Electroacupuncture Alleviates Diabetic Neuropathic Pain and Downregulates p-PKC and TRPV1 in Dorsal Root Ganglions and Spinal Cord Dorsal Horn

Diabetic neuropathic pain (DNP) is a common complication of diabetes. Streptozotocin (STZ)-induced changes of protein in dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) are critical for DNP genesis. However, which proteins change remains elusive. Here, the DNP model was established by a single intraperitoneal injection of STZ, accompanied by increased fasting blood glucose (FBG), decreased body weight (BW), and decreased paw withdrawal latency (PWL). Proteins change in L4-L6 DRGs and SCDH of rats were detected. Western blot and immunofluorescence results showed that expression levels of phosphorylated protein kinase C (p-PKC), transient receptor potential vanilloid-1 (TRPV1), Substance P (SP) and calcitonin gene-related peptide (CGRP) in the DRG and the SCDH of rats were increased after STZ injection. A preliminary study from our previous study showed that 2 Hz electroacupuncture (EA) effectively alleviates DNP. However, the analgesic mechanism of EA needs further elucidation. Here, EA at the bilateral Zusanli (ST36) and KunLun (BL60) acupoints was applied for one week, and to investigate the effect on DNP. EA reversed thermal hyperalgesia in DNP rats and downregulated the expression of p-PKC, TRPV1, SP, and CGRP in DRG and SCDH.


Introduction
Diabetes is a common metabolic disease [1], and the incidence of diabetes is on the rise [2]. Hyperglycemia can induce metabolic, microvascular lesions, and cause various acute and chronic neuropathy conditions [3]. Diabetic neuropathic pain (DNP) is a major complication of diabetes [4][5][6], which is mainly characterized by spontaneous pain, paresthesia and hyperalgesia, leading to a decrease in the quality of life of patients [7][8][9]. Te mechanisms underlying DNP still remain unclear, and need further elucidation to produce the efectiveness of some conventional treatment options for DNP.
Dorsal root ganglion (DRG) neurons are the primary aferent nerve cells for trunk and extremity nociception.
DRGs are implicated in transmitting and accommodating sensations and receiving and communicating nociception, and they play an important role in the mechanism of pain. Pain signals are transmitted from DRGs to the spinal cord dorsal horn (SCDH) [10,11]. Neurons in the central processes of the horn and neurons in the DRG form the primary synapse, in which SCDH plays a role in relaying and processing sensory information. Terefore, DRGs and SCDH are key sites for studying neuropathic pain mechanisms. Previous studies report that several DRG pain-related ion channels, receptors and neuropeptides such as Ca 2+ channels, Na + channels, phosphorylated protein kinase C (p-PKC), transient receptor potential vanilloid-1 (TRPV1) [12], calcitonin gene-related peptide (CGRP), and substance P (SP) [13] are implicated in the transmission of pain. Preliminary studies indicate that p-PKC, TRPV1, SP, and CGRP in DRG play fundamental roles in acute neurogenic infammation [12]. However, the changes in p-PKC, TRPV1, SP, and CGRP expression in DRG and SCDH in DNP model have not been systematically studied.
Although clinical drugs are used to alleviate DNP, clinical studies have failed to prove the efectiveness of treatment with less adverse efects [14,15]. Electroacupuncture (EA) therapy is an efective option for chronic pain, including DNP treatment [16], which combines electrical stimulation with the use of acupuncture needles [17][18][19]. Our previous study showed that 2 Hz EA was more efective than 100 Hz EA in relieving DNP [20]. However, the precise mechanism of 2 Hz EA on DNP has not been fully elucidated.
Te present study sought to explore the efect of STZ administration on expressions of p-PKC, TRPV1, SP, and CGRP in DRG and SCDH. Tese fndings will provide a basis for understanding the mechanism of DNP. Moreover, the efect of 2 Hz EA treatment on the expression levels of p-PKC, TRPV1, SP, and CGRP in DRGs and SCDH of DNP rats was explored.

Animals.
Male Sprague-Dawley rats (180 ± 20 g) were used in the present study. Rats were assigned to fve groups and lived in separate cages. Animals had free access to food and water. Rats were maintained in a controlled environment (20-24°C and 40-60%) with 12-h light/dark cycles at the Animal Laboratory Center of Zhejiang Chinese Medical University (SYXK (zhe) 2018-0012). Experiments were conducted after acclimatization of animals for a week. All experimental procedures were conducted according to animal management regulations. Te Animal Welfare Committee of Zhejiang Chinese Medical University approved all protocols in the present study (IACUC-20190805-04).

Establishment of the DNP Rat
Model. Rats were fasted for 16 hours and STZ (65 mg/kg, S0130, Sigma) dissolved in sodium citrate bufer (0.1 mol/L, pH 4.5) was administered into rats intraperitoneally [21,22]. Rats in the Control group received the same volume of the vehicle. Fasting blood glucose (FBG) was determined 3 days after STZ injection. Rats with FBG >13.9 mmol/L [23,24] and thermal nociceptive sensitivity were used as the criteria for a DNP rat model. Animals that met these criteria were used in subsequent experiments.

Experimental Procedures.
Te experiment was split into two phases. Te efect of STZ on inducing diabetic neuropathic pain was evaluated in the frst phase. Rats were randomly assigned to two groups: (1) Control group (n = 10, all rats were sacrifced and tissues were harvested after 3 weeks of experiment); (2) STZ group (n = 30, 10 rats were killed and tissues were harvested after 1 week, 2 weeks and 3 weeks of experiment). Expression levels of p-PKC, TRPV1, SP, and CGRP in lumbar 4-6 SCDH and DRGs were determined by western blot (WB) or immunofuorescence (IF) analysis. In the second phase, the analgesic efect of EA on DNP and whether p-PKC, TRPV1, SP, and CGRP are implicated in this efect was explored. Rats were randomly assigned to three groups (n = 8): (1) Control group; (2) STZ group; and (3) STZ + EA group. Rats in the STZ + EA group were administered with EA daily for a week from the 2 weeks. Tissues were harvested after treatment for western blot and immunofuorescence analysis. Expression levels of p-PKC, TRPV1, SP, and CGRP in lumbar 4-6 SCDH and DRGs were determined by WB or IF.

Determination of Fasting Blood Glucose and Body Weight.
Rats were fasted for 8 h and weighed. Blood was obtained from the tail and analysis of FBG was performed using ACCU-CHEK Performa blood glucose meter (Roche Diagnostics GmbH, Germany) a day before administration of STZ and 1 week, 2 weeks, and 3 weeks after STZ injection.

Assessment of Termal
Hypersensitivity. Paw withdrawal latency (PWL) analysis was conducted using the plantar test (37370, Ugo Basile, Italy). Rats were acclimatized in the Plexiglas cubicles (11.5 cm × 17 cm × 14 cm) on the glass plate for at least 30 min, before evaluation. Te cut-of time was set at 30 s, and the radiant heat was set to 40, to avoid damage of rat tissue. Te light beam was turned of and the timing stopped when the rat raised its paw. Te experiment was conducted 3 times per rat with an interval of 5 min between replicates. PWL was calculated as the average of the latencies in seconds.
2.6. EA Treatment. Rats in STZ + EA group received EA treatment once a day for one week. Rats that received EA were not anesthetized, but immobilized gently with a selfmade retainer. Te selected acupoints were bilateral Zusanli (ST36, 5 mm below the fbular head and 1 mm outside the anterior tibial edge) and Kunlun (BL60, depression between the lateral ankle joint and achilles tendon of the hind limb) points. Te acupuncture needles (0.25 mm × 13 mm, Hua Tuo, Suzhou Medical Appliance Factory, Jiangsu Province) were carefully inserted into the acupuncture points, and then the acupuncture needles were inserted at a depth of 3 mm for the Kunlun point and 7 mm for the Zusanli point, and then connected to the HANS acupoint electrical stimulation device (Hans-200A, Jisheng Medical Technology, Beijing, China) for 30 minutes. Te HANS acupoint electrical stimulation device was set at 1 mA and 2 Hz. Rats in the other groups underwent the same sedation process without EA stimulation.

Immunofuorescence Analysis.
Rats were anesthetized with sodium pentobarbital (80 mg/kg, i.p) and transcardially perfused with 4°C saline followed by 4% paraformaldehyde. Te spinal cord and DRGs from L4 to L6 were harvested, postfxed in 4% paraformaldehyde for 4 h, and then dehydrated in 15% sucrose solution for 24 h and 30% for 48 h. Tissue sections were prepared using a frozen microtome (30 μm thickness for the spinal cord and 10 μm thickness for DRGs) and subsequently fxed onto glass slides. Sections were rinsed thrice with 1 × TBST for 10 min for each rinse, then blocked with 10% donkey serum for 1 h at 37°C. Sections were incubated with diluted guinea pig anti-TRPV1 (1 : 200; ACC-030-GP, Alomone, Israel) antibodies mixed with rabbit anti-SP (1 : 1500; ab67006, Abcam, UK) or rabbit anti-CGRP (1 : 800; #14959, Cell Signaling Technology) antibodies overnight at 4°C. Tissues slices were washed 6 times in 1 × TBST, for 10 min per wash, then incubated with Goat Anti-Guinea pig IgG H&L (ALexa Fluor ® 488) (1 : 600; ab150185, Abcam, UK) and Goat Anti-Rabbit IgG H&L (Alexa Fluor ® 594) (1 : 800; ab150084, Abcam, UK) for 1 h at 37°C. Tissue sections were sealed with antifade solution. Te sections were then imaged under an Imager M2 microscope (ZEISS, Germany). Te scale bar for SCDH slices was 100 μm and the objective magnifcation was 10×. Te scale bar for DRG slices was 50 μm and the objective magnifcation was 20×. Te mean fuorescence intensity of SP and CGRP in SCDH was determined by Image J and the number of SP, CGRP, and TRPV1 positive cells in DRGs was evaluated. Tree sections were selected for each rat and three rats were analyzed for each group.
2.9. Statistical Analysis. Statistical analysis was conducted using SPSS 22.0 software. Data were presented as mean-± standard error of the mean (SEM). Independent t-test was carried out to compare two groups and one-way ANOVA followed by LSD or Dunnett's post hoc tests were used for the comparison of three or more groups. P < 0.05 was considered statistically signifcant.

Termal Hyperalgesia in a Rat Model of STZ-Induced
Diabetes. Te experimental design for the frst phase is given in Figure 1(a). Te FBG in the STZ group was higher than the Control group on 1, 2, and 3 weeks (Figure 1(b), P < 0.01, respectively). Te BW in the STZ group was lower than the Control group on 1, 2, and 3 weeks (Figure 1(c), P < 0.01, respectively). Te PWL in the STZ group was lower than the Control group on 2 and 3 weeks (Figure 1(d), P < 0.01, respectively). Tese results revealed that the DNP model was successfully established on day 14 after STZ injection.

p-PKC, TRPV1
, SP, and CGRP are Increased in the SCDH after STZ Injection. Te expression levels p-PKC, TRPV1, SP, and CGRP in SCDH were determined to explore the efect of STZ on SCDH (Figures 4 and 5). WB results indicated that the p-PKC protein was increased from one to three weeks (Figure 4(c), P < 0.01, P < 0.01, P < 0.01), and TRPV1 protein was increased from two to three weeks ( Figure 4(d), P < 0.05, P < 0.01). IF results showed SP and CGRP increased from one to three weeks (Figures 4(g) and 4(h), P < 0.01, respectively). Moreover, IF results demonstrated the coexpression of SP/TRPV1 and CGRP/TRPV1 in the SCDH (Figures 5(a) and 5(b)).

EA Alleviates Termal Hyperalgesia in a Rat Model of STZ-Induced DNP.
Te experimental design is given in Figure  6(a). Te FBG in the STZ group was increased, and the BW in the STZ group was decreased at 1, 2, and 3 weeks ( Figures  6(b) and 6(c), P < 0.01, respectively). Te PWL in the STZ group decreased at 2 and 3 weeks, indicating the successful establishment of DNP in rats ( Figure 6(d), P < 0.01, respectively). Te rats in the STZ + EA group were treated with EA from the 15th day to the 21st day. EA reduced STZinduced thermal hyperalgesia in DNP rat models in the third week ( Figure 6(d), P < 0.01). However, EA did not produce an efect on FBG and BW in DNP rats in the third week (Figures 6(b) and 6(c), P > 0.05, respectively).

EA Reduces Expression of p-PKC, TRPV1
, SP, and CGRP in the DRG of DNP Rats. Further WB and IF analyses were conducted to explore the efect of EA treatment on p-PKC, TRPV1, SP, and CGRP expression levels in L4-L6 DRGs of DNP rats (Figure 7). WB analysis showed p-PKC and

EA Reduces Expression of p-PKC, TRPV1, SP, and CGRP in the SCDH of DNP Rats.
Further WB and IF analyses were conducted to explore expression levels of p-PKC, TRPV1, SP, and CGRP in SCDH of DNP rats after EA treatment ( Figure 9). WB results indicated that p-PKC and TRPV1 expression increased remarkably. EA treatment decreased the increased expressions of p-PKC and TRPV1 (Figure 9(c), P < 0.01, P < 0.05; Figure 9(d), P < 0.01, P < 0.01). IF results indicated that STZ injection signifcantly increased the mean intensity of SP and CGRP in L4-6 SCDH (Figures 9(g) and 9(h), P < 0.01). Notably, EA stimulation remarkably reduced the mean intensity of SP and CGRP in L4-6 SCDH ( Figure 9(g), P < 0.01; Figure 9(h), P < 0.05).

Discussion
In the current study, we investigated the changes of p-PKC, TRPV1, SP, and CGRP protein in DRG and SCDH in STZinduced neuropathic pain. Te results showed that the expressions of p-PKC, TRPV1, SP, and CGRP were increased in L4-6 DRG and SCDH, and TRPV1 was coexpressed with SP, and TRPV1 was also coexpressed with CGRP. We then examined the efect of 2 Hz EA on the thermal hyperalgesia of DNP model rats. In total, 2 Hz frequency of EA was applied for 30 minutes every day after DNP model establishment, from days 15 to 21. Results indicated that 2 Hz EA produced antiallodynic efect on DNP model rats, and EA efectively reduced overexpression of the p-PKC, TRPV1, SP, and CGRP marker proteins. STZ is a glucosamine-nitrosourea that can selectively destroy pancreatic islet β-cells in mammals [27] and is commonly used in establishing diabetes models [28]. In this study, FBG increased and BW decreased remarkably starting at 1 week after STZ injection. PWL decreased remarkably starting at 2 weeks after STZ injection, indicating the successful establishment of the DNP model, which consisted with our previous research [29].
DRGs and SCDH play vital roles in many neuropathic pain [30][31][32]. DRG receives pain signals and transmits them   Evidence-Based Complementary and Alternative Medicine to the SCDH [33,34]. Many changes of protein in DRG and SCDH are involved in neuropathic pain [35][36][37]. Previous studies showed that PKC is involved in the transmission of neuropathic pain including DNP [14,38,39]. PKC is a phospholipid-dependent serine/ threonine kinase family. Tis family comprises 13 isoenzymes that can be activated by extracellular signals [40]. Te active state of PKC is p-PKC, which is a phosphorylated state [41,42] and is implicated in various roles [43]. TRPV1 is a nonselective ligand-gated cationic channel assembled as a  Evidence-Based Complementary and Alternative Medicine homotetramer and widely distributed in SCDH and DRGs [12,44,45]. TRPV1 receives various pain-causing stimuli such as noxious heat and diverse chemical irritants or toxins [46][47][48]. TRPV1 is an efective target for control of neuropathic pain [49]. A previous study reported that the expression of p-PKC and TRPV1 in neurogenic infammation was signifcantly upregulated in DRGs [12]. Tis is consistent with the results of the present study. In the current study, WB analysis showed an increase in p-PKC and TRPV1 expression levels in DRGs and SCDH of DNP rats. SP and CGRP are coexpressed in primary sensory nerves. IF results showed that the number of TRPV1-positive and CGRP-positive in DRG were increased signifcantly starting 1 week after STZ injection. SP-positive, TRPV1/CGRPpositive, and TRPV1/SP-positive cells in DRG were signifcantly increased starting 2 weeks after STZ injection. P-PKC, CGRP, and SP in SCDH are signifcantly elevated starting from the frst week, while TRPV1 in SCDH was signifcantly increased from the 2 weeks. Tis may be why thermal hyperalgesia developed at 2 weeks rather than 1 week after STZ injection. Sensory nerves endings are released to transmit pain signals when they are activated by stimuli [50]. SP and CGRP are expressed after activation of TRPV1 [51]. In the present study, immunofuorescence double staining was performed to explore colocalization of TRPV1 with SP and CGRP and to verify the upregulation of SP and CGRP expression in DNP. Te fndings indicated that STZ injection induces expression of p-PKC, TRPV1, SP, and CGRP in DRGs and SCDH upregulated. Currently, clinical studies have failed to prove the effectiveness of treatment with less adverse efects for patients with neuropathic pain [52]. A previous study reported that berberine blocks PKC channels to inhibit TRPV1 activation, thus improving DNP [14]. EA is a combination of acupuncture and electric current and is an efective approach for relieving neuropathic pain [53]. A previous study reported  that 2 Hz EA has better analgesic efects than 100 Hz EA [20]. Te analgesic efect of 2 Hz EA has also been demonstrated in other pain models [54,55]. Numerous studies have shown that EA intervention on ST36 and BL60 in rats can alleviate diferent types of neuropathic pain [56][57][58]. Te preliminary study of our research group showed that the intervention of EA of ST36 and BL60 can efectively alleviate diabetic neuropathic pain [59,60]. Tus, in the present study, the acupoints of ST36 and BL60 were selected to study the analgesic mechanism of EA. EA intervention in rats with neck-incision pain upregulated thermal pain thresholds and downregulated CGRP and SP expression in the dorsal aspect of the cervical spinal cord [61]. In addition, EA ameliorated nociceptive sensitization in rats with chronic pain and reduced TRPV1 expression on DRG [56]. EA treatment improved thermal hyperalgesia. EA treatment signifcantly reduced the overexpression of p-PKC, TRPV1, SP, and CGRP in SCDH and DRGs of DNP rats. Tese fndings all support that EA may be a promising therapeutic option for DNP. However, further clinical studies are needed to comprehensively evaluate the therapeutic potentials of EA on DNP patients.

Conclusion
In conclusion, p-PKC, TRPV1, SP, and CGRP in DRGs and SCDH were signifcantly elevated after STZ-induced neuropathic pain. EA treatment alleviates STZ-induced DNP, which may be associated with downregulation of p-PKC, TRPV1, SP, and CGRP in DRGs and SCDH. However, the specifc mechanism of action of EA was not explored in the current study. Further studies should be conducted to determine the role of p-PKC/TRPV1 in DNP.

Data Availability
Key data are included in the diagrams and the main text. Datasets used and analyzed in this study are available upon request from the corresponding author.

Ethical Approval
All animal experiments and studies were approved by the Animal Care and Welfare Committee of Zhejiang University of Traditional Chinese Medicine, China (approval no. IACUC-20190805-04).

Conflicts of Interest
Te authors declare no conficts of interest for this work.