Data Sheet 1_Modified ZhuJing pill protects retinal pigment epithelium against oxidative stress-induced epithelial–mesenchymal transition through Nrf2-mediated Akt/GSK3β pathway.docx

BackgroundThe modified ZhuJing pill (mZJP) has been widely used in China as a classical prescription for treating retinal diseases for years. Our preliminary experiment showed that mZJP exerted an antioxidant effect in treating dry age-related macular degeneration (AMD). Nevertheless, the specific mechanism underpinning the impact of mZJP on dry AMD remains obscure.
MethodsThe chemical metabolites of mZJP were qualitatively analyzed using LC-Q-TOF-MS. Dry AMD model mice were used to assess the efficacy of mZJP through optical coherence tomography (OCT), fundus autofluorescence (FAF), and immunofluorescence. Epithelial–mesenchymal transition (EMT) in OxLDL-induced ARPE-19 cells was evaluated by monitoring cellular integrity and quantifying EMT-related markers. Cell migration capacity was determined via wound healing and transwell assays. To investigate molecular mechanisms, cells were transfected with Nrf2 siRNA and analyzed through Western blotting, immunofluorescence, and migration assays under Nrf2 inhibition.
ResultsA total of 113 major metabolites were identified in mZJP. Our findings revealed that mZJP alleviated retinal pathological alterations and inhibited EMT progression. Furthermore, mZJP upregulated Nrf2 and HO-1 expression levels while downregulating Akt and GSK-3β phosphorylation levels. Notably, the EMT-suppressing effect of mZJP was significantly attenuated upon Nrf2 silencing, as evidenced by enhanced cell migration, decreased epithelial marker expression (E-cadherin), increased mesenchymal marker expression (vimentin and α-SMA), suppression of the Nrf2 pathway, and activation of the Akt/GSK3β pathway.
ConclusionOur study suggested that RPE protection by mZJP against oxidative stress induced EMT through Nrf2 activation and inhibition of the Akt/GSK3β pathway. MZJP could be a potential candidate drug for the treatment of dry AMD.

背景：加味朱晶丸（modified ZhuJing pill, mZJP）多年来作为经典方剂在国内广泛用于视网膜疾病的临床治疗。本团队前期实验证实，加味朱晶丸在治疗干性年龄相关性黄斑变性（dry age-related macular degeneration, AMD）中发挥抗氧化作用。然而，加味朱晶丸干预干性年龄相关性黄斑变性的具体分子机制仍未明确。
方法：采用液相色谱-串联四极杆飞行时间质谱（LC-Q-TOF-MS）对加味朱晶丸的化学代谢物进行定性分析。构建干性年龄相关性黄斑变性模型小鼠，通过光学相干断层扫描（optical coherence tomography, OCT）、眼底自发荧光（fundus autofluorescence, FAF）及免疫荧光实验评估加味朱晶丸的疗效。采用氧化低密度脂蛋白（OxLDL）诱导ARPE-19细胞构建上皮间质转化（Epithelial–mesenchymal transition, EMT）模型，通过监测细胞完整性及定量检测EMT相关标志物的表达水平，评估上皮间质转化情况。采用划痕愈合实验与Transwell小室实验检测细胞迁移能力。为探究分子机制，使用核因子E2相关因子2小干扰RNA（Nrf2 siRNA）转染细胞，在Nrf2抑制条件下，通过蛋白质印迹法、免疫荧光实验及迁移实验进行相关分析。
结果：本研究共在加味朱晶丸中鉴定出113种主要代谢物。实验结果显示，加味朱晶丸可减轻视网膜病理损伤，并抑制上皮间质转化进程。此外，加味朱晶丸可上调核因子E2相关因子2（Nrf2）与血红素氧合酶1（HO-1）的表达水平，同时下调蛋白激酶B（Akt）与糖原合成激酶3β（GSK-3β）的磷酸化水平。值得注意的是，当Nrf2被沉默后，加味朱晶丸的上皮间质转化抑制作用显著减弱，具体表现为细胞迁移能力增强、上皮标志物E-钙粘蛋白（E-cadherin）表达下调、间质标志物波形蛋白（vimentin）与α-平滑肌肌动蛋白（α-SMA）表达上调、Nrf2通路受到抑制以及Akt/GSK3β通路激活。
结论：本研究证实，加味朱晶丸通过激活Nrf2通路并抑制Akt/GSK3β通路，抑制氧化应激诱导的上皮间质转化，从而保护视网膜色素上皮（retinal pigment epithelium, RPE）。加味朱晶丸有望成为干性年龄相关性黄斑变性的潜在治疗候选药物。