MAPK信号转导途径在蜜蜂毒诱致的炎症与疼痛中的作用机制研究

发布时间：2018-08-12 14:00

【摘要】： 研究背景:外周组织损伤所致的炎症既伴随有疼痛的产生(持续性自发痛、痛敏及触痛等)同时又有明显的红、肿、热等炎症反应。我们以往的研究发现,大鼠后足底中心皮下注射蜜蜂毒溶液不仅能产生明显的持续自发痛反应、热及机械性痛敏反应[1-5],而且在注射部位有明显的红肿等炎症发应[1-5]。已有大量的研究表明MAP(mitogen-activated protein kinase)与疼痛及炎症的发生有关,且ERK1/2和P38在组织和神经损伤诱致的疼痛和热及机械痛敏中的作用各不相同。目的:通过观察周围局部预先给予ERK1/2、P38和MEK的抑制剂对皮下注射蜜蜂毒诱致的持续自发疼痛反应、机械性痛敏以及炎症反应,用以探讨MAPK信号转导途径在蜜蜂毒诱致的炎症与疼痛中的作用机制。这些研究可能为临床炎性疼痛治疗提供新的理论资料和思路。材料和方法:应用蜜蜂毒(bee venom, BV)作为化学致痛剂皮下注射模型来研究MAPK在大鼠的自发痛反应、机械痛敏和炎症反应中的作用。实验采用雄性SD大鼠,体重250～300克。分别预先10分钟给予大鼠一侧后肢足底皮下注射不同剂量的ERK1/2抑制剂PD98059,P38抑制剂SB202190和MEK1/2抑制剂U0126,其后注射侧后肢足底皮下注射蜜蜂毒(0.2mg/50　l)。而对照组皆注射PD98059,SB202190和U0126的溶媒(50μl/只,DMSO:PBS=3:7)。同时为了排除药物的全身效应,分别提前10分钟在蜜蜂毒注射的对侧皮下注射相应的较高剂量的PD98059(100ug),SB202190(100ug/50μl)和U0126(10ug/50μl)。观察大鼠在蜜蜂毒注射后1个小时内自发痛行为(缩足反射),每5分钟为一个计时点。蜜蜂毒注射前及注射后2小时用von-Frey机械刺激器纤维检测注射侧鼠爪机械刺激反应阈值( paw withdrawal mechanical threshold ,PWMT)以及应用YLS-7A足趾容积测量仪测量注射前及蜜蜂毒注射后3小时注射侧鼠爪的体积。结果: (1)对自发痛的影响:同侧及对侧SB202190处理组:对蜜蜂毒诱致的自发缩足反射无任何影响;同侧PD98059处理组:对蜜蜂毒诱致的自发缩足反射有明显的抑制作用,且在后期(26-60 min)更加明显,无明显剂量依赖性,对侧PD98059处理组:对蜜蜂毒诱致的自发缩足反射无影响;同侧U0126处理组:在早期及晚期低剂量的U0126对自发缩足反射皆有易化作用,中等剂量1ug对自发缩足反射无影响,高剂量10ug对自发缩足反射在早期无影响,在晚期有明显抑制作用,对侧U0126处理组:对蜜蜂毒诱致的自发缩足反射无影响。 (2)对痛敏的影响:同侧及对侧PD98059处理组:对蜜蜂毒诱致的机械性刺激阈值的下降则无任何影响;同侧SB202190处理组:蜜蜂毒诱致的机械性刺激阈值的下降发生翻转,各个剂量之间无明显差异即无明显剂量依赖,而对侧SB202190处理对蜜蜂毒诱致的机械性刺激阈值的下降则无任何影响;同侧及对侧U0126处理对蜜蜂毒诱致的机械性刺激阈值的下降无任何影响。 (3)对炎症的影响:同侧或对侧PD98059处理组:对由蜜蜂毒引起的鼠爪体积增加无明显影响;同侧SB202190处理组:高剂量(100ug)时能够明显抑制蜜蜂毒注射所引起的鼠爪体积的增加,而低剂量(1ug和10ug)及对侧SB202190处理组:对由蜜蜂毒引起的鼠爪体积增加无明显影响;同侧或对侧U0126处理组:对由蜜蜂毒引起的鼠爪体积增加无明显影响。结论: (1)MEK1/2—MAPK信号转导途径介导蜜蜂毒诱致的持续自发缩足行为,且其表达水平的高低对蜜蜂毒诱致的持续自发缩足行为有关:一定水平的MEK1/2表达可能抑制而不是易化蜜蜂毒诱致的自发痛行为,而高水平的MEK1/2表达可能促进蜜蜂毒诱致的自发痛行为。P38—MAPK信号转导途径不参与介导蜜蜂毒诱致的持续自发痛行为。ERK1/2—MAPK信号转导途径参与蜜蜂毒诱致的持续自发缩足反射。 (2)外周P38—MAPK信号转导途径参与蜜蜂毒诱致的机械性痛敏,但无剂量依赖性。MEK1/2—ERK1/2 MAPK信号转导途径不参与蜜蜂毒诱导的机械性痛敏的发生。 (3) ERK-MEK MAPK信号转导途径不参与蜜蜂毒诱致的炎症反应,而P38参与蜜蜂毒诱致的炎症反应,并且有剂量依赖性。
[Abstract]:BACKGROUND: Inflammation caused by peripheral tissue injury is accompanied by pain (persistent spontaneous pain, hyperalgesia, tenderness, etc.) and obvious inflammation such as red, swollen, and hot. Our previous studies have shown that subcutaneous injection of bee venom solution into the center of the hind plantar of rats can not only produce significant persistent spontaneous pain, but also produce significant thermal and mechanical effects. A large number of studies have shown that MAP (mitogen-activated protein kinase) is associated with pain and inflammation, and ERK1/2 and P38 play different roles in tissue and nerve injury-induced pain, heat and mechanical hyperalgesia.
OBJECTIVE: To investigate the mechanism of MAPK signaling pathway in inflammation and pain induced by honeybee venom by observing the effects of ERK1/2, P38 and MEK inhibitors on spontaneous pain, mechanical hyperalgesia and inflammation induced by subcutaneous injection of honeybee venom. To provide new theoretical information and ideas.
Materials and Methods: Bee venom (BV) was used as a subcutaneous injection model to study the effects of MAPK on spontaneous pain, mechanical pain and inflammation in rats. Male SD rats weighing 250-300 grams were given different doses of ERK1/2 subcutaneously for 10 minutes. PD98059, P38 inhibitor SB202190 and MEK1/2 inhibitor U0126 were injected subcutaneously into the plantar of hind limbs (0.2mg/50l). The control group was injected with PD98059, SB202190 and U0126 (50ml/rat, DMSO:PBS=3:7). In order to exclude the systemic effect of the drug, the contralateral subcutaneous injection was performed 10 minutes earlier. Higher doses of PD98059 (100ug), SB202190 (100ug/50ugl) and U0126 (10ug/50ugl) were used to observe the spontaneous pain behavior (foot retraction) in rats within 1 hour after bee venom injection, and the time point was 5 minutes. The mechanical stimulator fibers were used to detect the mechanical stimulus response threshold of rats'paws before and 2 hours after bee venom injection. Paw withdrawal mechanical threshold (PWMT) and YLS-7A toe volume meter were used to measure the paw volume before and 3 hours after bee venom injection.
Result:
(1) Effect on spontaneous pain: ipsilateral and contralateral SB202190 treatment group: no effect on the spontaneous systolic reflex induced by honey bee venom; ipsilateral PD98059 treatment group: the spontaneous systolic reflex induced by honey bee venom was significantly inhibited, and in the later period (26-60 minutes) more obvious, no significant dose-dependent, contralateral PD98059 treatment group: on the venom induced by honey bee venom Ipsilateral U0126 treatment group: low dose U0126 had facilitation effect on spontaneous shrinkage reflex in early and late stage, medium dose 1ug had no effect on spontaneous shrinkage reflex, high dose 10ug had no effect on spontaneous shrinkage reflex in early stage, but had obvious inhibition effect in late stage. The spontaneous retraction reflex had no effect.
(2) The effect on pain sensitivity: ipsilateral and contralateral PD98059 treatment group: no effect on the decrease of mechanical stimulation threshold induced by bee venom; ipsilateral SB202190 treatment group: the decline of mechanical stimulation threshold induced by bee venom reversed, and there was no significant dose dependence between different doses, while the ipsilateral SB202190 treatment on bee. The decrease of mechanical stimulation threshold induced by bee venom had no effect, and the decrease of mechanical stimulation threshold induced by bee venom was not affected by ipsilateral and contralateral U0126 treatments.
(3) Inflammation: ipsilateral or contralateral PD98059 treatment group: no significant effect on the increase of paw volume caused by bee venom; ipsilateral SB202190 treatment group: high dose (100ug) can significantly inhibit the increase of paw volume caused by bee venom injection, while low dose (1ug and 10ug) and contralateral SB202190 treatment group: on the increase of paw volume caused by bee venom The same side or contralateral U0126 treatment group had no significant effect on the increase of rat paw volume caused by bee venom.
Conclusion:
(1) MEK1/2-MAPK signal transduction pathway mediates persistent spontaneous foot shrinkage induced by honeybee venom, and its expression level is related to persistent spontaneous foot shrinkage induced by honeybee venom: a certain level of MEK1/2 expression may inhibit but not predispose spontaneous pain induced by honeybee venom, while high level of MEK1/2 expression may promote honeybee venom. P38-MAPK signal transduction pathway was not involved in the mediation of persistent spontaneous pain induced by bee venom. ERK1/2-MAPK signal transduction pathway was involved in the persistent spontaneous contraction reflex induced by bee venom.
(2) Peripheral P38-MAPK signal transduction pathway is involved in mechanical hyperalgesia induced by bee venom, but not dose-dependent. MEK1/2-ERK1/2 MAPK signal transduction pathway is not involved in mechanical hyperalgesia induced by bee venom.
(3) ERK-MEK MAPK signaling pathway did not participate in the inflammation induced by bee venom, but P38 was involved in the inflammation induced by bee venom in a dose-dependent manner.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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