乳香—没药通过抑制TRPV1缓解小鼠神经病理性疼痛的研究

发布时间：2018-09-18 21:53

【摘要】：神经病理性疼痛在临床上属于一种难治性的慢性疼痛病症,因其高发病率和低治愈率日益受到国内外多学科的关注。在神经病理性疼痛的机制研究中,TRPs通道受到广泛关注,尤其是主要表达在背根神经节(dorsal root ganglia,DRG)和三叉神经节(n.trigminus,TG)小直径神经元上的TRPV1(瞬时感受器电位香草酸受体1,Transient Receptor Potential Vanilloid 1),被认为与疼痛的关系尤为密切,是炎症和疼痛通路的门控分子,在神经病理性疼痛发生、发展过程中具有重要作用。TRPV1被认为是治疗急、慢性疼痛药物研发最具前景的作用靶点之一。乳香和没药在疼痛的治疗中表现出鲜明的特色和良好的效果,二者在临床上常被作为一组药对使用,来获得协同镇痛的效果,但是其镇痛的机制尚不清楚。为了揭示乳香和没药的镇痛机制,本论文建立了小鼠坐骨神经慢性压迫模型(chronic constriction injury,CCI),观察乳香-没药水提液(A combined water extract of frankincense andmyrrh,WFM)对CCI所致神经病理性疼痛痛觉敏化的影响,并围绕TRPV1开展乳香-没药水提液缓解神经病理性疼痛的机制研究。目的:探讨乳香-没药水提液在小鼠神经病理性疼痛模型中的镇痛作用和相关的神经机制。方法:(1)C57BL/6小鼠,雄性,6-8周,8只,灌胃给予乳香-没药水提液(7.5 g/kg),分别于给药前、给药后1、2、3、4、5小时50℃水浴浸尾,记录小鼠出现甩尾的时间。(2)16只小鼠随机分为生理盐水组(Saline)和WFM(7.5 g/kg),提前3小时分别灌胃给予生理盐水和WFM,然后右脚掌注射辣椒素(3 μg/mouse),录像并统计小鼠舔脚的次数和时间。(3)72只小鼠随机分为6组(每组12只):①空白(na1ve)组:正常野生型(wild type mice,WT)小鼠+蒸馏水(vehicle);②假手术(sham)组:假手术WT小鼠+蒸馏水;③坐骨神经慢性压迫模型(CCI)组:CCIWT小鼠+蒸馏水;④乳香-没药水提液低剂量(WFM-L)组:CCIWT小鼠+WFM-L(WFM,1.5g/kg/day);⑤乳香-没药水提液高剂量(WFM-H)组:CCIWT小鼠+WFM-H(WFM,7.5g/kg/day);⑥加巴喷丁(GBPT)组:CCI WT小鼠+ GBPT(GBPT,0.2 g/kg/day)。每组小鼠从第7天开始灌胃给蒸馏水或乳香-没药水提液或加巴喷丁,连续10天,到第16天结束,同组小鼠每天灌胃给药的体积是等量的,且由同一人操作。(4)6组小鼠分别于术后第1、3、5、7、10、13、16天称体重、VonFrey测机械缩足阈值(MWT)、热辐射仪测热缩足反射潜伏期(TWL)。(5)取小鼠腰4、5及骶部的DRG,采用免疫荧光染色、实时荧光定量-聚合酶链式反应(q-PCR)、蛋白质免疫印迹(Western blotting)观察各组小鼠TRPV1表达活性变化。(6)取小鼠腰4、5及骶部的DRG,通过钙离子成像技术观察各组小鼠DRG神经元TRPV1生理活性的变化。结果:(1)灌胃给予乳香-没药水提液后小鼠对伤害性热刺激的敏感性显著下降,第3、4、5小时与对照相比都有显著差异。(2)乳香-没药水提液灌胃处理后,脚掌注射辣椒素所致小鼠的舔脚次数和时间较生理盐水组都显著减少。(3)CCI小鼠体重略有下降,各组小鼠的体重无显著性差异。(4)从术后第5天开始小鼠的机械缩足阈值和热缩足反射潜伏期都显著下降,以第7天左右下降最明显,第7天开始热缩足反射潜伏期略有回升,械缩足阈值仍在继续下降。第16天的机械缩足阈值和热缩足反射潜伏期统计结果显示:naive组和sham组没有显著变化;与sham组相比,CCI组则显著下降(P0.001);与CCI组相比WFM-L组、WFM-H组、GBPT组,机械缩足阈值和热缩足反射潜伏期都显著升高(P0.05或P0.001)。(5)免疫荧光染色统计结果表明:与sham组相比CCI组TRPV1阳性神经元比例显著增加(P0.001),而WFM-H组、GBPT组与CCI组相比,TRPV1阳性神经元的比例显著减少(P0.05或P0.001)。(6)TRPV1基因和蛋白的相对定量结果发现:与sham组相比CCI组TRPV1的表达显著增加(P0.001)而WFM-H组、WFM-L组、GBPT组与CCI组相比显著减少(P0.01或P0.05)。(7)钙成像实验发现:与sham组相比CCI组DRG神经元对TRPV1激动剂辣椒素(capsaicine)的反应大小和比例都显著增加(P0.001),而WFM-H组、GBPT组DRG神经元对辣椒素反应的比例与CCI组相比显著下降(P0.05或P0.001),WFM-L组也有下降的趋势,但没有显著性差异;WFM-H组、WFM-L组、GBPT组DRG神经元对辣椒素反应的大小与CCI组相比都有显著下降(P0.001)。结论:WFM通过抑制TRPV1的表达和活性缓解了 CCI诱导的小鼠神经病理性疼痛。
[Abstract]:Neuropathic pain (NP) is a refractory chronic pain disorder in clinic, which has attracted more and more attention from many disciplines at home and abroad because of its high incidence and low cure rate. TRPV1 (Transient Receptor Potential Vanilloid 1) on small-diameter neurons of the menstrual ganglion (TG) is thought to be closely related to pain, and is a gated molecule in inflammatory and pain pathways, which plays an important role in the development of neuropathic pain. Frankincense and myrrh have shown distinct characteristics and good effects in the treatment of pain. They are often used as a group of drugs in clinic to achieve synergistic analgesic effect, but the mechanism of analgesia is still unclear. In order to reveal the analgesic effect of frankincense and myrrh Mechanisms: A combined water extract of frankincense and myrrh (WFM) was used to observe the effects of chronic constriction injury (CCI) on the sensitization of neuropathic pain induced by CCI in mice, and a frankincense-myrrh (WFM) aqueous extract was developed around TRPV1 to relieve neuropathic pain. Objective: To investigate the analgesic effect of Ruxiang-myrrh aqueous extract on neuropathic pain model in mice. Methods: (1) C57BL/6 mice, male, 6-8 weeks, 8 mice, were given Ruxiang-myrrh aqueous extract (7.5 g/kg) by gastric lavage, and were soaked in water bath at 50 C for 1,2,3,4,5 hours before and after administration respectively. (2) Sixteen mice were randomly divided into saline group (Saline) and WFM (7.5 g/kg), given saline and WFM three hours earlier, then injected capsaicin (3 ug/mouse) into the right paw, and recorded the times and time of licking feet. (3) 72 mice were randomly divided into 6 groups (12 mice in each group): blank (na1ve) group: normal field Wild type mice (WT) mice + distilled water (vehicle); sham operation (sham) group: sham operation WT mice + distilled water; chronic sciatic nerve compression (CCI) group: CCIWT mice + distilled water; frankincense-myrrh water extract low dose (WFM-L) group: CCIWT mice + WFM-L (WFM, 1.5g/kg/day); high dose of mastic-myrrh water extract (WFM-H) group: CCI WFM-H group: CCIWT small. Rats + WFM-H (WFM, 7.5 g/kg/day); _Gabapentin (GBPT) group: CCI WT mice + GBPT (GBPT, 0.2 g/kg/day). Each group of mice was given distilled water or frankincense-myrrh aqueous extract or gabapentin from the 7th day for 10 consecutive days until the end of the 16th day. The same group of mice were given the same amount of gabapentin daily and operated by the same person. Weight was weighed on the 1st, 3rd, 5th, 7th, 10th, 13th and 16th day after operation, mechanical foot shrinkage threshold (MWT) was measured by Von Frey, and latency of foot shrinkage reflex (TWL) was measured by thermal radiometer. Results: (1) The sensitivity of mice to noxious thermal stimulation was significantly decreased after administration of frankincense-myrrh aqueous extract by intragastric administration, and there were significant differences between the 3, 4 and 5 hours after administration of frankincense-myrrh aqueous extract and the control group. (3) The weight of CCI mice decreased slightly, and there was no significant difference among the groups. (4) From the 5th day after operation, the mechanical shrinkage threshold and the latency of heat shrinkage reflex of mice decreased significantly, to about 7 days. The mechanical shrinkage threshold and thermal shrinkage latency showed no significant changes on the 16th day. Compared with sham group, CCI group decreased significantly (P 0.001); WFM-L group, WFM-H group, GBPT group. The percentage of TRPV1 positive neurons in CCI group was significantly higher than that in sham group (P 0.001), while that in WFM-H group and GBPT group was significantly lower than that in CCI group (P 0.05 or P 0.001). Compared with sham group, the expression of TRPV1 in CCI group was significantly increased (P 0.001), while that in WFM-H group, WFM-L group, GBPT group and CCI group was significantly decreased (P 0.01 or P 0.05). (7) Calcium imaging showed that compared with sham group, the response of DRG neurons to capsaicine in CCI group was significantly increased (P 0.0). The percentage of DRG neurons responding to capsaicin in WFM-H, GBPT and WFM-L groups decreased significantly (P 0.05 or P 0.001), but there was no significant difference between WFM-H, WFM-L, GBPT and CCI groups. Conclusion: WFM can inhibit TRPV1 by inhibiting the response of DRG neurons to capsaicin in WFM-H, WFM-L and GBPT groups (P 0.001). Expression and activity alleviated CCI induced neuropathic pain in mice.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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