固本清目方对实验性干性年龄相关性黄斑变性小鼠的干预作用

Effect of Guben Qingmu Formula on experimental dry age-related macular degeneration in mice

目的观察固本清目方对实验性干性年龄相关性黄斑变性C57BL/6J小鼠的干预作用。方法4月龄C57BL/6J小鼠40只,用慢性光照联合氢醌诱导建立视网膜变性模型。随机分为中药组、模型组,每组20只。中药组小鼠每日给予0.6 kg/kg固本清目方0.3 ml灌胃1次;模型组小鼠每日给予同体积蒸馏水灌胃1次。同时取正常未干预C57BL/6J小鼠20只为正常组,常规饲养作为对照。分别于干预_(14 d)及_(28 d)采用视网膜电图(ERG)、HE染色观察各组小鼠视网膜功能和结构改变,TUNEL法观察视网膜细胞凋亡情况,免疫荧光法检测Fas及其配体FasL的表达和分布,RT-PCR检测Fas、FasL mRNA的表达。结果(1)ERG:在干预_(14 d)和_(28 d)时,①中药组与正常组比较,10Hz闪烁光反应(t_(14 d)=7.360,P=0.000;t_(28 d)=10.651,P=0.000)、最大反应(t_(28 d)=13.229,P=0.016)振幅较正常组降低,差异有统计学意义,而其他项目均无统计学意义(P>0.05);②中药组与模型组比较,视杆细胞反应(t_(14 d)=2.937,P=0.022;t_(28 d)=4.687,P=0.002)、最大反应(t_(14 d)=3.153,P=0.016)、暗适应震荡电位(t_(14 d)=3.597,P=0.009;t_(28 d)=3.371,P=0.012)、视锥细胞反应(t_(14 d)=2.988,P=0.020;t_(28 d)=4.176,P=0.004)振幅均升高,差异均有统计学意义,而其他项目均无统计学意义(P>0.05)。(2)组织病理学:在干预_(14 d)和_(28 d)时,HE染色显示,中药组小鼠视网膜色素上皮(RPE)细胞排列较规整,色素均匀分布于细胞内,Bruch膜结构相对完整,模型组小鼠RPE层呈现萎缩样改变;感光细胞计数比较,中药组较模型组显著增加(t_(14 d)=6.346,P=0.000;t_(28 d)=5.827,P=0.001),而与正常组比较差异均无统计学意义(P>0.05)。(3)细胞凋亡:在_(14 d)和_(28 d)时,中药组细胞凋亡率较模型组均显著降低(t_(14 d)=2.714,P=0.030;t_(28 d)=3.231,P=0.014)。(4)Fas和FasL:在_(14 d)和_(28 d)时,Fas、FasL蛋白于正常组均有少量表达,主要分布在RPE细胞层;中药组表达范围同正常组相似,而模型组除RPE层外,在感光细胞内核层亦可见FasL阳性表达。RT-PCR结果显示,干预_(14 d)和_(28 d)时,中药组与模型组比较Fas和FasLmRNA表达均降低(14 d:t_(Fas)=7.153,t_(FasL)=8.372,均P=0.000;28 d:t_(Fas)=11.204,t_(FasL)=7.011,均P=0.000),差异均有统计学意义。而与正常组比较差异均无统计学意义(P>0.05)。结论固本清目方对实验性干性年龄相关性黄斑变性小鼠具有保护作用,其机制与抑制视网膜细胞凋亡密切相关。

OBJECTIVE To observe the intervention effect of Guben Qingmu Formula on experimental dry age-related macular degeneration in C57BL/6J mice.METHODS Forty 4-month-old C57BL/6J mice were induced by chronic light combined with hydroquinone to establish the retinal degeneration model.They were randomly divided into Traditional Chinese Medicine(TCM)group and model group,with 20 mice in each group.Mice in the TCM group were treated with 0.6 kg/kg Guben Qingmu Formula(0.3 ml)by gavage once a day,and mice in the model group were given the same volume of distilled water by gavage once a day.At the same time,another 20 normal non-intervened C57BL/6J mice were taken as the normal group and routinely fed as the control.On the 14th and 28th day of intervention,the changes of retinal function and structure were observed by electroretinogram(ERG)and HE staining,the apoptosis of retinal cells was evaluated with TUNEL staining,and the expression and distribution of Fas and its ligand Fas-Ligand(FasL)were detected by immunofluorescence,and the mRNA expression of Fas and FasL were detected by RT-PCR.RESULTS(1)ERG:At 14th and 28th day of intervention:①Compared with the normal group,10 Hz scintillation light response(t_(14 d)=7.360,P=0.000;t_(28 d)=10.651,P=0.000)and maxium response(t_(28 d)=13.229,P=0.016)in the TCM group were significantly lower than those in the normal group,however other items had no statistical significance(P>0.05);②Compared with the model group,b wave amplitudes of rod cell response(t_(14 d)=2.937,P=0.022;t_(28 d)=4.687,P=0.002),maximum response(t_(14 d)=3.153,P=0.016),dark apaptation shock potential(t_(14 d)=3.597,P=0.009;t_(28 d)=3.371,P=0.012)and cone cell response(t_(14 d)=2.988,P=0.020;t_(28 d)=4.176,P=0.004)increased with significant differences,while other items had no statistical significance(P>0.05).(2)Histopathology:At 14th and 28th day of intervention,HE staining showed that retinal pigment epithelial(RPE)cells in the TCM group were arranged regularly,pigments were evenly distributed in the cells,Bruch membrane structure was relatively complete,and the RPE layer in the model group showed atrophic changes.Compared with the model group,the number of photoreceptor cells in the TCM group increased significantly(t_(14 d)=6.346,P=0.000;t_(28 d)=5.827,P=0.001),but there was no significant difference compared with the normal group(P>0.05).(3)Apoptosis:At 14th and 28th day of treatment,the apoptosis rate of the TCM group was significantly lower than that of the model group(t_(14 d)=2.714,P=0.030;t_(28 d)=3.231,P=0.014).(4)Fas and FasL:At 14th and 28th day of treatment,Fas and FasL proteins were expressed in a small amount in the normal group,mainly in the RPE cell layer.The expression range of the TCM group was similar to that of the normal group,and FasL positive expression was also observed in inner nuclear layer in the model group except RPE layer.The results of RT-PCR showed that both Fas and FasL mRNA expressions were lower than those in the model group at 14th and 28th day of treatment 14 d:t_(Fas)=7.153,t_(FasL)=8.372,both P=0.000;28 d:t_(Fas)=11.204,t_(FasL)=7.011,both P=0.000).However,when compared with the normal group,there was no significant difference(P>0.05).CONCLUSIONS Guben Qingmu Formula has protective effect on experimental dry age-related macular degeneration,and its mechanism is closely related to the inhibition of retinal cell apoptosis.