The present study was approved by the Institutional Ethics Committee of Nanjing Medical University (approval no. 1706017; Nanjing, China). The present study was conducted using adult male Sprague-Dawley rats (age, 6–8 weeks; weight, 180–240 g; Qinglongshan Animal Center). Rats were housed individually with free access to food and water, and were acclimated to the environment with a temperature of 23–25°C, a humidity of 40–60% and a 12-h light/dark cycle for at least 1 week before the experiment. After the experiment, all rats were euthanized by decapitation under deep anesthesia with 4–5% sevoflurane.

FS was induced in rats according to a previously described method (13). Briefly, in a cylinder (diameter, 30 cm; height, 50 cm) containing water at 24–26°C to a height of 20 cm, the rats were forced to swim for 3 consecutive days, for 10 min on the first day and then for 20 min on the subsequent 2 days. After each FS session, the rats were carefully dried and rewarmed. Inflammatory pain was induced via a single injection of CFA (50 µl; MilliporeSigma) into the right hind paw. FS stress has previously been reported to increase pain-like behaviors in the hind paw (14). In the present study, FS stress-induced mechanical hyperalgesia in rats in an inflammatory state was established using FS stress for 3 days, followed by a single CFA injection. Control rats were injected with 50 µl deionized water. To verify the efficacy of FS stress-induced mechanical hyperalgesia in rats in an inflammatory state, 6 rats were used in each group.

For serum corticosterone detection, 300 µl blood was collected from the tail vein under 2–3% sevoflurane anesthesia 2 h after FS stress, and the plasma was separated and stored at −80°C until the ELISA was performed. A commercially available ELISA kit (cat. no. ab108821; Abcam) was used to measure serum corticosterone levels according to the manufacturer's instructions. The OD value was acquired at 450 nm using a multi-function microplate reader (MD Spectramac M3; Molecular Devices, LLC). Different rat groups were measured across days 1–3, with three rats assessed at each time point. For control rats, the blood was collected at the approximately same time as for rats in the other groups.

Mechanical pain was examined using von-Frey filaments. Briefly, the rats were individually placed in a transparent Plexiglass chamber for 30 min. Thereafter, a series of von Frey filaments (0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0 and 15 g; Danmic Global, LLC) were vertically applied to the central plantar of the hind paw to evoke a flinch response using an up-down method (15). The paw withdrawal threshold was detected five times with an interval of 5 min, and the mean value of the last three results was calculated as the mechanical withdrawal threshold.

Hsc70 and TRPV1 expression levels in the DRG were examined by western blot, as previously described (16). After the pain behavioral test, the rats were sacrificed by decapitation under 4–5% sevoflurane anesthesia and L4-5 DRG was collected for western blot analysis. Briefly, the DRG was separated and homogenized in RIPA lysis buffer (cat. no. bl504a; Biosharp Life Sciences), and the resulting supernatant was collected. After protein quantification using the Bradford assay, 20 µg proteins were separated by SDS-PAGE 8–12% on gels and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated with primary antibodies against Hsc70 (1:500; cat. no. ab51052), TRPV1 (1:200; cat. no. ab305299) overnight at 4°C, and GAPDH (1:1,000; cat. no. ab8245) (all from Abcam) was used as an internal control. After washing in Tris-buffered saline-0.05% Tween three times, the membranes were incubated with horseradish peroxidase-labeled immunoglobulin G (1:5,000; cat. no. bl003A; Biosharp Life Sciences) for 1 h at room temperature. The bands were detected using a luminescent imaging system (G:BOX Chemi XR5; Syngene) and the blots were analyzed using ImageJ software (v. 1.53a; National Institutes of Health).

In the DRG, Hsc70 and TRPV1 expression levels were also detected by IF analysis. After sacrifice, the rats were transcardially perfused with 4% paraformaldehyde. The L4-5 DRG was removed, fixed in the same fixative at 4°C for 6 h, and thereafter embedded in paraffin. For IF staining, the DRG tissue (3 µm) was mounted on glass slides. After sequential dewaxing, hydration and microwave antigen retrieval in sodium citrate for 20 min, the sections were blocked with 5% bovine serum albumin (cat. no. 4240GR500; Biofroxx; neoFroxx GmbH) at 37°C for 1 h. After overnight incubation with a rabbit antibody against Hsc70 (1:200; cat. no. ab51052) or a mouse antibody against TRPV1 (1:200; cat. no. ab203103) (both from Abcam) at 4°C, the sections were incubated with fluorescein isothiocyanate-conjugated (1:200; cat. no. BL033A; Biosharp Life Sciences) or tetramethylrhodamine-conjugated (1:100; cat. no. 115-025-003; Jackson ImmunoResearch Europe Ltd.) secondary antibodies for 1 h at 37°C. Nuclear DNA was labelled with DAPI. The obtained IF sections were scanned and stored using an OlyVIA system (Olympus VS200; Olympus Corporation).

RT-qPCR detection of miR-3120 and Hsc70 expression in the DRG was performed. Briefly, total RNA was extracted from the DRG with ice-cold TRIzol^® reagent (cat. no. 15596-026; Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, RNA concentration and purity were determined using an ultraviolet photometer (Shimadzu UV-2450; Shimadzu Corporation) at 260 and 280 nm; a result of 1.8–2.1 was considered an appropriate purity. Using 2 µg total RNA as a template, cDNA was synthesized using a reverse transcriptase reagent kit (cat. no. RR036B; Takara Bio, Inc.) according to the manufacturer's instructions. qPCR amplification was performed using the One Step TB Green PrimeScript RT-PCR Kit II (cat. no. RR086B; Takara Bio, Inc.). The cycling conditions were: 95°C for 5 min, followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec. The 2^−ΔΔCq method was used to calculate the gene expression relative to the reference gene (17).

For detection of miR-3120, Bulge-loop™ miRNA qPCR Primer Sets specific for miR-3120 were designed by Nanjing KeyGen Biotech Co., Ltd. The primer sequences for miR-3120 and its internal control U6, as well as for Hsc70 and its internal control GAPDH, which were used for qPCR, are shown in Table I. To examine stress-induced changes in miR-3120 expression, the same rats used for serum corticosterone detection were assessed. Briefly, three rats were used to detect serum corticosterone levels at each time point, after which they were sacrificed, the DRG was dissected and miR-3120 expression was detected.

Cy3-labeled miR-3120 agomir, which encodes the red fluorescence protein Cy3 and miR-3120, as well as its negative control (NC; Cy3-labeled agomir NC), were produced by Nanjing KeyGen Biotech Co., Ltd. For the DRG injection, the paraspinal muscles were carefully separated along the vertebrae under 2–3% sevoflurane anesthesia and 4 mg/kg lidocaine was intraincisionally injected. Part of the bone covering the right L5 DRG was then removed to completely expose the ganglion. miR-3120 agomir or agomir NC (0.5 nM dissolved in RNAase-free deionized water to 5 µl) was slowly injected into the L5 DRG using a Hamilton microliter syringe with a 30-gauge needle 3 days before CFA injection in rats in the CFA + miR-3120 agomir or CFA + agomir NC groups.

Cellular distribution of miR-3120 in the DRG was examined using FISH according to a previously described method (18). Briefly, paraffin-embedded DRG slides were prepared using the same method as that described for IF analysis. After sequential dewaxing, hydration and permeabilized with 0.4% Triton X-100 for 15 min at room temperature, the slides were washed twice with 2X saline sodium citrate (SSC). The oligonucleotide probe was denatured for 5 min at 75°C in a water bath, and the slides were incubated with the pre-hybridization solution (cat. no. AR0152; Boster Biological Technology) at 42°C for 30 min, followed by 40 nM probe at 42°C overnight. After sequential washing with 2, 1, 0.5 and 0.1X SSC, the slides were counterstained with Hoechst 33258 (cat. no. C1017; Beyotime Institute of Biotechnology). The sections were scanned and stored using the OlyVIA system (Olympus VS200) with the required images exported when necessary. In addition, the cellular distribution of miR-3120 and Hsc70 was examined in the DRG using FISH and IF analysis, respectively. After FISH and IF staining, the colocalization, as well as the size of miR-3120- and Hsc70-labeled cells was analyzed using ImageJ software.

Full-length Hsc70 (Gene ID: 24468; Transcript: NM_024351.2) or scramble oligonucleotides were subcloned into the GV280 lentiviral vector (Nanjing KeyGen Biotech Co., Ltd.) to construct a lentiviral vector overexpressing Hsc70 (LV-Hsc70; 1×10⁸ TU/ml) and an LV-scramble, respectively. According to a previously reported method (19), lentiviral vector (10 µl) was intrathecally microinjected into the L5-6 intervertebral space of rats through the skin using a 30-gauge needle attached to a Hamilton microliter syringe under 2–3% sevoflurane anesthesia 1 week before CFA injection in the CFA + agomir + LV-Hsc70 and CFA + agomir + LV-scramble groups.

Statistical analyses were performed using GraphPad Prism 8.0 software (Dotmatics). Data are shown as the mean ± SEM. One-way ANOVA was used to assess the changes in the mechanical withdrawal threshold at each time point among the groups. In addition, one-way ANOVA was used to assess differences in the results of ELISA, western blotting, IF analysis and RT-qPCR among the groups. If a significant difference was observed, the Bonferroni post hoc test was applied. P<0.05 was considered to indicate a statistically significant difference.

In the present study, CFA hind paw injection was performed in rats to induce inflammatory pain (CFA rats). In addition, FS stress was performed for 3 days before CFA injection to evoke mechanical hyperalgesia to establish an SIH rat model (FS + CFA rats). A schematic diagram of the model is presented in Fig. 1A. To verify the efficiency of FS stress in rats, serum corticosterone levels were examined using ELISA. In control rats, the average serum corticosterone level was 10.1±0.5 ng/ml. After 3 days of FS stress, the average corticosterone concentration significantly increased to 12.3±0.3 ng/ml (Fig. 1B). Moreover, CFA hind paw injection quickly decreased the paw withdrawal threshold, which was further exacerbated by 3 days of FS stress (Fig. 1C). All of these observations indicated the successful establishment of FS stress-induced mechanical hyperalgesia in rats in an inflammatory state.

Hsc70 participates in CFA-induced inflammatory pain (6); however, its involvement in FS stress-induced mechanical hyperalgesia remains largely unknown. Therefore, the expression of Hsc70 in the DRG was detected using western blot and IF analysis. The results showed that Hsc70 protein expression was increased by ~1.9-fold in CFA; however, this increase was partially inhibited in FS + CFA rats (Fig. 2A). Consistent with the results of western blot, the results of IF analysis suggested a similar change in Hsc70 expression (Fig. 2B). However, FS stress alone did not significantly influence Hsc70 expression.

TRPV1 protein expression was increased by ~1.6-fold in CFA rats, which was further increased to ~2-fold after 3 days of FS stress (Fig. 2C). IF analysis exhibited a similar expression change (Fig. 2D). Notably, TPRV1 is a well-known nociceptor in pain perception. Moreover, to verify the etiological role of Hsc70 in FS stress-induced mechanical hyperalgesia, LV-Hsc70 was intrathecally injected into FS + CFA rats to increase the expression of Hsc70 the day before FS. The efficiency of intrathecal LV-Hsc70 was verified using RT-qPCR after 1 week (Fig. 2E). In the presence of intrathecal LV-Hsc70, TRPV1 expression in the DRG was partially inhibited (Fig. 2F) and the mechanical withdrawal threshold was increased (Fig. 2G) compared with that in the FS + CFA group. These results suggested that Hsc70 and TRPV1 may have an essential role in FS stress-induced mechanical hyperalgesia in rats in an inflammatory state.

As an endogenous ligand of Hsc70, miR-3120 is located in the neuronal cell body, and it targets and inhibits Hsc70 (10). To explore the potential regulatory role of miR-3120 for Hsc70, miR-3120 expression was examined by RT-qPCR in the DRG following FS stress. The results showed that 3 days of FS stress significantly upregulated the expression levels of miR-3120 by ~2.6-fold (Fig. 3A). Moreover, FS stress for 1 day transiently downregulated miR-3120 expression. This downregulation was consistent with that observed for serum corticosterone; however, the underlying reason remains unclear.

The cellular distribution of miR-3120 and Hsc70 was then examined in the DRG using FISH and IF analysis, respectively. After double fluorescence staining, the colocalization of miR-3120 and Hsc70 was analyzed, as well as the diameter of the labeled cells. miR-3120 and Hsc70 were both expressed on the cell surface and in the cytoplasm, and a number of miR-3120 and Hsc70 double-labelled neurons were observed (Fig. 3B). Cell size distribution histograms for miR-3120 (red) and Hsc70 (green) showed that more than half of the miR-3120 and Hsc70 double-labeled neurons were medium and large-sized neurons (cross-sectional area >600 µm²) (Fig. 3C). These results indicated that miR-3120 and Hsc70 may act on the same population of neurons.

To explore the participation of miR-3120 in FS stress-induced mechanical hyperalgesia, miR-3120 agomir was injected into the DRG in CFA rats. Agomirs are specially labeled and chemically modified double-stranded small RNA molecules that simulate endogenous miRNAs to regulate the biological functions of target genes. Compared with miRNA mimics, agomirs can enrich in target cells, presenting higher stability in vivo and resulting in more effective interference (20).

The efficiency of the miR-3120 agomir was examined using fluorescence detection and RT-qPCR. The results showed that the injection of Cy3-labeled miR-3120 agomir encoding both the red fluorescence protein Cy3 and miR-3120 into the L5 DRG induced red fluorescence in all sizes of DRG neurons as early as 48 h after injection (Fig. 4A). Although DRG injection of agomir NC also presented red fluorescence (Fig. 4A), RT-qPCR showed that miR-3120 agomir, but not agomir NC, significantly increased the expression levels of miR-3120 at 72 h after DRG injection compared with those in the control group (Fig. 4B), collectively verifying the efficiency of miR-3120 agomir after DRG injection.

After verifying the efficiency of DRG injection of miR-3120 agomir, the miR-3120 agomir or its NC was injected into the DRG 3 days before CFA injection. Western blotting showed that miR-3120 agomir, but not its NC, significantly upregulated TRPV1 expression by ~2-fold in CFA rats (Fig. 5A). Meanwhile, miR-3120 agomir significantly enhanced CFA-induced mechanical pain, which was similar to FS stress-induced behavioral changes detected in CFA rats (Fig. 5B). These results suggested that miR-3120 may participate in FS stress-induced mechanical hyperalgesia.

To further investigate whether Hsc70 is involved in miR-3120 agomir-induced TRPV1 expression and behavioral changes, miR-3120 agomir and LV-Hsc70 were injected into the DRG 3 days and intrathecally injected 1 week before CFA injection, respectively. As shown by western blotting and behavioral testing, miR-3120 agomir-induced TRPV1 expression and mechanical hyperalgesia in CFA rats were significantly abolished in the presence of intrathecal injection of LV-Hsc70 but not LV-scramble (Fig. 6A and B).