基于VEGF-C/VEGFR-3信号通路电针预处理促淋巴管再生修复淋巴水肿的作用机制研究

Effect of electroacupuncture pretreatment on mediating VEGF-C/VEGFR-3 signaling pathway to promote lymphatic vessel regeneration and repair lymphedema

目的观察电针预处理足三里联同阴陵泉穴对淋巴管内皮细胞(lymphatic endothelial cells,LECs)增殖、迁移、小管形成等生物学行为的影响及其相关效应及机制,为淋巴管再生治疗淋巴水肿提供理论依据,探讨电针预处理对淋巴水肿的修复作用可能机制。方法SD大鼠随机将其分为空白组、假手术组、模型组、连续波电针组、疏密波电针组,每组10只。假手术组具体操作与模型组同,但不进行淋巴管结扎以及淋巴结摘除,模型组和电针组先将左侧腹股沟皮肤沿圆周切开,然后连同周围的脂肪垫同腹股沟淋巴结一起切除,用10-0尼龙线将染色的淋巴管在结扎前和结扎后区域仔细缝合,并用周围的脂肪垫完全切除及摘掉腘窝淋巴结。电针组选取双侧“足三里穴、阴陵泉穴”进行电针干预,每次20min,每日1次,造模前连续干预7d。HE染色观察大鼠损伤近端皮肤形态及结构的病理改变;免疫组织化学法检测术后7d大鼠腿部损伤远端组织LYVE-1阳性淋巴管并进行计数;WB技术检测大鼠腿部损伤远端组织的、VEGFR3、VEGF-C、LYVE-1及IL-6的蛋白表达;PCR技术检测大鼠腿部损伤远端组织的VEGFR3、VEGF-C、LYVE-1的mRNA表达;ELISA技术检测血液中IL6、TNF-α水平表达情况。结果与假手术组比较,模型组大鼠术后LYVE-1、VEGF-C、VEGFR3表达及IL-6的表达升高(P<0.05)。与模型组比较,电针组大鼠VEGFR3、VEGF-C、LYVE-1表达升高,IL-6表达降低(P<0.05)。结论电针预处理可能通过增加LYVE1、VEGF-C、VEGFR3表达,降低IL-6等炎性反应,促进病变组织内淋巴管生成,修复受损的淋巴循环。

Objective To observe the effect of electroacupuncture pretreatment of Yinlingquan combined with Zusanli Point on biological behavior of LECs proliferation,migration,tubule formation and other related effects and mechanisms,so as to provide theoretical basis for lymphatic tube regeneration treatment of lymphedema.To explore the possible mechanism of repairing lymphedema by electroacupuncture preconditioning.Methods SD rats were randomly divided into blank group,sham operation group,model group,continuous wave electroacupuncture group and dense-wave electroacupuncture group,with 10 rats in each group.In the sham operation group,the left groin skin was cut around the circumference and then removed along with the surrounding fat pad.The specific operation was the same as that in the model group,but lymphatic vessel ligation and lymph node extraction were not performed.In the model group and the electroacupuncture group,the left groin skin was first cut along the circumference,and then the surrounding fat pads were removed together with the inguinal lymph nodes.The stained lymph vessels were carefully sutured before and after ligation with 10-0 nylon thread,and the popliteal lymph nodes were completely resected and removed with the surrounding fat pads.In the electroacupuncture group,bilateral"Zusanli point and Yinlingquan point"were selected for electroacupuncture intervention,20min each time,once a day,and continuous intervention was performed for 7 days before modeling.HE staining was used to observe the pathological changes of the morphology and structure of the injured proximal skin.Immunohistochemistry was used to detect the expression of LYVE1 in the distal leg injury tissue of 7-day-old rats after surgery.The protein expressions of LYVE1,VEGF-C,VEGFR3 and IL-6 in the distal tissue of the injured rat leg were detected by WB.mRNA expressions of LYVE1,VEGF-C and VEGFR3 were detected by PCR.The levels of IL6 and TNF-αin blood were detected by ELISA.Results compared with sham operation group,the expressions of LYVE1,VEGFR-C,VEGFR3 and IL-6 in model group were increased after operation(P<0.05).Compared with model group,the expression of LYVE1,VEGF-C and VEGFR3 in electroacupuncture group was increased,while the expression of IL-6 was decreased(P<0.05).Conclusion Electroacupuncture preconditioning may increase the expression of LYVE1,VEGF-C and VEGFR3,reduce IL-6 and other inflammatory responses,promote the formation of lymphatic vessels in pathological tissues,and repair the damaged lymphatic circulation.