枸杞子-丹参诱导穆勒细胞重编程治疗视网膜色素变性的实验研究

Experimental study on treatment of retinitis pigmentosa by inducing Müller cell reprogramming with Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma

探讨滋阴活血明目药对枸杞子-丹参(Lycii Fructus-Salviae Miltiorrhizae Radix, LFSMR)抑制穆勒细胞(Müller cell,MC)胶质化,促进MC重编程转分化为视网膜神经细胞治疗视网膜色素变性(retinitis pigmentosa, RP)的效应及机制。将12只C57小鼠作为正常对照组,48只转基因RP(rd10)小鼠随机分为模型组、阳性对照组和LFSMR低、高剂量组,每组12只。HE染色检测视网膜病理改变,视网膜电图检测视网膜功能,眼底光学相干断层扫描检测视网膜厚度并进行眼底照相,激光散斑灌注成像检测视网膜局部血流,数字PCR检测视网膜神经细胞相关基因表达,免疫荧光法检测视网膜神经细胞相关蛋白表达。LFSMR可明显改善rd10小鼠视网膜病理改变;LFSMR可明显增加小鼠视网膜a波和b波振幅;LFSMR可显著增加小鼠视网膜厚度;LFSMR可明显恢复RP病变过程小鼠眼底损伤;LFSMR可显著提升RP病变过程小鼠视网膜血流量;LFSMR可显著抑制RP发病过程胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)mRNA表达,上调性别决定域Y链蛋白2(sex determining region Y box protein 2, SOX2)、双链复合蛋白6(paired box protein 6, Pax6)、视紫红质(rhodopsin)、蛋白激酶C-α(protein kinase C-α, PKCα)、突触融合蛋白(syntaxin)、胸腺细胞抗原1.1(thymic cell antigen 1.1, Thy1.1)mRNA表达;LFSMR可显著抑制GFAP蛋白表达,提升SOX2、Pax6、rhodopsin、PKCα、syntaxin、Thy1.1蛋白表达。LFSMR可逆转rd10小鼠视网膜病理改变,改善视网膜功能和眼底表现,增加视网膜厚度,提升视网膜局部血流量,发挥治疗RP的效应。LFSMR的作用机制可能与抑制穆勒细胞胶质化,促进穆勒细胞重编程转分化为各类视网膜神经细胞有关。

This study aims to explore the effect of Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma(LFSMR),a drug pair possesses the function of nourishing Yin,promoting blood circulation,and brightening the eyes,in treating retinitis pigmentosa(RP)by inhibiting the gliosis of Müller cells(MCs)and inducing their reprogramming and differentiation into various types of retinal nerve cells.Twelve C57 mice were used as the normal control group,and 48 transgenic RP(rd10)mice were randomly divided into the model group,positive control group,and low and high dose LFSMR groups,with 12 mice in each group.HE staining was used to detect pathological changes in the retina,and an electroretinogram was used to detect retinal function.Retinal optical coherence tomography was used to detect retinal thickness and perform fundus photography,and laser speckle perfusion imaging was used to detect local retinal blood flow.Digital PCR was used to detect gene expression related to retinal nerve cells,and immunofluorescence was used to detect protein expression related to retinal nerve cells.LFSMR could significantly improve the pathological changes,increase the amplitude of a and b waves,increase the retinal thickness,restore retinal damage,and increase retinal blood flow in mice with RP lesions.LFSMR could also significantly inhibit the mRNA expression of the glial fibrillary acidic protein(GFAP)during the pathogenesis of RP and upregulate mRNA expression of sex determining region Y box protein 2(SOX2),paired box protein 6(Pax6),rhodopsin,protein kinase C-α(PKCα),syntaxin,and thymic cell antigen 1.1(Thy1.1).LFSMR could significantly inhibit GFAP protein expression and enhance protein expression of SOX2,Pax6,rhodopsin,PKCα,syntaxin,and Thy1.1.It could also reverse the pathological changes in the retina of rd10 mice,improve retinal function and fundus performance,increase retinal thickness,enhance local retinal blood flow,and exert therapeutic effects on RP.The mechanism of action of LFSMR may be related to inhibiting the gliosis of MCs and promoting their reprogramming and differentiation into various types of retinal nerve cells.