Curcumin protects trabecular meshwork cells against hydrogen peroxide-induced oxidative stress and apoptosis via Nrf2-keap1 pathway

Purpose: To explore the effects and molecular mechanism of action of curcumin on trabecular meshwork cells (TMCs). Methods: TMCs were isolated from pig eyes and NF-E2-related factor 2 (Nrf2) was knocked down by siRNA transfection. The effect of curcumin on intracellular reactive oxygen species (ROS) was measured by a ROS-specific dye. Annexin V-FITC/propidium iodide (PI) double labeling was applied to determine apoptosis. The expressions of apoptosis-associated proteins and Nrf2-Kelch-like ECHassociated protein 1 (Keap 1) pathway activation were assessed by western blotting, while target gene transcription was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Results: Curcumin reduced the levels of intracellular ROS and apoptosis induced by H2O2, upregulated B-cell lymphoma-2 (Bcl-2), downregulated Bcl2-associated X (Bax), and activated caspase-3 and nine other proteins (p < 0.05). Curcumin reduced the expressions of Nrf2, HO-1, and NQO1, and increased Keap1 in H2O2-induced TMCs (p < 0.05). Moreover, Nrf2 knockdown partly reversed the effect of curcumin on ROS and apoptosis in TMCs induced by H2O2 (p < 0.05). Conclusion: Curcumin inhibited oxidative stress and apoptosis by Nrf2-Keap1 activation in TMCs. Curcumin is therefore a potential therapeutic agent for the management of glaucoma.


INTRODUCTION
Glaucoma, the second leading cause of blindness, affects more than 70 million people and is characterized by progressive degeneration of retinal ganglion cells (RGCs) [1]. Many risk factors for this disorder have been identified, including elevated intraocular pressure (IOP), oxidative stress, apoptosis, alterations of cytokines, and autophagy [2][3][4]. Elevated IOP, a crucial factor related to RGC death which results from oxidative stress or apoptosis, is associated with biochemical changes in the trabecular meshwork (TM) [5]. The clinical application of traditional Chinese medicines have resulted in improvements in treating glaucoma because it reduces IOP and modulates optic nerve nutrition and systemic conditions [6].
NF-E2-related factor 2 (Nrf2), a major protein of oxidative stress regulating, is sequestered by cytoplasmic Keap1 [7]. Numerous studies demonstrated that the Nrf2-Keap pathway attenuates oxidative and apoptotic responses in vivo and in vitro [8]. However, the specific effects of Nrf2 and its regulating manner in the treatment of glaucoma remain unclear. Curcumin, the major extract of turmeric, is derived from Curcuma longa and has been widely used in many countries for centuries [9]. Many studies have suggested that curcumin could be a therapeutic agent for ocular diseases [10].
However, the molecular mechanism of curcumin in treating glaucoma is unclear. In this study, we characterized the protective roles of curcumin on trabecular meshwork cells (TMCs) exposed to H 2 O 2 , and investigated the role of Nrf2-Keap1 in H 2 O 2 -induced oxidative stress and apoptosis.

Isolation and culture of TMCs
Primary porcine TMCs were obtained from fresh pig eyes, and cultured as previously described [11]. Briefly, the eyes were bisected along the equator. After lens, iris, vitreous, and ciliary body were removed from the anterior part, the trabecular tissue was scraped from the sclera. After digested by collagenase type IV and centrifuged, TMCs were cultured in low glucose Dulbecco's Modified Eagle's Medium (Gibco, Gathersburg, MD, USA) with 10 % fetal bovine serum, penicillin/streptomycin, sodium pyruvate, nonessential amino acids, L-glutamine, and amphotericin B. All reagents were purchased from Invitrogen (Carlsbad, CA, USA).
TMCs or si-Nfr2 TMCs were placed into a dark 96-well plate and treated with different concentrations of curcumin at 20, 50 and 100 μM at 37 °C for 1 h, and exposed to H 2 O 2 for another 24 h and stained by DCF-DA about 30 min in dark place. The DCF-DA fluorescence was measured with a flow cytometer (FACS Calibur) and the fluorescence was measured with a microplate reader (Thermo Fisher Scientific) with wavelength 488 nm (excitation) and 520 nm (emission).

Apoptosis assay
Quantitative assay of the apoptosis was evaluated by annexin V/FITC detection kit (BD Biosciences). TMCs or si-Nfr2 TMCs were seeded onto a black 96-well plate and treated with curcumin at 20, 50, and 100 μM at 37 °C for 1 h, and exposed to H 2 O 2 for another 24 h and harvested for quantitation of apoptosis. The cells were blocked in binding buffer, stained with annexin V /PI for 15 min. The cells were then immediately analysed by fluorescence-activated cell sorting.

Protein extraction and western blotting
The treated cells were lysed in cold RIPA buffer with a protease and phosphatase inhibitor (Sigma-Aldrich) and centrifuged. Protein concentrations were measured by Pierce BCA Protein Assay Kit (Thermo Scientific) and diluted to equal concentrations with PBS and 5× SDS-PAGE sample loading buffer, then heated to 95 °C. Equal amounts of protein were loaded and separated on Invitrogen NuPAGE ™ Bis-Tris gels (10 %), and transferred onto PVDF membranes (Millipore). The membranes were incubated with primary antibodies (1:1,000 dilution) and secondary antibody (1:10,000 dilution). Protein expressions were detected by an ECL system and quantified using Imaging Systems analysis software (VersaDocMp5000).

RNA extraction and RT-PCR
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies,). Equal amounts of RNA were synthesized to cDNA using GoScript ™ (Promega).The target gene expression was detected by a SYBR Green PCR kit (Promega). The primers are shown in Table 1.

Statistical analysis
Data were analysed using one-way analysis of variance (ANOVA) with Tukey's tests using Prism 6 software. Data are presented as mean ± standard deviation (SD) and p < 0.05 was considered statistically significant.

Curcumin reduced H 2 O 2 -induced oxidative stress in TMCs
The intracellular ROS were measured by DCF-DA. As shown in Figure 1A, the level of intracellular ROS of H2O2 treated cells was about two times higher than control group (p < 0.01), which was inhibited by curcumin in concentration-dependent manner.

Curcumin reduced H 2 O 2 -induced cell apoptosis in TMCs
Hence, the anti-apoptosis effect of curcumin was confirmed by annexinV/PI staining. As shown in Figure 1 B, the apoptotic cells (Q2 (late apoptosis), An + /PI + ;Q3 (early apoptosis), An + /PI -) were increased in the H 2 O 2 -treated group (from 3.15 ± 8.23 % in the control to 43.7 ± 14.4 % in H 2 O 2 group). When pre-treated with curcumin, apoptotic cells were remarkably decreased (p < 0.01) in a concentration-dependent manner. To further explore the mechanism of curcumin on apoptosis, the apoptosis-related proteins were measured by western blotting. As shown in Figure 1 C, curcumin increased the expression of Bcl-2 (anti-apoptosis protein), and decreased the  expressions of caspase-3, caspase-9, and Bax (pro-apoptosis protein), in concentrationdependent manner. The results demonstrate that curcumin reducedH 2 O 2 -induced apoptosis in TMCs.

Curcumin reduces Nrf2, NQO1, and HO-1 expression, and increases Keap1 in H 2 O 2induced TMCs
The gene and protein expressions of Nrf2, Keap1, NQO1, and HO-1 in H 2 O 2 -induced TMCs were investigated. As shown in Figure 2 A, gene expressions of Nrf2, HO-1, and NQO1 were increased and Keap1 was decreased, indicating activation of the Nrf2-keap pathway. Curcumin significantly inhibited H 2 O 2 -induced genetic transcription dysregulation in a concentrationdependent manner. In parallel, protein expression was consistent with the genetic transcription results (Figure 2 B).

Nrf2 knockdown reverses the effect of curcumin on the Nrf2-Keap1 signaling pathway
To further characterize the role of Nrf2 in the regulation of curcumin on the expression of Nrf2-Keap1 pathway target proteins, Nrf2 siRNA was used to to knockdown the expression of Nrf2 in TMCs. The transfection efficiency of the knockdown was confirmed by western blotting and RT-PCR analysis. The Nrf2 mRNA and protein expression in the si-Nrf2 group were decreased when compared with the sc-Nrf2 group (Figure 3), and the expression of target genes and proteins changed accordingly. The expressions of Nrf2, HO-1, and NQO1 were much lower and Keap1 much higher of si-Nrf cells after treated by H 2 O 2 , which were reversed by curcumin in concentration-dependent manner. The protein expressions (Figure 3 B) were consistent with the genetic transcription results (Figure 3 A).

Nrf2 knockdown reverses the effect of curcumin on oxidative stress
The si-Nrf TMCs were also used to determine the effect of Nrf2 on curcumin against H 2 O 2 -induced oxidative stress. The levels of intracellular ROS in the si-Nrf2 group increased when compared with the sc-Nrf2 group (Figure 4 A), which was greatly enhanced by H 2 O 2 in Si-Nrf cells. The intracellular ROS of H 2 O 2 -treated si-Nrf cells were decreased by curcumin in a concentrationdependent manner.

Nrf2 knockdown reverses the effect of curcumin on apoptosis
Si-Nrf TMCs were also used to determine the effect of Nrf2 on curcumin against H 2 O 2 -induced apoptosis. The amount of apoptotic cells of the si-Nrf group was higher than that of the sc-Nrf group, and greatly enhanced by H 2 O 2 in si-Nrf cells. Curcumin inhibited H 2 O 2 -induced apoptosis of si-Nrf cells in a concentration-dependent manner. To further confirm the results of apoptosis, the apoptosis-related proteins, Flow cytometry analysis of reactive oxygen species (ROS)in si-Nrf2 TMCs exposed to 1 mM H2O2. Intracellular ROS were determined by 2, 7-dichlorofluorescein diacetate. (B) Flow cytometry analysis of si-Nrf2 TMC apoptosis by double staining with annexinV/propidium iodide. (C) Western blotting analysis of Bcl-2, Bax, caspase 3, and caspase-9; * p < 0.05 and ** p < 0.01 vs. control; # p < 0.05 and ## p < 0.05 vs. H2O2 alone group.
caspase-3, caspase-9, Bcl-2, and Bax were measured both in sc-Nrf and si-Nrf cells. The expressions of caspase-3, caspase-9, and Bax were increased and Bcl-2 decreased in the si-Nrf group compared with the sc-Nrf group, and the effects were enhanced by H 2 O 2 in si-Nrf cells.
Curcumin decreased the expression of caspase-3, caspase-9, and Bax and increased Bcl-2 in a concentration-dependent manner.

DISCUSSION
Oxidative stress is one of the risk factors for glaucoma, and many studies have reported that local oxidative radicals are a determining factor in the pathogenesis of glaucoma [12]. Long-term oxidative damage may lead to the progressive loss of TMCs and glaucoma. Himori et al reported a close relationship between clinical ocular parameters and systemic oxidative stress parameters in 73 patients with normal-tension glaucoma [13]. Their study indicated that skin autofluorescence and 8-hydroxy-2'deoxyguanosine, two markers of oxidative stress, may contribute to the pathogenesis of glaucomatous optic neuropathy.
Curcumin, a natural compound that has many biological properties including anti-inflammation, antiviral, antifungal, and anticancer activities, has been suggested to be an effective treatment for several ocular diseases, such as glaucoma [12]. Some studies have reported that curcumin exhibited antioxidant properties in neurodegenerative diseases, and was associated with activation of the Nrf2 cytoprotective pathway [14]. This study indicate that curcumin decreased the level of ROS in H 2 O 2 -induced TMCs and was associated with the activation of the Nrf2 signaling pathway, which were consistent with the results of previous studies.
Apoptosis is a physiological cell death mechanism that plays a crucial role in glaucoma. It is thought that the ultimate cause of irreversible blindness in this disease is RGC apoptosis [15]. The application of a new imaging technique called "detection of apoptosis retinal cells" (DARC) has shown that the level of RGC apoptosis is predictive of disease severity in patients with glaucomatous neurodegeneration [16]. Moreover, treatment with agents that inhibit RGC apoptosis attenuate glaucoma [17,18]. Apoptosis is initially characterized by a series of morphological changes, including cellular shrinkage, membrane blebbing, and nuclear chromatin condensation. Apoptotic programs are related to two pathways and involve the activation of a series of caspases [19]. The upstream caspase for the intrinsic pathway (also known as the membrane receptor-mediated pathway) is caspase-9 and the downstream effector is caspase-3 [20].
The intrinsic pathways are closely-regulated by the Bcl-2 family, which includes anti-apoptotic members (Bcl-2 and Bcl-Xl) and pro-apoptotic members (Bax and Bak) [21]. This results indicate that curcumin treatment prevent H 2 O 2 -induced TMCs apoptosis, with downregulation of cleaved caspase-3, caspase-9, and Bax, and upregulation of Bcl-2 protein. More importantly, analyses of apoptosis showed that high concentrations of curcumin (50 and 100 μM) not only decreased the apoptosis of TMCs, but also of necrosis cells, indicating that curcumin did not cause secondary necrosis and immune responses.
Nrf2, which is sequestered by Keap1, is an important transcription factor under oxidative stress. Nrf2-Keap1 has been reported to play an important role in the regulation of oxidative stress and the apoptosis of RGCs [22]. Lycium barbarum polysaccharides protect the retina after ischemia-reperfusion, partially by activation of Nrf2 [23]. Berberine could ameliorate methotrexate-induced oxidative stress and apoptosis by activating the Nrf2/HO-1 pathway and PPARγ in vivo [24]. Curcumin has similar effects on oxidative damage. Dai et al reported that curcumin inhibited quinocetone-induced oxidative stress, apoptosis, and inflammation via Nrf2/HO-1 and the NF-kB pathway in human hepatocytes [25]. However, the Nrf2-mediated molecular mechanism of curcumin remains unclear.
In the present study, the results showed that curcumin activated the Nrf2-Keap1 signaling pathway and regulated target gene expression in H 2 O 2 -induced TMCs, resulting in alleviation of both oxidative stress and apoptosis in TMCs. Moreover, Nrf2 knockdown partly reversed the inhibitory effect of curcumin on ROS and apoptosis in H 2 O 2 -induced TMCs. These results strongly suggest that curcumin inhibit oxidative stress and apoptosis by Nrf2-Keap1 signaling pathway. Overall, the results indicate that curcumin has a protective effect against H 2 O 2induced oxidative stress and apoptosis of TMCs via the Nrf2-Keap1 signaling pathway.

CONCLUSION
The results of the present study provide the experimental basis for the possible clinical use of curcumin in the management of certainly inflammatory diseases, as well as the possibility of its development as a therapeutic agent for the treatment of glaucoma.

Acknowledgement
This work was supported by funding entitled "Studies on the molecular mechanism of the treatment of San Xiao Nei Zhang Fang for diabetic retinopathy" (grant no. JDZX2014Y10), and funding entitled "Studies on the mechanism of prevention and treatment of San Xiao Nei Zhang Fang for the diabetic retinopathy based on the IRE1 pathway" (grant no. JDZX2014Z09).