黄芪甲苷干预碘酸钠诱导的光感受器退行性病变的效应及相关机制

Effects and mechanisms of astragaloside A treatment on sodium iodate-induced photoreceptor degeneration

目的观察并初步探讨黄芪甲苷(AS-A)干预碘酸钠(NaIO_(3))诱导的光感受器退行性病变的效应及相关机制。方法6~8周龄健康雄性C57BL/6J小鼠60只,随机分为正常对照组(NC组)、NaIO_(3)组、AS-A组,每组20只。建模前30 min,AS-A组按100 mg/kg的剂量腹腔注射100μl AS-A;30 min后,NaIO_(3)组、AS-A组小鼠按30 mg/kg的剂量腹腔注射100μl NaIO_(3)。其后,AS-A组小鼠每天早晚间隔12 h给药2次,直到实验结束。建模后1 d,紧密连接蛋白(ZO-1)染色观察视网膜色素上皮(RPE)细胞结构;实时荧光定量聚合酶链反应(qPCR)检测各组小鼠视网膜趋化因子配体2(Ccl2)、白细胞介素1β(Il-1β)、混合谱系激酶结构域样蛋白(Mlkl)、受体相互作用蛋白激酶3(Ripk3)、肿瘤坏死因子(Tnf)等基因的mRNA相对表达量。建模后3 d,免疫组织化学染色观察各组小鼠视网膜中离子钙接头蛋白1(Iba1)、神经胶质纤维酸性蛋白(GFAP)阳性表达;原位末端标记(TUNEL)法检测各组小鼠视网膜光感受器细胞的死亡情况。建模后4 d,采用眼底彩色照相、光相干断层扫描(OCT)检查观察各组小鼠眼底形态;苏木精-伊红染色(HE)观察各组小鼠视网膜形态结构。两组间比较采用独立样本t检验;三组间比较采用单因素方差分析。结果眼底彩色照相、OCT检查发现,NaIO_(3)组小鼠眼底可见大量散在黄白色玻璃膜疣样结构,RPE层出现大量强反射区;AS-A组RPE层中强反射区数量减少。ZO-1染色结果显示,NaIO_(3)组RPE细胞膜上ZO-1大量丢失;AS-A组ZO-1均匀分布于RPE细胞膜上。HE染色结果显示,NaIO_(3)组RPE层可见圆形黑色沉积物,光感受器内外节结构严重受损,外核层(ONL)细胞层核数显著减少;AS-A组RPE层色素整齐,光感受器内外节结构完整,ONL细胞核层数显著增加。TUNEL检测结果显示,NaIO_(3)组ONL可见大量TUNEL阳性细胞核,AS-A组视网膜ONL中TUNEL阳性细胞核数量显著减少,差异有统计学意义(t=2.66,P<0.05)。qPCR检测结果显示,与AS-A组比较,NaIO_(3)组视网膜中Mlkl、Ripk3、Ccl2、Il-1β、Tnf mRNA相对表达量显著升高,差异有统计学意义(F=39.18、10.66、53.51、41.40、24.13,P<0.001)。免疫组织化学染色结果显示,与NC组、AS-A组比较,NaIO_(3)组视网膜中GFAP阳性表达显著升高,差异有统计学意义(F=9.62,P<0.05)。结论AS-A能有效抑制NaIO_(3)诱导的光感受器退行性病变,其效应部分与抑制光感受器死亡和神经炎症反应,维护视网膜稳态相关。

Objective To investigate the effect of astragaloside A(AS-A)on the photoreceptor degeneration induced by sodium iodate(NaIO_(3))and its related mechanism.Methods Sixty healthy male C57BL/6J mice,aged 6-8 weeks,were randomly divided into normal control(NC)group,NaIO_(3)group,and ASA group,with twenty mice in each group.30 min before modeling,AS-A group mice were intraperitoneally injected with 100μl AS-A at a dose of 100 mg/kg body weight.30 min later,mice in NaIO_(3)group and AS-A group were intraperitoneally injected with 100μl NaIO_(3)at a dose of 30 mg/kg body weight.Subsequently,AS-A group mice were administered AS-A twice daily at 12 h intervals until the end of the experiment.On day 1 postmodeling,zonula occludens-1(ZO-1)immunohistochemistry was performed to observe the structure of retinal pigment epithelium(RPE)cells;real-time quantitative polymerase chain reaction(qPCR)was conducted to detect the mRNA expression of various retinal chemokine ligand-2(Ccl2),interleukin-1 beta(Il-1β),mixed lineage kinase domain-like protein(Mlkl),receptor-interacting protein kinase 3(Ripk3),and tumor necrosis factor(Tnf).On day 3 post-modeling,immunohistochemistry was performed to observe the expression of ionized calcium binding adaptor molecule 1(Iba1)and glial fibrillary acid protein(GFAP)in the retina;TdT-mediated dUTP nick-end labeling(TUNEL)assay was used to detect photoreceptor cell death in each group.On day 4 post-modeling,fundus morphology of mice in each group was observed by fundus color photography and optical coherence tomography(OCT).Hematoxylin-eosin staining(HE)was used to observe the morphological structure of the retina in each group.Inter-group comparisons between two groups were conducted using independent samples t-test,while comparisons among three groups were performed using one-way ANOVA.Results Fundus color photography and OCT examination showed that a large number of scattered yellowwhite subretinal nodular structures in the fundus of NaIO_(3)group mice,and a large number of strong reflection areas in the RPE layer.The number of strong reflection areas in the RPE layer was reduced in the AS-A group.Immunohistochemical analysis of ZO-1 showed that ZO-1 was largely lost on the RPE cell membrane in that NaIO_(3)group;whereas in the AS-A group,ZO-1 was evenly distributed on the RPE cell membrane.HE staining results showed circular black deposits were visible in the RPE layer of the NaIO_(3)group,and the inner and outer segments of photoreceptors were severely damaged,with a significant decrease in the number of outer nuclear layer(ONL)cell nuclei;whereas in the AS-A group,the RPE layer pigments were orderly,the inner and outer segments of photoreceptors were intact,and the number of ONL cell nuclei significantly increased.The results of TUNEL staining show that numerous TUNEL-positive cell nuclei were observed in the ONL of the retina in the NaIO_(3)group,while the number of TUNEL-positive cell nuclei in the ONL of the retina was significantly reduced in the AS-A group,with statistically significant differences(t=2.66,P<0.05).The analysis of qPCR data showed that compared with the AS-A group,the relative expression levels of Mlkl,Ripk3,Ccl2,Il-1βand Tnf mRNA in the retina were significantly increased in the NaIO_(3)group,with statistically significant differences(F=39.18,10.66,53.51,41.40,24.13;P<0.001).Immunohistochemical staining results showed that compared with NC group and AS-A group,the positive expression of GFAP in retina of NaIO_(3)group was significantly increased,and the difference was statistically significant(F=9.62,P<0.05).Conclusion AS-A antagonizes NaIO_(3)-induced photoreceptor degeneration in part by inhibiting photoreceptor cell death and neuroinflammation.Meanwhile,AS-A treatment protects against NaIO_(3)-triggered perturbation of retinal homeostasis.