补肾活血方调控VEGF-C/VEGFR-3信号促进淋巴管成熟和功能对骨性关节炎的影响

Osteoarthritis (OA) induces destruction and dysfunction of joints, or even extremity disability，and has no cure so far. In our previous studies, we found that there are close relationships between the maturation and function of lymphatic vessels and the pathological process of osteoarthritis. In and around the OA joints, there are accumulation of inflammatory substances, arthroedema, and destruction of articular cartilage and subchondral bone, accompanying with the reduction of mature lymphatic vessels and decrease of lymphatic clearance (Shi J, et al. Arthritis Rheumatol. 2014); with the use of "BushenHuoxue decoction" in OA patients and mice, the joint swelling and pain could be alleviated, and joint inflammation reduced, with vascular endothelial growth factor-C (VEGF-C) expression improved, but the mechanism is still unknown. We hypothesize that "BushenHuoxue decoction" promote the lymphatic maturation and function via regulation the VEGF-C/VEGFR-3 signaling which can accelerate the clearance of inflammatory substances and clastic enzymes to relieve inflammation and joint damage on OA. By using lymphatic function detection techniques such as whole-slide imaging system (WSIS), indocyanine green near-infrared (ICG-NIR), and cytology and biology techniques in meniscal-ligamentous injury OA (MLI-OA) models and lymphatic smooth muscle cells（LSMC）, from the VEGF-C/VEGFR-3 pathway, we will try to find out the effects of the "BushenHuoxue decoction" regulation the lymphatic maturation and function on joint inflammation and prognosis in osteoarthritic mice and cells, which to provide evidence for clinical treatment of OA.

骨性关节炎（OA）致关节破坏，功能障碍，甚则残疾。现无有效治疗。我们前期研究发现：淋巴管成熟与功能与OA病理过程密切相关，OA关节周围成熟淋巴管减少、清除功能下降(Shi J, et al. Arthritis Rheumatol.2014)，致炎性物质堆积，关节水肿，软骨及骨破坏；而补肾活血方可显著减轻OA关节肿痛，增加VEGF-C表达，其机制仍不清楚。据此我们假设，补肾活血方可调控VEGF-C/VEGFR-3信号促进淋巴管成熟与功能，加快炎性物质清除，缓解炎症，减轻关节损伤。本研究拟利用半月板韧带损伤OA模式小鼠及其淋巴管平滑肌细胞，采用全切片成像系统（WSIS）淋巴管定量技术、近红外线-吲哚菁绿（NIR-ICG）淋巴功能检测技术和细胞学，分子生物学等技术，从VEGF-C/VEGFR-3通路，探索“补肾活血”中药调控淋巴管增生与功能对OA炎症程度及转归的影响，为临床治疗OA提供证据。

骨性关节炎（OA）是临床最常见炎性疾病，以肿痛显著和关节破坏等为特征。目前仍缺乏有效药物。研究已证实OA关节肿胀与淋巴回流障碍密切相关，关节周围淋巴管功能障碍与中医“痹症”表型类似；“补肾活血方”具抗炎，消肿，延缓OA关节退变等确切疗效，但其作用机理尚未阐明，本研究旨在探讨其治疗OA可能的淋巴机制。. 本研究先完善原代淋巴管平滑肌细胞（LSMCs）培养方法，制备补肾活血中药含药血清并确定其最佳时效与量效；使用IL-1β（10ng/ml）建立了OA炎症LSMCs模型并评价，证实其可有效模拟OA炎症不同阶段；再依据中药血清药理学结果使用已制备的各含药血清对炎症LSMCs体系进行干预，观察其对不同炎症阶段LSMCs的作用效应，结果显示：补肾活血方及其部分单体可改善炎症LSMCs增殖率（MTT），降低炎性因子IL-1β、TNF-α，增加促淋巴管增生成熟因子VEGFC、AKT1，及抗凋亡因子Bcl2、Foxo1等基因mRNA及蛋白表达含量（QRT-PCR及Western blot），提示补肾活血方可通过调控VEGFC信号通路分子及其靶基因表达，改善炎症LSMCs生物学活性，缓解其炎症反应。再以半月板-韧带损伤OA模鼠为研究对象，采用补肾活血方浓缩煎剂灌胃，检测结果证实：补肾活血方可显著减缓OA小鼠关节肿胀；降低OARSI评分，减少关节软骨面丢失（H&E及Safranin-O 染色）；增加成熟淋巴管数量及比例（双免疫荧光组织化学染色，全切片成像系统）；同时提高ICG清除率、改善淋巴回流（NIR-ICG）；基因芯片证实其较广泛的基因谱调控作用（上调基因为56条，下调119条，共175条）；分子生物学检测证实：炎症因子IL-1β、TNF-α及分解酶类MMP13、ADAMTS4/5基因表达均不同程度下降；而VEGF-C/VEGFR-3信号通路分子及其靶基因中，VEGFC、VEGFR-3、AKT1、GSK3、 Bcl2及Foxo1基因mRNA及蛋白表达则不同程度增加。提示补肾活血方可增加成熟淋巴管分布及比例，促进OA淋巴管回流功能，改善关节炎症，从而延缓OA关节损害。. 综上，补肾活血方可调控VEGF-C/VEGFR-3信号通路分子及其靶基因表达，促进淋巴管成熟，增加淋巴管回流，从而加快炎症介质清除，减轻关节炎症，达到“除痹”效果，为OA的淋巴治疗提供了新思路。