary antibody for 1 h. The images were captured with a laser scanning confocal microscope (LSCM, Olympus FV3000, Japan). The antibodies used are listed in Table 3.

Table 3 The antibodies used in this studyRNA fluorescent in situ hybridization (RNA-FISH)

The fluorescence-conjugated probe for Anxa10-203 and Mc1r RNA was synthesized from GenePharma (Shanghai, China). After permeabilizing with 0.5% Triton, the tissue was incubated in a prehybridization solution then followed by a hybridization solution containing a 20 uM probe overnight at 37℃. the slides were finally washed with SSC solution. A LSCM (Olympus FV3000, Japan) was used to acquire the images.

Western blot (WB) analysis

The samples were homogenized in a lysis buffer with protease inhibitors. The proteins were separated on 10% SDS/PAGE gel and then transferred to PVDF membranes. The membranes were blocked by 10% non-fat milk and incubated with the primary antibodies at 4℃ overnight, and further incubated with a secondary antibody. The antibodies used are listed in Table 3. Quantification of immunoblots was measured by ImageJ. The relative expression level of the target protein in all groups were normalized to α-Tubulin.

RNA pull-down

The RNAs of the Anxa10-203, Mc1r, and four Anxa10-203 sequence segments were in vitro transcribed (Vazyme, China) and biotinylated (Thermo Scientific, USA). The Pierce magnetic RNA-protein pull-down kit (Thermo Scientific, USA) was used. Briefly, the biotinylated RNA was bonded to streptavidin magnetic beads (Thermo Scientific, USA). The TG lysates from CCI-ION mice were added into RNA-conjugated beads and incubated overnight at 4℃ with agitation. The RNA-interacting proteins were eluted and then analyzed by liquid chromatography-mass spectrometry (LCMS) and/or Western Blot. The input of the antisense RNA was defined as positive control and negative control separately.

RNA immunoprecipitation (RIP)

Magna RIP Kit (Millipore, USA) was used to conduct the RNA-binding protein immunoprecipitation assay according to the manufacturer’s instructions. Briefly, the TG of CCI-ION mice was lysed by RIP lysis buffer. The magnetic beads conjugated with anti-DHX30 antibody were incubated with the tissue lysates on a rotator at 4℃ overnight. The magnetic beads conjugated with anti-IgG served as a negative control. The RNA was eluted from the beads, and the expression of Anxa10-203 and Mc1r was analyzed by RT-qPCR.

Nascent protein synthesis assay

Click-iT Plus OPP Protein Synthesis Assay Kit (Invitrogen, USA) was applied. The Anxa10-203 oe+siDHX30 was the experimental group, and the Anxa10-203 oe+siNC served as the control group. The OPP working solution was added to the culture medium and incubated for 30 minutes. Then the cells were fixed and permeabilized. After washing, the OPP reaction cocktail was added and incubated for 30 minutes at room temperature. Images were acquired by LSCM (Olympus FV3000, Japan) and the relative fluorescence unit was assessed by a microplate reader (SpectraMax iD3, Molecular Devices, USA).

Whole-cell patch-clamp recording

The electrophysiology was recorded by an Axopatch-700B amplifier and a Digidata 1440 digitizer (Molecular Devices, USA). The MC1R agonist BMS-470539 (BMS, 100 nM, MedChemExpress, China) was used, and the extracellular solution without BMS was defined as control [28]. The extracellular solution consisted of (in mM) NaCl 150, KCI 5, CaCl2 2.5, MgCl2 2, HEPES 10, and D-glucose 10 (pH = 7.4 by NaOH, 330 mOsm/L). The intracellular pipette solution contained (in mM) KCl 140, CaCl2 1, MgCl2 2.5, HEPES 10 and D-glucose 10, EGTA 11, Mg-ATP 5 (pH = 7.2 by NaOH, 310 mOsm/L). The resting membrane potential (RMP) was recorded for 3 min. The currents from -120 pA to 170 pA with an increment of 10 pA were injected into TGNs to evoke action potential (AP) [29]. The rheobase current was defined as the minimum current required to evoke the first AP. The AP threshold was obtained at dV/dt= 10 mV/ms. The action potential-related parameters including AP latency, the AP amplitude, AP half-width, afterhyperpolarization (AHP) time, and AHP amplitude are measured.

Statistical analysis

Data were expressed as mean ± standard deviation. A two-tailed student’s t-test was conducted to compare the differences between the two groups. A repeated-measures ANOVA was used to detect the effect on HWT from both treatment effect and time effect, followed by one-way ANOVA for the difference between groups at each time point. Two-way ANOVA was applied to detect the expression level of genes from both treatment and time effect, followed by one-way ANOVA for differences between groups at each time point. And one-way ANOVA with the least significant difference (LSD) test was applied to analyze the differences among multiple groups. Statistical analyses were performed using GraphPad Prism software (v8.0.1, United States). P < 0.05 was considered as a statistical significance.