Introduction

Diabetes is a leading health burden worldwide [1] and affects about 415 million people, with the number predicted to rise to 700 million by 2045 [2]. In diabetics, hyperglycemia and vascular impairment contribute to the neuronal dysfunction in the peripheral nervous system [3, 4]. In view of the high prevalence of diabetes worldwide, diabetic neuropathic pain (DNP) has become a relatively frequent disease [5, 6] that affects 11–21% of diabetics and is characterized by aberrant pain sensation, including hyperalgesia, spontaneous pain, and allodynia [7,8,9]. Despite being the major symptom of diabetic neuropathy, the pathogenesis of DNP is still not fully understood.

P2X receptors are ligand-gated ion channels assembled into homotrimers or heterotrimers [10]. There are 7 known P2X receptors with 35–48% homology. P2X receptors are generally activated by adenosine 5′-triphosphate (ATP) and are involved in various physiologic and pathophysiologic processes, including inflammation and pain [11]. Since the identification of the link between injection of adenosine compounds into human skin blisters and pain initiation in 1977 [12], numerous studies have found that the activation of P2X receptors causes pain-related reactions in animals [13,14,15,16]. We previously reported the relationship between pain sensitization and P2X3 in dorsal root ganglia (DRG) of DNP rats [17, 18]. However, systematic studies of the involvement of other P2X receptors in DNP are lacking.

DNP is usually managed by using analgesics, like duloxetine, pregabalin, and opioids [19, 20], but these are not always effective and have significant side effects. Clinically, electroacupuncture (EA) is employed to treat various chronic pain disorders [21,22,23,24] and has potential use in DNP treatment [25]. However, the mechanisms underlying the effect of EA against DNP are not clear. Here, we used a rat model of streptozocin (STZ)-triggered DNP, and examined mRNA expression of P2X1–2 and P2X4–7 receptors in DRG at diverse times after STZ administration. Western blot (WB) and immunofluorescence (IF) were then used to investigate the protein expression of receptors with elevated mRNA content. The influences of EA on DNP rats and the expression of P2X4 and P2X7 in DRG were evaluated.

Materials and methods

Animals

Male Sprague–Dawley rats (200 ± 20 g) were bought from Shanghai Slac Laboratory Animal Co., Ltd (SCXK (hu) 2017–0005) and housed at Animal Laboratory Center of Zhejiang Chinese Medical University (SYXK (zhe) 2018–0012). Five rats per cage were housed under controlled conditions (20–24 °C, 40–60% relative humidity, and 12 h light/dark cycles) and free access to food and water. The rats were adapted to the new environment for 1 week before. Animal experiment protocols were approved by the animal welfare committee of Zhejiang Chinese Medical University (IACUC-20190805–04).

Generation of the DNP rat model

Rats were fasted for 16 h before intraperitoneal (i.p.) injection with 65 mg/kg of STZ (S0130, Sigma) in citrate buffer (0.1 mol/L, pH 4.5) [26, 27]. Rats in DNP group, DNP + normal saline group (DNP + NS group), DNP + TNP-ATP 50 nmol group, and DNP + TNP-ATP 100 nmol group were intraperitoneally inoculated with STZ, while control group (Ctrl group) and control + normal saline group (Ctrl + NS group) rats received an equal volume of vehicle. Fasting blood glucose (FBG) was measured 7 days after STZ inoculation. Rats with FBG content > 13.9 mmol/L [28] and thermal hyperalgesia considered to be successful DNP models.

Experimental groups

There were 3 experiment types. In experiment type 1, rats were assigned at random into a Ctrl group (n = 16), with tissues being collected 21 days after STZ injection, and a DNP group (n = 56). Of those in the DNP group, 16 rats were sacrificed for tissues 7 days after STZ administration, 16 were sacrificed 14 days after administration, 16 were sacrificed at 21 days after administration and tissues collected, four rats died, and four were modeled unsuccessfully. Body weight (BW), FBG, and paw withdrawal latency (PWL) were examined as per the schedule on Fig. 1a. Changes in mRNA and protein levels of P2X1–2 and P2X4–7 receptors in DRG were assessed at various DNP stages.

Fig. 1
figure 1

Establishment of the DNP rat model. a Schematic representation of the process of establishing the DNP rat model. b Time course effect of STZ injection on BW. c Time course effect of STZ injection on FBG. d Time course effect of STZ injection on PWL. e Normalized area under the curve (AUC) analysis of d. AUC was standardized to the Ctrl group. Data are given as mean ± SD, n = 16 per group. **P < 0.01 vs. Ctrl group

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In experiment type 2, the involvement of P2X4 and P2X7 in DNP was assessed by treating the rats with TNP-ATP (2’,3’-O-(2,4,6-trinitrophenyl)-adenosine 5’-triphosphate), a nonspecific antagonist of P2X receptors. Twenty-four rats were randomly divided into a Ctrl + NS, a DNP + NS, a DNP + TNP-ATP 50 nmol, and a DNP + TNP-ATP 100 nmol group, 6 rats per group. One rat died and two were modeled unsuccessfully. Rats in DNP + TNP-ATP 50 nmol and DNP + TNP-ATP 100 nmol group were treated with reduplicative administrations from 15 to 21 days after STZ injection, while other groups received an equal volume of normal saline (NS). The BW, FBG, and PWL of all rats were recorded as outline in Fig. 5a.

Experiment type 3 assessed the influences of EA on DNP and P2X4, P2X7 in DRG. Rats were randomly divided into a Ctrl, a DNP, and a DNP + EA group (16 rats for each group). Four rats were modeled unsuccessfully. Rats in DNP + EA group received EA treatment from 15 to 21 days after STZ injection, once a day. BW, FBG, and PWL tests were done as outline in Fig. 6a and tissues from all group rats were collected 21 days post administration of STZ.

Fasting blood glucose and body weight measurement

FBG was measured using a glucometer (ACCU-CHEK Performa, Roche Diagnostics GmbH, Germany) at 1 day before STZ administration and 7, 14, and 21 days after STZ administration. BW was taken after starving the rats for 12 h followed by blood collection from the tail vein for FBG measurement.

Paw withdrawal latency test

PWL was evaluated by measuring hind paw withdrawal from a heat stimulus using a plantar test (37370, Ugo Basile, Italy). PWL tests in experiment types 1 and 3 were done at 1 day before STZ inoculation and then 7, 14, and 21 days after STZ injection (Fig. 1a, Fig. 6a). In experiment type 2, the PWL test was done as outline in Fig. 5a. Before testing, rats were acclimated to individual Plexiglas cubicles on a glass plate for at least 30 min. The cutoff time was then set at 30 s and the radiant heat at 40 to avoid injury to rats. The light beam was switched off and timing stopped upon paw withdrawal. Each rat was tested independently thrice, at 5-min intervals, and PWL given by mean latencies, expressed in seconds.

RT-qPCR analysis

After deep anesthesia using sodium pentobarbital (80 mg/kg, i.p.), bilateral DRGs from L4 to L6 of rats were collected and stored in trizol (Invitrogen Corporation, USA) at − 80 °C, followed by total RNA extraction according to trizol manufacturer instructions. Next, cDNA was generated from 2 μg of total RNA with the RevertAid First Strand cDNA synthesis kit (#K1622, Thermo, USA) and stored at − 80 °C. Gene expression was then analyzed by real-time quantitative polymerase chain reactions (RT-qPCRs) on an ABI Stepone plus RT-PCR system using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene. The following primers were used: GAPDH: forward 5′-CTGGAGAAACCTGCCAAGTATG-3′; reverse 5′-GGTGGAAGAATGGGAGTTGCT-3′; P2X1: forward 5′-GGTGGGAGTCATTTTCCGTCT-3′; reverse 5′-CTGATGAGGTCACTTGAGGTCTG-3′; P2X2: forward 5′-GGGCAGTGTAGTCAGCATCAT-3′; reverse 5′-TCAGAAGTTCCATCCTCCACC-3′; P2X4: forward 5′-GAAAAGGGCTACCAGGAAACG-3′; reverse 5′-TCAGGAATCTCTGGACAGGTGC-3′; P2X5: forward 5′-GGTTTGTGCTGTCTCTGTTCG-3′; reverse 5′-CCCAAGCATCGTGGTGTTAGT-3′; P2X6: forward 5′-TGCCTTAGATACCTGGGACAAC-3′; reverse 5′-GAGCAGTCAGAGCCTTTCGT-3′; P2X7: forward 5′-CTGGCTACAACTTCAGATACGC-3′; reverse 5′-GCCAAACCGAAATAGGACAGG-3′. The reaction was done in a final reaction volume of 15 μL comprised of 7.5 μL FastStart Universal SYBR Green Master (04,913 914 001, Roche, Germany), 1.5 μL sense and anti-sense primers (400 nM), 2 μL template cDNA, and 4 μL ribonuclease/deoxyribonuclease-free water. The reactions program was 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s. The reaction was then held at 60 °C for 60 s and of 60–95 °C melting curves generated. Each sample was analyzed in triplicate. Primer specificity was evaluated using melting curves and gel electrophoreses analysis. The 2−∆∆Ct method was adopted to determine relative expression levels.

Western blot analysis

After deep anesthesia with sodium pentobarbital (80 mg/kg, i.p.), bilateral L4–L6 DRGs were collected and kept at − 80 °C. To extract protein, tissues were minced using ophthalmic scissors on ice in RIPA Lysis Buffer (P0013B, Beyotime, China) enriched with protease inhibitors (P1050, Beyotime, China), sonicated, the homogenate cleared by centrifugation at 12,000 rpm at 4 °C for 20 min, and then collected the supernatants. The supernatants were diluted with 2 × loading buffer, protein concentration determined using BCA Protein Assay Kit (23225, Thermo Fisher, USA), and then denatured at 100 °C for 3 min. Proteins (20 μg) of each sample were fractionated with SDS-PAGE gels electrophoresis, transfer-embedded onto polyvinylidene difluoride membranes, and blocked with 5% nonfat milk in 1 × TBST (pH 7.5) for 1 h. They were then inoculated overnight with rabbit anti-P2X2 (1:1000, APR-003, Alomone, Israel), rabbit anti-P2X4 (1:1000; GTX54851, Gene Tex, USA), rabbit anti-P2X7 (1:1000; APR-004, Alomone, Israel), and β-actin (HRP conjugate, 1:5000; #12262, Cell Signaling Technology, USA) at 4 °C. After rinsing thrice in 1 × TBST, 10 min each, the membrane for β-actin was visualized by chemiluminescence (ECL Plus; Beyotime, China), while the membranes for P2X2, P2X4, and P2X7 were incubated with HRP-linked antibody (1:5000; #7074, Cell Signaling technology, USA) for 2 h at room temperature and then washed with 1 × TBST and developed by chemiluminescence. Band intensities were quantified on an Image Quant LAS 4000 system. Target protein contents were normalized against β-actin content. Image J software was adopted to analyze the optical densities of the target proteins and β-actin. And the relative expression amount of the target protein was obtained from the ratio of gray value of target protein to β-actin.

Immunofluorescence

Rats were anesthetized using sodium pentobarbital (80 mg/kg, i.p.) and transcardially perfused with saline (4 °C) followed by 4% paraformaldehyde. The L4–L6 DRGs were then removed and post-fixed in 4% paraformaldehyde for 4 h. They were then dehydrated with 15% sucrose solution for 24 h and 30% for 48 h (until DRGs sank into the sucrose solution). The DRGs were then sectioned at 10 µm using a frozen microtome and placed on glass slides. After that, sections were rinsed thrice with 1 × TBST (pH 7.4), 10 min per wash, and blocked using 10% donkey serum in 1 × TBST for 1 h at 37 °C. They were then incubated overnight with rabbit anti-P2X4 (1:200, APR-002, Alomone, Israel) and rabbit anti-P2X7 (1:500, GTX104288, Gene Tex, USA) at 4 °C. They were washed 6 times with 1 × TBST (10 min each) and then incubated with Alexa Fluor 488 donkey anti-rabbit IgG (1:400, Jackson, 711–545-152) for 1 h at 37 °C. They were then sealed using antifade solution and cover glass and imaged on an Imager M2 microscope (ZEISS, Germany). The number of positive cells was calculated. Three sections per rat (3 rats from each group) were analyzed.

Drug administration

TNP-ATP (2464, Tocris, USA) was diluted to desired concentrations in freshly 0.9% NS before administration. TNP-ATP was administered into DNP + TNP-ATP 50 nmol (50 μL, 50 nmol) and DNP + TNP-ATP 100 nmol groups (50 μL, 100 nmol) through subcutaneous injection at dorsum of foot (left hind paw) from D15 to D21, once a day. Ctrl + NS and DNP + NS group rats received an equal volume of NS.

EA treatment

Rats in DNP + EA group were treated with 2 Hz EA daily for 7 consecutive days. The acupuncture needle (0.25 mm * 13 mm, Hua tuo, Suzhou Medical Appliance Manufactory, Jiangsu, China) was inserted into bilateral Zusanli (ST36, posterior lateral to the knee joint and about 5 mm below the capitulum fibulae) and Kunlun (BL60, depression between lateral malleolus and Achilles tendon of the hind limb) acupoints of rats. Then the needles were connected to HANS acupoint electrical stimulation device (Hans-200A, Jisheng Medical Technology, Beijing, China) for 30 min, setting with 1 mA and 2 Hz. Rats in other groups received same calming procedure without EA.

Statistical analysis

Data were given as mean ± standard deviation (SD) and analyzed on SPSS 22.0 (IBM, USA). Comparisons between 2 groups were done using independent sample t tests. Comparisons among 3 (or over 3) groups were done using one-way ANOVA followed by LSD or Dunnett’s post hoc tests. For the behavioral tests, two-way repeated-measures ANOVA was used, followed by Bonferroni’s post hoc test, with P < 0.05 signifying statical significance.

Results

Establishment of a STZ-induced model of DNP

A type 1 diabetes model was developed via i.p. injection of high-dose STZ (65 mg/kg) and development of pain sensitization determined by behavioral testing (Fig. 1a). Compared to the Ctrl group, DNP rats exhibited remarkably higher FBG and lower BW 7 days after STZ injection (Fig. 1b,c, P < 0.01, respectively). PWL of DNP rats was remarkably reduced on D14 and this phenomenon remained to the end of the experiment (Fig. 1d, P < 0.01, respectively). Area under the curve (AUC) analysis revealed that PWL was remarkably lower in DNP group in contrast with the Ctrl group (Fig. 1e, P < 0.01). These data indicated that the DNP model was successfully established on D14.

Change in mRNA content of P2X receptors at various DNP stages

RT-qPCR analysis of P2X1-2, and P2X4-7 mRNA expression in rat DRGs at different stages after STZ administration revealed that in contrast with the Ctrl group, P2X2 and P2X4 were remarkably upregulated on D7 (Fig. 2b,c, P < 0.01, respectively), while P2X7 was remarkably elevated on D14 (Fig. 2f, P < 0.01). However, in contrast with the Ctrl group, P2X2 mRNA expression lost its difference on D21 (Fig. 2b, P > 0.05), while P2X4 and P2X7 mRNA expression remained consistently higher compared to Ctrl group (Fig. 2c,f, P < 0.01, respectively).

Fig. 2
figure 2

P2X mRNA content in DRG of the rat DNP model at various timepoints a–f mRNA content of P2X1, P2X2, P2X4, P2X5, P2X6, and P2X7 in DRG of rats from various groups. Data are given as mean ± SD, n = 5–6 per group. *P < 0.05, **P < 0.01 vs. Ctrl group

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Expression of P2X2, P2X4, and P2X7 in rat DRG at various DNP stages

Next, we used WB and IF to evaluate the protein content of P2X2, P2X4, and P2X7 in DRG from DNP rats (Figs. 3 and 4). WB analysis revealed that in contrast with the Ctrl group, the contents of P2X4 and P2X7 were markedly elevated on D21 (Fig. 3d,f, P < 0.01, respectively), while P2X4 was also evaluated on D14 (Fig. 3d, P < 0.05). However, P2X2 protein content did not differ remarkably in DRG of DNP rats in contrast with the Ctrl group (Fig. 3b, P > 0.05). IF analysis revealed that in contrast with Ctrl group, DNP rats had significantly higher P2X4 expression on D7, which persisted to D21 (Fig. 4b, P < 0.01, respectively), while P2X7 expression in DRG was elevated on D14 and persisted to D21 (Fig. 4c, P < 0.01, respectively).

Fig. 3
figure 3

Protein content of P2X2, P2X4, and P2X7 in rat DRG after STZ injection at various timepoints. a Representative images of P2X2 protein content in DRG. b Relative P2X2 protein level in DRG of rats from various groups. c Representative images of P2X4 protein content in DRG. d Relative P2X4 protein level in DRG of rats from various groups. e Representative images of P2X7 protein content in DRG. f Relative P2X7 protein level in DRG of rats from various groups. Data are given as mean ± SD, n = 5 per group. *P < 0.05, **P < 0.01 vs. Ctrl group

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Fig. 4
figure 4

Immunofluorescence analysis of P2X4 and P2X7 levels in DRG of DNP rats at various timepoints. a Representative images of P2X4 and P2X7 staining in DRGs from various groups. b Quantification of P2X4 positive cells in DRG of rats from various groups. c Quantification of P2X7 positive cells in DRG of rats from various groups. Scale bars = 50 μm. Data are given as mean ± SD, n = 3 per group. **P < 0.01 vs. Ctrl group

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Effects of single and multiple TNP-ATP injections in different doses on DNP rats

We verified the involvement of P2X4 and P2X7 in the process of DNP via single and multiple TNP-ATP subcutaneous injections at dorsum of foot and then measured the PWL changes (Fig. 5). At 15 days after STZ administration, TNP-ATP was administered into rats at left hind paw with two doses (50 nmol, 100 nmol) and BW, FBG, and PWL were measured as outline in Fig. 6a. This analysis illustrated that in contrast with the DNP + NS group, BW and FBG in DNP rats were not affected by TNP-ATP at both doses (Fig. 5b,c P > 0.05, respectively). Next, we examined PWL at 0.5 h, 1 h, and 1.5 h after TNP-ATP administration on D15 and found that in contrast with the DNP + NS group, 100 nmol TNP-ATP increased PWL at 0.5 h after injection (Fig. 5d, P < 0.05), but 50 nmol of TNP-ATP did not change the PWL (Fig. 5d, P > 0.05). To determine if repetitive TNP-ATP administrations affect PWL, we treated the DNP rats with TNP-ATP (50 nmol and 100 nmol) daily from D15 to D21 (Fig. 5a). This analysis illustrated that in contrast with DNP + NS group, repetitive administrations of 100 nmol TNP-ATP remarkably increased the PWL of DNP rats on D21 (Fig. 5e, P < 0.01), but 50 nmol dose did not affect PWL in DNP rats (Fig. 5e, P > 0.05).

Fig. 5
figure 5

Effects of various TNP-ATP doses on PWL in DNP rats. a Schedule for TNP-ATP treatment. b Time course effect of STZ and TNP-ATP on BW. c Time course effect of STZ and TNP-ATP on FBG. d Effect of the single administration of 50 and 100 nmol TNP-ATP on PWL in DNP rats. e Effect of the multiple administrations of 50 and 100 nmol TNP-ATP on PWL in DNP rats. Data are given as mean ± SD, n = 6 per group. *P < 0.05, **P < 0.01 vs. Ctrl + NS group. #P < 0.05, ##P < 0.01 vs. DNP + NS group

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Fig. 6
figure 6

Effect of EA on PWL in DNP rats. a Schedule of DNP rat model establishment and EA treatment. b Time course effect of STZ and EA treatment on BW. c Time course effect of STZ and EA treatment on FBG. d Analgesic effects of EA on PWL. e Normalized AUC analysis for D14 to D21 of d. Data are given as mean ± SD, n = 16 per group. *P < 0.01 vs. Ctrl group. ##P < 0.01 vs. DNP group

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EA relieves thermal hyperalgesia in DNP rats

Next, we treated STZ-induced DNP rats with EA for 7 consecutive days as outline in Fig. 6a. This analysis revealed that in contrast with the Ctrl group, rats in DNP and DNP + EA groups exhibited lower PWL at 14 days after STZ administration (Fig. 6d, P < 0.01, respectively). EA treatment for 7 days increased PWL in contrast with DNP rats on D21 (Fig. 6d, P < 0.01). AUC analysis of PWL from D14 to D21 exhibited the overall impact of EA on thermal hyperalgesia in DNP rats (Fig. 6e, P < 0.01). In contrast with the DNP group, EA did not affect BW and FBG in DNP rats (Fig. 6b,c, P > 0.05, respectively).

EA reduces the mRNA level of P2X4 and P2X7 in DRG of DNP rats

RT-qPCR analysis of P2X4 and P2X7 mRNA expression in DRG after EA treatment for 7 days revealed that both remarkably upregulated in DRG of DNP rats in contrast with Ctrl group (Fig. 7a,d, P < 0.01 for P2X4, P < 0.05 for P2X7), and that EA treatment reduced their mRNA expression in contrast with the DNP group (Fig. 7a,d, P < 0.05, respectively).

Fig. 7
figure 7

Effect of EA on the mRNA levels of P2X4 and P2X7 in DRG. a mRNA level of P2X4 in DRG of rats from various groups. b mRNA level of P2X7 in DRG of rats from various groups. Data are given as mean ± SD, n = 5–6 per group. *P < 0.05, **P < 0.01 vs. Ctrl group. #P < 0.05 vs. DNP group

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EA reduces P2X4 and P2X7 expression in DRG of DNP rats

Next, we used WB and IF to assess if EA treatment for 7 days affects the protein content of P2X4 and P2X7 in DRG of DNP rats (Figs. 8 and 9). WB analysis revealed that in contrast with the Ctrl group, P2X4 and P2X7 protein expressions were elevated in DNP group (Fig. 8b,d, P < 0.01 for P2X7, P < 0.05 for P2X4), while EA treatment reduced P2X4 and P2X7 protein expression in DNP + EA group in contrast with DNP group (Fig. 8b,d, P < 0.05, respectively). IF analysis revealed that DNP rats had higher P2X4 and P2X7 expression levels in DRG in contrast with Ctrl group (Fig. 9b,c, P < 0.01, respectively), and that EA treatment markedly inhibited their expression in contrast with DNP group (Fig. 9b,c, P < 0.01, respectively).

Fig. 8
figure 8

Effect of EA on the protein level of P2X4 and P2X7 in DRG. a Representative images of P2X4 protein level in DRG. b Relative P2X4 protein level in DRG of rats from various groups. c Representative images of P2X7 protein level in DRG. d Relative P2X7 protein level in DRG of rats from various groups. Data are given as mean ± SD, n = 5 per group. *P < 0.05, **P < 0.01 vs. Ctrl group. #P < 0.05 vs. DNP group

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Fig. 9
figure 9

Effect of EA treatment on P2X4 and P2X7 levels in DRG. a Representative IF images of P2X4 and P2X7 in DRG from various groups. b Quantification of P2X4 positive cells in rat DRGs from various groups. c Quantification of P2X7 positive cells in rat DRGs from various groups. Scale bars = 50 μm. Data are given as mean ± SD, n = 3 per group. **P < 0.01 vs. Ctrl group. ##P < 0.01 vs. DNP group

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Discussion

STZ-induced diabetes recapitulates various aspects of human diabetes, entailing insulin deficiency, weight loss, and hyperglycemia [29, 30], and is also widely used in the investigation of the mechanisms underlying DNP and potential therapies. Here, we generated a rat model of DNP by intraperitoneally injecting with a single large dose of STZ (65 mg/kg). Our findings showed that at 7 days after STZ administration, the FBG of the DNP group rats increased and the BW decreased. And the PWL in DNP group rats reduced at 14 days post STZ administration, indicating successful generation of DNP rat model.

Chronic neuropathic pain often begins with peripheral nerve injury. Once the initial irritant injury occurs, a cascade of inflammatory mediators will be activated peripherally and centrally [31]. The DRG, which is located at the junction of the peripheral and central nervous system, is an ideal target for pain control [32]. P2X receptors are ligand-gated ion channels (Na+, K+, and Ca2+) which regulate rapid response [33]. These receptors can be activated by ATP, leading to intercellular communication and inflammatory transmission [34]. Ionotropic P2X receptors are reported to be involved in pain signaling under physiologic conditions [35]. Until now, the relationship between P2X3, P2X4, P2X7, and pain sensitization is clearer than other subtypes [36,37,38]. However, it is not clear if P2X1, P2X2, P2X5, and P2X6 receptors in DRG are involved in neuropathic pain.

P2X1 receptor has been implicated in the development of thrombotic inflammatory diseases [39]. The ATP-activated P2X2, P2X4, and P2X6 receptors in the 2nd layer of the spinal cord are involved in the transmission of nociceptive information [40], while in spinal the P2X2 receptor also mediates neuropathic pain, which can be affected by drugs [41]. In addition, P2X5 has been implicated in peripheral pain [42]. We have previously shown that in DRG, P2X3 takes part in the development of DNP [17, 18]. Here, analysis of the mRNA content of P2X1–2 and P2X4–7 receptors in DRG at different DNP stages revealed that the mRNA levels of P2X2, P2X4, and P2X7 receptors were increased in DNP rats. Both WB and IF results showed that P2X4 and P2X7 protein contents were increased but P2X2 did not change remarkably. These results are consistent with previous findings [43, 44].

TNP-ATP is a nonspecific P2X receptor antagonist that inhibits all P2X receptors [45, 46]. Intrathecal injection of TNP-ATP is reported to block tactile allodynia triggered by L5 nerve injury and to inhibit P2X4 activation in microglia [47]. TNP-ATP has also been shown to inhibit chicken P2X7 receptor and chicken P2X7 receptor-associated current has been shown to have similar properties to those P2X7 receptors in other animals [48]. To determine if the P2X4 and P2X7 in DRG were involved in thermal hyperalgesia in DNP rats, we administered single and multiple injections of various doses of TNP-ATP into the dorsum of the foot and found that single and multiple injections of TNP-ATP at high dose (100 nmol), but not the low dose (50 nmol), reversed DNP-induced pain sensitization. Thus, TNP-ATP dose dependently reduced thermal hyperalgesia in DNP rats. Altogether, these findings illustrate that elevated expression of P2X4 and P2X7 may contribute to diabetes-induced hyperalgesia.

The extensive application of electroacupuncture has been proven by multifaceted research, especially in the aspect of analgesia [49,50,51,52]. Studies show that EA relieves pain by regulating signaling pathways and inflammatory cytokines [53, 54]. However, it is not clear if EA influences hyperpathia in DNP rats by regulating P2X4 and P2X7 in DRG. Because the DNP rat model was successfully established at 14 days after STZ injection, we performed EA treatment from 15 to 21 days after STZ injection. The results show that EA increased the PWL in DNP rats and reduces the expression of P2X4 and P2X7 in DRG. Our data indicate that EA may relieve DNP by regulating P2X4 and P2X7 in DRG.

Conclusions

In summary, our results indicate that activation of P2X4 and P2X7 in L4–L6 DRGs contributes to diabetes-induced hyperalgesia and that EA may ameliorate pain hypersensitivities in a rat model of DNP by suppressing P2X4 and P2X7 upregulation in DRG. Our findings highlight the therapeutic potential of EA in clinical DNP management.