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. 2022 Jan 13;19(1):123–133. doi: 10.1007/s11302-021-09828-0

Long non-coding RNA MSTRG.81401 short hairpin RNA relieves diabetic neuropathic pain and behaviors of depression by inhibiting P2X4 receptor expression in type 2 diabetic rats

Mengyun Sun 1, Mingming Zhang 1, Haoming Yin 2, Hongcheng Tu 3, Yuqing Wen 1, Xingyu Wei 4, Wenhao shen 5, Ruoyu Huang 5, Wei Xiong 5, Guodong Li 1, Yun Gao 1,6,✉
PMCID: PMC9984665  PMID: 35022948

Abstract

Patients with diabetic neuropathic pain (DNP) experience immense physical and mental suffering, which is comorbid with other mental disorders, including major depressive disorder (MDD). P2X4 receptor, one of the purinergic receptors, is a significant mediator of DNP and MDD. The present study aimed to identify the roles and mechanisms of MSTRG.81401, a long non-coding RNA (lncRNA), in alleviating DNP and MDD-like behaviors in type 2 diabetic rats. After administration with MSTRG.81401 short hairpin RNA (shRNA), the model + MSTRG.81401 shRNA group demonstrated increased mechanical withdrawal threshold, thermal withdrawal latency, open-field test, and sucrose preference test; however, immobility time on the forced swimming test decreased. MSTRG.81401 shRNA administration significantly decreased the expression of the P2X4 receptor, tumor necrosis factor-α, and interleukin-1β in the hippocampus and spinal cord in the model + MSTRG.81401 shRNA group. Simultaneously, MSTRG.81401 shRNA administration downregulated phosphorylation of ERK1/2 in the hippocampus and spinal cord. Thus, lncRNA MSTRG.81401 shRNA can alleviate DNP and MDD-like behaviors in type 2 diabetic rats and may downregulate the expression of P2X4 receptors in the hippocampus and spinal cord of rats.

Keywords: lncRNA, Diabetic neuropathic pain, Major depressive disorder, Hippocampus, Spinal cord, P2X4 receptor

Introduction

Diabetic neuropathic pain (DNP) is a well-known complication of diabetes, which may occur even in the early stages of diabetes [1]. Despite significant advances in diabetes and neurology, the causative mechanisms for DNP remain unclear. DNP may be comorbid with other mental disorders, including major depressive disorder (MDD). MDD is often attributed to factors such as the environment and personality of the individual, including diseases of the immune system, which disrupt the integrity pathway of the frontal striatum, amygdala, and hippocampus [2]. MDD and DNP are often comorbid, with high incidence rates, as indicated in some studies [3–5].

Long non-coding RNA (lncRNA) has been implicated in pain, and research suggested that they can relieve pain and have a strong relationship with MDD [6–11]. P2X4 receptor, a type of purinergic receptors, is widely found in the central nervous system [12, 13]. Studies have shown that P2X4 receptors play an important role in the pathophysiology of the central nervous system, including injury, inflammation, pain, depression, and anxiety [12, 14–16]. In our study, we used high-throughput microarrays to detect the differences in lncRNA expression profiles in the hippocampal tissues of DNP and MDD rats as compared to normal rats. We found that lncRNA MSTRG.81401 [Gene_id: MSTRG.81401; Rat(chr17)] was significantly increased and may be involved in DNP and MDD. Furthermore, we used a bioinformatics prediction platform (http://service.tartaglialab.com/page/ largeRNAs_group) to predict the binding force between the RNA and protein and found that MSTRG.81401 had high P2X4 binding force fragments (Fig. 1).

Fig. 1.

Fig. 1

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The heat map predicted by the basic tool in the catRAPID online website, which showed that MSTRG.81401 and the amino acids of P2X4 protein have a strong combination ability

The present study aimed to investigate whether the treatment of MSTRG.81401 with short hairpin RNA (shRNA), a lncRNA, could regulate the central nervous system P2X4 receptor expression and affect the comorbidity of DNP and MDD in type 2 diabetic rats.

Materials and methods

Animals and treatments

Male Sprague–Dawley rats (200–220 g) were provided by the Centre of Laboratory Animal Science at Nanchang University. The procedures were approved by the Animal Care and Use Committee at Nanchang University Medical School and were performed according to the ethical guidelines for pain research in animals of the International Association for the Study of Pain. Rats were housed under controlled conditions (25 °C and 60% humidity), with freely available food and water. Six rats were housed in each cage.

The rats were randomly divided into four groups: normal control group (control group), DNP + MDD group (model group), DNP + MDD + MSTRG.81401 shRNA group (model + MSTRG.81401 shRNA group), and DNP + MDD + scramble shRNA group (model + scramble shRNA group). The rats were fed with a high glucose and high-fat diet for 4 weeks and injected intraperitoneally (i.p.) with streptozotocin; rats with a fasting blood glucose > 7.8 mmol/L were selected as type 2 diabetic rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests were used to select DNP rats. Additionally, chronic unpredictable mild stimulation was given for 4 weeks. Sucrose preference (SPT), forced swimming (FST), and open-field (OFT) tests were used to select comorbid DNP and MDD rats. The effective MSTRG.81401 shRNA interfering sequence was screened using quantitative real-time polymerase chain reaction (Q-PCR) technology. On day 1, an equal amount of MSTRG.81401 shRNA and scrambled shRNA were injected into the lateral ventricle of the rats using a brain stereotactic instrument [17]. The ratio of shRNA to transfection reagent was 2:1 (14 μl:7 μl in detail), according to the transfection reagent instruction and only one injection was given. Each experiment below was repeated three or more times.

Mechanical withdrawal test

The mechanical withdrawal threshold (MWT) was used to observe the withdrawal responses to mechanical stimulation, which was induced using the BME-404 electronic mechanical stimulator (provided by the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences). The end-face diameter of the test needle, the pressure measurement range, and the pressure measurement resolution of the stimulator were 0.6 mm, 0.1–50 g, and 0.05 g, respectively. Before evaluation, each rat was placed in a clean glass box positioned on the sieve of a metal frame for an adaptive period of at least 30 min. The test needle touched the place between the third and fourth metatarsus of the hind paws until the rat attempted to withdraw its paw. The computer recorded the pressure values automatically. The stimulus alternated between the left and right hind paws at 5-min intervals. The MWT was calculated as the mean of three consecutive stable values, expressed in rams, and determined by one observer [17].

Thermal withdrawal test

Thermal withdrawal latency (TWL) was evaluated by measuring the latency to hind paw withdrawal from a thermal stimulus, which was administered using the BME-410C Thermal Paw Stimulation System (provided by the Institute of Biomedical Engineering of Chinese Academy of Medical Sciences). Before evaluation, each rat was placed on a glass plate in a transparent, square, bottomless acrylic box, for an adaptive period of at least 30 min. A beam of radiant heat was oriented at the plantar surface of the rat’s paws. The activation of the beam simultaneously activated a timer and the cutoff time for heat stimulation was 30 s. The light beam was switched off when the animal lifted its paw, and the timing was over. The time on the screen of the apparatus was designated as the TWL and was expressed in seconds. The hind paw withdrawal was tested in triplicate, and hind paws were alternated at 5-min intervals [17].

Sucrose preference test

Before the test, rats were fasted for 24 h and then placed in separate cages. Two identical water bottles, one containing 100 mL of 1% sucrose in water and another containing 100 mL of pure water, were placed in each cage. SP was evaluated by measuring the levels of sugar water and pure water consumption in 1 h. The SP rate was calculated as the ratio of sugar water/total liquid consumption × 100%. The SPT reflects a lack of pleasure, which is a symptom of MDD [17].

Forced swimming test

Rats were placed in an 80-cm-tall glass cylinder with a 40-cm inner diameter. The water temperature was approximately 20 °C and the water depth was 30 cm. The immobility time (IT) of rats in the water (i.e., when rats stopped struggling and floated in a fixed shape) and the swimming time of each rat were recorded for 5 min and expressed in seconds [17].

Open-field test

Before the test, rats were placed in the dark for 30 min to adapt to the environment. Then, they were placed in a black box that measured 40 × 60 × 50 cm. Each rat was placed gently in the middle of the box, and the distance navigated by the animal was recorded using a Canon Powershot A610 camera (Canon Inc., Tokyo, Japan) during a 5-min session. The recorded videos were analyzed and processed using MATLAB (MathWorks Co., Natick, MA, USA) to determine the total distance traveled, expressed in centimeters. The apparatus was cleaned with a 10% ethanol solution before the next animal was introduced into the box [17].

Quantitative real-time PCR

Rats were anesthetized by i.p. injection of 10% chloral hydrate (batch no. 050101; Shanghai Xingya Medical Co., Ltd., Shanghai, China). Hippocampi and L4/5 spinal cord were isolated immediately after sacrifice, flushed with ice-cold phosphate-buffered saline (PBS), and stored in RNA storage solution at – 20 °C until further use. All instruments were treated with diethyl pyrocarbonate before use. Total RNA was isolated from the hippocampus and spinal cord using the TRIzol Total RNA Reagent (TransGen Biotech Co., Ltd., Beijing, China). Complementary DNA synthesis was performed with 2 μg total RNA using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fischer Scientific, Waltham, MA, USA). The primers were designed using Primer Express 3.0 Software (Applied Biosystems, Foster City, CA, USA) and Q-PCR was performed using the SYBR® Green Master Mix in the ABI PRISM®7500 Sequence Detection System (Applied Biosystems). The expression of each gene was quantified using the ΔΔCT method, with CT as the threshold cycle. The relative levels of target genes normalized to the sample with the lowest CT are presented as 2−ΔΔCT [18].

The Q-PCR primer sequences were as follows:graphic file with name 11302_2021_9828_Figa_HTML.jpg

Western blotting

After rats were anesthetized, the hippocampus and spinal cord were separated and flushed with PBS. Tissues were positioned in the spherical portion of a 2-mL homogenizer and homogenized in RIPA lysis buffer (50 vmM Tris–Cl, pH 8.0, 150 vmM NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P40, 0.02% sodium deoxycholate, 100 vmg/mL phenylmethylsulfonyl fluoride, and 1 mg/mL aprotinin) containing protease inhibitors. Tissues were ground for 30 min on ice and centrifuged at 4 °C at 15,000 rpm for 10 min. The supernatants were collected, diluted with 6 × loading buffer, and heated to 95 °C for 10 min. The protein concentration was calculated with the BCA Protein Assay Kit and the samples were kept at − 20 °C until they were removed for use. Proteins in samples from each group (20 μg) were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, using Bio-Rad electrophoresis device (Bio-Rad Laboratories, Hercules, CA, USA), and transferred onto polyvinylidene fluoride membranes. Polyvinylidene fluoride membranes were blocked with 5% nonfat dry milk in 1 × TBST (tris-buffered saline and Tween 20) for 2 h at room temperature, followed by incubation with antibodies against P2X4 (1:500; Alomone Labs, Jerusalem, Israel), β-actin (1:1,000; Beijing Zhongshan Biotech Co., Beijing, China), rabbit anti-interleukin (IL)-1β (1:500, Boster Biological Technology, Pleasanton, CA, USA), rabbit anti-tumor necrosis factor (TNF)-α (1:500, AF7014; Affinity Biosciences, Cincinnati, OH, USA), extracellular signal-regulated kinases 1/2 (ERK1/2; 1:1,000; Cell Signaling Technology Inc., Boston, MA, USA), and phosphorylated (p)-ERK1/2 (1:1,000; Cell Signaling Technology Inc.) at 4 °C overnight. Membranes were washed three times with 1 × TBST (10 min each) and incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary goat anti-rabbit immunoglobulin G (IgG) and goat anti-mouse IgG antibodies (1:2000; Beijing Zhongshan Biotech Co.) in blocking buffer and washed again three times with 1 × TBST (10 min each). Labeled proteins were then visualized with enhanced chemiluminescence on a Bio-Rad system. Band intensities were quantified using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA), and the intensities of target proteins were normalized against the respective β-actin internal controls [18].

Double-immunofluorescence labeling

Rats were anesthetized with 10% chloral hydrate and were transcardially perfused with 4% paraformaldehyde (PFA). The hippocampus and spinal cord were removed and fixed in 4% PFA at 4 °C for 2 h at room temperature. The tissues were immersed in 30% sucrose solution (in 4% PFA) for 24 h at 4 °C for dehydration; solutions were changed every 8 h. Tissues were cut into 12-μm or 8-μm-thick slices in a cryostat (Leica Biosystems Inc., Concord, ON, Canada). The sections were placed at 37 °C for 2 h and then stored at – 20 °C until they were taken out for use.

Before staining, the parts sat at room temperature for a while, rinsed with 0.01 M PBS three times (5 min each), incubated with 0.3% Triton X-100, and rewashed with PBS three times (5 min). Then, slices were incubated in 10% goat serum for 1 h at 37 °C, followed by incubation with the diluted antibodies (rabbit anti-P2X4, 1:100, Alomone Labs; mouse anti-glial fibrillary acidic protein (GFAP), 1:200, MilliporeSigma, Burlington, MA, USA) overnight at 4 °C. The secondary antibodies were tetramethyl rhodamine isothiocyanate (Affinity Biosciences) conjugated to pure goat anti-rabbit IgG and fluorescein isothiocyanate conjugated to pure goat anti-mouse IgG, and the incubation was performed in 0.01 M PBS for 1 h at 37 °C. The slides were washed with PBS prior to the application of coverslips and then examined under a fluorescence microscope (Olympus, Tokyo, Japan). The mean optical density value was calculated by ImageJ software and used to describe the staining intensity [17].

Statistical analysis

Statistical analyses were performed on a computer using SPSS (version 21.0; IBM, Armonk, NY, USA). All results are expressed as the mean ± standard error. Statistical significance was determined using one-way analyses of variance, followed by Fisher’s post hoc test for multiple comparisons. P < 0.05 was considered significant.

Results

Effect of MSTRG.81401 shRNA on MWT, TWL, SPT, FST, and OFT of each group

Compared with the control group, MWT, TWL, SPT, OFT, and FST values in the model group were decreased, while FST immobility time was increased (P < 0.01). MWT, TWL, SPT, and OFT values of the model + MSTRG.81401shRNA group increased compared with the model group, while FST immobility time decreased (P < 0.01; Figs. 2 and 3).

Fig. 2.

Fig. 2

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Effects of MSTRG.81401 shRNA on thermal withdrawal latency (TWL) (A), mechanical withdrawal threshold (MWT) (B), and sucrose preference test (SPT) (C) of comorbid diabetic neuropathic pain (DNP) and major depressive disorder (MDD) rats. Data are presented as the mean ± standard error of the mean. n = 6, *P < 0.05, **P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. model group

Fig. 3.

Fig. 3

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Effects of MSTRG.81401 shRNA on sucrose preference test (SPT) (A), forced swimming test (FST) (B) and open-filed test (OFT) (C) of comorbid DNP and MDD rats. Data are presented as the mean ± standard error of the mean. n = 6, *P < 0.05, **P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. model group

Effects of MSTRG.81401 shRNA on the expression of P2X4 receptor in the hippocampus and spinal cord of each group

The expression of MSTRG.81401 in the hippocampus and spinal cord was measured by Q-PCR. After administration of MSTRG.81401 shRNA in the model rats, the expression of MSTRG.81401 in the hippocampus and spinal cord declined significantly compared to the model rats treated with scramble shRNA (P < 0.01).

The expression of P2X4 in the hippocampus and spinal cord was measured by Q-PCR and Western blot. These results suggest that P2X4 mRNA and protein expression in the model group were higher compared with the control group (P < 0.01). The P2X4 mRNA and protein expression in the model + MSTRG.81401 shRNA group was significantly decreased compared to the model group (P < 0.01). No differences in the expression of P2X4 mRNA and protein were found between the model and the model + scramble shRNA groups (P > 0.05). These results revealed that MSTRG.81401 shRNA treatment may decrease the upregulated expression of the P2X4 receptor in the hippocampus and spinal cord of DNP and MDD rats (Figs. 4 and 5).

Fig. 4.

Fig. 4

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Effects of MSTRG.81401 shRNA mRNA (A, C) and P2X4 receptor mRNA (B, D) expression in the hippocampus and the spinal cord of rats of each group. Values are presented as the means ± standard error of the mean. n = 6, **P < 0.01 vs. control group or model + MSTRG.81401 shRNA group; ##P < 0.01 vs. model group

Fig. 5.

Fig. 5

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Effects of MSTRG.81401 shRNA on the expression of the protein of P2X4 in the hippocampus (A, B) and the spinal cord (C, D) of rats of each group. Values are presented as the means ± standard error of the mean. n = 6, **P < 0.01 vs. control group; ##P < 0.01 vs. model group

Effects of MSTRG.81401 shRNA on the expression levels of IL-1β and TNF-α in the hippocampus and spinal cord of each group

Compared with the control group, the expression of TNF-α and IL-1β in the hippocampus and spinal cord of the model group was significantly increased (P < 0.05). Moreover, the expression of TNF-α and IL-1β in the model + MSTRG.81401 shRNA group was lower than in the model group (P < 0.05). There was no significant difference between the model and the model + scramble shRNA groups (P > 0.05). These results indicate that MSTRG.81401 shRNA treatment may reduce the upregulated expression levels of IL-1β and TNF-α proteins in the hippocampus and spinal cord of each group (Fig. 6).

Fig. 6.

Fig. 6

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Effects of MSTRG.81401 shRNA on the expression of the protein of TNF-αand IL-1β in the hippocampus (A, B, C) and spinal cord (D, E, F) of rats of each group. Values are presented as the means ± standard error of the mean. n = 6, **P < 0.01 vs. control group; ##P < 0.01 vs. model group

Effects of MSTRG.81401 shRNA on the co-expression levels of P2X4 and GFAP in the hippocampus and spinal cord of each group

The co-expression of P2X4 and GFAP in the hippocampus and spinal cord of each group was assayed via double immunofluorescence. Double-immunofluorescence staining results showed that the P2X4 receptor was co-expressed with GFAP in the astrocytes of the hippocampus and spinal cord. The co-expression levels of the P2X4 receptor and GFAP in model group rats were increased compared to those in the control group (P < 0.05). The co-expression levels of the P2X4 receptor and GFAP in the model + MSTRG.81401 shRNA group were decreased as compared to those in the model group (P < 0.05). No significant differences were found between the model and model + scramble shRNA groups (P > 0.05). These results demonstrate that MSTRG.81401 shRNA treatment reduces the upregulated co-expression levels of the P2X4 receptor and GFAP in the hippocampus and spinal cord of DNP and MDD rats (Figs. 7 and 8).

Fig. 7.

Fig. 7

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Effects of MSTRG.81401 shRNA on the co-expression of P2X4 and glial fibrillary acidic protein (GFAP) in the hippocampus of rats with comorbid DNP and MDD. The green signal represents GFAP staining with fluorescein isothiocyanate (FITC), and the red signal indicates P2X4 staining with tetramethyl rhodamine isothiocyanate (TRITC). Scale bar = 100 μm. Values are presented as the means ± standard error of the mean. n = 6, **P < 0.01 vs. control group; ##P < 0.01 vs. model group

Fig. 8.

Fig. 8

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Effects of MSTRG.81401 shRNA on the co-expression of P2X4 and glial fibrillary acidic protein (GFAP) in the spinal cord of rats with comorbid DNP and MDD. The green signal represents GFAP staining with fluorescein isothiocyanate (FITC), and the red signal indicates P2X4 staining with tetramethyl rhodamine isothiocyanate (TRITC). Scale bar = 100 μm. Values are presented as the means ± standard error of the mean. n = 6, **P < 0.01 vs. control group; ##P < 0.01 vs. model group

Effects of MSTRG.81401 shRNA on the phosphorylation of ERK1/2 in the hippocampus and spinal cord of each group

The expression of p-ERK1/2 in the hippocampus and spinal cord of the model and model + scramble shRNA groups were significantly higher than the control group (P < 0.05), while the expression of p-ERK1/2 in the hippocampus and spinal cord of the model + MSTRG.81401 shRNA group was significantly lower than the model group (P < 0.05; Fig. 9).

Fig. 9.

Fig. 9

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Effects of MSTRG.81401 shRNA on the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) in the hippocampus (A, B, C) and spinal cord (D, E, F) of rats with comorbid DNP and MDD. Data are presented as the mean ± standard error of the mean. n = 6, **P < 0.01 versus control group; ##P < 0.01 versus model group

Discussion

DNP is a common neurological complication in diabetes [19], and many diabetic patients have varying degrees of depression [20]. Psychological changes in diabetic patients can affect insulin secretion, thus accelerating glucose metabolism and causing patients to develop symptoms of MDD. The MDD occurrence often reduces compliance with diabetes treatment, consequently causing poor glycemic control and low quality of life [21]. Compared to other complications, DNP had the greatest association with MDD in diabetic patients [22, 23]. The current clinical treatments are not ideal, because the mechanisms involved in comorbid DNP and MDD are unclear and require further research. In the present study, we established a comorbid DNP and MDD rat model to investigate potential comorbid molecules.

lncRNA, more than 200 nucleotides long with no protein-coding or limited capacities, was identified to be abnormally expressed in the spinal cord, dorsal root ganglion, hippocampus, and prefrontal cortex under chronic neuropathic pain conditions [24, 25]. We administered lncRNA MSTRG.81401 shRNA to comorbid DNP and MDD rats and found that this relieved DNP and MDD-like behaviors. We speculated that MSTRG.81401 is involved in the occurrence and development of chronic neuropathic pain and MDD and may be a novel target for treatment.

P2X4 receptor may play a key role in regulating a variety of neural and behavioral functions, including pain, depression, and hippocampal plasticity [26–28]. In the present study, the expression of the P2X4 receptor in the hippocampus and spinal cord of the model group was higher as compared to the control group, and the co-expression of the P2X4 receptor and GFAP in the hippocampus and spinal cord in model rats was increased as compared to that in control rats. Treatment with MSTRG.81401 shRNA reversed the increases in the P2X4 receptor of the hippocampus and spinal cord in the model group to a normal level. Thus, we speculate that MSTRG.81401 affects the expression of the P2X4 receptor in the hippocampus and spinal cord and facilitates the progress of comorbid DNP and MDD.

Increased inflammatory factors, such as IL-1β and TNF-α, are found both in DNP and MDD [17, 29]. There is evidence that increased P2X4 receptor expression is associated with increased levels of inflammatory cytokines [30–32]. In the present study, the protein and mRNA expression levels of IL-1β and TNF-α in the hippocampus and spinal cord in the model group were higher than in the control group. However, after treatment with MSTRG.81401 shRNA, the upregulation of IL-1β and TNF-α in the hippocampus and spinal cord were decreased.

To further explore the molecular mechanisms involved, we examined the phosphorylation of ERK in the hippocampus and spinal cord. Investigations of previous studies conducted in our lab found that increased levels of ERK phosphorylation in the hippocampus and spinal cord contribute to the complications of pain and depression [17, 18]. In this present study, the expression of p-ERK1/2 in the hippocampus and spinal cord of model rats was significantly higher than in the control rats, while MSTRG.81401 shRNA treatment significantly suppressed these elevated levels. Thus, we hypothesize that rats with comorbid DNP and MDD may have elevated P2X4 receptor expression in the hippocampus and spinal cord, which causes high levels of IL-1β and TNF-α and is related to enhanced p-ERK1/2 in the hippocampus and spinal cord of model rats.

In conclusion, lncRNA MSTRG.81401 shRNA can alleviate pain and MDD behaviors of comorbid DNP and MDD rats. We believe this may be because lncRNA MSTRG.81401 shRNA can reduce P2X4 receptor expression, IL-1β and TNF-α levels, and ERK1/2 phosphorylation in the hippocampus and spinal cord. Although the exact molecular mechanism requires further research, lncRNA MSTRG.81401 may be an efficient novel target for the treatment of comorbid DNP and MDD.

Author contribution

M.S. conducted the experiments with assistance from M.Z., H.Y., H.T., Y.W., X.W., W.S., R.H., and W.X. Experimental design, data analysis and interpretation, and writing were done by M.S., G.L., and Y.G. We thank Prof. Shangdong Liang and Prof. Guodong Li for assisting us in the experimental design. All authors reviewed the manuscript and approved the final version for publication.

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 81760152, 82160161, 81860199, 81970749).

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical approval

The procedures were approved by the Animal Care and Use Committee at Nanchang University Medical School and were performed according to the International Association for the Study of Pain’s ethical guidelines for pain research in animals.

Conflict of interest

The authors declare no competing interests.

Footnotes

Mengyun Sun, Mingming Zhang, and Haoming Yin are joint first authors.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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