基于Th17/Treg阴阳消长平衡的清热益气通窍法干预CRS黏膜重塑的研究

As the common refractory disease in otolaryngological department, chronic rhinosinusitis（CRS）has strong impacts on patients. Mucosa tissue remodeling has close associations with refractoriness and discontented outcome of CRS. Both pro-inflammatory Th17 and anti-inflammatory Treg, two respects of unity of opposites, constitute dynamic equilibrium system. Th17/Treg imbalance would lead to mucosa remodeling in CRS. We showed that clear away Heat and supplement Qi method to relieve stuffy nose has ability of ameliorating mucosa remodeling of CRS in earlier researches, but had no chance to illuminate the concrete mechanism due to limitations of research direction and funds. It is implied that clear away Heat and supplement Qi method to relieve stuffy nose could down-regulate expression of cytokine IL-17 secreted by Th17 but up-regulate expression of E3 Ubiquitin Ligase CHIP related to Treg regulation. We speculate that clear away Heat and supplement Qi method to relieve stuffy nose may have impacts on the key to differentiation and function of Th17/Treg , not only to restore equilibrium of Th17/Treg but also ease mucosa remodeling. It is worth to expound how such therapeutic method ameliorates mucosa remodeling in CRS from the perspective in Th17/Treg balance. Hence,we plan to adopt histopathological and molecular biological analysis to observe the effects of this therapeutic method on the equilibrium, differentiation and function of Th17/Treg.

慢性鼻—鼻窦炎（CRS）是耳鼻喉科常见难治病。鼻窦黏膜重塑在CRS炎症迁延及难治性方面有重要意义。Th17与Treg间构成了符合阴阳对立统一性的消长平衡体系。Th17/Treg平衡失常是CRS黏膜重塑的重要原因。在前期科学基金研究中，我们发现清热益气通窍治疗能够改善CRS鼻窦黏膜重塑，惜未阐明机制，甚憾。但部分研究结果显示：清热益气通窍治疗能够抑制IL-17这一由Th17分泌的细胞因子表达，促进CHIP这一参与Treg调节的E3泛素连接酶表达。故推测：清热益气通窍法改善CRS鼻窦黏膜重塑的机制，可能与其影响Th17及Treg分化和功能的关键信号网络，恢复Th17/Treg的消长平衡有关。因此，本研究拟应用分子生物学等研究手段，观察清热益气通窍法对CRS模型鼻窦黏膜Th17/Treg平衡及相关分化和功能调控网络的影响，从Th17/Treg间阴阳消长平衡角度，阐明该法改善CRS黏膜重塑的机制。

慢性鼻-鼻窦炎（chronic rhinosinusitis，CRS）是耳鼻喉科常见难治病，鼻窦黏膜重塑在CRS炎症迁延及难治方面有重要意义。Th17与Treg细胞间的平衡失常是CRS黏膜重塑的重要原因。课题组前期研究发现清热益气通窍治疗对鼻窦黏膜上皮重塑有一定影响，但未深入探讨。故本项目拟建立小鼠CRS模型，应用病理学及实时荧光定量PCR、免疫印记等分子生物学研究手段，从Th17/Treg相关分化及功能调控网络的角度，深入探索中医清热益气通窍疗法改善CRS鼻窦黏膜重塑的可能机制。研究发现清热益气通窍疗法能够显著降低CRS小鼠模型鼻窦黏膜组织病理指数评分、上皮损伤评分、鼻窦黏膜基底膜厚度、鼻窦黏膜黏液腺增生评分、黏膜纤维化评分；显著降低鼻窦黏膜局部和外周血异常升高的Th17细胞，促进鼻窦黏膜局部和外周血Treg细胞的恢复；降低CRS小鼠鼻窦黏膜TGF-β1、IL-2、IL-6、IL-17、IL-21、IL-23含量，增加IL-10含量；抑制鼻窦黏膜组织TGFβR2、gp130、STAT3、Smad2、RORγ基因表达，促进STAT5、Smad3、RUNX1、RUNX3、Foxp3基因表达；抑制黏膜STAT3、SMAD2磷酸化，促进SMAD3磷酸化；抑制RORγ、MMP-2、MMP-9蛋白表达，促进FOXP3蛋白表达。以上发现提示：清热益气通窍疗法能够改善CRS时的鼻窦黏膜上皮损伤、基底膜增厚、黏液腺增生、黏膜纤维化等重塑关键指标，其机制与：①影响细胞因子IL-6、IL-6RA、IL-21、IL-23产生，减少STAT3基因表达及磷酸化，抑制JAK2/STAT3通路活化；降低细胞因子TGF-β1生成及TGFβR2基因表达，减少Smad2基因表达及蛋白磷酸化，抑制TGF-β1/Smad2通路活化，进而减少RORγ基因和蛋白表达，抑制CD4+T细胞分化为Th17细胞，减少其分泌IL-17等促炎细胞因子，及由IL-17诱导的MMP-2、MMP-9表达。②增加Smad3基因表达及蛋白磷酸化，改善TGF-β1/Smad3/FOXP3通路活化，促进CD4+T细胞向Treg细胞分化和分泌IL-10，抑制过度炎症反应，改善鼻窦黏膜炎症损伤有关。从Th17/Treg平衡的角度揭示了中医清热益气通窍疗法改善CRS时鼻窦黏膜重塑的可能机制。