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Exp Eye Res. 2016 May;146:318-29. doi: 10.1016/j.exer.2016.02.013. Epub 2016 Mar 16.

Heme oxygenase-1 (HO-1) protects human lens epithelial cells (SRA01/04) against hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis.

Author information

1
Department of Ophthalmology, The Chinese People's Liberation Army General Hospital, Beijing, China.
2
Department of Ophthalmology, The Tianjin Eye Hospital, Tianjin, China.
3
Department of Burn and Plastic Surgery, Peking University First Hospital, Beijing, China.
4
Department of Ophthalmology, The Chinese People's Liberation Army General Hospital, Beijing, China; Medical School, Nankai University, Tianjin, China.
5
Department of Ophthalmology, The Chinese People's Liberation Army General Hospital, Beijing, China. Electronic address: weishihui706@hotmail.com.
6
Department of Ophthalmology, The Chinese People's Liberation Army General Hospital, Beijing, China. Electronic address: zhaohuili650@hotmail.com.

Abstract

OBJECTIVES:

This study aimed to investigate the protective role of heme oxygenase-1 (HO-1) in H2O2-induced oxidative stress and apoptosis in human lens epithelial cells (hLEC; SRA01/04).

METHODS:

SRA01/04 cells were exposed to a hydrogen peroxide (H2O2) concentration gradient and inducers of HO-1 such as cobalt protoporphyrin (CoPP) and zinc protoporphyrin (ZnPP), respectively. In addition, an RNA silencing experiment was conducted to investigate the HO-1 function in this study. A Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Western blot and ELISA were used to detect the level of HO-1 expression. In our study, hLECs were exposed to 400 μM hydrogen peroxide (H2O2) for 24 h with or without pretreatment with 10μΜ CoPP or 10μΜ ZnPP, respectively. Double immunofluorescence staining was used for cell identification and the qualitative expression of HO-1. Expression of HO-1 was monitored using Western blot and ELISA. Intracellular reactive oxygen species (ROS) were detected by flow cytometry analyses; commercial enzymatic kits were used to measure the levels of glutathione (GSH), as well as superoxide dismutase (SOD). The proportion of cell apoptosis was quantified by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The expression of caspase family (-8, -3) proteins was measured by Western blot analysis.

RESULTS:

HO-1 significantly restored the cell viability under H2O2 injury via reducing the generation of ROS and increasing the levels of SOD and GSH activity. Moreover, HO-1 also inhibited H2O2-induced caspase-8 and caspase-3 proteins, thus significantly reducing the apoptosis of SRA01/04. An RNA silencing experiment demonstrated the increased resistance of LECs to oxidative stress specifically for increased levels of HO-1.

CONCLUSIONS:

These findings suggested that HO-1 protects human lens epithelial cells from H2O2-induced oxidant stress by upregulating antioxidant enzyme activity, reducing ROS generation, and thus inhibiting caspase family-dependent apoptosis.

KEYWORDS:

Antioxidation; Apoptosis; Cataract; Heme Oxygenase-1 (HO-1); Human lens epithelial cells (SRA01/04); Oxidative stress

PMID:
26992777
DOI:
10.1016/j.exer.2016.02.013
[Indexed for MEDLINE]

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