Purpose: To observe the protective effects and underlying mechanisms of dl-3-n-butylphthalide (NBP) against H₂O₂-induced oxidative damage in retinal Müller cells. Methods: Cultured human Müller cell line (MIO-M1line) were exposed to H₂O₂ for 2 hours. Cell survival was evaluated by Calcein AM cell viability assay. Dichlorofluorescein diacetate (DCFDA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells. Mitochondrial membrane potential detection (JC-1) was used to observe cell membrane potential change and early apoptosis. Cell apoptosis was detected by Hoechst33258 staining. The expressions of Nrf2, HO-1 were documented by cell Immunofluorescence staining and Western blot analysis. Results: NBP effectively improved the survival ability of Müller cells shown by MTT assay. NBP effectively alleviated the morphological and apoptotic changes induced by H₂O₂ stimulation by Calcein AM assay, HE staining, Hoechst 33258, JC-1 staining. H₂O₂ induction increased the expression level of ROS, whereas, the treatment with NBP could remarkably lower the expression level of ROS. Cell immunofluorescence staining indicated that the fluorescence staining intensity of HO-1 in the NBP group was significantly higher than that in the control group. While the western blotting results showed that the expression level of HO-1 could be increased by NBP in a time-dependent manner. The translocation of Nrf2 in nuclei was observed within 2 h and Nrf2 was identified in nuclei for up to 48 h. Conclusions: Our study demonstrated that NBP had a protective effect on H₂O₂-induced cytotoxicity in retinal Müller cells in vitro and that it was a potent activator of Nrf2 and HO-1signaling.

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