清营汤对川崎病小鼠TNF-α/NF-κB/MMPs通路调控的研究

Kawasaki disease (KD) is defined as "febrile disease" in traditional Chinese medicine. With increasing of its prevalence over years, its coronary complication has becoming the leading cause of acquired heart disease in children. Although standard western medicine therapy has improved its prognosis, coronary complication still occurs in 7% to 10% children with KD. Searching for new medicines has becoming a challenge in the management of these children. "Qingying decoction" is a well known traditional Chinese medicine formula for cleaning "Qi" and cooling "Ying", and has been proved to be effective on treatment of KD in children. By using murine models of KD which have been established in our previous projects as study subjects, zymography, electrophoretic mobility shift assay (EMSA), immunohistochemistry and Western Blotting as study methods, this study sought to evaluate the changes of serum levels of cytokines including TNF-α, the expression and activation of NF-kB and IκB-α protein in peripheral monocytes, thyroid gland, cardiac tissue and coronary artery and coronary endothelial cells, the expression levels of matrix metalloproteinases and their inhibitors in thyroid gland, cardiac tissue and coronary artery in murine models of KD treated with Qingying decoction by intragastrical way, and controls treated by intravenous immunoglobulin. At the same time, clinical presentations, cardiac function assessed by echocardiography and coronary arterial pathological damage were recorded. Thus this study would try to evaluate the therapeutic mechanism of Qingying decoction in treatment of KD on TNF-α/NF-κB/MMPs pathway, and provide scientific evidences for treatment of KD by traditional Chinese medicine.

川崎病(KD)属中医温病，发病率逐年增高，其冠状动脉并发症已成为儿童最常见的后天性心脏病，探索新疗法是急需解决的儿科难题。临床已证实传统方剂清营汤可清气凉营，有改善急性KD患儿临床症状、减轻冠状动脉损伤的作用。本项目拟利用课题组已建立的小鼠KD疾病模型为对象，利用明胶酶谱法、凝胶电泳迁移率（EMSA)、免疫组织化学和蛋白免疫印迹（Western Blotting）等检测手段，以常规人血丙种球蛋白注射为对照，检测不同剂量清营汤对模型小鼠不同时期外周血TNF-α等细胞因子水平，血液单核细胞及胸腺、心脏、冠状动脉内皮细胞的核因子-κB（NF-κB）蛋白的活性及表达水平；胸腺、心脏、冠状动脉基质金属蛋白酶系列活性及表达水平，同时观察小鼠表观征象，超声测定心功能、病理检测冠动病变严重程度，在TNF-α/NF-κB/MMPs通路探查清营汤对KD冠状动脉炎的治疗机制，为进一步探索中药治疗KD奠定基础。

川崎病(KD)属中医温病，发病率逐年增高，其冠状动脉并发症已成为儿童最常见的后天性心脏病，探索新疗法是急需解决的儿科难题。临床已证实传统方剂清营汤可清气凉营，有改善急性KD患儿临床症状、减轻冠状动脉损伤的作用。本项目利用课题组前期项目已建立的小鼠KD疾病模型为对象，利用明胶酶谱法、凝胶电泳迁移率（EMSA)、免疫组织化学和蛋白免疫印迹（Western Blotting）等检测手段，以常规人血丙种球蛋白注射为对照，检测不同剂量清营汤对模型小鼠不同时期外周血TNF-α等细胞因子水平，血液单核细胞及胸腺、心脏、冠状动脉内皮细胞的核因子-κB（NF-κB）蛋白的活性及表达水平；胸腺、心脏、冠状动脉基质金属蛋白酶系列活性及表达水平，同时观察小鼠表观征象，超声测定心功能、病理检测冠动病变严重程度，发现改良的清营汤可以改善川崎病小鼠的急性期临床征象，减轻冠状动脉炎性细胞浸润，减轻冠状动脉扩张，抑制冠状动脉瘤的形成，对川崎病模型有一定的治疗作用。清营汤干预通过调低急性期NF-κBp65、MMP-9/TIMP-1的蛋白表达及水平及活性，降低MMP活性，达到减轻冠状动脉的损伤的治疗目的，但对TNF-α等细胞因子影响不大，其机制需进一步探索。研究还鉴定了清营汤的可能有效的大分子单体，并探索了丹酚酸的治疗效果，为进一步探索治疗川崎病冠状动脉炎的中央单分子或者潜在新药奠定了一定的基础。