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.

目的 探讨电针对糖尿病神经痛(DNP)大鼠脊髓小胶质细胞和离子通道型嘌呤能受体P2X4(P2X4R)表达的影响.方法 将20只健康雄性SD大鼠随机选取6只作为对照组,其余14只予以腹腔注射STZ(65 mg/kg)以诱导1型糖尿病(T1DM)神经痛模型,将12只造模成功的DNP大鼠随机分为模型组和电针组.电针组从造模后14 d开始予以2 Hz电针干预,选取双侧足三里和昆仑穴,每次30 min,每日1次,连续干预7 d;余组仅予相同固定,不作干预.观察各组大鼠的体质量、空腹血糖、热痛阈变化,采用Western blot法检测脊髓小胶质细胞Iba1和P2X4R蛋白的表达.结果 STZ注射14 d后模型组空腹血糖上升(P<0.01),热痛阈下降(P<0.01),说明DNP大鼠造模成功;与对照组比较,模型组大鼠脊髓Iba1和P2X4R表达明显增加(P<0.05);与模型组比较,电针组Iba1、P2X4蛋白表达明显减少(P<0.01,P<0.05).结论 低频电针对DNP模型大鼠有良好的镇痛作用,其作用机制可能与其有效抑制小胶质细胞和P2X4R蛋白表达有关.

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.

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.

目的 观察糖尿病神经痛(diabetic neuropathic pain,DNP)大鼠L4-L6 脊髓背角中小胶质细胞和脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)表达的变化及电针对其干预作用,探讨小胶质细胞和BDNF是否参与电针镇痛.方法 SD大鼠分为正常组(N组),模型组(M组),电针组( EA组),每组10只. EA组于第6周开始电针双侧足三里和昆仑穴,每次30 min,隔日1次,干预7次.分别检测各组大鼠的基础( base)、4周(4 weeks)、6周( 6 weeks)、8 周( 8 weeks)空腹血糖( fasting blood glucose, FBG)、热痛阈( thermal paw withdrawal latency,PWL)和机械痛阈(paw withdrawal thresholds,PWT)变化;于8 weeks应用免疫荧光法检测大鼠L4-L6 脊髓背角小胶质细胞和BDNF蛋白的表达.结果 (1) 6 weeks、8 weeks 时,M组和EA组FBG较N 组显著升高,EA组FBG较M组无明显变化;(2)6 weeks、8 weeks 时,M组PWL和PWT较N 组显著下降;8 weeks时,EA组PWL和PWT较M组显著升高;6 weeks时,EA组PWL和PWT与M组无明显差异;(3) M组大鼠L4-L6 脊髓背角CD11b (小胶质细胞标记物)和BDNF平均光密度值较N组显著升高,EA组大鼠L4-L6 脊髓背角CD11b(小胶质细胞标记物)和BDNF平均光密度值较M组显著下降.结论 脊髓背角小胶质细胞和BDNF可能参与了大鼠糖尿病神经痛的产生与维持,电针可能通过减少DNP大鼠脊髓背角小胶质细胞表达和BDNF的表达产生镇痛作用.

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.

慢性疼痛是临床常见的病症之一,给患者和社会都带来较大的负担,其发病机制较为复杂,因此,慢性疼痛的治疗一直是临床上的难题之一.三磷酸腺苷(ATP)是一种重要的疼痛信号物质,ATP可作用于P2X受体产生效应.近年来研究发现,P2X受体在神经病理性疼痛、炎性痛、癌痛及内脏痛等慢性疼痛的痛觉形成、传导和调节中起着重要作用,有望成为慢性疼痛治疗的新靶点.本文就P2 X介导慢性疼痛的研究进展进行了综述.

[目的]观察电针(electroacupuncture, EA)对糖尿病神经痛(diabetic neuropathic pain,DNP)大鼠L4～L6脊髓星形胶质细胞活化的影响. [方法]将26只雄性SD大鼠随机分为正常组(6只)和高脂高糖饲养组(20只),高脂高糖饲养组大鼠采用高脂高糖饲养联合单次链脲佐菌素注射建立DNP模型.将造模成功的大鼠分为DNP模型组、EA组,每组6只. EA组造模后7周开始以EA针刺"足三里""昆仑",每次30min,1次/d,持续1周,余组仅予相同固定,不作干预.各组于造模前和造模后5、7、8周分别测量大鼠机械缩足反射阈值(paw withdrawal threshold,PWT).造模后8周处死大鼠,以免疫荧光法检测大鼠L4～L6脊髓星形胶质细胞活化的标志物胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和N-甲基-D-天冬氨酸受体2B亚基磷酸化(phospho-N-methyl-D-aspartic acid receptor 2B subunit,p-NR2B)的表达. [结果]与正常组比较,造模后7周DNP模型组大鼠PWT明显降低(P＜0.01),出现痛觉过敏;造模后8周,EA组PWT较DNP模型组明显升高(P＜0.01).造模后8周,DNP模型组L4～L6脊髓背角GFAP和p-NR2B阳性表达高于正常组(P＜0.01,P＜0.01),EA组L4～L6脊髓中GFAP和p-NR2B阳性表达明显低于DNP模型组(P＜0.01,P＜0.05). [结论]EA对DNP具有镇痛作用,其机制可能是抑制脊髓背角星形胶质细胞活化和下调p-NR2B的表达.

钙调蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinase,CaMKⅡ)是一种多功能的丝氨酸/苏氨酸蛋白激酶,在神经元中大量存在,广泛参与疼痛调制.神经病理痛是一种由疾病或躯体感觉系统的损伤引起的慢性难治性疼痛.钙调蛋白激酶Ⅱ在中枢、外周神经病理痛、代谢型神经病理痛和药物引起神经病理痛等各种类型的神经病理痛的发生发展中发挥着重要的作用.本文拟将从钙调激酶Ⅱ介导的各型神经病理痛及其上下游的调控两个方面进行综述,以期为今后钙调蛋白激酶Ⅱ在神经病理痛领域的研究提供一定参考.