Curcumin Protects Trabecular Meshwork Cells From Oxidative Stress

Abstract

Purpose: Glaucoma is closely linked with oxidative stress and inflammation, and difficult to treat. Its occurrence frequently is contributed by the failure of the trabecular meshwork (TM). Curcumin is known as an antioxidative and anti-inflammatory substance, possessing the potential to treat glaucoma.

Methods: Using TM cells as the in vitro model system, we investigated the effects of curcumin on oxidative stress-induced markers for TM impairments, including cell death, production of intracellular reactive oxygen species (iROS), induction of proinflammatory proteins, activation of senescence marker, accumulation of carbonylated proteins, and apoptotic cell numbers.

Results: Curcumin treatment protected TM cells against oxidative stress-induced cell death. Curcumin treatment at concentrations between 1 and 20 μM reduced the production of iROS in H₂O₂-exposed TM cells in a dose-dependent manner. Further studies demonstrated that curcumin treatment (20 μM) significantly inhibited proinflammatory factors, including IL-6, ELAM-1, IL-1α, and IL-8, whereas it decreased activities of senescence marker SA-β-gal, and lowered levels of carbonylated proteins and apoptotic cell numbers.

Conclusions: Curcumin is capable of protecting TM cells against oxidative stress, shedding new light on potential treatment for glaucoma.

Glaucoma is an aging-related disease that causes irreversible blindness in millions of individuals worldwide.¹ One of the major risk factors for glaucoma is the elevated IOP. High IOP observed in glaucoma frequently is contributed by failure of the trabecular meshwork (TM).² The aqueous humor normally flows through the TM, from the anterior chamber to Schlemm's canal. Trabecular meshwork failure can lead to the occurrence of cell decay, reduction of outflow, and eventually increased IOP.² Thus, current treatments are focused mainly on lowering IOP.³ Better understanding of the mechanisms underlying TM failure could potentially promote clinical applications toward efficient treatments. 

Glaucoma has been linked with oxidative stress and inflammation.^4–6 Oxidative stress, usually caused by aging, has a deleterious impact on the TM. Treatment with hydrogen peroxide (H₂O₂) has been applied to simulate the effects of oxidative stress on primary TM cells.^7,8 Results reveal that oxidative stress causes cell death, produces intracellular reactive oxygen species (iROS), induces proinflammatory factors, activates senescence markers, accumulates carbonylated proteins, promotes proteasome activity, and increases the apoptotic cell numbers, which are hallmarkers of glaucoma. Thus, the H₂O₂-exposed TM cells are used as an in vitro model for glaucoma-related oxidative stress. 

Curcumin is the main ingredient of the spice turmeric (Curcuma longa), and has been administered as traditional medicine in Asia, mainly in China, India, and Iran.⁹ Curcumin has been known for decades as an antioxidative and anti-inflammatory substance.⁹ It has the ability to bind iron (Fe), manganese (Mn), and copper (Cu) to modulate the antioxidant capabilities.^9,10 Curcumin also is able to inhibit the production of nitric oxide and reactive oxygen species (ROS).^11,12 For thousands of years in Asia, curcumin has been applied in traditional medicine to use its anti-inflammatory ability. Modern science has discovered that curcumin modulates various important molecular targets, including transcription factors, proinflammatory enzymes, cell cycle regulators, cytokines and growth factors, receptors, adhesion molecules, antiapoptotic proteins, cell survival proteins, and drug resistance proteins.^13,14 In this context, curcumin may serve as a promising drug with the potential to treat glaucoma. Therefore, we investigated the protective roles of curcumin against oxidative stress in the H₂O₂-exposed TM cells. 

Methods and Materials

Cell Culture

Fresh porcine eyes were used to obtain primary porcine TM cells as previously described.⁷ Primary TM cells then were maintained at 37°C with 5% carbon dioxide (CO₂) in low glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Halethorpe, MD, USA) supplemented with sodium pyruvate, glutamine, penicillin/streptomycin, nonessential amino acids, amphotericin B, and 10% fetal bovine serum (FBS). This study was carried out in strict accordance with the ARVO Animal Statement for the Use of Animals in Ophthalmic and Vision Research. 

Oxidative Stress and Curcumin Treatment

Briefly, confluent TM cells were exposed to H₂O₂ in 10% FBS supplemented with DMEM to simulate the effects of chronic oxidative stress.⁷ Trabecular meshwork cells were treated with different concentrations of H₂O₂ (0.1, 0.2, 0.5, 1, and 2 mM), twice a day, for consecutive 4 days. To avoid acute stress responses, a 3-day recovery was allowed after the H₂O₂ exposure. Curcumin (Sigma-Aldrich Corp., St. Louis, MO, USA) was prepared at different concentrations (1, 5, 10, 20, 50, and 100 μM) in the DMEM media during the cell culture. 

3-(4,5 Dimethylthiazole-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay

Cell viability was measured by MTT assay at day 7 after seeding. A final concentration of 0.5 mg/ml MTT was added in the DMEM media for a total of 4 hours at 37°C. After incubation, the media were replaced with DMSO to dissolve the formed formazan crystals. The optical densities (ODs) of blue MTT-formazan was measured at 490 nm with a Microplate reader (PerkinElmer, Singapore) and normalized to the control. 

iROS Measurement

Intracellular ROS was determined by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Calbiochem, San Diego, CA, USA). In brief, the culture medium containing curcumin was discarded and cells were incubated with 10 μM of H₂DCFDA for 30 minutes in PBS. This procedure was followed by one round PBS wash, and another round incubation in media for 20 minutes. In the end, the cells were trypsinized and collected in PBS and were placed on ice until measurements. The cells then were analyzed by a fluorescence plate reader. 

Quantitative Real-Time PCR (RT-PCR)

RNA isolation was performed with the RNeasy mini kit (Qiagen, Inc., Valencia, CA, USA) for the subsequent synthesis of cDNA using the QuantiTect reverse transcription kit (Qiagen, Inc.). Real-time PCR was performed using SYBR Green Master Mix (Qiagen, Inc.). Samples were normalized to internal control β-actin. Primer sequences are listed as follows: IL-6 (forward primers 5′-GCT TCC AAT CTG GGT TCA AT-3′; reverse primers 5′-CTA ATC TGC ACA GCC TCG AC-3′), ELAM-1 (forward primers 5′-CCC ATG GAA CAC AAC CTG TGC ATT-3′; reverse primers 5′-AGC TTT ACA CGT TGG CTT CTT GCC-3′), IL-1α (forward primers 5′-AAG TGT TGA CAG GCC GTA TG-3′; reverse primers 5′-TAC CAG ACT TCG CTC CCT CT-3′), IL-8 (forward primers 5′-AAA CTG GCT GTT GCC TTC TT-3′; reverse primers 5′-ATT TAT GCA CTG GCA TCG AA-3′), and β-actin (forward primers 5′-AAG ATC AAG ATC ATC GCG CCT CCA-3′; reverse primers 5′-TGG AAT GCA ACT AAC AGT CCG CCT-3′).⁸ 

Measurement of β-Galactosidase (SA-β-gal) Activity

Culture medium containing curcumin was discarded. Activity of SA-β-gal was determined by flow cytometry using the fluorogenic substrate C12FDG (Molecular Probes, Eugene, OR, USA) as described previously.⁸ For each experiment, the average number of cells was 10,000 for each analyis. 

Proteasome Activity Assay

The activities of the proteasome subunits chymotrypsin-like (CT-L), trypsin-like (T-L) and caspase-like (PGPH) were determined by the fluorogenic peptides (Sigma-Aldrich Corp.) as described previously.⁸ 

Protein Extraction and Carbonylation Assay

Briefly, cells were collected, washed in PBS, and lysated in ×1 radioimmuniprecipitation assay (RIPA) buffer for protein extraction. The measurement of total protein carbonylation was performed using the OxyBlo Protein Oxidation Detection Kit (Chemicon International, Inc., Temecula, CA, USA). The manufacturer's instructions were followed to perform the comparative analysis. 

Apoptosis Examination

Cell apoptosis was analyzed by an Annexin V-FITC apoptosis detection kit (eBioscience, San Diego, CA, USA) following the manufacturer's instructions. In brief, TM cells were trypsinized after TSA or vehicle treatment, and resuspended in binding buffer. After washing, cells were incubated with Annexin V-FITC and propidium iodide followed by flow cytometry analysis. 

Statistical Analysis

Data were presented as mean ± SD, and obtained from at least three independent experiments. Statistical analyses were performed by the Student's t-test, which considered P values less than 0.05 as statistically significant. 

Results

Curcumin Treatment Protects TM Cells Against Oxidative Stress-Induced Cell Death

To simulate the effects of oxidative stress, TM cells were exposed to increasing concentrations of H₂O₂ (0.1, 0.2, 0.5, 1, and 2 mM). It was found that H₂O₂ (0.2, 0.5, 1, and 2 mM) significantly reduced cell viability in a dose-dependent manner (Fig. 1a). Meanwhile, we also evaluated potential cytotoxic effects of curcumin on TM cells by treating the cells with increasing concentrations of curcumin (1, 5, 10, 20, 50, and 100 μM). Results suggested that the concentration of curcumin less than 50 μM did not show any cytotoxicity (Fig. 1b). Furthermore, we examined the effects of different concentrations of curcumin against 0.2 or 1 mM H₂O₂-induced cell death. Treatments with curcumin at 5, 10, and 20 μM successfully inhibited cell death caused by 0.2 mM H₂O₂ exposure, which neither low (1 μM) nor high concentrations (50 and 100 μM) of curcumin could achieve (Fig. 1c). Similar results were observed in the cultures under 1 mM H₂O₂ treatment, where curcumin also exerted certain protective effects against H₂O₂-induced cell death (Fig. 1d). 

Figure 1

 

Curcumin treatment protects TM cells against oxidative stress-induced cell death. (a) Effects of H₂O₂ (0.1, 0.2, 0.5, 1, and 2 mM) exposure (a), curcumin (1, 5, 10, 20, 50, and 100 μM) without H₂O₂ exposure (b), with 0.2 mM H₂O₂ exposure (c), and with 1 mM H₂O₂ exposure (d) on TM cell viability. Cell viability was measured by MTT assay at day 7 after seeding. Data were presented as mean ± SD. *P < 0.05 and **P < 0.01 compared to control.

Effects of Curcumin Treatment on the Production of iROS in the H₂O₂-Exposed TM Cells

Elevated levels of iROS indicated oxidative stress. Exposure to H₂O₂ induced significantly higher iROS production in TM cells, while treatments with curcumin at 1, 5, 10, and 20 μM successfully decreased the amount of iROS in a dose-dependent manner, indicating curcumin was able to protect TM cells against oxidative stress by lowering iROS levels (Fig. 2). We chose 20 μM curcumin in the following experiments due to its optimal protective efficiency in TM cells. 

Figure 2

 

Effects of different concentrations of curcumin treatment (1–20 μM) on the production of iROS in TM cells when exposed by H₂O₂ (0.2 mM). Intracellular ROS were determined by H₂DCFDA. Data were presented as mean ± SD. **P < 0.01 compared to control, #P < 0.05 and ##P < 0.01 compared to H₂O₂ alone group.

Effects of Curcumin Treatment on the Induction of Proinflammatory Factors and Senescence Marker in the H₂O₂-Exposed TM Cells

Increased iROS has the potential to induce sustained activation of NF-κB, which promotes the production of proinflammatory factors. Expression levels of proinflammatory factors, including IL-6, ELAM-1, IL-1α, and IL-8, were analyzed by real time PCR. Under oxidative stress caused by H₂O₂ exposure, significantly induced levels of IL-6, ELAM-1, IL-1α, and IL-8 in TM cells were achieved. Treatment with 20 μM curcumin was able to downregulate the expression of these proinflammatory factors in the H₂O₂-exposed TM cells (Fig. 3). In addition, curcumin treatment (20 μM) in H₂O₂-exposed TM cells significantly inhibited the activities of senescence marker, SA-β-gal, when compared to the H₂O₂-exposed TM cells without curcumin (Fig. 4). 

Figure 3

 

Effects of curcumin treatment (20 μM) on the induction of proinflammatory factors in TM cells when exposed by H₂O₂ (0.2 mM). Expressions of proinflammatory factors, including IL-6, ELAM-1, IL-1α, and IL-8, were analyzed by RT-PCR. Beta-actin was used as internal control. Experiments were repeated in triplicate. Data were presented as mean ± SD. **P < 0.01 compared to control, ##P < 0.01 compared to H₂O₂ alone group.

Figure 4

 

Effects of curcumin treatment (20 μM) on the activities of senescence marker SA-β-gal in TM cells when exposed by H₂O₂ (0.2 mM). Activities of SA-β-gal were calculated by flow cytometry. Data were presented as mean ± SD. **P < 0.01 compared to control, ##P < 0.01 compared to H₂O₂ alone group.

Effects of Curcumin Treatment on the Accumulation of Carbonylated Proteins and Proteasome Activity in the H₂O₂-Exposed TM Cells

Protein carbonylation, a type of protein oxidation, is promoted by iROS. Western blot analysis for oxidized proteins indicated that the levels of carbonylated proteins following oxidative stress were significantly lower in the curcumin-treated H₂O₂-exposed TM cells when compared to the H₂O₂ group (Fig. 5a). Semiquantitative analysis of the expression of oxidized proteins confirmed the reduction (Fig. 5b). To investigate whether the decreased oxidized proteins were associated with increased proteasomal activities, we analyzed proteolytic activities of the proteasome subunits, including CT-L, T-L, and PGPH. No significant changes in activities of these three proteasome subunits were observed (Fig. 5c). 

Figure 5

 

Effects of 20 μM curcumin treatment on the accumulation of carbonylated proteins and proteasome activity in TM cells when exposed by H₂O₂ (0.2 mM). (a) Representative images of Western blot analysis for oxidized proteins in the experimental groups. (b) Semiquantitative analysis of the expression of oxidized proteins in (a). (c) Proteolytic activities of the proteasome subunits in the three experimental groups, including CT-L, T-L, and PGPH. Data were presented as mean ± SD. **P < 0.01 compared to control, ##P < 0.01 compared to H₂O₂ alone group.

Curcumin Reduced the Apoptotic Cell Numbers in H₂O₂-Exposed TM Cells

Apoptotic cells were identified with Annexin V staining, and were further measured by flow cytometry (Fig. 6a), which indicated that H₂O₂-exposed TM cells had more apoptotic cells than control cells in the absence of H₂O₂ exposure. On the other hand, H₂O₂-exposed TM cells treated with 20 μM curcumin had reduced apoptotic cell numbers. Statistical analysis further confirmed the findings, indicating that 20 μM curcumin treatment significantly reduced the apoptotic cell numbers in H₂O₂-exposed TM cells (Fig. 6b). 

Figure 6

 

Curcumin (20 μM) reduced the apoptotic cell numbers in the TM cells under H₂O₂ (0.2 mM) treatment. (a) Apoptotic cells were identified with Annexin V staining and were measured further by flow cytometry. (b) The percentage of apoptotic cells in control, H₂O₂, and cotreatment of H₂O₂ and curcumin groups. Data were presented as mean ± SD. **P < 0.01 compared to control, ##P < 0.01 compared to H₂O₂ alone group.

Discussion

Our findings suggested that the concentrations of curcumin less than 50 μM does not significantly induce cytotoxicity in primary TM cells, which is consistent with previous reports.¹⁵ Sameermahmood et al.¹⁶ demonstrated that human retinal endothelial cell (HREC) treated with 30 μM of curcumin inhibited SDF-1α-induced cell migration through blockage of Ca²⁺ influx and reduction of PI3K/AKT signaling. Premanand et al.¹⁷ also indicated 10 μM of curcumin induced the HRECs apoptosis partly by the control of iROS production. Bian et al.¹⁸ reported that 20 μM of curcumin effectively reduced glycated human serum albumin-induced IL-8 and MCP-1 secretion in human RPE cells. In addition, several other publications further suggested curcumin doses between 10 and 20 μM are effective in RPE cells.^19–21 

Oxidative stress has been known to contribute to the TM failure in glaucoma. H₂O₂ exposure of cultured TM cells caused excessive iROS release through mitochondria. The production of iROS in turn results in sustained stress response, which contributes to the malfunction of TM in glaucoma.⁷ Meanwhile, iROS also significantly promotes NF-κB activity, which has been reported to activate the expressions of inflammatory markers in glaucoma.²² The inflammatory markers upregulated by NF-κB activation including IL-6, ELAM-1, IL-1α, and IL-8⁷ were analyzed in this study. In addition, the accumulation of senescent cells and reduction of the cell numbers are well-known outcomes of oxidative stress in glaucoma.²³ Activity of SA-β-gal was applied as a senescence marker in our study, while iROS-promoted protein carbonylation, a type of protein oxidation, was monitored as well. Our results showed that curcumin treatment in H₂O₂-exposed TM cells was able to effectively downregulate the aforementioned markers induced by oxidative stress. 

Curcumin is an antioxidative substance that possesses the ability to reduce inflammation. It has been reported recently that curcumin-treated L02 cells exhibited less cytotoxicity and genotoxicity induced by quinocetone, confirming the ability of curcumin to scavenge excessive iROS and resist oxidative stress.²⁴ Trabecular meshwork cells under chronic oxidative stress produce sustained amount of iROS through the mitochondria. Jat et al.²⁵ found that curcumin could elevate endogenous glutathione levels, thus protecting the mitochondrial integrity and enhancing the mitochondrial defense system to attenuate oxidative stress. Our results also confirmed that the levels of iROS were significantly lowered in the curcumin-treated group in comparison with the H₂O₂-treated group. Therefore, the effects of curcumin on the reduction of iROS may be considered as the major contribution to the relief of oxidative stress. The activation of iROS induced by chronic oxidative stress in TM cells frequently upregulates inflammatory markers, including IL-6, ELAM-1, IL-1α, and IL-8. Long et al.²⁶ revealed that curcumin ameliorated inflammation by targeting NF-κB pathway, leading to the downregulation of IL-6. Kumar et al.²⁷ reported that pretreatment of human umbilical vein endothelial cells with curcumin for 1 hour completely blocked the expression of ELAM-1. Das and Vinayak²⁸ demonstrated that curcumin was able to inhibit carcinogenesis by suppressing proinflammatory cytokine IL-1α through AP-1 in mice. McFarlin et al.²⁹ found that oral supplementation with curcumin effectively reduced inflammatory biomarkers, such as IL-8. Consistent with these reports, our data indicated curcumin was a potent anti-inflammatory substance, with the capabity to inhibit oxidative stress induced inflammatory markers in TM cells, including IL-6, ELAM-1, IL-1α, and IL-8. 

In addition to the protective roles of curcumin against oxidative stress-induced oxidation and inflammation, curcumin also demonstrated a potent antiapoptotic effect after oxidative insults. Our results showed that curcumin treatment inhibited SA-β-gal activity, and decreased the apoptotic cell numbers, indicating curcumin protected TM from oxidative stress by preserving its celluar intergrity and viability. Lin et al.³⁰ found that curcumin exerted its antiapoptotic and anti-inflammatory functions through maintaining CISD2 levels in neural cells, indicating a possible molecular mechanism. 

Conclusions

In summary, we reported that curcumin treatment protects the TM cells from oxidative stress-induced impairments. A concentration of 20 μM curcumin is optimal with the best efficacy. Our findings may lead to potential clinical applications to preserve TM for glaucoma treatment, using curcumin to attenuate oxidative stress. 

Acknowledgments

Disclosure: C. Lin, None; X. Wu, None