基于外泌体介导的microRNA126转运探讨补肾益精法对系统性硬皮病的血管保护机制

Systemic sclerosis often merge vascular disease, the prognosis and outcome of systemic sclerosis depends on the range and severity of vascular damage .Tonifying kidney and Boosting essence treatment is the therapy for systemic sclerosis from Prof. Deng, who recommended treating this disease on the theory of “Lung-Spleen-Kidney Correlation”, and “tonifying kidney and boosting essence at the first place, strengthening spleen and nourishing lung at the supplement place”. Our previous study have indicated that this therapy not only has anti-fibrotic effect,but also improve the patient's microcirculation and increase the number of capillaries in skin lesions.It can also inhibited the transition from endothelial to mesenchymal cells in vitro.MicroRNA126 has the function consistent with Tonifying kidney and Boosting essence treatment, which can be transfer by exosome.Therefore, we hypothesized that the therapeutic mechanisms of Tonifying kidney and Boosting essence treatment on proliferative vasculopathy in systemic sclerosis based on extracellular Exosome-Shuttled microRNA126.We will utilize the techniques such as PCR, Western Blot and immunohistochemical staining etc to clarify the mechanism of Tonifying kidney and Boosting essence treatment on proliferative vasculopathy in systemic sclerosis.The result of this study can provide a scientific evidence for the clinical application of Tonifying kidney and Boosting essence treatment, benefit the development of new targets and methods for the prevention and treatment of related vascular diseases.

系统性硬皮病常合并血管病变，血管损害的范围和程度常影响其预后和结局。补肾益精法是邓铁涛教授辨治硬皮病的经验治法，主张从“肺脾肾相关理论”辨治硬皮病、“补肾益精为主，健脾养肺为辅”。我们前期研究发现，补肾益精法不仅具有抗纤维化的功效，还可抑制血管内皮细胞-间质转化（EndMT）过程，增加皮损区毛细血管数量，改善硬皮病患者微循环。microRNA126具有与补肾益精法一致的血管保护功能，可通过外泌体转运介导血管修复。因此，我们提出研究假说：补肾益精法对系统性硬皮病的血管保护作用，可能通过调节外泌体对microRNA126的转运实现？本研究拟运用PCR、Western Blot、免疫组化染色、细胞行为学检测等技术进一步深入探讨补肾益精法对系统性硬皮病血管保护的作用途径和机制，为补肾益精法的临床应用提供科学依据，为相关血管损伤性疾病的防治提供新的靶点和思路。

研究背景：系统性硬皮病常合并血管病变，多由血管损害所致，系统性硬皮病的预后和结局常取决于血管损害的范围和严重性。microRNA126（miR-126）是血管内皮细胞内特异性高表达的小分子miRNA，可由内皮细胞释放的外泌体转运靶向目标细胞，具有抑制内皮细胞EndMT和促进血管修复、再生的功能。系统性硬皮病患者内皮细胞外泌体中miR-126的表达情况及功能尚不明确。. 研究方法：采用高通量测序分析检测系统性硬皮病患者与正常对照者血清外泌体中小分子RNA尤其是miR-126的表达情况；采用PCR、NTA、电镜示踪、RNA干预等技术对外泌体RNA分子间的互作机制进行进一步研究；在细胞、动物层面运用补肾益精法干预，采用PCR、Western Blot、免疫组化染色、细胞行为学检测等技术，观察补肾益精法对外泌体功能的影响及对miR-126相关信号通路的调节作用。. 研究结果：Akt/mTOR信号通路是与miR-126相关的促血管再生修复、抑制内皮细胞EndMT的重要通路。补肾益精法通过抑制Akt、mTOR的磷酸化水平抑制内皮细胞EndMT进程，促进血管再生修复，但对外泌体miR-126表达无影响。补肾益精法可通过影响外泌体的功能促进内皮细胞的增殖及成管能力，促进硬皮病小鼠模型的皮肤血管修复。与正常对照者相比，SSc患者血清外泌体中miR-126表达水平显著上调，外泌体miR-126可通过拮抗LncRNA JPX20提升VEGFR2、Ang-1等促血管生成因子表达水平，促进血管生成。. 研究结论：补肾益精中药复方通过调节Akt/mTOR信号通路中Akt、mTOR的磷酸化水平发挥血管保护作用，而非通过促进外泌体miR-126表达促进血管修复与再生，其血管保护作用可经外泌体介导。研究结果为补肾益精法治疗系统性硬皮病血管损伤的进一步机制研究提供了一定基础，为补肾益精法治疗硬皮病血管损伤的应用及新药开发提供了科学依据。内皮细胞来源外泌体中显著高表达的miR-126可通过靶向LncRNAJPX20促进血管修复再生，LncRNAJPX20及其相关靶向通路可能成为硬皮病治疗的新靶点。