麻风杆菌通过Endocan调节Th1/Th2细胞因子表达影响麻风病的进程

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae), whose outcome is closely related to host immune status. Leprosy reactions are acute inflammatory processes occurred during the chronic course of leprosy due to changes in immune status, which may cause or aggravate the disability without prompt and proper treatment. However, the pathogenesis of leprosy reaction has not been fully elucidated. Our preliminary study found that the endothelial cell specific molecule -1 (ESM-1/endocan) levels in the sera of leprosy patiens are higher than those in the sera of controls, and the secretion of endocan from peripheral blood mononuclear cells (PBMC) can be regulated by M. leprae sonicate, while Th1/Th2 balance in PBMC can be modulated by endocan. Further the differences of endocan and cytokines in the skin lesions and sera among different types of leprosy patients and reaction will be detected in in situ experiment and evaluated to explore the relationship between endocan, cytokines and leprosy progression. Secondly, human umbilical vein endothelial cells (HUVECs) are infected with M. leprae in vitro, and transcription factor inhibitors-treated group is also set up and then the variations in expression levels of endocan are detected to clarify the effect of M. leprae on synthesis and secretion of endocan and the signaling pathways involved. Use siRNA to knock down the expression of endocan and then analyze its effect on the secretion of Th1/Th2 cytokines by PBMC. Explore the variation and role of endocan in the course of leprosy along with the possible mechanism.

麻风病是由麻风杆菌引起的、与机体免疫状态密切相关的慢性传染病，在其慢性进程中会发生麻风反应，如不及时治疗将引起畸残或畸残加重。但麻风反应的致病机制尚未完全阐明。我们前期发现麻风患者血清中endocan水平较高，且麻风抗原刺激PBMC可调节endocan分泌，而endocan刺激PBMC可调节Th1/Th2细胞因子的表达。本课题拟通过1）在体实验：观察各型麻风病和反应患者皮损和血清中endocan和细胞因子的变化，初步探讨endocan、细胞因子与麻风病进程的关系。2）细胞培养实验：用麻风杆菌体外感染HUVECs，并设立转录因子抑制剂处理组，检测endocan的变化以明确麻风杆菌对endocan合成和分泌的作用及参与其中的信号通路；分析siRNA沉默endocan对麻风患者PBMC分泌Th1/Th2型细胞因子的影响，探究endocan在麻风病发生发展中的变化和作用及可能机制。

多药联合化疗显著改善麻风病治疗和患者康复前景，目前治疗中存在的主要问题是发生“反应”。因此须要深入理解麻风反应的发病机制并发现新的早期诊断标志物和治疗靶标。研究内容包括：（1）观察各型麻风病和反应患者皮损和血清中endocan和细胞因子的变化；（2）用和不用多种信号通路抑制剂处理HUVECs，比较麻风杆菌抗原刺激后endocan表达水平的变化；（3）Endocan特异性RNAi慢病毒载体感染HUVEC，麻风杆菌抗原刺激endocan表达降低的HUVEC，用该HUVEC上清液刺激PBMC，检测PBMC中Th1/Th2细胞因子的变化。结果发现：（1）患者血浆中endocan水平显著低于对照者，麻风反应患者血浆中endocan水平显著高于无反应患者；患者血浆中IL-4和IL-6水平显著高于对照者；TNF-α的水平在患者和对照者间无显著性差异，但麻风反应患者血浆中TNF-α水平显著高于无反应患者。在患者皮损切片中，endocan主要表达于表皮的角质形成细胞，真皮内浸润的组织细胞和部分淋巴细胞，汗腺、皮脂腺和毛囊的漏斗部以及毛细血管的内皮细胞；结核样型麻风一端的患者皮损中endocan和TNF-α的表达高于瘤型麻风一端患者，麻风反应者皮损中endocan和TNF-α的表达高于未发生反应者。（2）用不同的麻风杆菌抗原刺激HUVECs后发现：脂阿拉伯甘露糖刺激HUVEC后，endocan显著上调，IL-6显著下调；(超)声波裂解产物、胞浆馏分刺激HUVEC后，endocan显著下调；γ辐照处理的麻风杆菌和天然双糖-辛基-人血清白蛋白刺激HUVEC后，endocan变化不显著。分别用六种信号通路的抑制剂处理HUVEC，用LAM刺激处理过的HUVEC，发现LAM通过MEK-ERK和P38 MAPK信号通路上调endocan表达；同时通过NF-κB 信号通路上调HUVEC中TNF-α表达，通过P38 MAPK信号通路下调IL-6表达。（3）Endocan对PBMC中IL-6、IL-10和TNF-α的表达有促进作用。综上所述，麻风反应患者血浆和皮损中endocan的水平升高，提示endocan可能作为麻风反应的预测因子。麻风杆菌LAM通过P38 MAPK信号通路上调endocan表达，endocan增加PBMC中IL-6、IL-10和TNF-α的表达，从而促进麻风反应的发生。