Effects of different concentrations of tetramethylpyrazine, an active constituent of Chinese herb, on human corneal epithelial cell damaged by hydrogen peroxide

Abstract

AIM

To discuss the effects of different concentrations of tetramethylpyrazine (TMP), an active constituent of Chinese herb, on damaged Shandong human corneal epithelial cell (SDHCEC) induced by hydrogen peroxide.

METHODS

We detected the combined effects of TMP with concentrations ranging from 4 mg/mL to 0.03 mg/mL and 800 µM hydrogen peroxide on SDHCEC. The methyl thiazolyl tetrazolium (MTT) assay was processed at 3, 6 and 12h separately while the detection of cell apoptosis at 6h only by flow cytometry.

RESULTS

The viability of SDHCEC with 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL TMP joint with 800 µM hydrogen peroxide at 3h and 6h was significantly higher than that with 800 µM hydrogen peroxide only, P<0.05. However, except 0.25 mg/mL, TMP with other concentrations joint with 800 µM hydrogen peroxide at 12h could not significantly inhibit decreased SDHCEC viability induced by 800 µM hydrogen peroxide. At 12h, TMP of 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL could significantly inhibit SDHCEC early apoptosis induced by 800 µM hydrogen peroxide, most remarkable at 0.25 mg/mL TMP, P<0.05.

CONCLUSION

Our results suggested that hydrogen peroxide can induce apoptosis related damage to SDHCEC. TMP can protect SDHCEC from the damage, and the protective effects may be associated with its anti-apoptosis mechanism.

INTRODUCTION

In recent years, reports about the effects of oxidative stress in biomedical fields are abundant, such as aging, infertility, tumor, diabetes, neurodegenerative disease, and so on^[1]. Eyes, which are continuously exposed to light, air, chemicals in the environment, etc. are vulnerable to oxidative stress. Researchers have proved that oxidative damage are related to many eye diseases, such as age-related macular degeneration, senile cataract, actinic keratoconjunctivitis induced by ultraviolet radiation, and so on. Of course, the corneal injury^[2]–^[4] which we are going to talk about. Especially for corneal epithelium, which is directly exposed to outside ambient, the antioxidants mainly exist in the tear film and epithelial tissue itself^[5]–^[9].

Active oxygen species, such as hydroxyl group, superoxide anion, hydrogen peroxide, etc. are produced by cell metabolism process. Though activity of hydrogen peroxide itself is not strong, it can be decomposed into hydroxyl radical of perfect antioxidant effect^[10]. Besides, hydrogen peroxide is a stable medium oxidant produced biologically, which has been widely applied in studies. Recent evidence showed that active oxygen species can induce cell apoptosis via mitochondrial function damage, and there into hydrogen peroxide may attack some enzymes, consume adenosine triphosphate (ATP), decrease the level of nicotinamide adenine dinucleotide phosphate, and finally lead to cell apoptosis^[11]. Recently, several studies showed that hydrogen peroxide can lead to injury of corneal epithelial cell and corneal endothelium^[2],^[4]. However, there are no reports found about corneal epithelial cell apoptosis induced by hydrogen peroxide.

Traditional Chinese medicine represents a worldwide resource for potential treatments of many diseases. Recent studies show that tetramethylpyrazine (TMP), extracted from the Chinese herbal medicine Ligusticum wallichii franchat (chuan xiong in Chinese), is a significant anti-lipid-peroxidation, anti-free radical, anti-apoptosis^[12]–^[15] and calcium antagonist agent^[16]. And TMP has been widely used in clinic for treatment of cardiovascular and cerebrovascular diseases and various diseases of retina^[17],^[18]. Therefore, TMP is a potentially useful treatment for ocular surface disorders. In the present study, we aimed to observe effects of different concentrations of TMP against damage of human corneal epithelial cells induced by hydrogen peroxide from the aspects of antiapoptotic in vitro, which no one ever discussed before.

SUBJECTS AND METHODS

Our study followed the principles outlined in the Declaration of Helsinki (2008).

Cell Culture

Shandong human corneal epithelial cells (SDHCEC) (provided by Prof. Zhi-Chong Wang in Zhongshan Ophthalmic Center of Sun Yat-sen University) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose supplemented with 10% heat-inactivated fetal bovine serum, 100 000 U/L penicillin, 100 mg/L streptomycin, 2 g/L sodium bicarbonate, 0.01 mg/L human epidermal growth factor, 0.292 g/L L-Glutamine, 5 mg/L insulin, 5 mg/L human transferrin, 400 ng/L hydrocortisone, 1× nonessential amino acids in CO₂ incubator (37°C, 5% CO₂; Thermo Fisher Scientific, America). We firstly observed the influence of hydrogen peroxide with different concentrations (1600 µM, 800 µM, 400 µM, 200 µM, 100 µM) on SDHCEC to choose an appropriate concentration. And then we detected the combined effects of TMP with different concentrations (4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.06 mg/mL, 0.03 mg/mL) and 800 µM hydrogen peroxide on SDHCEC, while simultaneously we observed TMP with different concentrations (4 mg/mL, 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.06 mg/mL, 0.03 mg/mL) alone on SDHCEC. The methyl thiazolyl tetrazolium (MTT) assay was processed at 3, 6 and 12h separately while the detection of cell apoptosis with annexin-V-FITC and propidium iodide (PI) double staining at 6h only.

Cell Viability Assay

Cell viability analysis was based on the capacity of mitochondrial enzymes to transform MTT to MTT formazan. The formazan production would be decreased when mitochondrial redox function was impaired or cellular reactive oxygen species (ROS) was enhanced. Therefore, MTT assay may be used as an index of cell viability responses to experimental interventions. In short, cells were cultured in 96-well plates (0.5×10⁵ cells/mL, 200 µL) for 24h and treated as described above. We set normal control wells in which we cultured cells only with serum-free high-glucose DMEM and positive control wells in which we cultured cells with serum-free high-glucose DMEM containing hydrogen peroxide (terminal concentration was 800 µM). After the treatment with different medium conditions for 3, 6 and 12h separately, 20 µL of MTT (5 mg/mL) was added to each well and cells were incubated at 37°C for 4h. Then culture medium with dye was removed and 200 µL of dimethyl sulfoxide (DMSO) per well was added for formazan solubilization. The absorbance of converted dye was measured at wavelengths of 570 nm and 630 nm using Multiskan MK3 (Thermo Fisher Scientific, America) and the difference values were used to calculate the cell viability. The computational formula: absorptance (tested well)/absorptance (normal control well)×100%.

Analysis of Cell Apoptosis by Flow Cytometry

The apoptotic rate of SDHCEC was detected using an Annexin-V FITC/PI apoptosis detection kit. After the drug treatment, cells were harvested with 0.25% trypsinase, washed with serum-free high-glucose DMEM, removed supernatant after centrifugating at 1500 r for 10min and double-stained with Annexin-V FITC and PI in the dark at room temperature for 15min. After that, samples were analyzed using a FACSaria flow cytometer (Becton, Dickinson and Company, America).

Statistical Analysis

Statistical analysis was performed using SPSS version 16.0 (SPSS, Inc, Chicago, IL, USA). A one-way analysis of variance followed by either the Bonferroni or Dunnett T3 test were used to compare the mean value of all items. A significance level was set at P<0.05.

RESULTS

The viability of SDHCEC with 1600 µM and 800 µM hydrogen peroxide at 3, 6 and 12h was significantly lower than that in normal controls, progressing with time, while the viability of SDHCEC with 400 µM hydrogen peroxide at 12h was lower than that in normal controls as well, P<0.01 or P<0.05 (Figure 1).

Figure 1. Influence of hydrogen peroxide with different concentrations on SDHCEC viability detected by MTT.

^aP<0.01 vs normal controls; ^bP<0.05 vs values at 6h at the same group.

The viability of SDHCEC with 4 mg/mL and 2 mg/mL TMP at 3, 6 and 12h was significantly lower than that in normal controls, P<0.001, while other concentrations less than or equal to 1 mg/mL showed no significance (Figure 2).

Figure 2. Influence of TMP with different concentrations on SDHCEC viability detected by MTT.

^aP<0.001 vs normal controls.

The viability of SDHCEC with 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL TMP joint with 800 µM hydrogen peroxide at 3, 6h was significantly higher than that with 800 µM hydrogen peroxide only, most remarkable at 0.25 mg/mL TMP, P<0.05. However, except 0.25 mg/mL TMP, TMP with other concentrations joint with 800 µM hydrogen peroxide at 12h could not protect SDHCEC from decreased viability induced by 800 µM hydrogen peroxide. The viability of SDHCEC with 4 mg/mL and 2 mg/mL TMP joint with 800 µM hydrogen peroxide at all the three time points was significantly lower than that with 800 µM hydrogen peroxide only, P<0.05 (Figure 3).

Figure 3. Influence of TMP with different concentrations on decreased SDHCEC viability induced by 800 µM hydrogen peroxide detected by MTT.

^aP<0.05 vs 800 µM H₂O₂.

Cell apoptosis at 6h was assayed by flow cytometry. SDHCEC detected by Annexin-V FITC/PI apoptosis kit can be classified into normal cells (Q1), early apoptotic cells (Q2), late apoptotic cells (Q3) and necrotic cells (Q4). Our results showed that hydrogen peroxide of 1600 µM and 800 µM could induce SDHCEC early apoptosis, while 1600 µM hydrogen peroxide could induce SDHCEC late apoptosis and necrosis, P<0.001 (Figure 4).

Figure 4. Influence of hydrogen peroxide with different concentrations on the apoptotic rate of SDHCEC detected by flow cytometry.

^aP<0.001 vs normal controls in early apoptosis; ^bP<0.001 vs normal controls in late apoptosis; ^cP<0.001 vs normal controls in early necrosis; ^dP<0.001 vs normal controls in all abnormal cells.

Compared with normal control group, TMP of 4 mg/mL could induce SDHCEC early apoptosis, late apoptosis and necrosis separatly while TMP of 2 mg/mL could only induce SDHCEC early apoptosis, P<0.001 or P<0.01. There was no significance between TMP with concentrations less than or equal to 1 mg/mL and normal controls, P>0.05 (Figure 5).

Figure 5. Influence of TMP with different concentrations on the apoptotic rate of SDHCEC detected by flow cytometry.

^aP<0.001 vs normal controls in early apoptosis; ^bP<0.001 vs normal controls in late apoptosis; ^cP<0.01 vs normal controls in early necrosis; ^dP<0.001 vs normal controls in all abnormal cells.

TMP of 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL at 6h could significantly inhibit SDHCEC early apoptosis induced by 800 µM hydrogen peroxide, most remarkable at 0.25 mg/mL TMP, P<0.001 or P<0.05. TMP of 4 mg/mL could significantly increase SDHCEC early apoptosis induced by 800 µM hydrogen peroxide (Figures 6, 7).

Figure 6. Effects of TMP with different concentrations on SDHCEC apoptosis induced by 800 µM hydrogen peroxide detected by flow cytometry.

^aP<0.001 vs normal controls in early apoptosis; ^a1P<0.05 vs 800 µM H₂O₂ in early apoptosis; ^bP<0.001 vs normal controls in late apoptosis; ^cP<0.001 vs normal controls in early necrosis; ^dP<0.001 vs normal controls in all abnormal cells; ^d1P<0.05 vs 800 µM H₂O₂ in all abnormal cells.

Figure 7. The images of SDHCEC about the effects of TMP with different concentrations on SDHCEC apoptosis induced by 800 µM hydrogen peroxide detected by flow cytometry.

CONCLUSION

Our results showed that hydrogen peroxide of 1600 µM and 800 µM at 3, 6 and 12h could significantly decrease SDHCEC viability, inducing cell early apoptosis. Similarly, previous studies have showed that hydrogen peroxide could induce oxidative damage and apoptosis of various cells, such as endothelial cells, pheochromocytoma 12 cells, cardiac muscle cells, endometrial carcinoma cells, human embryonic stem cells, retina cells, and so on^[11]–^[15],^[19]–^[21]. In our study, TMP of 4 mg/mL and 2 mg/mL could induce SDHCEC oxidative damage while TMP with concentrations less than or equal to 1 mg/mL showed no significant influence on SDHCEC. TMP of 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL joint with 800 µM hydrogen peroxide at 3h and 6h could significantly inhibit oxidative damage induced by hydrogen peroxide, most remarkable at 0.25 mg/mL TMP. Results by flow cytometry assay also showed that TMP of 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.06 mg/mL joint with 800 µM hydrogen peroxide at 6h could significantly inhibit cell apoptosis induced by hydrogen peroxide, indicating that TMP could protect SDHCEC from oxidative damage induced by hydrogen peroxide, which might be related to anti-apoptosis effect of TMP. The results were similar to reports that TMP can protect damaged cells via anti-oxidation and/or anti-apoptosis effects. Cheng et al^[13] reported in 2007 that TMP could inhibit oxidative damage of PC12 cells induced by hydrogen peroxide in vitro, which was related to anti-apoptosis effect of TMP. Li et al^[14] reported in 2009 that TMP could inhibit oxidative damage of human umbilical vein endothelial cells induced by hydrogen peroxide in vitro via anti-oxidation and anti-apoptosis effects. Ou et al^[11] also reported in 2010 the similar effect of TMP and its extract. Some researches about eye diseases also showed similar effects. Yang et al^[12] proved that TMP could protect retina cells from oxidative damage induced by hydrogen peroxide, which was related to anti-oxidation and anti-apoptosis effects of TMP. Our previous studies also showed that TMP could inhibit lens opacity induced by sodium selenite via anti-oxidation and calcium antagonistic effects in vivo and in vitro^[22],^[23].

MTT results at 12h showed that except 0.25 mg/mL TMP, TMP with other concentrations could not significantly increase the viability of SDHCEC damaged by 800 µM hydrogen peroxide, indicating that persistence of hydrogen peroxide led to irreversible damage of SDHCEC.

In conclusion, our results showed that TMP could protect damaged SDHCEC induced by hydrogen peroxide, which might be associated with anti-apoptosis mechanism of TMP. The study provides a basis for local application of TMP on cornea correlated diseases.