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Inhibition of endoplasmic reticulum stress by 4-phenylbutyrate alleviates retinal inflammation and the apoptosis of retinal ganglion cells after ocular alkali burn in mice

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Abstract

Objective

Retinal ganglion cell (RGC) apoptosis is one of the most severe complications that causes permanent visual impairment following ocular alkali burn (OAB). Currently, very few treatment options exist for this condition. This study was conducted to determine the effect of 4-phenylbutyric acid (4-PBA) on endoplasmic reticulum (ER) stress after OAB using a well-established OAB mouse model.

Methods

Ocular alkali burn was induced in C57BL/6 mouse corneas using 1 M NaOH. 4-PBA (10 mg/kg; 250 μL per injection) or saline (250 μL per injection) was injected intraperitoneally once per day for 3 days before the establishment of the OAB model. The apoptosis of retinal ganglion cells (RGCs) was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and the histological damage was examined by hematoxylin and eosin and immunofluorescence assay on retinal flat mounts. The key inflammatory response and the expression of ER stress-related markers in the retinal tissues were assessed by real-time PCR, western blotting and histologic analyses.

Results

4-PBA significantly alleviated the apoptosis of RGCs and prevented the structural damage of the retina, as determined by the evaluation of RGC density and retinal thickness. Inhibition of ER stress by 4-PBA decreased the expression of vital proinflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 beta; and suppressed the activation of retinal microglial cells and nuclear factor-kappa B (NF-κB). 4-PBA reduced the expression of the ER stress molecules, glucose-regulated protein 78, activated transcription factor 6, inositol-requiring enzyme-1 (IRE1), X-box-binding protein 1 splicing, and CCAAT/enhancer-binding protein homologous protein, in the retinal tissues and RGCs of OAB mice.

Conclusions

The present study demonstrated that the inhibition of ER stress by 4-PBA alleviates the inflammatory response via the IRE1/NF-κB signaling pathway and protects the retina and RGCs from injury in an OAB mouse model. Such findings further suggest that 4-PBA might have potential therapeutic implications for OAB treatment.

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Abbreviations

RGCs:

Retinal ganglion cells

OAB:

Ocular alkali burn

4-PBA:

4-Phenylbutyric acid

ER stress:

Endoplasmic reticulum stress

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Acknowledgements

This work is supported by National Natural Science Foundation of China (81974135, 81900851) and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology (30306020240020130, 3030902113030).

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Author notes

  1. Yanqiao Huang and Miner Yuan contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, 510060, China

    Yanqiao Huang, Miner Yuan, Fang Duan, Yao Yang, Bingsheng Lou & Xiaofeng Lin

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  1. Yanqiao Huang
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  2. Miner Yuan
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  3. Fang Duan
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Correspondence to Xiaofeng Lin.

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Ethical approval

All experiments followed the US National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals developed by the US National Academy of Sciences and were approved by the Administration Committee of Experimental Animals, Guangdong Province, China.

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Supplementary file1 (DOCX 2641 KB)

11_2022_1565_MOESM2_ESM.jpg

Supplementary file2 Effect of three different concentrations of 4-PBA on RGC apoptosis in the retinal tissues of OAB mice. A Representative retinal cryosection images stained with TUNEL (red fluorescence) and DAPI (blue) in the saline treatment group, 10 mg/kg/d 4-PBA treatment group, 40 mg/kg/d 4-PBA treatment group, and 80 mg/kg/d 4-PBA treatment group on day one after OAB. B Quantitative analysis of the TUNEL+ cells in the different groups in the central retina, mid-peripheral retina, and peripheral retina. Data are expressed as mean ± SEM (*P<0.05, **P<0.01, ***P<0.001). Scale bar = 20 μm. OAB, ocular alkali burn; 4-PBA, 4-phenylbutyric acid; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer (JPG 11530 KB)

11_2022_1565_MOESM3_ESM.jpg

Supplementary file3 The mRNA transcription and protein levels of the ER stress markers and inflammatory factors within 24 hours after OAB. The expression levels of the ER stress-related molecules, GRP78 (A) and CHOP (B), and inflammatory cytokines, TNF-α (C) and IL-1β (D), in the OAB retinal tissues at 6, 12, and 24 hours after OAB were analyzed by RT-PCR. Representative gel images and quantification graphs for GRP78 (E) and CHOP (F). The optical density of each band was normalized by the relative β-tubulin level. Data are expressed as mean ± SEM (*P < 0.05, **P < 0.01). Ctrl, control; OAB, ocular alkali burn (JPG 3199 KB)

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Huang, Y., Yuan, M., Duan, F. et al. Inhibition of endoplasmic reticulum stress by 4-phenylbutyrate alleviates retinal inflammation and the apoptosis of retinal ganglion cells after ocular alkali burn in mice. Inflamm. Res. 71, 577–590 (2022). https://doi.org/10.1007/s00011-022-01565-3

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