基于CXCL12/CXCR4生物轴研究密蒙花总黄酮抑制碱烧伤角膜新生血管形成的分子机制

Corneal neovascularization (CNV) can lead to vision loss, and this condition can be caused by infection, chemical burns, or contact lens wear. CNV is typically associated with inflammation or hypoxic stress. To date, the exact pathogenesis of CNV is yet unclear, and thus it is still lack of precise and effective clinical prevention or treatment for CNV. All clinical treatment were not efficacy and resulted that CNV become one of the main reasons of blindness.. Stromal-derived factor-1 (SDF-1 or CXCL12) is a CXC chemokine. It was first cloned from a bone marrow-derived stromal cell line and was later identified as a pre-B-cell growth stimulating factor (PBSF). CXCL12 is broadly expressed in a variety of tissue types where it acts as a potent chemoattractant for immature and mature hematopoietic cells. CXCR4 is the main receptor for CXCL12, The CXCL12–CXCR4 biological axis has been associated with tumor invasion, metastases and cancer angiogenesis. Previous studies have shown that the signaling pathway of CXCL12/CXCR4 plays a critical role in ocular neovascular diseases such as corneal neovascularization, choroidal neovascularization, diabetic retinopathy and hypoxia-induced retinopathy. Blocking CXCL12/CXCR4 signaling reduce the vascular endothelial growth factor (VEGF) expression and neovascularization in alkali-induced injuried cornea..According to TCM, it was found that Buddleja Officinalis could eliminate vascular proliferation in data. Some studies proved that Buddleja Officinalis could inhibit vascular endothelial cell proliferation in anaerobic condition, and downstream signaling,such as VEGF and others factors. We found that the Buddleja Officinalis has active effec on alkali-burn corneal neovascularization; reduce the level of VEGF in blood serum, anterior chamber aqueous humor and corneal tissue homogenate of mice. But its detailed mechanism remains unclear. Therefore, we speculate that Buddleja Officinalis eliminate blood vessel on inhibition blood endothelial cells proliferate by administrate CXCL12/ CXCR4 signaling. This study focused on the effect of Buddleja Officinali flavone on alkali burn induced corneal neovascularization via CXCL12/CXCR4 signaling pathways with modern molecular biological research, and interpreted the mechanism of action of Buddleja Officinalis eliminate blood vessel on inhibition corneal neovascularization. Meanwhile, if this mechanism is confirmed, it would be a new strategy for the treatment of both corneal neovascularization and other ocular neovascular disease such as choroidal neovascularization, diabetic retinopathy in traditional Chinese medicine.

角膜新生血管（CNV）是一种常见致盲眼部病变，确切的发病机制尚不清楚，主要与炎症和缺氧有关，临床疗效有限，已属眼科难治性疾病之一。研究表明CXCL12/CXCR4在形成过程发挥重要作用，通过诱导产生血管内皮生长因子（VEGF），调节血管增生。抑制CXCR4可以治疗CNV。在前期研究中，我们发现密蒙花有“消目中赤脉” 记载和抑制新生血管形成的作用，动物实验显示密蒙花可以减少碱烧伤诱导CNV生成，下调VEGF表达，但其具体作用通路尚不明确，推测与调控CXCL12/CXCR4信号通路有关。为深入研究其作用机制，本项目拟采用现代分子生物学技术和细胞学技术，研究其信号通路和作用靶点，阐明密蒙花对CXCL12/CXCR4信号通路的调控作用及机理，为中医中药治疗角膜新生血管和眼部其他新生血管性疾病（如脉络膜新生血管和视网膜新生血管等）开辟新的治疗途径奠定实验基础，并为中药密蒙花的传承、发展提供实验佐证。

初步揭示了密蒙花治疗CNV的分子机理：我们再一次证实氢氧化钠诱导小鼠角膜新生血管（CNV）动物模型稳定、重复性好，可作为碱烧伤CNV的动物模型。经中药密蒙花治疗后，小鼠角膜新生血管面积缩小，角膜组织中CXCL12和CXCR4 mRNA表达下降，PI3K、ERK、P38和AKT等相关蛋白表达下降。细胞实验方面，我们建立CXCL12转染人角膜上皮细胞模型，发现蒙花苷下调CXCL12转染人角膜上皮细胞分泌VEGF量，下调CXCL12转染人角膜上皮细胞VEGFA 和CXCR4 mRNA表达。再一次证实了中药密蒙花可以有效地治疗碱烧伤CNV，其分子机理是通过调控CXCL12/CXCR4生物轴达到抑制CNV。. 初步阐述了密蒙花“消目中赤脉”的科学内涵：目中赤脉是病变状态下出现的目中血络，病因多为“风”、“火”、“燥”，主要病机是肝郁，肝郁则脉络受损，即“木郁”。密蒙花性味甘，微寒，入足厥阴肝经，甘以“润肝”、“消风”，寒以“清肝热”，既能祛风散邪，清肝泻火，外散内清解肝郁，又能退翳明目治目昏。具有清热泻火，养肝明目，退翳的功效，发挥清泻肝火、养血补肝、疏风升发、退翳明目作用，使得肝热清、肝燥润、肝血养、肝郁解，同时，密蒙花轻清上浮，将肝血中清净之血上达于目而目明，即“达之”。据此，我们认为密蒙花发挥“木郁达之”作用实现“消目中赤脉”功效。. 理论创新：我们在阐述密蒙花“消目中赤脉”科学内涵的基础上，传承创新，认为角膜混浊和角膜血管新生的病机主要是玄府闭逆，气机升降出入失司，目失濡养，神光不能发越而成。据此，认为“开通玄府”为治疗“目中赤脉”的根本大法，研究发现细辛具有开通玄府的作用。结合密蒙花消目中赤脉作用，我们总结出以细辛、密蒙花为主的“明目消翳散”治疗角膜血管翳，并申请了国家发明专利，专利受理号为：202210985812.0。临床上，我们口服明目消翳散+中药密蒙花熏洗治疗碱烧伤所致的角膜新生血管，初步研究结果表明具有较好疗效，证实其正确性。