Effect of dl-3-n-Butylphthalide on apoptosis of retinal müller cells induced by hydrogen peroxide_Chinese Journal of Ocular Fundus Diseases

Authors：

Xing Xiaoli , Huang Liangyu , Su Ruihong , Liu Xun , Zhao Jinzhi , Gao Meizi , Zhang Xiaomin , Li Xiaorong ,  Dong Lijie

Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, College of Optometry and Ophthalmology, Tianjin 300384, China;

Corresponding author：

Dong Lijie, Email: aitaomubang@126.com

Keywords：

Butylphthalide; Oxidative stress; Apoptosis; Hydrogen peroxide; Müller cells

DOI：

10.3760/cma.j.issn.1005-1015.2018.05.014

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ObjectiveTo observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H₂O₂).MethodsHuman retinal Müller cells cultured in vitro were divided into normal control group, model group (H₂O₂ group) and experimental group (H₂O₂+NBP group). The cells in the H₂O₂ group and H₂O₂+NBP group were cultured with 200 μmol/L H₂O₂ for 2 h. Then the culture solution of the H₂O₂ group replace with complete medium and the H₂O₂+NBP group replace with complete medium containing 1 μmol/L NBP. The normal control group was a conventional cultured cells. Müller cells were identified by immunofluorescence staining. Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes. MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention. Hoechst33258 staining was used to observe the apoptosis. LIVE/DEAD ^® cell activity/cytotoxicity kit was used to detect cell viability. Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells. One-way ANOVA combined with Dunnett statistical method were used for data analysis.ResultsHE staining showed that the number of cells in H₂O₂+NBP group was higher than that in H₂O₂ group. MTT assay showed that after 24 h and 48 h of NBP intervention, the differences in cell viability between the normal control group and the H₂O₂ group, the H₂O₂ group and the H₂O₂+NBP group were statistically significant (t=28.96, 3.658, 47.58, 20.33; P＜0.001, 0.022). The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H₂O₂+NBP group was crescent-shaped and the nuclear fragmentation was reduced, and the blue fluorescence of the remaining cells was uniform. The LIVE/DEAD ^® cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H₂O₂ group increased significantly, and the number of viable cells with green fluorescence decreased significantly. In the H₂O₂+NBP group, the number of viable cells with green fluorescence increased, and the number of dead cells with red fluorescence decreased. The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H₂O₂ group was significantly enhanced; the green fluorescence intensity of H₂O₂+NBP group was lower than that of H₂O₂ group.ConclusionNBP alleviates H₂O₂-induced apoptosis of human retinal Müller cells by inhibiting ROS production.

Citation： Xing Xiaoli, Huang Liangyu, Su Ruihong, Liu Xun, Zhao Jinzhi, Gao Meizi, Zhang Xiaomin, Li Xiaorong, Dong Lijie. Effect of dl-3-n-Butylphthalide on apoptosis of retinal müller cells induced by hydrogen peroxide. Chinese Journal of Ocular Fundus Diseases, 2018, 34(5): 481-486. doi: 10.3760/cma.j.issn.1005-1015.2018.05.014 Copy

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1. 高凤, 季敏, 吴继红, 等.视网膜Müller细胞的生理功能及在青光眼病理中的作用[J]. 生理学报, 2013, 65(6): 654-663.Gao F, Ji M, Wu JH, et al. Roles of retinal Müller cells in health and glaucoma[J]. Acta Physiologica Sinica, 2013, 65(6): 654-663.
2. Gentsch J, Keindorf K, Schwarze D, et al. Neuronal glutathione supply by Müller cells during oxidative stress[J]. Neuron Glia Biology, 2007, 2: 74.
3. Feng L, Sharma A, Niu F, et al. TiO₂-nanowired delivery of DL-3-n-butylphthalide (DL-NBP) attenuates blood-brain barrier disruption, brain edema formation, and neuronal damages following concussive head injury[J]. Mol Neurobiol, 2018, 55(1): 350-358. DOI: 10.1007/s 12035-017-0746-5.
4. Peng Y, Xu S, Chen G, et al. l-3-n-Butylphthalide improves cognitive impairment induced by chronic cerebral hypoperfusion in rats[J]. J Pharmacol Exp Ther, 2007, 321(3): 902-910. DOI: 10.1124/jpet.106.118760.
5. Zheng B, Zhou Y, Zhang H, et al. Dl-3-n-butylphthalide prevents the disruption of blood-spinal cord barrier via inhibiting endoplasmic reticulum stress following spinal cord injury[J]. Int J Biol Sci, 2017, 13(12): 1520-1531.DOI: 10.7150/ijbs.21107.
6. Xiong N, Huang J, Chen C, et al. Dl-3-n-butylphthalide, a natural antioxidant, protects dopamine neurons in rotenone models for Parkinson’s disease[J]. Neurobiol Aging, 2012, 33(8): 1777-1791.DOI: 10.1016/j. neurobiolaging.2011.03.007.
7. Wang YG, Li Y, Wang CY, et al. L-3-n-Butylphthalide protects rats’ cardiomyocytes from ischaemia/reperfusion-induced apoptosis by affecting the mitochondrial apoptosis pathway[J]. Acta Physiol (Oxf), 2014, 210(3): 524-533. DOI: 10.1111/apha.12186.
8. Yu J, Zhong Y, Shen X, et al. In vitro effect of adenosine A2A receptor antagonist SCH 442416 on the expression of glutamine synthetase and glutamate aspartate transporter in rat retinal Müller cells at elevated hydrostatic pressure[J]. Oncology Reports, 2012, 27(3): 748-752. DOI: 10.3892/or.2011.1565.
9. Yang XD, Cen ZD, Cheng HP, et al. L-3-n-butylphthalide protects HSPB8 K141N mutation-induced oxidative stress by modulating the mitochondrial apoptotic and nrf2 pathways[J]. Front Neurosci, 2017, 11: 402. DOI: 10.3389/fnins.2017.00402.
10. Zha BS, Zhou H. ER stress and lipid metabolism in adipocytes[J/OL]. Biochem Res Int, 2012, 2012: 312943[2012-02-12].http://dx.doi.org/10.1155/2012/312943. DOI: 10.1155/2012/312943.
11. Li J, Wang JJ, Yu Q, et al. Inhibition of reactive oxygen species by Lovastatin downregulates vascular endothelial growth factor expression and ameliorates blood-retinal barrier breakdown in db/db mice: role of NADPH oxidase 4[J]. Diabetes, 2010, 59(6): 1528-1538.DOI: 10.2337/db09-1057.
12. Jing G, Wang JJ, Zhang SX. ER stress and apoptosis: a new mechanism for retinal cell death[J/OL]. Exp Diabetes Res, 2012, 2012: 589589[2011-12-14]. http://dx.doi.org/10.1155/2012/589589. DOI: 10.1155/2012/589589.
13. Zhang YP, Zhang Y, Xiao ZB, et al. CFTR prevents neuronal apoptosis following cerebral ischemia reperfusion via regulating mitochondrial oxidative stress[J]. J Mol Med(Berl), 2018, 98(7): 611-620.DOI: 10.1007/s00109-018-1649-2.
14. Wang F, Ma J, Han F, et al. DL-3-n-butylphthalide delays the onset and progression of diabetic cataract by inhibiting oxidative stress in rat diabetic model[J/OL]. Sci Rep, 2016, 6: 19396[2016-01-13].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725374/pdf/srep19396.pdf.DOI: 10.1038/srep19396.

Chinese Journal of Ocular Fundus Diseases

Effect of dl-3-n-Butylphthalide on apoptosis of retinal müller cells induced by hydrogen peroxide

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