补肾益精方对先天性视网膜色素变性RCS大鼠感光细胞凋亡的抑制作用

目的　探讨补肾益精方对先天性视网膜色素变性RCS大鼠感光细胞凋亡的影响。方法　将先天性视网膜色素变性模型RCS大鼠24只随机分为补肾益精方组及蒸馏水组，分别给予补肾益精方药液及蒸馏水灌胃，12只健康SD大鼠常规饲养作为正常组。在给药7 d及28 d后HE染色观察各组大鼠视网膜组织病理学变化，TUNEL法检测细胞凋亡情况，实时荧光定量PCR检测视网膜中睫状神经营养因子、脑源性神经营养因子以及碱性成纤维细胞生长因子表达情况。结果　HE染色切片光学显微镜下正常组SD大鼠视网膜结构清晰，层次分明，各层细胞排列整齐。蒸馏水组大鼠视网膜内核层及外核层均较正常组明显变薄，细胞排列稀疏，可见空泡样改变，感光细胞数减少，且随鼠龄增加减少日益显著。补肾益精方组大鼠视网膜内核层及外核层亦较正常组变薄，但与蒸馏水组相比增厚，感光细胞数较后者增多，细胞排列也较为整齐。给药7 d及28 d时补肾益精方组每个高倍视野感光细胞数分别为140±9和80±9，蒸馏水组分别为113±8和44±6，补肾益精方组较蒸馏水组明显增多，差异有统计学意义（均为P<0.05）。TUNEL检测结果显示给药7 d及28 d时补肾益精方组感光细胞凋亡率分别为31.67±5.39%及29.68±4.31%，蒸馏水组分别为50.34±5.21%及44.02±7.17%，补肾益精方组较蒸馏水组均明显降低，差异均有统计学意义（均为P<0.05）。实时荧光定量PCR结果显示给药7 d及28 d补肾益精方组视网膜睫状神经营养因子表达均较蒸馏水组增加（均为P<0.05），给药7 d时补肾益精方组视网膜脑源性神经营养因子表达较蒸馏水组明显增加（P<0.05），28 d时两组差异无统计学意义（P>0.05）；给药7 d及28 d两组碱性成纤维细胞生长因子的表达差异均无统计学意义（均为P>0.05）。结论　补肾益精方对RCS大鼠感光细胞凋亡有明显的抑制作用，其作用机制可能与补肾益精方促进视网膜分泌神经营养因子有关。

Effects of Bu Shen Yi Jing Fang on photoreceptor cell apoptosis in RCS rat with inherited retinal degeneration

Objective　To observe the effects of Bu Shen Yi Jing Fang on photoreceptor cell apoptosis in royal college of surgeons rat （RCS） with inherited retinal degeneration and investigate the mechanisms.Methods　Totally 24 RCS rats were randomly divided into Bu Shen Yi Jing Fang group and distilled water group.The rats were gavaged with either Bu Shen Yi Jing Fang solution or distilled water every day.And 12 healthy SD rats were fed regularly as the normal control.On day 7，day 28 after treatment，HE staining and TdT-mediated dUTP nick end labeling （TUNEL） method were used to detect retina histopathological changes and cell apoptosis rate.Quantitative real time polymerase chain reaction （q-PCR） was used to determine the effects of Bu Shen Yi Jing Fang on the expression of ciliary neurotrophic factor （CNTF），brain derived neurotrophic factor （BDNF） and basic fibroblast growth factor （bFGF）.Results　The slides processed with HE staining were observed under light microscope.The retina structure in normal SD rats was clear and well arranged，and the cells in each layer were in good order.The inner nuclear layer and outer nuclear layer in distilled water group were markedly thinner than those in the normal group.Cells arrange sparsely and vacuoles change could be observed in the cells.The number of photoreceptor cells decreased，which aggravated with the rat age.The inner nuclear layer and outer nuclear layer in Bu Shen Yi Jing Fang group were thinner than those in the normal group too，but thicker when compared with that of distilled water group.The number of photoreceptor cell was more than the later，and arranged more orderly.On day 7 and day 28 after treatment，the number of photoreceptor cells in Bu Shen Yi Jing Fang group were 140±9 and 80±9，while its number in distilled water group was 113±8 and 44±6 respectively.The photoreceptor number in Bu Shen Yi Jing Fang group was significantly more than that of the distilled water group （both P<0.05）.It was shown by TUNEL detection that the apoptosis rates of photoreceptor cell in Bu Shen Yi Jing Fang group were （31.67±5.39）% and （29.68±4.31）% on day 7 and day 28 after treatment，while the apoptosis rates in distilled water group were （50.34±5.21）% and （44.02±7.17）% respectively.The apoptosis rates in Bu Shen Yi Jing Fang group were significantly lower than those of distilled water group （both P<0.05）.It was shown by q-PCR detection that expression of CNTF in Bu Shen Yi Jing Fang group was significantly higher than that of distilled water group both on day 7 and day 28 after treatment （both P<0.05）.On day 7 after treatment，expression of BDNF in Bu Shen Yi Jing Fang group was significantly higher than that of distilled water group （P<0.05）.However，there was no significant difference between the two groups on day 28 after treatment （P>0.05）.And the difference of bFGF expression between the two groups was not significantly different both on day 7 and day 28 after treatment （both P>0.05）.Conclusion　Bu Shen Yi Jing Fang can protect the apoptosis of photoreceptor cells in RCS rats significantly，and the mechanism may be related to the promotion of retinal neurotrophic factor expression.