SHP2抑制剂NSC-87877对炎性疼痛的抑制作用及其机制

目的探讨蛋白酪氨酸磷酸酶SHP2(src homology-2 domain-containing phosphatase 2)的抑制剂NSC-87877对炎性疼痛的影响及其机制;方法小鼠左后足底皮下注射完全佛氏佐剂(Complete Freund's Adjuvant;CFA)建立疼痛模型;鞘内给予NSC-87877前后,测定缩足阈值;随后分离左侧L4-L5节段脊髓背角,免疫印迹法检测N-methyl-D-aspartate(NM-DA)型谷氨酸受体的表达;结果 SHP2广泛分布于脊髓背角;且SHP2抑制剂NSC-87877(3μg)能够抑制CFA诱发的痛觉超敏,而对正常动物的痛行为无作用。虽然NSC-87877不影响NMDA受体NR2B和NR2A亚基的总蛋白含量,但却能够特异性降低CFA组动物NR2B的突触表达水平;结论 SHP2抑制剂NSC-87877通过逆转NR2B的突触表达亢进,对炎性疼痛产生明显的抑制作用。     

鼠尾草属药用植物抑制蛋白酪氨酸磷酸酶1B活性初探

An in vitro screening model was applied to test the inhibitory activities of 17 Salvia species on protein tyrosine phosphatase 1B (PTP1B).  Root methanol extracts from wild-collected Salvia species were  analyzed using this model.  Most of the samples tested showed positive activities on human PTP1B.  The   inhibition rates of Salvia crude extracts varied from 9.76% to 100% at 30 μg·mL^-1, with the most convincing  effects coming from Salvia evansiana and Salvia castanea.  HPLC analysis revealed seven components shared by Salvia samples could be related to the inhibitory activities.
     

脊髓背角PKC在慢性炎性疼痛中的作用及其机制

目的探讨蛋白激酶C(protein kinase C,PKC)的抑制剂和激动剂对痛觉超敏的影响及其分子机制。方法小鼠后趾皮下注射完全弗氏佐剂(complete Freund's adjuvant,CFA)建立炎性疼痛模型;鞘内给予PKC抑制剂白屈菜赤碱(chelerythrine,CHE)或激动剂Phorbol 12-myristate 13-acetate(PMA)前后,测定小鼠缩足阈值;随后立即分离脊髓背角,免疫印迹法检测NMDA(N-methyl-D-aspartate)型谷氨酸受体的突触表达。结果 PKC抑制剂CHE在缓解炎性痛觉超敏的同时,明显翻转脊髓NMDA受体NR2B亚基的突触表达亢进;而正常小鼠鞘内给予PKC激动剂PMA,可模拟CFA的效应,即:诱发痛觉超敏,并特异性增加NR2B亚基的突触含量。结论 PKC通过调节脊髓NMDA受体NR2B亚基的突触表达,参与炎性疼痛的形成。     

蛋白酪氨酸磷酸酶1B及其小分子抑制剂的研究进展

蛋白酪氨酸磷酸酶1B（PTP-1B）是胰岛素和瘦素信号传导通路的负调节因子。目前,作为糖尿病和肥胖症治疗的新靶点,PTP-1B抑制剂的研究引起了广泛的关注。现从PTP-1B简介、生理功能以及抑制剂的结构性质等方面进行综述。     

榅桲果实及种子不同提取部位的蛋白酪氨酸磷酸酶1B抑制活性筛选

目的探讨榅桲果实和种子的甲醇提取物及其不同溶剂提取部位对蛋白酪氨酸磷酸酶1B(PTP1B)的抑制作用,筛选抗糖尿病活性部位。方法利用PTP1B体外筛选系统,对榅桲果实和种子不同提取部位进行细致筛选,确定其降糖活性部位。结果榅桲果实和种子各部位均有不同程度抑制PTP1B活性的作用,其中榅桲种子乙酸乙酯部位、榅桲果实二氯甲烷部位和榅桲果实水部位的体积分数为50%的乙醇溶液洗脱部位对PTP1B抑制作用强,其半数抑制质量浓度(IC50)分别为0.844,0.423和0.395mg·L-1,初步推测榅桲果实及种子提取物具有抗2型糖尿病的潜力。结论榅桲种子乙酸乙酯部位、榅桲果实二氯甲烷部位和榅桲果实水部位的体积分数为50%的乙醇溶液洗脱部位是主要的抗糖尿病活性部位。     

急性阿片耐受大鼠脊髓NMDA受体NR2A及NR2B亚基表达的改变

目的：了解大鼠短期多次应用芬太尼能否发生急性阿片耐受以及急性阿片耐受大鼠脊髓NMDA受体NR2A和NR2B亚基表达的改变。方法：24只体重为200-220g的雄性SD大鼠随机分为3组（n=8）：对照组（C）,生理盐水组（S）及芬太尼组（F）。F组大鼠给予皮下注射芬太尼30μg/kg,共4次,每两次注射之间间隔15min,S组大鼠以生理盐水代替芬太尼,C组大鼠未给药。给药前及给药结束后每30min以Von-Frey仪测定各组大鼠的机械刺激缩足阈值（paw withdrawal threshold,PWT）。当F组大鼠的PWT恢复到给药前基础水平时,各组大鼠均给予腹腔注射吗啡5mg/kg。随后仍每30min测定各组大鼠的PWT,直至F组大鼠的PWT再次回到基础水平。对各组大鼠不同时间点的PWT进行组内和组间比较。另24只体重为2000-220g的雄性SD大鼠分组及给药方法同前（分为C^＊、S^＊及F^＊组）,当F＊组大鼠的PWT首次恢复到基础值时,不给予吗啡,处死各组大鼠,取脊髓,以Western blotting方法测定NMDA受体NR2A及NR2B亚基的蛋白表达水平。结果：连续4次皮下注射芬太尼（F组）后大鼠首先表现为PWT较基础值显著升高,随后PWT降低到基础值以下,然后逐渐恢复至基础值水平,此时皮下注射吗啡后,吗啡的镇痛效果显著低于其他两组大鼠（S组,C组）。F^＊组大鼠脊髓的NR2B亚基表达水平显著高于C^＊组及S^＊组,各组大鼠脊髓的NR2A亚基表达水平的差异无统计学意义。结论：短期应用芬太尼可导致大鼠发生急性阿片耐受。急性阿片耐受大鼠的脊髓NMDA受体NR2B亚基表达水平显著升高,NR2A亚基表达水平无明显变化。     

外周组织损伤对脊髓AMPA受体突触表达的影响及其机制

目的研究外周组织损伤对脊髓背角AMPA受体突触表达的影响及其分子调节机制。方法小鼠足底皮下注射完全弗氏佐剂(CFA),建立炎性疼痛模型;24 h后分离L4～L5脊髓背角,提取"富含突触后致密质(PSD)"的亚细胞结构,免疫印迹法检测AMPA受体GluR2亚基突触表达的变化,并观察cAMP依赖性蛋白激酶(PKA)在调节GluR2突触含量中的作用。结果 CFA在引发痛觉超敏的同时,脊髓背角GluR2的突触含量明显升高。虽然PKA抑制剂H-89并不影响正常小鼠的痛阈及GluR2的突触含量,但H-89却能有效缓解炎性痛觉超敏,同时完全翻转CFA诱发的GluR2亚基在突触中的过量表达。结论外周组织损伤通过激活脊髓PKA,明显提高AMPA受体GluR2亚基在脊髓突触中的含量,可能是慢性炎性疼痛形成的重要机制。     

糖尿病神经痛大鼠脊髓背角NMDA受体表达的变化

目的探讨糖尿病神经病理性痛(DNP)大鼠脊髓背角N-甲基-D-天冬氨酸(NMDA)受体表达的变化。方法 SD雄性大鼠24只,4周龄,体重130～150g,随机分为2组,DNP组(D组,n=15)腹腔注射链尿佐菌素(STZ)50mg/kg制备成DNP模型,正常对照组(C组,n=9)给予等容量0.9%氯化钠溶液。给予STZ或0.9%氯化钠溶液后3、5、7周时分别取D组(5只)和C组(3只)大鼠,测量体重,采集静脉血样,测定空腹血糖浓度,然后测定机械缩足阈值,取脊髓组织,采用免疫组织化学法分别测定脊髓背角NR1和NR2B亚单位表达水平。结果与C组比较,在各时间点,D组体重下降,血糖升高,机械缩足阈值明显降低(P<0.05),脊髓背角NR1和NR2B亚单位免疫阳性细胞数表达明显增加(P<0.05)。结论脊髓背角NMDA受体在脊髓水平参与了大鼠糖尿病神经病理性痛的形成。     

蛋白酪氨酸磷酸酶SHP-1在哮喘小鼠肺组织的表达及其与气道炎症关系

目的 探讨蛋白酪氨酸磷酸酶SHP-1在哮喘小鼠肺组织的表达及其与哮喘气道炎症的关系.方法 实验分为3组,随机将30只BALB/c小鼠分为对照组(N)、哮喘组(A)、原矾酸钠干预组(S),每组10只.哮喘组采用OVA腹腔注射致敏、长时间反复OVA雾化吸入复制慢性哮喘动物模型.对照组采用等量生理盐水腹腔注射,生理盐水雾化吸入.原矾酸钠组早期同哮喘模型组一样采用OVA腹腔注射及OVA雾化吸入,在动物处死前末周采用原矾酸钠溶液腹腔注射.末次激发后24h内处死动物,支气管肺泡灌洗液(BALF)作细胞计数测小鼠的白细胞总数及分类,免疫组化测IL-4R的表达,逆转录PCR测小鼠肺组织SHP-1 mRNA的表达.结果 细胞总数A组(7.35±0.589)×105/ml与N组(1.35±0.369)×105/ml比较差异有统计学意义(P＜0.05),S组(7.55±0.925)×105/ml与N组差异有统计学意义(P＜0.05),A组与S组比较差异无统计学意义(P＞0.05).IL-4R IOD A组(149.221±11.154)与N组(92.676±16.414)比较差异有统计学意义(P＜0.05),S组(150.337±10.832)与N组差异有统计学意义(P＜0.05),A组与S组比较差异无统计学意义(P＞0.05).SHP-1 mRNA含量积分光密度A组(0.0763±0.018)与N组(2.728±0.101)比较差异有统计学意义(P＜0.05),S组(0.064±0.011)与N组比较差异有统计学意义(P＜0.05),A组与S组差异无统计学意义(P＞0.05).各组肺组织中IL-4R与SHP-1 mRNA均呈负相关,相关系数分别是A组r=-0.963,S组r=-0.909,N组r=-0.956.结论 SHP-1 mRNA的表达与哮喘小鼠炎性细胞及IL-4R的表达呈负相关.     

饮食诱导肥胖大鼠蛋白酪氨酸磷酸酶1B表达的变化

目的 观察饮食诱导肥胖大鼠骨骼肌、肝脏、胰腺蛋白酪氨酸磷酸酶1B表达变化,探讨蛋白酪氨酸磷酸酶1B在肥胖胰岛素抵抗发生巾的作用.方法 雄性SD大鼠20只,随机均分为正常饲养(NC)组和高脂饲养(HF)组,喂养8周,比较两组大鼠体重、胰岛素抵抗指数(HOMA-IR)及骨骼肌、肝脏、胰腺蛋白酪氨酸磷酸酶1B的表达.结果 HF组大鼠体重比NC纰显著增加,并出现胰岛素抵抗(HOMA-IR 8.58±3.37 vs.3.46±2.21)(P＜0.01).HF组大鼠骨骼肌、肝脏、胰腺蛋白酪氨酸磷酸酶1R表达量较NC组显著增加(P＜0.01).结论 高脂饮食可诱导大鼠肥胖伴胰岛素抵抗,其胰岛素抵抗的形成可能与蛋白酪氨酸磷酸酶1B表达水平的升高有关.     

蛇床子素通过抑制髓核致炎大鼠脊髓背角CXCL1/CXCR2的表达发挥抗炎镇痛作用

目的研究蛇床子素(osthole,Ost)对大鼠髓核源性神经根痛的抗炎镇痛作用。方法雄性SD大鼠60只随机分为3组:髓核致炎组(NP组,n=36)、Sham组(n=12)、Blank组(n=12),检测术前和术后3、7、14、21、28 d的50%MWT;q-PCR检测术后各时间点脊髓背角CXCL1、CXCR2和TNF-α的mRNA水平,Western blot检测术后各时间点脊髓背角CXCL1/CXCR2的蛋白水平。另外雄性SD大鼠64只髓核致炎后随机均分为4组:生理盐水组(NP+NS),DMSO组(NP+DMSO),Ost组(NP+Ost),CXCL1中和抗体组(NP+AF-515),于术后d 3经硬膜外注射相应药物50μL。给药前后多个时点均检测50%MWT。于术后d 7取L5脊髓背角,通过Western blot检测各组脊髓背角CXCL1/CXCR2的蛋白水平。结果NP组术后各时间点的50%MWT较Sham组均降低(P<0.05)。NP组术后各时间点脊髓背角CX-CL1、CXCR2和TNF-α的mRNA水平均较Sham组升高(P<0.05),且NP组术后各时间点脊髓背角CXCL1/CXCR2的蛋白水平较Sham组升高(P<0.05)。NP+Ost组给药后50%MWT比给药前升高(P<0.05),NP+AF515组在给药后3 h 50%MWT升高,持续到给药后12 h(P<0.05)。NP+Ost组术后7d脊髓背角CXCL1/CXCR2蛋白表达降低(P<0.05)。结论脊髓背角CXCL1/CXCR2可能参与了大鼠髓核源性神经根痛的发生发展,蛇床子素通过抑制脊髓背角CXCL1/CXCR2的表达而缓解髓核致炎大鼠神经根性疼痛。     

Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice

BACKGROUND AND PURPOSE: Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. EXPERIMENTAL APPROACH: We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. KEY RESULTS: Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. CONCLUSIONS AND IMPLICATIONS: The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.     

Involvement of protein kinase C and Src tyrosine kinase in acute tolerance to ethanol inhibition of spinal NMDA-induced pressor responses in rats

Background and purpose:

The present study was carried out to examine the role of protein kinases in the development of acute tolerance to the effects of ethanol on spinal N-methyl-D-aspartate (NMDA) receptor-mediated pressor responses during prolonged ethanol exposure.

Experimental approach:

Blood pressure responses induced by intrathecal injection of NMDA were recorded. The levels of several phosphorylated residues on NMDA receptor NR1 (GluN1) (NR1) and NMDA receptor NR2B (GluN2B) (NR2B) subunits were determined by immunohistochemistry and Western blot analysis.

Key results:

Ethanol inhibited spinal NMDA-induced pressor responses at 10 min, but the inhibition was significantly reduced at 40 min following continuous infusion. This effect was dose-dependently blocked by chelerythrine [a protein kinase C (PKC) inhibitor, 1–1000 pmol] or PP2 (a Src family tyrosine kinase inhibitor, 1–100 pmol) administered intrathecally 10 min following ethanol infusion. A significant increase in the immunoreactivity of phosphoserine 896 of NR1 subunits (pNR1-Ser896) and phosphotyrosine 1336 of NR2B subunits (pNR2B-Tyr1336) was found in neurons of intermediolateral cell column during the development of tolerance. Levels of pNR1-Ser896 and pNR2B-Tyr1336 were also significantly increased in lateral horn regions of the spinal cord slices incubated with ethanol for 40 min in vitro. The increases in pNR1-Ser896 and pNR2B-Tyr1336 levels were inhibited by post-treatment with chelerythrine and PP2, respectively, both in the in vivo and in vitro studies.

Conclusions and implications:

The results suggest that activation of PKC and Src tyrosine kinase during prolonged ethanol exposure leading to increases in the levels of pNR1-Ser896 and pNR2B-Tyr1336 may contribute to acute tolerance to inhibition by ethanol of NMDA receptor function.     

运动训练对脊髓损伤大鼠脊髓内BDNF及其受体TrkB表达的影响

目的 研究运动训练对脊髓损伤（SCI）大鼠脊髓内脑源性神经营养因子（BDNF）及其酪氨酸激酶受体B（TrkB）表达的影响,探讨运动训练促进脊髓损伤功能恢复的可能机制。方法 24只成年雌性SD大鼠随机分为假手术组、损伤对照组和运动训练组。采用通用型脊髓打击器建立大鼠T10脊髓损伤模型。损伤后7天起,对SCI大鼠进行4周运动训练,假手术组和损伤对照组不进行运动训练。损伤前及损伤后第1、2、3、4、5周采用BBB评分评定运动功能。运动训练结束后(即损伤后5周)取大鼠T12～L1节段脊髓,免疫组织化学检测脊髓内BDNF和TrkB表达及分布,Western blot检测脊髓内BDNF和TrkB蛋白含量。结果 运动训练组和损伤对照组BBB评分均较损伤后第1、2周明显提高,运动训练组较损伤对照组增加更为显著（P<0.05）。BDNF免疫反应阳性产物多分布于脊髓前角,脊髓后角及中央管周围也有出现;运动训练组BDNF阳性染色颗粒增多,平均光密度值较假手术组及损伤对照组均显著增加（P<0.05）。TrkB免疫反应阳性产物于脊髓前角、后角、中央管周围等处均出现较多分布;运动训练组TrkB阳性染色颗粒增多,平均光密度值较假手术组及损伤对照组均显著增加（P<0.05）。Western blot结果显示运动训练组大鼠脊髓内BDNF及TrkB的表达较假手术组、损伤对照组均明显增加(P＜0.05)。结论 运动训练能诱导脊髓损伤大鼠脊髓内BDNF及其受体TrkB表达,促进SCI大鼠运动功能恢复。     

蛇床子素对髓核致炎神经根痛大鼠的保护作用

目的 研究蛇床子素通过抑制脊髓背角胞外信号调节激酶(ERK)/丝裂原活化蛋白激酶(MAPK)通路对髓核致炎神经根痛大鼠的保护作用.方法 将80只大鼠随机分为对照组(n=20)、模型组(n=20)及低(n=20)、高剂量实验组(n=20).模型组及低、高剂量实验组大鼠均用手术在特定椎节(L5-6)左侧建立髓核致炎神经根痛...     

Regulation of NMDA receptor subunit expression and its implications for LTD, LTP, and metaplasticity

NMDA-type glutamate receptors (NMDARs) mediate many forms of synaptic plasticity. These tetrameric receptors consist of two obligatory NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. In the neonatal neocortex NR2B-containing NMDARs predominate, and sensory experience facilitates a developmental switch in which NR2A levels increase relative to NR2B. In this review, we clarify the roles of NR2 subunits in synaptic plasticity, and argue that a primary role of this shift is to control the threshold, rather than determining the direction, for modifying synaptic strength. We also discuss recent studies that illuminate the mechanisms regulating NR2 subunits, and suggest that the NR2A/NR2B ratio is regulated by multiple means, which may control the ratio both locally at individual synapses and globally in a cell-wide manner. Finally, we use the visual cortex as a model system to illustrate how activity-dependent modifications in the NR2A/NR2B ratio may contribute to the development of cortical functions.     

IL-36Ra抑制炎性痛小鼠脊髓A1型星形胶质细胞极化

目的评价不同剂量IL-36Ra对炎性痛小鼠痛行为以及脊髓A1型星形胶质细胞极化的影响。方法雄性C57BL/6小鼠32只,采用足底注射完全弗氏佐剂(CFA)建立炎性痛模型。随机将小鼠分为CFA+生理盐水组、CFA+IL-36Ra 50 ng组、CFA+IL-36Ra 100 ng组以及CFA+IL-36Ra 200 ng组,每组8只。各组小鼠在造模后d 1~d 7,每日1次鞘内给药。同时,分别在造模前以及造模后d 1、3、5、7检测4组小鼠机械刺激缩足阈值(mechanical withdraw threshold, MWT)和辐射热刺激缩爪潜伏期(PWL)的变化。以逆转录聚合酶链反应检测IL-36Ra对CFA小鼠脊髓A1、A2型星形胶质细胞标志物表达水平的影响。以免疫组化法检测各组小鼠脊髓背角A1型星形胶质细胞标志物C3与GFAP共表达水平的变化。结果炎性痛小鼠造模后患侧MWT、PWL均明显下降,IL-36Ra(100、200 ng)可显著改善小鼠的机械性痛觉超敏与热痛觉过敏;且在治疗7 d后,IL-36Ra可降低CFA小鼠脊髓星形胶质细胞激活标志物GFAP、Lcn2的表达水平,抑制星形胶质细胞激活;同时,IL-36Ra(100、200 ng)可下调CFA小鼠脊髓A1型星形胶质细胞标志物Serping1、 H2-T23的mRNA水平,但IL-36Ra各个剂量对A2型星形胶质细胞标志物表达没有明显作用;此外,IL-36Ra还可抑制脊髓背角A1型星形胶质细胞标志物C3表达。结论 IL-36Ra可能通过抑制CFA小鼠脊髓A1型星形胶质细胞极化,进而改善小鼠炎性痛痛行为。