IL-37抑制Rac1阻止单纯疱疹病毒性角膜炎中HSV-1复制的机制

HSV-1 infection leading to herpes simplex keratitis (HSK) is one of the most serious infectious eye diseases in the world. It is also the main cause of the blinding keratopathy. However, the mechanism of HSK is still unclear and therapeutic approach is limited. Our recent study found that IL-37 expression significantly increased in the cornea of the HSK patients as compared to normal ones. HSV-1-infected corneal epithelial cells（HCEC）could also induce elevated IL-37. According to previous studies of malignancies, IL-37 may suppress cancer invasion by inhibiting Rac1 activation and our preliminary experiment showed that Rac1 inhibitor 6-thioguanine potently inhibited HSV-1 . Our other experiments showed that the Rac1 inhibitors NSC23766 and 6-thioguanine could significantly reduce HSV-1 infection in vitro assay. In the parallel experiments, the therapeutic index of 6-thioguanine in the inhibition of HSV-1 replication was significantly higher than the commercial drugs, acyclovir and ganciclovir. In order to investigate the role of IL-37 in the HSK，we detected them in the corneal epithelial cells of clinical specimens. We also used HSV-1-infected corneal epithelial cells (HCEC) to explore the biological roles of IL-37 and Rac1 in the HSV-1 infection. In vitro assay showed that Rac1 signaling pathway supported HSV-1 replication. Rac1 inhibitors and Rac1 siRNA could significantly reduce HSV-1 replication in a dose dependent manner. The current research proposal aims to understand the roles of IL-37 and Rac-1 in HSV-1 pathogenesis, to identify potential therapeutic targets and to explore potential application of the newly found drug, 6-thioguanine.

HSV-1感染眼睛引起单纯疱疹病毒性角膜炎（HSK）是危害严重的感染性眼病之一，然而HSK致病机制尚不清楚，治疗方法有限。我们研究发现，HSK患者的角膜上皮组织IL-37明显升高；体外实验中HSV-1感染角膜上皮细胞（HCEC）诱导IL-37升高。在肿瘤细胞里，IL-37抑制Rac1限制肿瘤细胞迁移。我们推测IL-37抑制Rac1阻止HSV-1复制。预实验显示在HSV-1感染过程中Rac1上调，而Rac1 siRNA 和 Rac1抑制剂 NSC23766减少HSV-1复制。对比实验表明，Rac1抑制剂6-thioguanine抑制HSV-1复制的效果好于阿昔洛韦和更昔洛韦。本课题拟通过临床标本研究IL-37在HSK致病过程中的调控作用；利用HSV-1感染HCEC探究IL-37在HSV-1感染中的作用；利用HSK动物模型确认Rac1作用，为治疗HSK提供一个新的靶点和理论基础。

HSV-1感染角膜引起病毒性角膜炎（herpes simplex keratitis, HSK），会造成角膜炎症和新生血管，反复发作的HSK会导致视力损害，甚至致盲。由于阿昔洛韦耐药性的日益普遍、激素类药物的副作用及手术易复发等问题，开发治疗HSK的新方法迫在眉睫。IL-37是一种新型抗炎因子，在人体组织中广泛分布，异常的血清IL-37水平与多种炎症性疾病密切相关，但其在病毒学感染疾病中的生物学效应报道很少。此外，IL-37有5种异构体（IL-37a-e），IL-37b可靶向抑制Rac1活性。其中Rac1活性抑制剂硫鸟嘌呤（6-Thioguanine, 6-TG）是一种嘌呤类似物。我们课题组研究发现：（1）临床数据显示，HSK患者血清和泪液中IL-37水平显著低于健康对照者。（2）在体内和体外实验发现,HSV-1感染显著促进Rac1活性，并发现硫鸟嘌呤靶向抑制Rac1活性，显著减少HSV-1复制。在机制上，发现IL-37b是与细胞内的rac1直接结合，靶向抑制Rac1活性，从而干预HSV-1复制。4）此外，用硫鸟嘌呤处理HSV-1感染的HCEC细胞后，病毒mRNA和蛋白的表达明显下降，且使用同等浓度的硫鸟嘌呤和阿昔洛韦处理后，硫鸟嘌呤对病毒蛋白的下调更加明显。（5）我们还通过药物协同实验发现，硫鸟嘌呤和阿昔洛韦的协同指数（CI）小于1，表明两者联合有协同作用。（6）另外，硫鸟嘌呤对阿昔洛韦耐药株也有明显的抑制作用。（7）我们通过BALB/c小鼠角膜接种HSV-1，成功建立小鼠HSK模型，在临床症状、角膜病理、组织的病毒载量等方面，与临床HSK病变相一致。硫鸟嘌呤治疗后，小鼠角膜病变得到改善，HE显示角膜病理变化有所减轻，角膜病毒载量明显下降，表明硫鸟嘌呤对HSK有很好的治疗效果。我们还使用新西兰兔评估硫鸟嘌呤凝胶对兔子角膜无毒性作用，都显示硫鸟嘌呤凝胶在眼表使用的安全性。