染料木素拮抗雌激素作用缓解咀嚼肌疼痛的机制研究

There are considerable controversies about the effect of estrogen on the pain in the masticatory muscle and temporomandibular joint of women with temporomandibular disorders (TMD) and the available treatments for these chronic pain conditions are ineffective. Animal studies indicate increasing estrogen level enhances the sensitivity of nociceptors in masseter muscle to NMDA receptor agonists. Theoretically, phytoestrogens can relieve pain by reducing the magnitude of fluctuations in estrogenic effect over the menstrual cycle. However, the effect of phytoestrogens on acute and chronic masticatory muscle pain and underlying mechanisms are not clarified. Our previous findings demonstrated that high level of estrogen decreases the mechanical threshold in masseter muscle and amplifies the hyperalgesia response of female rats; genistein （a soy phytoestrogen）relieves glutamine-induced acute masseter muscle pain. The hypotheses of this study are: elevation of estrogen can enhance occlusal interference-induced chronic hyperalgesia in masticatory muscle of female rats，and genistein can antagonize this action. Ovariectomized rats will be used to establish models of masseter muscle pain with high level of estrogen. Genistein, antagonists of estrogen receptors, or tyrosine kinase inhibitor will be administered. Methods of animal ethology, electrophysiology, immunohistochemistry, and western blotting will be used to explore the action of genistein and its mechanisms from different levels of animal, cell and molecule. It is high-safe and novel to intervene chronic masticatory muscle pain with soy phytoestrogens. Results of this study will provide scientific evidences for future clinical studies to use genistein for the alleviation of masticatory muscle pain.

雌激素对女性TMD咀嚼肌痛和颞下颌关节痛的影响一直存在争论，且对这类慢性疼痛缺乏有效的治疗。动物研究发现雌激素水平升高能增加咬肌伤害感受器对NMDA受体兴奋剂的敏感度，理论上植物雌激素能通过降低雌激素效应的周期性波动缓解疼痛，但国内外缺乏植物雌激素治疗咀嚼肌急、慢性疼痛效果和机制的研究。我们前期实验证明高水平雌激素可增强雌性大鼠咬肌痛敏反应，初步观察到染料木素（大豆植物雌激素）可缓解谷氨酸诱发的咬肌急性痛敏。本研究假设：高水平雌激素可加重咬合干扰致雌性大鼠咀嚼肌慢性痛敏，染料木素能拮抗此效应。研究采用去卵巢大鼠建立高雌激素水平的咬肌痛敏模型，给予染料木素、雌激素受体拮抗剂或酪氨酸激酶抑制剂，通过行为学、电生理、免疫荧光、蛋白定量手段，从动物、细胞、分子水平分析染料木素的作用机制。大豆植物雌激素安全性高，研究结果将开辟治疗慢性咀嚼肌痛的新途径，为临床探索染料木素治疗咀嚼肌疼痛提供科学依据。

咀嚼肌疼痛的女性患病率较男性高，提示雌激素（17β-estradiol，E2）可能在疼痛中发挥重要作用。染料木素（genistein，Gen）作为植物雌激素可逆转E2对谷氨酸诱导的咬肌急性痛敏的影响，能否逆转E2在实验咬合干扰（experimental occlusal interference，EOI）诱导的咬肌慢性痛敏中的作用尚不清楚。. 本项目用行为学测试探究EOI致大鼠咀嚼肌机械痛敏的性别差异，结果表明EOI诱发雌性大鼠的咬肌痛敏较雄性大鼠强；雌性大鼠动情前期的咬肌、颞肌机械刺激反应阈值相比其他动情阶段最低。通过行为学、膜片钳电生理、western blot及免疫荧光等方法明确E2通过上调三叉神经节（trigeminal ganglion，TG）神经元兴奋性与瞬时受体电位香草酸亚型1（transient receptor potential vanilloid 1，TRPV1）表达，以及上调三叉神经脊束核（spinal trigeminal tract，SpⅤ）、海马CA3区神经元磷酸化细胞外信号调节激酶（phospho-extracellular signal regulated kinase1/2，p-ERK1/2）表达，加重EOI致大鼠咬肌痛敏。在此基础上探索Gen拮抗E2对EOI致咬肌痛敏的作用及潜在机制，Gen预处理组显著下调TG中TRPV1表达，降低TG中TRPV1阳性神经元比例，下调SpV中p-ERK1/2表达，机械刺激反应阈值均较E2+EOI组高。但染料木苷（genistin，Gin）预处理组作用低于Gen预处理组。结果证实，Gen有部分拮抗E2对EOI致咬肌痛敏的作用，Gen通过拮抗E2受体、抑制酪氨酸激酶等机制主要在TG、SpV发挥作用。本研究为植物雌激素可能用于慢性咀嚼肌疼痛的治疗提供实验室依据，为临床上女性多见的TMD相关疼痛提供新的治疗策略。