胡黄连保护光损伤小鼠视网膜结构与功能的研究

Protective effects of Picrorhiza Scrophulariiflora on retinal morphological structure and visual function in light-damaged mice

目的探讨胡黄连对光损伤诱导的光感受器细胞退行性病变的保护效应。方法将BALB/c小鼠随机分为正常对照组(No Light)、光损伤模型组(Light_Vehicle)、胡黄连低剂量组(Light_HHL_L)、胡黄连中剂量组(Light_HHL_M)和胡黄连高剂量组(Light_HHL_H)5组,每组6只。其中正常对照组和光损伤模型组均以每20 g小鼠体质量200μL纯水溶剂灌胃,胡黄连低(167 mg/kg)、中(500 mg/kg)、高(1.5 g/kg)剂量组小鼠接受每20 g小鼠体质量200μL胡黄连水煎液灌胃治疗。连续给药7 d,给药第4天所有小鼠开始接受暗适应,第7天给药后0.5 h进行光损伤造模。光照后第7天利用光学相干断层扫描(OCT)影像学检查及苏木素-伊红(HE)染色,观察小鼠视网膜形态结构变化,并分析视网膜外核层(ONL)厚度变化;利用视网膜电图(ERG)观察小鼠视网膜a、b波振幅变化,并分析其视网膜功能改变;利用免疫组织化学法对视紫红质(Rhodopsin)和中波敏感视蛋白(M-opsin)进行染色,观察视杆细胞和视锥细胞的病理改变情况;利用TUNEL法对视网膜细胞凋亡进行原位观察。结果正常对照组视网膜各层形态结构完整,ONL厚度正常,细胞核排列整齐,ERG暗视a波和b波振幅绝对值随刺激强度增加而增加,Rhodopsin和M-opsin表达正常,视网膜各层未见凋亡细胞。与正常对照组比较,光损伤模型组视网膜结构明显受损,ONL厚度显著变薄(P<0.01),细胞核排列疏松紊乱,ERG暗视a波和b波振幅显著降低(P<0.05),Rhodopsin和M-opsin表达明显降低,ONL可见大量凋亡细胞。与光损伤模型组比较,胡黄连高剂量组视网膜形态结构维持完好,ONL细胞核排列整齐,胡黄连有效抑制ONL厚度变薄(P<0.01),显著抑制ERG暗视a波和b波振幅降低(P<0.05),胡黄连干预后Rhodopsin和M-opsin表达明显增加,ONL几乎未见凋亡细胞。结论胡黄连可显著抑制光损伤诱导的光感受器细胞凋亡,对光损伤小鼠视网膜形态结构和视功能具有显著的保护效应。

Objective To investigate the protective effects of Picrorhiza scrophulariiflora(PS)on photoreceptor cell degeneration induced by light injury.Methods BALB/c mice were randomly divided into five groups:normal control group(No Light),light damage group(Light_Vehicle),low-dose PS treatment group(Light_HHL_L),medium-dose PS treatment group(Light_HHL_M)and high-dose PS treatment group(Light_HHL_H),with 6 mice in each group.The normal control group and the light damage group were gavaged with 200μL pure water solution per 20 g of murine body mass.The low(167 mg/kg),medium(500 mg/kg)and high(1.5 g/kg)dose groups of PS were received decoction of PS with 200μL per 20 g of murine body mass.The mice were treated for continuous 7 days.All mice began to receive dark adaptation on the 4 th day.On the 7 th day,the PS treatment groups and light damage group were exposed to bright light for 30 min after intragastric administration.Seven days after illumination,the optical coherence tomography(OCT)imaging was performed to detect the thickness of retinal outer retinal nucleus layer(ONL)in vivo.The hematoxylin-eosin(HE)staining was used to observe the changes of retinal morphology and structure.Electroretinogram(ERG)was used to analyze the amplitude changes of a wave and b wave,which reflected the retinal function.The immunohistochemical assessment of Rhodopsin and M-opsin protein were performed to observe the rod and cone cells.Retinal apoptosis cells were observed in situ by TUNEL assay.Results The retinal morphology and structure were intact and the cell nuclei were neatly arranged in the normal control group,and the amplitudes of a wave and b wave in scotopic ERG increased with stimulus intensity,and the expressions of rhodopsin and m-opsin were normal.No apoptotic cells were found in all layers of the retina.Compared with the normal control group,the retinal structure was significantly damaged and the thickness of retinal ONL was significantly thinner(P<0.01)in the light damage group.The cell nuclei in ONL were loosely arranged and disordered,and the amplitudes of a and b waves of scotopic ERG were significantly reduced(P<0.05).The expression of rhodopsin and M-opsin decreased significantly.A large number of apoptotic cells were observed in ONL.Compared with the light damage group,the retinal morphology and structure remained intact and the ONL nuclei were arranged neatly in the high dose PS treatment group.PS could effectively inhibit the thinning of ONL thickness(P<0.01)and the decrease of a wave and b wave(P<0.05).The expressions of Rhodopsin and M-opsin were significantly increased in the PS treatment groups and few apoptotic cells were observed in ONL.Conslusion The apoptosis of photoreceptor cells induced by light injury is significantly inhibited by the intervention of PS,and PS has a significant protective effect on the retinal morphological structure and visual function in bright light-damaged mice.