滋阴明目方对视网膜色素变性rd10小鼠PERK-ATF4信号通路的影响

Effects of Ziyin Mingmu Formula on PERK-ATF4 signaling pathway in rd10 mice with retinitis pigmentosa

目的研究滋阴明目方对视网膜色素变性小鼠的影响及其可能机制。方法将60只rd10小鼠随机分为模型组(等量生理盐水)、维生素A(Vitamin A,VitA)组[VitA 750 IU/(kg·d)]及滋阴明目方低、中、高剂量组[滋阴明目方水煎液13.5、27、54 g/(kg·d)],12只C57BL/6小鼠作为空白组(等量生理盐水),均灌胃干预28 d。HE染色观察视网膜组织病理改变,TUNEL法检测视网膜组织凋亡,微滴式数字PCR和免疫荧光双染检测视网膜组织蛋白激酶样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)、活化转录因子4(activating transcription factor 4,ATF4)mRNA和蛋白的表达。结果与空白组比较,模型组小鼠视网膜明显萎缩、变薄,视网膜色素上皮(retinal pigment epithelial,RPE)细胞不可见,外核层消失,视网膜厚度减小(P<0.01),小鼠视网膜细胞数量明显减少,存在大量凋亡细胞;PERK、ATF4 mRNA和蛋白表达量升高(P<0.01)。与模型组比较,VitA组和滋阴明目方各剂量组视网膜状况改善,厚度均增加(P<0.01);细胞数量增多,凋亡细胞减少;PERK、ATF4 mRNA表达量降低(P<0.01)。与模型组比较,VitA组和滋阴明目方中、高剂量组PERK、ATF4蛋白表达量降低(P<0.01);滋阴明目方低剂量组ATF4蛋白表达量降低(P<0.01)。与滋阴明目方低剂量组比较,滋阴明目方高剂量组视网膜厚度增加(P<0.05),滋阴明目方中、高剂量组ATF4蛋白表达量及PERK mRNA表达量降低(P<0.01)。与VitA组相比,滋阴明目方高剂量组ATF4蛋白表达量降低(P<0.05),滋阴明目方中、高剂量组PERK mRNA表达量降低(P<0.01)。结论滋阴明目方可改善小鼠视网膜的形态,减少视网膜组织的凋亡,其分子机制可能与调控PERK-ATF4信号通路有关。

Objective To study the effects and the possible mechanisms of Ziyin Mingmu Formula(ZYMMF)on mice with retinitis pigmentosa(RP).Methods Sixty rd10 mice were randomized into model group(equal volume of saline),Vitamin A(VitA)group[VitA 750 IU/(kg·d)],and low-,medium-,and high-dose ZYMMF groups[ZYMMF decoction 13.5,27,54 g/(kg·d)].Twelve C57BL/6 mice were used as the blank group(equal volume of saline).All groups were administered by gavage for 28 days.HE staining was used to observe the pathological changes in retinal tissue,TUNEL assay was used to check the apoptosis in retinal tissue,and droplet digital PCR and immunofluorescence double staining were used to examine the mRNA and protein expressions of protein kinase RNA-like endoplasmic reticulum kinase(PERK)and activating transcription factor 4(ATF4)in retinal tissue.Results Compared with the blank group,the model group showed significant retinal atrophy and thinning,with the retinal pigment epithelial(RPE)cells being invisible,the outer nuclear layer disappearing,and a decrease in retinal thickness(P<0.01),along with a significant reduction in the number of retinal cells and the presence of a large number of apoptotic cells;the mRNA and protein expression levels of PERK and ATF4 increased(P<0.01).Compared with the model group,the VitA group and each dose group of ZYMMF showed improvement in retinal condition and increased thickness(P<0.01);the number of cells increased and the number of apoptotic cells decreased;the mRNA expression levels of PERK and ATF4 decreased(P<0.01).Compared with the model group,the protein expression levels of PERK and ATF4 in the VitA group and the medium-and high-dose ZYMMF groups decreased(P<0.01);the protein expression level of ATF4 in the low-dose ZYMMF group was reduced(P<0.01).Compared with the low-dose ZYMMF group,the high-dose ZYMMF group showed an increase in retinal thickness(P<0.05),and the medium-and high-dose ZYMMF groups showed a decrease in protein expression of ATF4 and mRNA expression of PERK(P<0.01).Compared with the VitA group,the high-dose ZYMMF group showed a decrease in protein expression of ATF4(P<0.05),and the medium-and high-dose ZYMMF groups exhibited a decrease in mRNA expression of PERK(P<0.01).Conclusion ZYMMF can improve the morphology of mouse retina and reduce the apoptosis of retinal tissue,and its molecular mechanism may be related to the regulation of PERK-ATF4 signaling pathway.