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.

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.

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.

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.

目的:观察电针对糖尿病周围神经痛(DPNP)大鼠空腹血糖、热痛阈、脊髓背角缓激肽B1受体(B1R)和磷酸化蛋白激酶C(p-PKC)的影响.方法:高脂高糖饲养联合腹腔注射STZ构建DPNP大鼠模型,选取足三里穴、昆仑穴予以电针干预,每日1次,每次30 min,持续7d.检测各组大鼠空腹血糖和热痛阈,Western Blot检测大鼠腰段脊髓背角B1R和p-PKC的蛋白表达水平.结果:第49天后,与正常组比较,模型组空腹血糖显著升高(P＜0.01),热痛阈显著降低(P＜0.01);电针干预后,与模型组比较,电针组热痛阈显著提高(P＜0.01),B1R和p-PKC的蛋白表达显著下降(P＜0.01).结论:电针能显著下调DPNP大鼠脊髓背角B1R和p-PKC的蛋白表达,该作用可能参与电针缓解DPNP的过程.

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 研究铁皮石斛(Dendrobium officinale Kimura et Migo)的体外抗氧化活性及通过测量经络电压探索铁皮石斛的归经作用.方法 测定铁皮石斛汤剂的氧化还原电位值、pH值、总抗氧化能力.召集20名健康志愿者按随机数字表法分为2组,试验组(服用铁皮石斛汤剂,8g铁皮石斛粉冲泡于80 mL水中),对照组(饮用等量水),每组10名.服用药物前后进行人体经络电压的测量,选取12条经络上(肺经、肝经、脾经、肾经、胃经、心经、大肠经、胆经、心包经、三焦经、小肠经、膀胱经)的合穴和原穴作为经络电压的测量点,均以合穴为正极,原穴为负极,记录经络电压的变化.结果 通过pH的测定结果可以看出铁皮石斛呈现弱酸性.与蒸馏水比较,铁皮石斛氧化还原电位降低,抗氧化能力及总抗氧化能力升高(P＜0.01).在经络电压测定中,与对照组比较,试验组大肠经、胆经、心包经、三焦经、小肠经和膀胱经的电压升高,肺经、肝经、脾经、肾经、胃经和心经的电压则无显著变化.结论 铁皮石斛对可以显著增加大肠经、胆经、心包经、三焦经、小肠经和膀胱经所属的六腑经络的电压,对肺经、肝经、脾经、肾经、胃经和心经所属的五脏经络的电压则无显著作用.经络电压的变化规律与铁皮石斛的抗氧化性具有一定的关联.

超氧化物歧化酶(SOD)是生物体内产生的一种主要抗氧化酶,能够针对性清除呼吸代谢产生的超氧阴离子自由基.牛奶中自身含有SOD,SOD也被作为食品添加剂加入乳制品中,用于提高产品的抗氧化性,具有保健的作用.为了研究SOD强化奶片的抗氧化性,以及从对人体经络电压作用的角度研究其功能,对奶片(1.00 g奶粉)和SOD强化奶片(0.99 g奶粉+0.01 g 2.53×106 U/g SOD酶)分别进行氧化-还原电位(ORP)、DPPH自由基清除能力、SOD活力检测.召集20名健康志愿者,随机分为两组进行双盲试验,奶片组合服1片奶片,SOD强化奶片组则含服1片SOD强化奶片.实时记录志愿者左侧12条经络,并且在监测的第25 min开始含服,含服前、后一共记录85 min.实验结果显示,与奶片相比,SOD强化奶片的SOD活性和还原性(ORP值降低)显著增加,在质量浓度为1.250 mg/mL与1.562 mg/mL时,可增加对DPPH自由基的清除率,在3.125 mg/mL时则降低对DPPH自由基的清除率.经络电压监测结果显示:针对肺经和胆经经络电压,奶片没有影响,而SOD强化奶片有影响;针对大肠经、脾经、小肠经、膀胱经、三焦经和肝经经络电压,奶片有影响,SOD强化奶片的影响更强;针对胃经、心经、肾经和心包经经络电压,奶片和SOD强化奶片都有一定的影响,且作用相似.

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.

目的 观察电针对糖尿病神经痛(diabetic neuropathic pain,DNP)大鼠脊髓背角核转录因子 κB(nuclear factor kappa B,NF-κB)p65蛋白的干预作用,以探讨电针对糖尿病神经痛的部分镇痛机制.方法 将30只SD大鼠用完全随机分组法分为正常组(normal group)和造模型,分别用常规饲料和高脂高糖饲料喂养5周,喂养5周后造模组大鼠予以单次腹腔注射链脲佐菌素(streptozotocin,STZ).将造模组中糖尿病神经痛模型成功的大鼠再进一步随机分为模型组(DNP group)和模型+电针组(DNP+EA group).穴位选取双侧"足三里、昆仑穴",频率为2 Hz,强度为1 mA干预15 min后2 mA干预15 min,于第7周开始,每天1次,干预7次.观察各组大鼠的血糖、机械痛阈及热痛阈变化.采用苏木精-伊红(hematoxylin-eosin,HE)染色观察大鼠坐骨神经形态的变化,采用免疫印记法检测大鼠脊髓背角NF-κB p65蛋白的表达水平.结果 高脂高糖喂养5周后予以STZ单次腹腔注射2周后大鼠血糖上升,机械痛阈、热痛阈下降,表明大鼠DNP模型造模成功;电针可上调DNP大鼠的机械痛阈和热痛阈,但对血糖没有明显的干预作用.HE染色结果显示DNP大鼠坐骨神经有髓神经纤维排列紊乱,轴索肿胀,髓鞘密度不均匀,空泡变性,电针干预对DNP大鼠坐骨神经形态无明显的作用.免疫印迹结果显示:DNP大鼠脊髓背角NF-κB p65蛋白表达明显增多,电针可下调DNP大鼠脊髓背角NF-κB p65蛋白表达.结论 低频电针对DNP模型大鼠有良好的镇痛效果,其作用可能与其抑制糖尿病DNP大鼠脊髓背角NF-κB p65蛋白表达有关.

目的 观察糖尿病神经病理痛(diabetic neuropathic pain,DNP)模型大鼠不同时期背根神经节(dorsal root ganglion,DRG)上磷酸化钙调激酶II(p-CaMK II)的表达情况.方法 1)取21只健康雄性SD大鼠一次性大剂量注射链脲佐菌素(STZ)建立DNP大鼠模型,分别观察造模前(Base),造模后7 d(Day 7),14 d(Day 14),21 d(Day 21),28 d(Day 28)热辐射刺激的缩足反应时间(paw withdrawal latency,PWL)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p-CaMKII阳性细胞表达情况.(2)将20只大鼠随机分为正常+生理盐水(Control+NS)组,模型+生理盐水(DNP+NS)组,模型+CaMKII抑制剂KN93组(DNP+KN93);STZ注射14 d后,DNP+KN93组予以足背注射KN93溶液,其余两组分别予以注射等量NS.结果 1)与正常组比较,DNP模型组大鼠D7 PWL无明显改变,Day 14、Day 21、Day 28 PWL显著降低.免疫荧光结果显示,与正常组相比,STZ注射7、14、21、28 d后,DNP大鼠L4 DRG上p-CaMKII的阳性细胞的表达显著升高,L5和L6 DRG上p-CaMKII的阳性细胞表达亦显著上升.(2)KN93干预前,DNP+NS组与DNP+KN93组PWL无显著差异,干预1 h后,与DNP+NS组相比,DNP+KN93组PWL明显升高.结论 糖尿病神经病理痛的产生和维持与DRG神经元p-CaMK II表达上调有关,足背注射CaMK II抑制剂KN93可抑制热痛觉过敏反应.

Diabetic neuropathic pain (DNP) is a troublesome diabetes complication all over the world. P2X3 receptor (P2X3R), a purinergic receptor from dorsal root ganglion (DRG), has important roles in neuropathic pain pathology and nociceptive sensations. Here, we investigated the involvement of DRG P2X3R and the effect of 2 Hz electroacupuncture (EA) on DNP. We monitored the rats’ body weight, fasting blood glucose level, paw withdrawal thresholds, and paw withdrawal latency, and evaluated P2X3R expression in DRG. We found that P2X3R expression is upregulated on DNP, while 2 Hz EA is analgesic against DNP and suppresses P2X3R expression in DRG. To evaluate P2X3R involvement in pain modulation, we then treated the animals with A317491, a P2X3R specific antagonist, or α β-me ATP, a P2X3R agonist. We found that A317491 alleviates hyperalgesia, while α β-me ATP blocks EA’s analgesic effects. Our findings indicated that 2 Hz EA alleviates DNP, possibly by suppressing P2X3R upregulation in DRG.