Diabetic neuropathic pain (DNP) is a frequent complication of diabetes. Calcium/calmodulin-dependent protein kinase II α (CaMKIIα), a multi-functional serine/threonine kinase subunit, is mainly located in the surface layer of the spinal cord dorsal horn (SCDH) and the primary sensory neurons in dorsal root ganglion (DRG). Numerous studies have indicated electroacupuncture (EA) takes effect in various kinds of pain. In this research, we explored whether CaMKIIα on rats' SCDH and DRG participated in DNP and further explored the mechanisms underlying the analgesic effects of EA. The DNP model in rats was successfully established by intraperitoneal injection of streptozotocin. Certain DNP rats were treated with intrathecal injections of KN93, a CaMKII antagonist, and some of the DNP rats received EA intervention. The general conditions, behaviors, the expressions of CaMKIIα and phosphorylated CaMKIIα (p-CaMKIIα) were evaluated. DNP rats' paw withdrawal threshold was reduced and the expressions of p-CaMKIIα in SCDH and DRG were upregulated compared with the Normal group, while the level of CaMKIIα showed no significance. KN93 attenuated DNP rats' hyperalgesia and reduced the expressions of p-CaMKIIα. We also found EA attenuated the hyperalgesia of DNP rats and reduced the expressions of p-CaMKIIα. The above findings suggest that p-CaMKIIα in SCDH and DRG is involved in DNP. The analgesic effect of EA in DNP might be related to the downregulation of p-CaMKIIα expression level. Our study further supports that EA can be an effective clinical treatment for DNP.

Diabetic neuropathic pain (DNP) is a diabetic complication that causes severe pain and deeply impacts the quality of the sufferer's daily life. Currently, contemporary clinical treatments for DNP generally exhibit a deficiency in effectiveness. Electroacupuncture (EA) is recognized as a highly effective and safe treatment for DNP with few side effects. Regrettably, the processes via which EA alleviates DNP are still poorly characterized. Transient receptor potential vanilloid 1 (TRPV1) and phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) are overexpressed on spinal cord dorsal horn (SCDH) in DNP rats, and co-localization is observed between them. Capsazepine, a TRPV1 antagonist, effectively reduced nociceptive hypersensitivity and downregulated the overexpression of phosphorylated CaMKIIα in rats with DNP. Conversely, the CaMKII inhibitor KN-93 did not have any impact on TRPV1. EA alleviated heightened sensitivity to pain caused by nociceptive stimuli and downregulated the level of TRPV1, p-CaMKIIα, and phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) in DNP rats. Intrathecal injection of capsaicin, on the other hand, reversed the above effects of EA. These findings indicated that the CaMKII/CREB pathway on SCDH is located downstream of TRPV1 and is affected by TRPV1. EA alleviates DNP through the TRPV1-mediated CaMKII/CREB pathway.

Abstract Background Sciatica and low back pain are prevalent clinical types of neuropathic pain that significantly impair patients' quality of life. Conventional therapies often lack effectiveness, making these conditions challenging to treat. Electroacupuncture (EA) is an effective physiotherapy for pain relief. Prior research has demonstrated a relationship between the frequency of neuropathic pain and the analgesic impact of EA stimulation. This work aimed to assess the analgesic effects of EA in a rat model of chronic compression of the dorsal root ganglion (CCD) and to understand the underlying processes. Methods We established a rat CCD model to simulate sciatica and low back pain. EA was applied to rats with CCD at various frequencies (2 Hz, 100 Hz, and 2/100 Hz). The paw withdrawal threshold (PWT) was measured to assess analgesic effects. Additionally, protein levels of the purinergic receptor P2X3 (P2X3) and the expression of nociceptive neuronal markers were analyzed using immunohistochemistry and western blot (WB) techniques. The study also measured levels of proinflammatory cytokines TNF-α and IL-1β in the dorsal root ganglion (DRG). The involvement of P2X3 receptors was further investigated using the P2X3 agonist, α,β-methylene ATP (α,β-meATP). Results CCD rats developed pronounced mechanical allodynia. EA stimulation at all tested frequencies produced analgesic effects, with 2/100 Hz showing superior efficacy compared to 2 Hz and 100 Hz. The expression of P2X3 was increased in ipsilateral DRG of CCD model rats. P2X3 were co-labeled with isolectin B4 (IB4) and transient receptor potential vanilloid (TRPV1), indicating their role in nociception. 2/100 Hz EA treatment significantly reduced mechanical allodynia and inhibited the overexpression of P2X3, TRPV1, substance P (SP), and calcitonin gene-related peptide (CGRP) in the ipsilateral DRG of CCD model rats. Additionally, EA reduced the levels of proinflammatory cytokines TNF-α and IL-1β in the ipsilateral DRG, indicating an anti-inflammatory effect. The P2X3 agonist α,β-me ATP attenuated the analgesic effect of 2/100 Hz EA in CCD rats. The WB and immunofluorescence results consistently demonstrated P2X3 inhibition contributed to the analgesic effects of 2/100 Hz EA on CCD-induced neuropathic pain. Conclusions Our findings suggest that 2/100 Hz EA alleviates neuropathic pain in rats by inhibiting the upregulation of P2X3 receptors in the ipsilateral DRG. This study backs up EA as a viable treatment option for sciatica and low back pain in clinical settings.

OBJECTIVE:Diabetic neuropathic pain (DNP) is one of the most prevalent symptoms of diabetes. The alteration of proteins in the spinal cord dorsal horn (SCDH) plays a significant role in the genesis and the development of DNP. Our previous study has shown electroacupuncture could effectively relieve DNP. However, the potential mechanism inducing DNP's genesis and development remains unclear and needs further research. METHODS:This study established DNP model rats by intraperitoneally injecting a single high-dose streptozotocin; 2 Hz electroacupuncture was used to stimulate Zusanli (ST36) and Kunlun (BL60) of DNP rats daily from day 15 to day 21 after streptozotocin injection. Behavioral assay, quantitative PCR, immunofluorescence staining, and western blotting were used to study the analgesic mechanism of electroacupuncture. RESULTS:The bradykinin B1 receptor (B1R) mRNA, nuclear factor-κB p65 (p65), substance P, and calcitonin gene-related peptide (CGRP) protein expression were significantly enhanced in SCDH of DNP rats. The paw withdrawal threshold was increased while body weight and fasting blood glucose did not change in DNP rats after the electroacupuncture treatment. The expression of B1R, p65, substance P, and CGRP in SCDH of DNP rats was also inhibited after the electroacupuncture treatment. CONCLUSION:This work suggests that the potential mechanisms inducing the allodynia of DNP rats were possibly related to the increased expression of B1R, p65, substance P, and CGRP in SCDH. Downregulating B1R, p65, substance P, and CGRP expression levels in SCDH may achieve the analgesic effect of 2 Hz electroacupuncture treatment.

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.

Diabetic neuropathic pain (DNP) is a common and destructive complication of diabetes mellitus. The discovery of effective therapeutic methods for DNP is vitally imperative because of the lack of effective treatments. Although 2 Hz electroacupuncture (EA) was a successful approach for relieving DNP, the mechanism underlying the effect of EA on DNP is still poorly understood. Here, we established a rat model of DNP that was induced by streptozotocin (STZ) injection. P2X4R was upregulated in the spinal cord after STZ-injection. The upregulation of P2X4R was mainly expressed on activated microglia. Intrathecal injection of a P2X4R antagonist or microglia inhibitor attenuated STZ-induced nociceptive thermal hyperalgesia and reduced the overexpression of brain-derived neurotrophic factor (BDNF), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the spinal cord. We also assessed the effects of EA treatment on the pain hypersensitivities of DNP rats, and further investigated the possible mechanism underlying the analgesic effect of EA. EA relieved the hyperalgesia of DNP. In terms of mechanism, EA reduced the upregulation of P2X4R on activated microglia and decreased BDNF, IL-1β and TNF-α in the spinal cord. Mechanistic research of EA's analgesic impact would be beneficial in ensuring its prospective therapeutic effect on DNP as well as in extending EA's applicability.

Objective:To observe any effect of electroacupuncture (EA) on the expression of phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) and phosphorylated cyclic adenosine monophosphate response element binding protein (p-CREB) in the spinal dorsal horns of diabetics experiencing neuropathic pain.Methods:Eight rats were randomly selected from 30 healthy male Sprague-Dawley rats as the normal group (N), and the remaining twenty-two rats were treated with a single high-dose intraperitoneal injection of streptozotocin (STZ) to establish a neuropathic pain model. The rats modeled successfully were randomly divided into a model group (M, n=8) and an EA group ( n=8). In the EA group, electroacupuncture was applied at the bilateral Hou san li and Kunlun acupoints starting on the 15th day after the STZ injection. The daily sessions lasted 30 minutes for 1 week. Body weight (BW), fasting blood glucose (FBG) and paw withdrawal latency (PWL) were observed before the STZ injection and on the 7th, 14th, and 21st days afterward. The expression of p-ERK1/2 and p-CREB in the dorsal horns of the rats′ spinal cords was detected using western blotting. The count of p-CREB-positive cells in the dorsal horns and their co-localization with neurons was detected using immunofluorescence. Results:In comparison with the N group, the average BW of the M group on the 7th, 14th and 21st days after the STZ injection was significantly lower, while the average FBG was significantly higher. There was no significant difference between the M and N groups in the average PWL on the 7th day after the STZ injection, but it had decreased significantly in the M group on the 14th and 21st days. Compared with the M group, the average PWL of the EA group was significantly longer on the 21st day after the injection. The expression of p-ERK1/2 and p-CREB protein in the spines of the M group was significantly higher than in the N group. p-CREB positive cells were more numerous in the M group compared with the N group, while in the EA group they were fewer. P-CREB was co-located with neurons in the spinal dorsal horn.Conclusion:EA can alleviate neuropathic pain effectively, perhaps by inhibiting the expression of p-ERK1/2 and p-CREB in the dorsal horns of the spinal cord.

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate), a nonspecific P2X1-7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4-L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.

Diabetic neuropathic pain (DNP) is frequent among patients with diabetes. We previously showed that P2X3 upregulation in dorsal root ganglia (DRG) plays a role in streptozotocin (STZ)-induced DNP but the underlying mechanism is unclear. Here, a rat model of DNP was established by a single injection of STZ (65 mg/kg). Fasting blood glucose was significantly elevated from the 1st to 3rd week. Paw withdrawal thresholds (PWTs) and paw withdrawal latencies (PWLs) in diabetic rats significantly reduced from the 2nd to 3rd week. Western blot analysis revealed that elevated p-CaMKIIα levels in the DRG of DNP rats were accompanied by pain-associated behaviors while CaMKIIα levels were unchanged. Immunofluorescence revealed significant increase in the proportion of p-CaMKIIα immune positive DRG neurons (stained with NeuN) in the 2nd and 3rd week and p-CaMKIIα was co-expressed with P2X3 in DNP rats. KN93, a CaMKII antagonist, significantly reduce mechanical hyperalgesia and thermal hyperalgesia and these effects varied dose-dependently, and suppressed p-CaMKIIα and P2X3 upregulation in the DRGs of DNP rats. These results revealed that the p-CaMKIIα upregulation in DRG is involved in DNP, which possibly mediated P2X3 upregulation, indicating CaMKIIα may be an effective pharmacological target for DNP management.

Abstract Management of diabetic neuropathic pain (DNP), a prevalent, refractory diabetic complication, remains a challenge. Previous research evidences have implicated microglia P2X4 receptor (P2X4R) in occurrence and development of DNP. To date, however, the specific mechanism of microglia P2X4R action in DNP needs to be further explored. We elucidated the role and underlying mechanism of microglia P2X4R in DNP. The DNP rat model was established by injection of streptozotocin (STZ). DNP rats developed thermal hyperalgesia from day 14. Western blot and immunofluorescence analyse revealed that microglia, P2X4R, p-p38 mitogen-activated protein kinase (p-p38 MAPK), and downstream targets brain-derived neurotrophic factor (BDNF), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were upregulated in the spinal cord. In addition, a P2X4R antagonist 5-BDBD and a microglia inhibitor minocycline (mino) not only alleviated DNP, but also suppressed P2X4R, microglia, p-p38 MAPK, BDNF, TNF-α and IL-1β levels in the spinal cord. Based on these findings, it was evident that central pain sensitization in DNP occurs via the microglia P2X4R/p38 signaling pathway, while p-p38 MAPK activates microglia to release BDNF, TNF-α and IL-1β in spinal cords. These results demonstrate that P2X4R/p38 signaling pathway in microglia residing in the spinal cord contribute to the establishment and long term maintenance of DNP and that these represent potential targets for pain therapy.

OBJECTIVE:To observe the occurrence time of neuralgia and the expression of purinergic ligand-gated ion channel 7 receptor (P2X7R) in the dorsal horn of the spinal cord after intraperitoneal injection of streptozotocin (STZ) in diabetic rats, and to explore the effect of electroacupuncture (EA) and pretreatment of EA on the heat pain threshold and expression of P2X7R in the spinal dorsal horn in rats with diabetic neuropathic pain (DNP), and to explore the possible mechanism of EA for DNP.METHODS:PartⅠ: Thirty male SD rats were randomly selected from 64 male SD rats as the control group; the remaining rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model, and 30 rats were successfully modeled as the model group. The control group and the model group were divided into three subgroups respectively at 7, 14 and 21 days, with 10 rats in each subgroup. Body mass, fasting blood glucose (FBG) and thermal pain threshold were recorded at 7, 14 and 21 days after injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot. PartⅡ: Eight SD rats were randomly selected from 35 male SD rats as the blank group, and the remaining 27 rats were given intraperitoneal injection of STZ (10 mg/mL) at a dose of 65 mg/kg to establish the diabetes model. The 24 rats with successful diabetes model were randomly divided into a DNP group, an EA group and a pre-EA group, 8 rats in each group. Fifteen to 21 days after STZ injection, the EA group received EA at "Zusanli" (ST 36) and "Kunlun" (BL 60), continuous wave, frequency of 2 Hz, 30 min each time, once a day; the intervention method in the pre-EA group was the same as that in the EA group. The intervention time was 8 to 14 days after STZ injection. The body mass, FBG and thermal pain threshold were recorded before STZ injection and 7, 14 and 21 days after STZ injection; the expression of P2X7R in spinal dorsal horn was detected by Western blot 21 days after injection.RESULTS:PartⅠ: Compared with the control group, in the model group, the body mass was decreased and FBG was increased 7, 14 and 21 days after STZ injection (P<0.01), and the thermal pain threshold was decreased 14 and 21 days after STZ injection (P<0.05), and the expression of P2X7R in spinal dorsal horn was increased 7, 14 and 21 days after STZ injection (P<0.05, P<0.01). PartⅡ: Compared with the blank group, in the DNP group, the body mass was decreased and fasting blood glucose were increased 7, 14 and 21 days after STZ injection (P<0.01). Compared with the DNP group, in the pre-EA group, the heat pain threshold was increased 14 and 21 days after STZ injection (P<0.05), while in the EA group, the heat pain threshold was increased 21 days after STZ injection (P<0.01), and the expression of P2X7R in the dorsal horn in the EA group and the pre-EA group was decreased (P<0.01).CONCLUSION:The diabetic neuropathic pain is observed 14 days after STZ injection. EA could not only treat but also prevent the occurrence of DNP, and its mechanism may be related to down-regulation of P2X7R expression in the dorsal horn of the spinal cord.

目的 本研究拟观察注射链脲佐菌素(streptozotocin,STZ)后大鼠不同时期背根神经节(dorsal root ganglion,DRG)上嘌呤受体(purinergic receptor subtype,P2X3)的表达情况及P2X3受体拮抗剂TNP-ATP对糖尿病神经痛(diabetic neuropathic pain,DNP)大鼠的干预作用.方法 (1)20只健康雄性SD大鼠随机选取6只选取作为正常组,其余14只大鼠予以腹腔注射STZ,剔除未成模的2只大鼠,分别观察造模前(Base),造模后7、14、21 d的机械缩爪阈值(paw withdrawal threshold,PWT)变化情况;并在上述各时间点取大鼠L4-L6 DRG,采用免疫荧光法检测L4-L6 DRG上P2X3阳性细胞表达情况.(2)将25只健康雄性SD大鼠随机选取6只作为正常+生理盐水(Control+NS)组,其余19只予以STZ注射,剔除未成模大鼠1只,成功建立DNP的大鼠随机分为模型+生理盐水(DNP+NS)组,模型+50 nmol P2X3抑制剂TNP-ATP组(DNP+50 nmol TNP-ATP),模型+100 nmol P2X3抑制剂TNP-ATP组(DNP+100 nmol TNP-ATP),每组6只;STZ注射14 d后,DNP+TNP-ATP组分别按照上述剂量予以足背注射TNP-ATP溶液,其余两组分别予以注射等量NS,观察注射后0.5、1、1.5 h大鼠PWT变化.同时观察连续注射7 d药物对大鼠PWT的影响.结果 (1)与正常组比较,模型组大鼠第7天、第14天及第21天空腹血糖显著升高;与正常组比较,模型组大鼠Day 7 PWT无明显改变,第14天、第21天PWT显著降低.免疫荧光结果显示,与正常组相比,STZ注射7、14、21 d后,DNP大鼠L4、L5 DRG上P2X3的阳性细胞的表达显著升高,STZ注射14、21 d后,DNP大鼠L6 DRG上P2X3的阳性细胞的表达显著升高.(2)TNP-ATP干预前,DNP+NS组与DNP+50 nmol TNP-ATP组、DNP+100 nmol TNP-ATP组PWT无显著差异;干预0.5 h后,与DNP+NS组、DNP+50 nmol TNP-ATP组相比,DNP+100 nmol TNP-ATP组PWT明显升高,效果持续至1 h.(3)连续注射7 d TNP-ATP后,DNP+100 nmol TNP-ATP组PWT较DNP+NS组与DNP+50 nmol TNP-ATP组明显升高.结论 腹腔注射STZ可成功建立DNP大鼠模型,背根神经节P2X3表达上调参与糖尿病神经痛的调节.

目的 观察电针对糖尿病神经痛大鼠背根神经节磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)表达的影响.方法 将20只健康雄性SD大鼠按随机数字表法分为空白组6只,造模组14只,造模组大鼠采用单次腹腔注射链脲佐菌素(65mg/kg)建立糖尿病神经痛模型,造模成功的12只大鼠按随机数字表法分为模型组和电针组,每组6只;电针组于2周后介入电针,取双侧足三里和昆仑穴,每天1次,每次30min,干预1周.空白组、模型组大鼠予以同电针组相同的固定.采用动态足底触觉仪检测大鼠造模前、造模后1、2、3周机械痛阈,采用免疫荧光法检测大鼠腰4至腰6背根神经节中p-p38MAPK阳性细胞表达.结果 与空白组比较,造模后1周模型组和电针组大鼠机械痛阈[(31.06±0.66)g、(32.51±0.84)g比(31.04±1.63)g,P>0.05]无显著性差异,造模后2、3周模型组大鼠机械痛阈[(25.20±1.03)g比(31.17±0.68)g,(18.29±2.01)g比(31.92±0.95)g,P均<0.05]均显著降低;与模型组比较,造模后3周电针组大鼠机械痛阈[(26.91±2.81)g比(18.29±2.01)g,P<0.05]显著升高.与空白组比较,模型组大鼠L4-L6背根神经节上的p-p38MAPK阳性细胞个数[(37.76±0.38)个比(21.49±1.57)个、(35.63±1.84)个比(22.49±2.66)个、(33.95±1.34)个比(21.79±1.09)个,P均<0.05]表达显著升高;与模型组比较,电针组大鼠腰4至腰6背根神经节上的p-p38MAPK阳性细胞个数[(26.69±1.35)个比(37.76±0.38)个、(23.54±3.22)个比(35.63±1.84)个、(26.38±2.65)个比(33.95±1.34)个,P均<0.05]表达显著降低.结论 电针对糖尿病神经痛模型大鼠有良好的镇痛效果,其机制可能与抑制背根神经节神经元p-p38MAPK表达相关.