明睛颗粒联合雷珠单抗对湿性年龄相关性黄斑变性纤维血管膜纤维化相关蛋白表达的影响

Mingjing Granules combined with leizumab on vascular membrane fibrosis associated proteins in wet age-related macular degeneration

关键词:  湿性年龄相关性黄斑变性  明睛颗粒  两阶段激光诱导  纤维血管膜  TGF-β1  纤维化相关蛋白

Key words:  wet age-related macular degeneration  Mingjing Granules  Two-stage laser induction  fibrovascular membrane  TGF-β1  fibrosis-associated protein

摘要:

[目的] 观察明睛颗粒联合雷珠单抗玻璃体腔注射对实验性湿性年龄相关性黄斑变性（nAMD）纤维血管膜以及纤维化相关蛋白表达的影响，阐释明睛颗粒治疗nAMD的作用机制，为临床上中药联合抗血管内皮生长因子（VEGF）药物治疗提供理论依据。[方法] 应用两阶段激光光凝的方法建立实验性nAMD纤维血管膜模型，将造模成功后的BN大鼠随机分为3组：模型组、抗VEGF组、明睛颗粒（MJKL）+抗VEGF组。模型组：予蒸馏水灌胃；抗VEGF组：予玻璃体腔注射雷珠单抗注射液；MJKL+抗VEGF组：予玻璃体腔注射雷珠单抗注射液，同时予明睛颗粒灌胃。10只BN大鼠不造模，常规饲养作为正常对照组。造模40 d后，采用眼底照相（FP）、眼底血管荧光造影（FFA）、光学相干断层扫描（OCT）、视网膜色素上皮（RPE）-脉络膜-巩膜铺片观察眼底病变形态、病变渗出面积及MD值；组织病理学观察视网膜结构的改变，免疫荧光法检测Collagen-1，Fibronectin，α-SMA，TGF-β1的表达及分布，qRT-PCR法检测Collagen-1mRNA，FibronectinmRNA，α-SMAmRNA和TGF-β1 mRNA的相对表达量。[结果] 两阶段激光造模后40d建立纤维血管膜模型；抗VEGF组在病变渗出面积、光密度值（MD）值、病变高度均较模型组显著降低（P<0.05），病变面积及视网膜结构损伤程度显著降低；而MJKL+抗VEGF组较抗VEGF组在病变渗出的MD值、病变高度更加降低（P<0.05），病变面积及视网膜结构损伤程度也明显降低。Collagen-1，Fibronectin，α-SMA，TGF-β1的荧光强度中模型组均较正常组明显升高（P<0.05），抗VEGF组均较模型组明显降低（P<0.05）；MJKL+抗VEGF组中Collagen-1，Fibronectin，TGF-β1的荧光强度较抗VEGF组明显降低（P<0.05）。Collagen-1mRNA，FibronectinmRNA，α-SMAmRNA和TGF-β1 mRNA的相对表达量中，模型组均较正常组明显升高（P<0.05），抗VEGF组均较模型组明显降低（P<0.05），MJKL+抗VEGF组均较抗VEGF组明显降低（P<0.05）。[结论] 明睛颗粒联合抗VEGF药物可以通过降低Collagen-1，Fibronectin，α-SMA，TGF-β1蛋白的表达，从而较单一应用抗VEGF药物可以更好地抑制实验性nAMD纤维血管膜的生长。

Abstract:

[Objective] To observe the effects of Mingjing Granules(MJKL) combined with lezumab intravitreal injection on the expression of vascular membrane and fibrosation-related proteins in experimental wet age-related macular degeneration(nAMD),explain the mechanism of action of MJKL in the treatment of nAMD,and provide theoretical basis for the clinical treatment of traditional Chinese medicine(TCM) combined with anti-vascular endothelial growth factor(VEGF) drugs. [Methods] Experimental nAMD fibrovascular membrane model was established by two-stage laser photocoagulation. BN rats were randomly divided into three groups:model group,anti-VEGF group,MJKL +anti-VEGF group. Model group:distilled water was given intragastric administration;Anti-VEGF group:intravitreous cavity was injected with leizumab injection;MJKL+ anti-VEGF group:Leizumab injection was injected into the vitreous cavity,and MJKL was given intragastric administration. Ten BN rats were not modeled and were fed as normal control group. After 40 days of model making,fundus lesion morphology,lesion exudation area and MD value were observed by fundus photography(FP),fundus angiography(FFA),optical coherence tomography(OCT),retinal pigment epithelium(RPE)-choroid-sclera film. The changes of retinal structure were observed by histopathology,the expression and distribution of collagenia-1,Fibronectin,α-SMA,TGF-β1 were detected by immunofluorescence,and collagenia-1 mRNA and Fibronectin mRNA were detected by qRT-PCR. The relative expression of α-SMA mRNA and TGF-β1 mRNA. [Results] The fibrovascular membrane model was established 40 d after two-stage laser modeling. The lesion lesion area,optical density(MD) value and lesion height in anti-VEGF group were significantly decreased compared with model group(P<0.05),and lesion area and retinal structural damage degree were significantly decreased. Compared with the anti-VEGF group,MJKL+ anti-VEGF group significantly reduced the MD value and lesion height in lesion exudation(P<0.05),and the lesion area and retinal structural damage degree were also significantly reduced. The fluorescence intensity of Collagen 1,Fibronectin,α-SMA and TGF-β1 in the model group was significantly higher than that in the normal group(P<0.05),while the fluorescence intensity in the anti-VEGF group was significantly lower than that in the model group(P<0.05). The fluorescence intensity of Collagen 1,Fibronectin and TGF-β1 in MJKL+ anti-VEGF group was significantly lower than that in anti-VEGF group(P<0.05). The relative expression levels of Collagen 1 mRNA,Fibronectin mRNA,α-SMA mRNA and TGF-β1 mRNA in the model group were significantly higher than those in the normal group(P<0.05),while those in the anti-VEGF group were significantly lower than those in the model group(P<0.05). MJKL+ anti-VEGF group was significantly lower than anti-VEGF group(P<0.05). [Conclusion] MJKL combined with anti-VEGF drugs can reduce the expression of Collagen 1,Fibronectin,α-SMA,TGF-β1 proteins,which can better inhibit the growth of experimental nAMD fiber vascular membrane than the single application of anti-VEGF drugs.