Animals

All procedures were approved by the Animal Ethics Committee of West China Hospital, Sichuan University, China (Protocol No.20230310052), and the protocols were performed under strict adherence to the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. Animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals published by the NIH. Wild-type C57BL/6J mice were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were housed in a conventional facility at 22 ± 2 °C under a 12 h light-dark cycle with free access to food and water.

Male C57BL/6J mice (~ 4 weeks of age, weighed 13–15 g) were randomly fed a high-fat diet (HFD) composed of 60% kcal per kg fat (D12492, Research Diets Inc, New Brunswick, NJ, USA), or a chow diet (CD) composed of 11.85% kcal per kg fat (SWC9101, Xietong Ltd., Nanjing, China) for 8 weeks. Body weight and nonfasting blood glucose levels were recorded weekly. PP2Cmfl/fl mice on a C57BL/6J background were purchased from Cyagen Biosciences Inc. (Guangzhou, China) and bred to produce littermates. Adult PP2Cmfl/fl mice (~ 8 weeks of age, weighed 20–25 g) of both sexes were intrathecally injected with 5 µL of pAAV9-hSyn-Cre (3 × 1013 VG/mL) to generate PP2Cm-cKO mice. Control mice (PP2Cm-ctrl) received the same volume of pAAV9-hSyn vector (Vigenebio, Jinan, China). After 3 weeks, the efficiency of PP2Cm deletion in DRG was validated using Western blotting analysis. The mice were randomly assigned to different groups. The experimenters were blinded to the group assignments and treatment conditions. At the end of the experimental protocol, the mice were anesthetized with 2% isoflurane and sacrificed to collect the bilateral lumbar 4–5 (L4-L5) DRGs.

Behavioral testing

On each testing day, the assay order was randomized, and experiments were carried out by blinded observers. Prior to testing, the mice were acclimated to the environment. Mechanical, thermal, and cold sensitivity tests were conducted at 30- to 60-minute intervals, allowing the mice to return to their home cages with access to food and water.

In the von Frey test [44], the mice were placed in an individual Plexiglas chamber on an elevated mesh grid floor. After 1-hour acclimation period, the mechanical paw withdrawal thresholds were assessed using von Frey filaments with an ascending order (mice: 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, and 1.4 g). Each filament was applied 5 times to the mid-plantar surface of hind paws for 5 s with a 30 s interval, starting with 0.008 g and ending with 1.4 g force. Abrupt paw raising, retracting, or licking was considered as a positive response. The absolute withdrawal thresholds were determined by the up-down method [27].

In the Hargreaves test [44], paw withdrawal latencies in response to thermal stimulation were examined with a radiant heat source (Model 37370; Ugo-Basile). In a quiet room, the mice were placed in a transparent chamber on a glass plate. After the mice adapted to the environment, the plantar surface of hind paws was stimulated with a radiant heat beam (IR = 35 mW/cm2). When the hind paws were moved, the light source automatically turned off, and the withdrawal latencies were recorded. Measurements were repeated 3 times with a 10-min interval. The average latencies were calculated. To avoid damage to the hind paws, a cutoff time of 20 s was used.

In the cold plate test [2], the mice were placed on a cold plate apparatus (Bioseb) set at 4 ˚C. Paw withdrawal latencies were defined as the time at which a positive response occurred (e.g., jumping, shaking, or licking of the feet). Measurements were repeated 3 times within a 10-min interval. A cutoff time of 50 s was applied to avoid potential tissue damage. Mice showing no response within 50 s were removed from the apparatus, and the time was recorded as 50 s.

Drugs and intrathecal injection

Compound BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid, sc-276559, Santa Cruz Biotechnology), an inhibitor of BCKDK, was diluted in the vehicle (5% DMSO, 10% cremophor EL, and 85% 0.1 M sodium bicarbonate, pH 9.0) and administered via oral gavage at a dose of 40 mg/kg body weight per day. A mouse neutralizing antibody for CCL5 (anti-CCL5, AF478-SP, R&D Systems) was reconstituted in saline and intrathecally injected at a dose of 2.5 µg/mouse in a volume of 5 µL, for 2 consecutive days [19]. The CCR5 specific antagonist maraviroc (M1971, AbMole) was dissolved in sterile DMSO and diluted with saline for single intrathecal (i.t.) injection at a dosage of 40 µg/mouse in 5 µL volume [12]. The TRPA1-selective antagonist Chembridge-5861528 (Chem-5861528, M7387, AbMole) was dissolved in sterile DMSO and diluted with saline for 20 µg/5 µL/mouse i.t. injection [34, 35]. Normal IgG and saline were used as controls. The Ccr5-directed small interfering RNA (siCcr5, sense: 5’-CAGUAGUUCUAAUAGACUATT-3’; antisense: 5’-UAGUCUAUUAGAACUACUGTT-3’), Trpa1-directed small interfering RNA (siTrpa1, sense: 5’-GCUAAGCUGUGUAAAUCAATT-3’; antisense: 5’-UUGAUUUACACAGCUUAGCTT-3’), and control small interfering RNA (siCtrl, sense: 5’-UUCUCCGAACGUGUCACGUdTdT-3’; antisense: 5’-ACGUGACACGUUCGGAGAAdTdT-3’) (Vigenebio, Jinan, China) were dissolved in RNase-free water and administered intrathecally at 2 µg/µL in a volume of 5 µL.

Measurement of branched-chain amino acid concentrations

Fresh bilateral L4-L5 DRG tissues from CD and HFD mice, PP2Cm-cKO and PP2Cm-ctrl mice, PP2Cm-cKO + Vehicle and PP2Cm-cKO + BT2 mice, were collected for subsequent BCAA concentration analysis. DRG tissue samples were homogenized in sterile water and mixed with equal volumes of acetonitrile. After centrifugation (at 15000 × g, 4 °C, for 10 min), the supernatant was collected and diluted for the detection of BCAA concentrations by liquid chromatography-tandem mass spectrometry (LC-MS, 1260–6460, Agilent).

Chromatographic separation was performed using a InfinityLab Poroshell HPH-C18 column (4.6 × 100 mm, 2.7 μm, Part no. 695975-702, Agilent), and involved isocratic condition with a flow rate of 0.3 mL/min and an injection volume of 1 µL. The column temperature was maintained at 30 °C. Mobile phase composition was 0.1% formic acid aqueous solution and acetonitrile (v/v = 95:5). Mass spectrometric acquisition was performed in the positive mode of electrospray ionization (ESI), with source temperature of 350 °C and capillary voltage of 3500 V. Multiple reaction monitoring (MRM) was employed as the scan mode. Standard chemicals, L-leucine, L-isoleucine, and L-valine (Sigma-Aldrich) were used to quantify their concentrations. A series of calibration standards were analyzed to generate concentration-response curves over a dynamic range. Three models were established using linear regression to back-calculate the concentrations of leucine, isoleucine, and valine in samples, respectively. The parameters of fragmentor, mass-to-charge ratio (m/z), and collision energy, as well as representative LC-MS traces in blank, standard, and sample, have been provided in Supplementary Fig S1.

RNA sequencing

Bilateral L4-L5 DRG tissues from 9 mice per group were excised for RNA sequencing (RNA-seq). Then, the DRGs from 3 mice were pooled into each sample. Total RNA was isolated using the TRIzol Reagent (15596026CN, Invitrogen/ThermoFisher Scientific), after which the concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (701-058112, ThermoFisher Scientific). Three micrograms of RNA were used for RNA sample preparation. mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations at elevated temperature in Illumina’s proprietary buffer. Sequencing libraries were constructed with the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina (NR606, Vazyme). The insert size for final cDNA library was 300–400 bp. We then performed 2 × 150 bp paired-end sequencing on the Illumina NovaSeq™ 6000 platform (Personal Biotechnology Co., Ltd., Shanghai, China) following the manufacturer’s recommended protocol. We obtained raw read counts on each gene of each sample and standardized gene expression using fragments per kilobase of transcript per million mapped reads (FPKM) method. Volcano plot, cluster heatmap, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted on Personalbio gene clouds under the following screening criteria: expression fold change > 1.5 and significant p value < 0.05.

Quantitative real-time PCR

Total RNA was isolated from frozen DRG tissues using TRIzol Reagent (15596026CN, Invitrogen/ThermoFisher Scientific), and cDNA was synthesized using iScript cDNA Synthesis Kit (170–8891, Bio-Rad). Quantitative PCR (qPCR) was performed using iTaq Universal SYBR Green Supermix (1725124, Bio-Rad) with template cDNA and primers for each gene in a Bio-Rad CFX96 real-time PCR system. The relative fold change in mRNA expression was calculated with the 2-ΔΔCt method with β-actin as the internal control. The primer sequences were listed in Supplementary Table S1.

Western blotting assay

Frozen DRG tissues were lysed with RIPA buffer supplemented with a protease inhibitor cocktail (4693116001, Roche), PMSF and Na2VO3. Protein samples (20–120 µg) were separated by SDS–PAGE and transferred onto PVDF membranes. The blots were blocked in 5% nonfat milk and incubated at 4 °C overnight with primary antibodies, including anti-PP2Cm (1:1000, DF4348, Affinity Biosciences), anti-CCR5 (1:1000, AF6339, Affinity Biosciences), anti-TRPA1 (1:1000, 19124-1-AP, Proteintech), anti-α-tubulin (1:5000, AF0524, Affinity Biosciences) and anti-GAPDH (1:5000, AF7021, Affinity Biosciences). After incubation with secondary antibody (1:10000, RGAR001, Proteintech) for 1 h at room temperature, the signals were visualized by a Novex™ ECL Chemiluminescent Substrate Reagent Kit (WP20005, ThermoFisher Scientific) and exposed using ChemiDoc XRS system (Bio-Rad) with Image Lab software. The band intensities were quantified with Image J software.

CCL5 enzyme-linked immunosorbent assay (ELISA)

DRG tissues were rapidly dissected and homogenized in 100 µL of DPBS containing protease inhibitors. The homogenate supernatants were isolated by centrifugation at 4000 × g for 10 min. CCL5 levels in the L4-L5 DRGs were measured using an ELISA kit (E-EL-M0009, Elabscience) according to the manufacturer’s protocol.

Immunohistochemistry

Mice were deeply anesthetized with 2% isoflurane and then transcardially perfused with phosphate buffered saline (PBS, pH 7.4) for 5 min, followed by 4% paraformaldehyde until muscle twitching stopped. The L4-L5 DRGs were fixed in 4% paraformaldehyde, followed by dehydration in an ascending ethanol series. Tissues were embedded in paraffin and cut into 5 μm sections for immunofluorescence staining. The sections were incubated at 4 °C overnight with the following primary antibodies: PP2Cm (1:100, DF4348, Affinity Biosciences), CCR5 (1:100, AF6339, Affinity Biosciences), TRPA1 (1:100, DF13269, Affinity Biosciences), Neurofilament 200 (NF200, 1:100, N0142, Sigma Aldrich), CGRP (1:100, ab81887, Abcam), and IB4 Alexa Fluor 488 (5 µg/µL, I21411, ThermoFisher Scientific). The sections, except for slices incubated with IB4 Alexa Fluor 488 antibodies, were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:400, ab150113, Abcam), or Fluor 594 goat anti-rabbit IgG (1:400, ab150080, Abcam) secondary antibody for 2 h. Fluorescence images were acquired by Olympus (VS200, Tokyo, Japan), and analysis was performed blindly on three sections per DRG using OlyVIA digital software and ImageJ software.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 8 software. All data were presented as the mean ± SEM unless otherwise stated. The intensities of the protein bands and immunofluorescence were quantified using ImageJ software. Body weight, nonfasting blood glucose levels, the behavioral thresholds in CD and HFD-fed mice, as well as in PP2Cm-ctrl and PP2Cm-cKO mice, fold changes in mRNA expression of Ccl5, Ccr5, Il-1β, Tnf-α, Trpa1, and Trpv1, CCL5 levels, and the immunofluorescence intensity were analyzed with two-tailed unpaired Student’s t-tests. BCAA levels, the fold changes in BCAA catabolic enzymes, and behavioral time course data for anti-CCL5, maraviroc, siCcr5, Chem-5,861,528 and siTrpa1 treatment in PP2Cm-cKO mice were analyzed with two-way repeated measures analysis of variance (ANOVA) with Bonferroni posttests to determine the significance of differences between groups at each individual time point. CCL5 levels after anti-CCL5 treatment, Ccr5 mRNA levels after siCcr5 injection, and Trpa1 mRNA levels after siCcr5 and siTrpa1 injection were analyzed with one-way ANOVA with Tukey’s multiple comparisons test. Differences were considered significant when P < 0.05.