雷公藤红素调控TLR4/MyD88/NF-κB信号通路抗角膜移植排斥机制研究

Corneal transplantation rejection（CTR), the main reason for the failure of allogeneic corneal transplants, is still a therapeutic challenge for ophthalmologists. Macrophages (Mφ), as the main inflammatory cells, play a key role in organ transplant rejection. Our preliminary show that celastrol prolonged rat corneal allograft survival through suppressing the macrophage infiltration. However, the exact mechanism involved in celastrol modulation of CTR remains unclear. Previous studies have suggested that TLR4/MyD88/NF-κB pathway plays a vital role in organ transplant rejection. TLR4 is highly expressed in macrophages. We also find that celastrol inhibits the activity of NF-κB in macrophages. Therefore, we speculate that TLR4/MyD88/NF-κB pathway play a pivotal role in the inhibiting effect of celastrol on macrophages in CTR. In this present study, we would like to develop celastrol-loaded positively charged micelles based on a triblock copolymer to better improve the hydrophilicity of celastrol and enhance the corneal penetration after topical application. And then we will further explore the potential mechanism of celastrol-mediated modulatory effects on macrophages in CTR. It is expected that this study might constitute a deep understanding of the underlying mechanism of CTR. It will be of help in offering new therapeutic options and explore a useful clinical therapeutic strategy for organ transplant rejection.

角膜移植排斥（CTR）是角膜移植失败的主要原因，治疗棘手。Mφ是器官移植排斥的主要效应细胞。我们前期研究发现雷公藤红素（Celastrol）通过调节Mφ减轻角膜移植排斥反应，具体机制尚不完全清楚。既往研究显示TLR4/MyD88/NF-κB信号通路在器官移植排斥中发挥重要作用，Mφ高表达TLR4，我们也发现Celastrol抑制MφNF-κB活性。因此我们大胆推测：TLR4/MyD88/NF-κB存在CTR中，Celastrol也许是通过TLR4/MyD88/NF-κB通路调控Mφ实现其抗CTR效应。本课题拟制备负载Celastrol的正离子纳米胶束，以增强Celastrol水溶性及角膜通透性，明确Celastrol在CTR中的作用及其调节Mφ抗CTR机制，有望加深对 CTR发生机理的理解；结合新兴的纳米技术，为Celastrol治疗器官移植排斥以及寻找新的治疗策略提供理论依据。

角膜移植术后排斥是角膜移植手术失败的主要原因。研究表明巨噬细胞（Mφ）在角膜移植排斥反应（CTR）中发挥关键作用。本项目旨在制备新型高效的抗CRG药物——雷公藤红素（Celastrol）正离子纳米胶束（CNMs），研究其抗CTR效果及其调控Mφ的机制。我们发现：成功制备的粒径约24nm和表面正电位+8.5mV的聚合物纳米胶束，载药量6.58%，小鼠结膜囊内单次点眼，30min已达角膜深层基质，60min胶束几乎达全层角膜基质。这种兼具小粒径和表面正电位，能高效负载 Celastrol并增加其表观溶解度，改善疏水性药物的理化性能，采用局部点眼的给药方式，能克服眼部的生物屏障，解决了疏水性药物的角膜通透性难题。在此基础上，对以下问题进行研究：1）局部给予CNMs，评估其对角膜植片存活的影响，评价其对角膜组织内浸润Mφ、细胞因子以及TLR4表达的影响；2）研究Celastrol对激活Mφ分泌细胞因子及TLR4/MyD88/NF-κB通路的调控。我们发现Wistar-SD大鼠CTR模型中，对照组平均排斥时间7.3±0.6天，CNMs明显延长角膜植片的存活，平均排斥时间为29.1±2.4天。术后第8天，在排斥的角膜植片内，对照组有大量高表达TLR4的Mφ浸润，而CNMs组浸润的Mφ明显减少，且TLR4的表达也明显减弱。且CNMs下调角膜植片内多种促炎因子和趋化因子（如TNF-α、IL-6、IFN-γ和MCP-1）的mRNA和蛋白表达。采用巯基乙酸诱导小鼠腹腔Mφ并鉴定，结果示获得的MφF4/80和Dextran双标阳性细胞率约98.5%±1.2%。与TLR4-/-敲除鼠相比，此方法诱导的野生型MφTLR4/MyD88/NF-κB信号被活化。CNMs下调MφTLR4的表达，将LPS诱导MφTLR4的比率由45.2±3.2%下调至26.7±2.3%。进一步研究显示，CNMs抑制LPS诱导Mφ多种促炎因子和趋化因子的分泌，抑制TLR4/MyD88/NF-κB通路。这提示我们Celastrol也许通过调控MφTLR4/MyD88/NF-κB通路实现其抗CTR效应。本研究制备的兼具小粒径正电荷的纳米胶束，增加药物的角膜透性，适合眼部用药，具有广泛的应用前景。本项目加深了对CNMs抑制CTR机制的理解，并为最终抗CTR提供新的治疗措施和有意义的理论依据。