芪灯明目胶囊对氧诱导视网膜病变小鼠视网膜血管的保护作用

Protective effect of Qideng Mingmu capsule on retinal vessels in mice with oxygen-induced retinopathy

目的研究芪灯明目胶囊对氧诱导视网膜病变(OIR)小鼠视网膜新生血管形成及重塑的影响。方法将36只出生后第7天的(P7)SPF级C57BL/6J幼鼠采用随机数字表法分为正常组、OIR组、芪灯明目胶囊组和阿帕替尼组,每组9只。正常组小鼠于正常环境下饲养,其余小鼠均在高氧环境中[氧浓度为(75±2)%]饲养5 d(P7~P12)后再在正常环境中饲养5 d(P12~P17)建立OIR模型。芪灯明目胶囊组和阿帕替尼组从P12开始分别给予芪灯明目胶囊(900 mg/kg)与血管内皮生长因子受体2抑制剂阿帕替尼(70 mg/kg)灌胃,1次/d,连续5 d。P17时,摘取小鼠眼球,制作眼球石蜡切片并行苏木精-伊红染色,计数各组小鼠突破内界膜的血管内皮细胞数目;制作视网膜铺片并行FITC-dextran荧光染色,计算视网膜无灌注区面积比、新生血管密度与总血管密度;采用免疫荧光染色法检测视网膜血管内皮细胞标志物CD31与周细胞标志物α-平滑肌肌动蛋白(α-SMA)的分布和荧光强度;采用免疫组织化学染色法检测视网膜缺氧诱导因子1α(HIF-1α)与血管内皮钙黏蛋白(VE-cadherin)的表达分布。结果正常组、OIR组、芪灯明目胶囊组和阿帕替尼组突破内界膜的血管内皮细胞数目分别为(2.83±4.40)、(37.33±5.43)、(23.83±6.79)和(14.00±9.34)个,总体比较差异有统计学意义(F=28.313,P<0.001),其中OIR组突破内界膜血管内皮细胞数明显多于正常组、芪灯明目胶囊组和阿帕替尼组,差异均有统计学意义(均P<0.05)。视网膜铺片结果显示,OIR组视网膜存在大片无灌注区域与新生血管芽,血管迂曲,分布紊乱,芪灯明目胶囊组和阿帕替尼组血管分布较OIR组更均匀,无灌注区面积和新生血管较OIR组减少。正常组、芪灯明目胶囊组和阿帕替尼组视网膜无灌注区面积比和新生血管密度均较OIR组降低,差异均有统计学意义(均P<0.05)。正常组、芪灯明目胶囊组和阿帕替尼组CD31免疫荧光强度和HIF-1α吸光度值明显低于OIR组,α-SMA免疫荧光强度和VE-cadherin吸光度值明显高于OIR组,差异均有统计学意义(均P<0.05)。结论芪灯明目胶囊能够抑制OIR小鼠视网膜新生血管形成,增加血管周细胞覆盖,缓解视网膜缺氧以及增加血管完整性,对OIR小鼠视网膜血管起到保护作用。

Objective To investigate the effect of Qideng Mingmu capsule on the formation and remodeling of retinal neovascularization in mice with oxygen-induced retinopathy(OIR).Methods Thirty-six postnatal day 7(P7)SPF grade C57BL/6J pups were divided into normal group,OIR group,Qideng Mingmu capsule group and apatinib group by random number table method,with 9 mice in each group.The mice in the normal group were raised in normal environment.The mice in the other three groups were fed in hyperoxic environment of(75±2)%oxygen concentration for 5 days from P7 to P12 and then were fed in normal environment for 5 days from P12 to P17 to establish the OIR model.From P12,mice in Qideng Mingmu capsule group and apatinib group were given intragastric administration of Qideng Mingmu capsule(900 mg/kg)and vascular endothelial growth factor receptor 2 inhibitor apatinib(70 mg/kg)respectively,once a day for 5 consecutive days.On P17,paraffin sections of mouse eyeballs were made and stained with hematoxylin-eosin to count the number of vascular endothelial cells that broke through the internal limiting membrane.The retinal slices were prepared and stained with FITC-dextran to quantify the retinal non-perfusion area,neovascularization density and total vascular density.The distribution and fluorescence intensity of retinal vascular endothelial cell marker CD31 and pericyte markerα-smooth muscle actin(α-SMA)were observed by double immunofluorescence staining.Immunohistochemical staining was used to detect the expression and distribution of retinal hypoxia inducible factor-1α(HIF-1α)and vascular endothelial cadherin(VE-cadherin).The use and care of animals were in accordance with the Regulations on the Management of Laboratory Animals issued by the Ministry of Science and Technology.This study was approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine(No.2019-30).Results The number of vascular endothelial cells breaking through the internal limiting membrane in normal group,OIR group,Qideng Mingmu capsule group and apatinib group were(2.83±4.40),(37.33±5.43),(23.83±6.79)and(14.00±9.34),respectively,with a statistically significant overall difference(F=28.313,P<0.001).There were more vascular endothelial cells breaking through internal limiting membrane in OIR group than in normal group,Qideng Mingmu capsule group and apatinib group,showing statistically significant differences(all at P<0.05).In the observation of mouse retinal slices,there were large non-perfusion areas,neovascularization buds and disordered distribution of blood vessels in OIR group.The distribution of blood vessels was more uniform and the areas of non-perfusion and neovascularization were smaller in Qideng Mingmu capsule group and apatinib group than in OIR group.The relative area of central retinal non-perfusion area and neovascularization density were significantly lower in normal group,Qideng Mingmu capsule group and apatinib group than in OIR group(all at P<0.05).The immunofluorescence intensity of CD31 and the absorbance value of HIF-1αwere significantly lower,and the immunofluorescence intensity ofα-SMA and the absorbance value of VE-cadherin were significantly higher in normal group,Qideng Mingmu capsule group and apatinib group than in OIR group(all at P<0.05).Conclusions Qideng Mingmu capsule can inhibit retinal neovascularization formation,increase vascular pericyte coverage,relieve retinal hypoxia and increase vascular integrity in OIR mice.It can protect the retinal vessels of OIR mice.