Introduction

Diabetes mellitus (DM) has always been one of the central issues in the medical community and its occurrence has been excessively increasing since the 21st century, and the development of DM will affect about one-tenth of the world’s adults [1,2]. Diabetic neuropathic pain (DNP) is a diabetic complication with common symptoms including tingling, numbness, nociceptive hyperalgesia, allodynia, and hypoalgesia [3,4]. It is because DNP causes so much suffering to patients, which is essential to find an efficient treatment with minimum side effects. While several possible mechanisms have been identified, the precise molecular mechanisms underlying DNP are still unclear yet.

Previous studies have shown that there are diverse views on the neurophysiological mechanisms associated with neuropathic pain, and one possible hypothesis is that calcium-associated cascade reactions lead to receptor damage and sensitization [5]. Studies have shown that different types of calcium signaling conduction processes have a prominent effect on the variation of Ca2+ concentration and Calcium/calmodulin-dependent protein kinase II (CaMKII) [6,7]. CaMKII is extensively seen in the central and peripheral nervous system. It has a major contribution to molecular memory and neuronal excitability by converting transitory changes in calcium concentration into changes in cellular activity [8,9]. CaMKII is formed by 12 catalytically active subunits [10], each encoded by 4 genes [11]. As a reaction to Ca2+ elevations induced by extracellular stimulation, CaMKII at the 286 threonine position (Thr286) is self-phosphorylated by the adjacent kinase domain and has Ca2+-independent activity [12,13]. Self-phosphorylation of CaMKIIα continues to activate enzymes and downstream pathways efficiently even after injury-induced calcium influx [14].

CaMKIIα is mainly located on the surface layer of spinal cord dorsal horn (SCDH) and the primary sensory neurons in dorsal root ganglion (DRG), which is essential for the transmission and processing of nociceptive signals [15,16]. Studies could confirm that CaMKIIα might be a possible new target for the therapy of long-term pain [17,18]. Thus, this study mainly concentrated on the mechanism of phosphorylated CaMKIIα (p-CaMKIIα) in SCDH and DRG of DNP rats.

Combining traditional acupuncture with modern electrical stimulation, electroacupuncture (EA) is already clinically recognized as an efficient supportive treatment for the relief of a multitude of pain conditions [19,20]. EA has been officially recognized by international organizations for its effectiveness and is utilized by millions of patients for pain relief and anti-inflammatory. Numerous studies are available to illustrate the analgesic action of EA on different neuropathic pain including DNP [21–24], however, the underlying mechanism has not been fully elucidated.

We hypothesized that p-CaMKIIα in SCDH and DRG was involved in the DNP process. Additionally, we hypothesized that the analgesic action of EA involved in DNP is accomplished through p-CaMKIIα in SCDH and DRG.

Materials and methods Animals

Sprague–Dawley (SD) rats (adult, male, 180–220 g) were bought from the Shanghai Laboratory Animal Center (SCXK (hu) 2017 - 0005). The animals were placed under temperature control at 25 ± 20°C, 12 h of illumination/darkness (light on at 6 a.m., light off at 6 p.m.), and 55% ± 5% humidity. They were provided with adequate food and water that they could obtain unfetteredly. All experimental methods and treatments are monitored by Zhejiang Chinese Medical University Animal Welfare Committee to ensure that all procedures meet the requirements.

Induction of the DNP model

In order to induct DM, following a 16-hour fast, the rats will be given intraperitoneal (i.p.) injections with a large dosage of streptozotocin (STZ) (65 mg/Kg) dissolved in 0.1M citric acid-sodium citrate buffer (pH = 4.5) as described before [25]. After 2 weeks of the injection, rats whose fasting blood glucose (FBG) inclusion criteria were more than 16.7 mmol/L and accompanied by a behavioral sensitivity to pain are considered successful DNP models. We performed STZ i.p. injection in a total of 70 healthy SD rats, and the DNP model was successfully established in 63 rats, (5 were excluded for not meeting the DNP standard, and 2 died during the experiment), with a modeling success rate of 90%. Besides, rats in the Normal group were intraperitoneally injected with the same dose of citric acid-sodium citrate buffer.

Experimental design

There were 3 experiment phases. In experiment phase 1, there were 8 rats in the Normal group, and the tissues were collected 28 days after citric acid-sodium citrate buffer injection. 32 rats were divided into the Model group and injected with STZ, of which 8 rats were sacrificed for tissues 7 days after administration, 8 rats were sacrificed for tissues 14 days after administration, 8 rats were sacrificed for tissues 21 days after administration, 8 were sacrificed at 28 days after administration and tissues collected. The paw withdrawal threshold (PWT), body weight (BW), and FBG of rats were recorded as outlined (Fig. 1a).

In experiment phase 2, there were 5 rats in the Normal group. 15 rats were given intraperitoneal injections with STZ, randomly divided into a Model, a Model + Vehicle, and a Model + KN93 group, 5 rats per group. The PWT, BW, and FBG of rats were recorded as outlined (Fig. 3a).

In experiment phase 3, there were 8 rats in the Normal group. 16 rats were given intraperitoneal injections with STZ and then randomly divided into a Model, a Model + EA group, 8 rats per group. The PWT, BW, and FBG of rats were recorded as outlined (Fig. 5a).

The changes in PWT in diabetic neuralgia rats after STZ administration were observed. Western blot analysis and immunofluorescence were operated to explore the role of p-CaMKIIα in SCDH and DRG of DNP rats. Furthermore, part of the rats were injected with KN93, a CaMKII antagonist, to confirm the participation of p-CaMKIIα in the DNP model [26]. Last, we came to evaluate the analgesic action of EA and to observe the expressions of CaMKIIα and p-CaMKIIα in DNP rats.

Diabetes-related physiological conditions

For each blood glucose measurement, the rats were fasted for 8 h without cutting off water, and then blood was taken at the end of the caudal vein for measurement with a glucometer (ACCU-CHEK Performa, Roche Diagnostics GmbH, Germany). Initial BW and FBG of rats were measured before STZ injection and once a week after STZ administration.

Behavioral experimental method

The von Frey hair test was conducted to access the PWT of rats, which were for the neuropathic pain assessment. The PWT was measured on base 1 day before STZ injection and once a week after STZ administration. The rats were housed on a wire grid and each was partially restricted by a clear acrylic-covered box. After 30 min acclimation, the method of up-down was used for measuring mechanical nociception. Von Frey hairs with increasing hardness were applied to the plantar area of the rats’ hind paws in order to induce paw withdrawal. The PWT was recorded in grams, which was equal to the von Frey hair hardness that caused 50% paw withdrawal [27]. Ensure that each manipulation is handled by the same operator to avoid handling errors.

Electroacupuncture treatment

The Model + EA group rats were DNP rats that accepted EA treatments. Kunlun (BL60) and Zusanli (ST36) were selected as targeting acupoints. Kunlun (BL60) was positioned between the outer ankle and Achilles tendon of the foot, and Zusanli (ST36) was positioned posterior lateral to the knee joint and about 5 mm below the caput fibulae. For the EA operation, the rats were first softly restrained to avoid struggling and after cleaning the skin of rats with alcohol cotton swabs, the acupuncture needles (0.25 mm × 13 mm) were quickly inserted into the bilateral acupoints about 1/2 inch deep. A HANS electrical stimulation device was connected to the acupuncture needles with parameters of 1 mA and 2 Hz. The stimulation was performed 30 min every other day, 2 weeks after the STZ injection. The respiratory condition, heartbeat and body temperature of rats were concerned. After treatment, all rats were allowed to move freely in the cage. Other rats underwent the same restraint without acupuncture or EA treatment.

Protein extraction and western blot analysis

After the animal experiments were completed, the rats were anesthetized with sodium pentobarbital solution (55 mg/Kg, i.p.). The rats were perfused transcardially with 0.9% saline, and the desired SCDH and L4-L6 DRG tissues were removed and cryopreserved. The tissues were ground and centrifuged (12 000 rpm, 20 min) using Ripa buffer under cryogenic conditions, and the supernatant was aspirated and BCA kit was used to measure protein concentrations.

8% SDS-PAGE gels were used to separate protein samples, then the protein was transferred to PVDF membrane. Mixed with defatted milk powder at 5% for 1 h at ambient temperature using Tris-buffered saline tween (TBST, pH = 7.5), and added p-CAMKIIα (1:1000, Abcam, ab5683) and GAPDH (1:5000, CST, 3683) after blocking the bands for 16 h at 4 °C. HRP-coupled secondary antibodies (1:5000, CST, 7074) were incubated at ambient temperature for 2 h. After membrane regeneration CAMKIIα (1:1000, CST, 50049) was added. Apply Image-J and Adobe Illustrator software to analyze the images. Using GAPDH as a normalized protein.

Immunofluorescence

Rats were in deep anesthesia using 55 mg/Kg concentration of sodium pentobarbital solution, and sequentially anesthetized using 0.9% saline and paraformaldehyde by transcardial perfusion. The SCDH and L4-L6 DRG tissues were harvested and stored in paraformaldehyde for fixation, and then stored at −80 °C after gradient dehydration using sucrose solution for 72 h at 4 °C until complete dehydration. The tissues were wrapped with optimum cutter temperature (OCT) and cut into SCDH sections of 30 μm thickness and DRG sections of 10 μm thickness using a frozen microtome, and the sections were fixed to glass slides. All slides were blocked with TBST (pH = 7.4) containing 10% normal donkey serum for an hour at ambient temperature, and the corresponding primary antibodies were incubated overnight at 4 °C, the primary antibodies used were p-CaMKIIα (1:400, Abcam, ab5683) and NeuN (1:400, Abcam, ab104224). The following day, slides were rinsed 6 times with TBST for 10 min each, and then incubated for an hour in darkness. Tissues were labeled with fluorescent secondary antibodies (Cy3-, Cy5-, or FITC-conjugated) and washed for 1 hour. Images were taken uniformly using Zeiss Structured Illumination Optical Section Microscope to generate fluorescent images. The dorsal horn of spinal cord was photographed, and the cell-rich parts in the same area of each DRG tissue were photographed, and 5–8 sections were photographed on each slide. There were three rats in each group, and 3 discontinuous spinal cord or DRG sections of each rat were randomly selected for quantification. The images were quantified using ImageJ software and then analyzed.

Intrathecal injection

Using 5% isoflurane to anesthetize the rat, a 2 cm long vertical skin cut was made at the mid-line of the lumbar region using a scalpel. A PE-10 polyethylene catheter filled with saline is trimmed to the desired length and gently inserted into the subarachnoid space. Keeping the catheter angle parallel to the spinal cord at all times, it is slowly advanced toward the head to the desired position within the spine, and successful catheter placement is evidenced by features such as tail-flicking during insertion. Leave the catheter in place for 2 cm outside the body and close the catheter port. After the animal was awake, the success of catheter placement was verified again with 2% lidocaine.

Drug treatment

KN93 (422708, Sigma-Aldrich) was dissolved in 100% dimethyl sulfoxide (DMSO, ST038, Beyotime). Then dilute the liquid with 0.9% saline to 50 nmol/L [28]. In the Model + KN93 group, each rat was intrathecally injected with 25 μL KN93 solution, and then injected with 25 μL saline to fully inject the drug into the subarachnoid space. The rats in the Normal, Model, and Model + Vehicle groups were injected with the same volumes of DMSO and saline miscible liquids.

Statistical analysis

Data were statistically analyzed using GraphPad Prism 8 software, and all outcomes were presented as mean ± SEM. The independent sample t test was used to compare the data between the two groups. One-way ANOVA or two-way ANOVA followed by LSD test and Dunnett’s T3 test were applied to compare the data between multiple groups based on the homogeneity of variance. P < 0.05 was considered statistically significant.

Results Establishment of DNP model

The experimental results are seen in comparison with the Normal group, the BW and the FBG increased (Fig. 1b and c) (BW: F(4, 70) = 73.02, P < 0.0001; FBG: F(4, 70) = 77.18, P < 0.0001) after intraperitoneal injection of STZ in the Model group rats. Furthermore, we observed the allodynia was aggravated after STZ intraperitoneal injection in the Model group rats, with a decrease in PWT since the 14 days after drug injection (Fig. 1d and e) (PWT: F(4, 70) = 7.617, P < 0.0001; Area under the curve of the PWT: P = 0.0004). It’s demonstrated that a high dose injection of STZ could succeed in inducing DNP model.

Fig. 1:

Changes of BW, FBG, and PWT of DNP rats at different points of time. (a) Experimental protocol. (b) Rats’ BW at different points of time. (c) Rats’ FBG at different points of time. (d) Rats’ PWT at different points of time. (e) Area under the curve of the PWT (n = 8 per group). Data are expressed as the mean ± SEM. **P < 0.01 vs. Normal group.

The expressions of p-CaMKIIα were upregulated both in DNP rats’ SCDH and DRG

We checked the level of p-CaMKIIα in rats’ SCDH by western blot assay (Fig. 2a), and it showed that the expression was upregulated after the injection of STZ from 7 to 28 days as expected (Fig. 2b) (D7: P = 0.1278; D14: P = 0.0151; D21: P = 0.0366; D28: P = 0.0016). Although there were also some upward trends in CaMKIIα, it was not statistically different (Fig. 2c) (D7: P = 0.5844; D14: P = 0.1874; D21: P = 0.1930; D28: P = 0.5117). The degree of CaMKIIα phosphorylation was obtained from the ratio of p-CaMKIIα to CaMKIIα (Fig. 2d) (D7: P = 0.1601; D14: P = 0.0490; D21: P = 0.0055; D28: P = 0.0308).

Fig. 2:

Expressions in SCDH and DRG of DNP rats at different points in time. (a) Representative western blot images of CaMKIIα and p-CaMKIIα in SCDH in each group (n = 5 per group). (b) The relative levels of p-CaMKIIα in SCDH in each group. (c) The relative levels of CaMKIIα in SCDH in each group. (d) The ratio of p-CaMKIIα and CaMKIIα in SCDH in each group. (e) Representative images of p-CaMKIIα in SCDH of DNP rats at different points in time after STZ injection (n = 3 per group, green represents p-CaMKIIα, red represents neurons. Scale bar 100 µm). (f) Representative images of p-CaMKIIα in DRG at different points in time after STZ injection (green represents p-CaMKIIα, red represents neurons. Scale bar 50 µm). (g) Average fluorescent intensity of p-CaMKIIα in SCDH of DNP rats at different points in time. (h) Quantification of the p-CaMKIIα ratio of positive cells in DRG of DNP rats in each group. Average fluorescent intensity of p-CaMKIIα in DRG of DNP rats at different points in time. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01 vs. Normal group.

To further verify whether p-CaMKIIα participated in the pain process of DNP in SCDH, we used immunofluorescence to assess the expression of various groups at different points in time. The results were further corroboration by the previous western blot assay findings that expressions of p-CaMKIIα were increased in SCDH in DNP rats at different points in time (Fig. 2e and g) (F (4, 10) = 62.68, P < 0.0001).

Next, to confirm whether p-CaMKIIα played a role in pain process of DNP in DRG, immunofluorescence was used to assess the expression in the DRG at different time points (Fig. 2f). As expected, we obtained similar conclusions that the expression in DRG of the Model group was elevated at different points of time compared with the Normal group, with a roughly trending increase (Fig. 2h) (ratio of p-CaMKIIα positive cells in DRG: F(4, 10) = 14.59, P = 0.0004; average fluorescent intensity of p-CaMKIIα in DRG: F(4, 10) = 11.22, P = 0.0010).

KN93 increased the PWT and decreased the expression of p-CaMKIIα in DNP rats

To elucidate the effects of p-CaMKIIα on DNP rats, we treated rats with KN93 every 2 days since 14 days after the injection of STZ. The results showed that in comparison with the Model group, BW and FBG of rats in the Model + KN93 group were no significant difference (Fig. 3b and c) (all P between the Model group and the Model + KN93 group > 0.05). However, after the injection of KN93, the PWT of rats was improved, and even after 2 weeks of KN93 dose, the PWT of the Model + KN93 group could approach the level of the Normal group (Fig. 3d and e) (PWT: F(12, 80) = 9.434, P < 0.0001; Area under the curve of the PWT: F(3, 16) = 26.62, P < 0.0001).

Fig. 3:

The effects of KN93 on DNP model rats at different time points. (a) Experimental protocol. (b) Rats’ BW at different points of time in each group. (c) Rats’ FBG at different points of time in each group. (d) Rats’ PWT at different points of time in each group. (e) The area under the curve of the PWT in each group (n = 5 per group). Data are expressed as the mean ± SEM. **P < 0.01 vs. Normal group. $$P < 0.01 vs. Model+ vehicle group. No significance (NS) vs. Model group.

We observed that KN93, besides reversing hyperalgesia, also reduced the expression of p-CaMKIIα at rats’ SCDH. We examined the level of p-CaMKIIα in SCDH in various groups by western blotting assay. The findings expressed that based on the success of DNP model, KN93 injection reduced the expression of SCDH in DNP rats (Fig. 4) (p-CaMKIIα/GAPDH: F(3, 16) = 19.04, P < 0.0001; CaMKIIα/GAPDH: F(3, 16) = 3.196, P = 0.0519; p-CaMKIIα/CaMKIIα: F(3, 16) = 13.43, P = 0.0001). Nociceptive hypersensitivity could be reversed by KN93, which further demonstrated the involvement of p-CaMKIIα in the DNP process.

Fig. 4:

The expression of p-CaMKIIα and CaMKIIα in the rats’ SCDH after KN93 intrathecal injection. (a) Representative western blot images of p-CaMKIIα and CaMKIIα in SCDH in each group. (b) The relative levels of p-CaMKIIα in SCDH in each group. (c) The relative levels of CaMKIIα in SCDH in each group. (d) The ratio of p-CaMKIIα and CaMKIIα in SCDH in each group (n = 5 per group). Data are expressed as the mean ± SEM. **P < 0.01 vs. Normal group. $P < 0.05, $$P < 0.01 vs. Model+ vehicle group. No significance (NS) vs. Model group.

Effects of EA on mechanical pain threshold and p-CaMKIIα expression in DNP rats

We explored the way by which EA is involved in the alleviation of DNP by observing changes in behavior and expression levels. To investigate the role of EA in DNP rats, we administered EA treatment every other day since day 14 after STZ injection. We observed that both BW and FBG of the animals were no significant difference between the Model + EA group and the Model group (Fig. 5b and c) (all P between the Model group and the Model + EA group > 0.05), while EA could reduce the nociceptive hypersensitivity in DNP rats (Fig. 5d and e) (PWT: F(8105) = 15.29, P < 0.0001; Area under the curve of the PWT: F(2, 21) = 28.61, P < 0.0001).

Fig. 5:

The effects of EA in SCDH for DNP rats. (a) Experimental protocol. (b) Rats’ BW in each group. (c) Rats’ FBG in each group. (d) Rats’ PWT in each group. (e) The area under the curve of the PWT (n = 8 per group). Data are expressed as the mean ± SEM. **P < 0.01 vs. Normal group. #P < 0.05, ##P < 0.01 vs. Model group.

We checked the level of p-CaMKIIα in SCDH by western blotting assay (Fig. 6a), it showed that the expression was downregulated after the EA treatment (Fig. 6b), while CaMKIIα was not statistically different (Fig. 6c). The degree of CaMKIIα phosphorylation was obtained from the ratio of p-CaMKIIα to CaMKIIα (Fig. 6d) (p-CaMKIIα/GAPDH: F(2, 12) = 15.81, P = 0.0004; CaMKIIα/GAPDH: F(2, 12) = 6.098, P = 0.0149; p-CaMKIIα/CaMKIIα: F(2, 12) = 6.032, P = 0.0154).

Fig. 6:

The expression of p-CaMKIIα and CaMKIIα in the rats’ SCDH and DRG with EA intervention. (a) Representative western blot images of CaMKIIα and p-CaMKIIα in SCDH in each group (n = 5 per group). (b) The relative levels of P-CaMKIIα in SCDH in each group. (c) The relative levels of CaMKIIα in SCDH in each group. (d) The ratio of p-CaMKIIα and CaMKIIα in SCDH in each group. (e) Representative images of p-CaMKIIα in SCDH of DNP rats from various groups (n = 3 per group). Green represents p-CaMKIIα, red represents neurons. Scale bar 100 µm). (f) Average fluorescent intensity of p-CaMKIIα in SCDH of rats in the Normal, Model, and Model + EA groups. (g) Representative images of p-CaMKIIα in DRG of DNP rats from various groups (green represents p-CaMKIIα, red represents neurons. Scale bar 50 µm). (h) Quantification of the p-CaMKIIα ratio of positive cells in DRG of DNP rats in the Normal, Model, and Model + EA groups. Average fluorescent intensity of p-CaMKIIα in DRG of rats in the Normal, Model, and Model + EA groups. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01 vs. Normal group. #P < 0.05, ##P < 0.01 vs. Model group.

To more systematically confirm the involvement of EA in the pain process of DNP rats via p-CaMKIIα in SCDH, we used immunofluorescence to assess the expression of different groups (Fig. 6e). We found compared with the Normal group, the expression of the Model group was upgraded. Compared to the Model group, the expression of the Model + EA group was decreased (Fig. 6f) (F(2, 6) = 26.68, P = 0.0010). This illustrated that EA can reverse nociceptive hypersensitivity in SCDH when the DNP rat model is established successfully.

To examine the participation of EA in the pain process of DNP rats through p-CaMkIIα in the DRG, we used immunofluorescence to assess the expressions in different groups (Fig. 6g). Similar results illustrated that EA can also reverse nociceptive hypersensitivity in DNP via p-CaMkIIα in DRG (Fig. 6h) (ratio of p-CaMKIIα positive cells in DRG: F(2, 6) = 12.30, P = 0.0075; average fluorescent intensity of p-CaMKIIα in DRG: F(2, 6) = 201.7, P < 0.0001).

Discussion

As the incidence of DM is growing all over the world, DNP, a common DM complication, has become more frequent and is gradually becoming one of the leading reasons for neuropathic pain [29]. Statistics showed 60% of patients with chronic DM are affected by DNP [30]. DNP is common in patients with chronic diabetes and causes great suffering in their lives, which may even develop into disability. The 10-year mortality rate is higher in patients with DNP than the painless ones [31]. The quality of health of patients with DNP is deeply affected by pain and causes great suffering in their daily lives [32–34]. The current clinical treatment for DNP is based on antidepressants and opioids, which have high side effects, so it is urgent to find efficient analgesics with low side effects [35]. Acupuncture is an age-old treatment that has been passed down for thousands of years, and its analgesic efficacy has been proven throughout history [36,37]. EA is a masterful combination between modern technology and classic acupuncture techniques, which has been extensively used in clinical practice as a proven therapy [38]. Thus, identifying EA analgesic mechanisms and applying them to clinical pain treatment is of positive significance in reducing patient suffering.

Recently, compared with the correlation researches on DNP, more studies emphasized the mechanism of CaMKII with cardiovascular disease and lipid metabolism caused by diabetes [39–43]. However, a key element of CaMKII has been recognized as a regulator of various cascades, which responded to a variety of different signal transmission pathways and increases [44]. Inhibition of TRPV2 in the hypothalamus of DM rats by trinostat treatment reversed the molecular actions of DM and increased CaMKII phosphorylation level, thereby reducing intracellular calcium ion influx [45].

Currently, DNP models are commonly used for mechanism exploration or drug development. Studies have shown that STZ injection can successfully establish a DNP model, so this model was used in this experiment to investigate the mechanism of EA to alleviate DNP. Our previous studies have found that the upregulation of p-CaMKIIα in DNP rats’ DRG may be an underlying mechanism of the development of DNP [46]. According to the experimental results, it was found that the expressions of p-CaMKIIα of DNP model group rats had increased in both SCDH and DRG compared to the normal group, while CaMKIIα had no statistical differences. This is consistent with our hypothesis that the CaMKIIα, as a subtype of the CaMKII family, is phosphorylated and the expressions of it were elevated in SCDH and DRG in DNP model rats, which is induced by STZ.

According to a study by Wong et al. [47], KN93, a selective CaMKII inhibitor, is bound directly to Ca2+/CaM instead of CaMKII. This combination would destroy the interaction between Ca 2+/CaM and CaMKII, thus successfully blocking the activation of CaMKII. Based on the research, KN93, a CaMKII inhibitor, improved mechanical pain thresholds after intrathecal injection into rats, while reducing the expression of p-CaMKIIα in SCDH and DRG. This further confirmed the involvement of p-CaMKIIα in pain regulation.

Furthermore, there is increasing evidence of EA treatment for people with diabetes with clinical implications [48,49]. In further studies of the mechanism of DNP, we used EA treatment to intervene in the DNP rats and observe the body weight, FBG, and PWT at different points in time after STZ injection. In this particular part of the study, we observed the changes in the expressions of p-CaMKIIα in both SCDH and DRG, as they are representative of central and peripheral nervous systems, respectively. We found that EA had a significant upregulation of pain threshold in DNP rats. Apart from that, the expression level of p-CaMKIIα was downgraded in SCDH and DRG in the Model + EA group. Accordingly, we hypothesized the antinociceptive role of EA on DNP might be associated with the downregulation of p-CaMKIIα expression in SCDH and DRG. Our study could help further explain the analgesic mechanism of EA and facilitate the development of EA as a DNP treatment in clinical practice. Of course, the mechanism of p-CaMKIIα involvement in EA still requires further validation, and how p-CaMKIIα is involved in EA to alleviate DNP also needs to be further explored, which will be the direction of our future research.

To summarize, our findings suggest p-CaMKIIα might participate in EA treatment of DNP from central and peripheral aspects, and the analgesia may be related to the reduction of p-CaMKIIα induced by EA treatment. Our research further explains the unclear analgesic mechanism of EA, while promoting the wider application of EA as a therapeutic modality targeting DNP.

Acknowledgements

The research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant No. LY22H270006, by Zhejiang Chinese Medical University under grant numbers 2022FSYYZZ109, and by National Natural Science Foundation of China No. 81774389.

Conflicts of interest

There are no conflicts of interest.

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