Coenzyme Q10, also known as ubiquinone, is a fat-soluble molecule that is a potent antioxidant and a vitamin-like substance. Coenzyme Q10 is an important component of the mitochondrial respiratory chain, and it is a compound found in a wide range of cells in living organisms and plays a very important role in the body[1] . Studies[2-3] have shown that coenzyme Q10 has an antioxidant effect, can recycle vitamin E, scavenge free radicals in the body, enhance the body's humoral and cellular immunity, have a certain killing effect on tumor cells in the body, and can significantly relieve fatigue, improve motor function, and have preventive and therapeutic effects on cardiovascular and cerebrovascular diseases.
In recent years, studies[4-5] have also found that coenzyme Q10 is effective in the treatment of retinal diseases in the elderly. The occurrence of many ophthalmic diseases, such as glaucoma and age-related macular degeneration, is related to a variety of factors, including retinal aging, elevated intraocular pressure (IOP), nutrition, heredity, the environment, mechanical damage, oxidative damage, etc. Oxidative damage plays a very important role in the occurrence of ophthalmic diseases in the elderly, and is therefore attracting more and more attention. However, whether the effect of coenzyme Q10 on retinal epithelial cells is direct or not, and its possible mechanism of action have not been fully revealed, and in-depth study of these issues will hopefully provide ideas for the diagnosis and treatment of such diseases.
In this study, we used human retinal pigment epithelial cells (RPE) as the research object, and intervened with coenzyme Q10, used the drug gradient experiment to clarify whether Q10 had a protective effect on RPE, and used molecular immunology and other techniques to analyze the possible mechanism of coenzyme Q10 from the perspective of cellular oxidative stress, damage, apoptosis, etc., to provide a test basis for the prevention and treatment of ophthalmic diseases in the elderly. We also used molecular immunology and other techniques to analyze the possible mechanism of coenzyme Q10 from the perspective of oxidative stress damage and apoptosis, so as to provide experimental basis for the prevention and treatment of ophthalmic diseases in the elderly.
1 Materials and Methods
1. 1 Cell processing
The human retinal pigment epithelial cell line RPE-19 (purchased from Sun Yat-sen University Ophthalmic Center) was cultured in DMEM culture medium containing 10% calf serum at 37 ℃ (5% CO2). The experiments were divided into four groups: the oxidative stress model group, cells were cultured in 200 μmol/L H2 O2 solution for 2 h; the experimental group was divided into two groups, 10 μmol/L coenzyme Q10 group and 1 μmol/L coenzyme Q10 group, cells of the two groups were cultured in 10 μmol/L and 1 μmol/L coenzyme Q10 for 48 h, then cultured in 200 μmol/L H2 O2 solution for 2 h; and cells of the two groups were cultured in 10 μmol/L and 1 μmol/L coenzyme Q10 for 48 h, then cultured in 200 μmol/L H2 O2 solution for 2 h. The cells in the normal control group were not treated with coenzyme Q10 and H2 O2.
1.2 Cell Activity Assay
The cell activity was detected by CCK-8 method, 10% CCK-8 working solution was added into 4 groups of cells, and a control group was set up at the same time, then incubated in the incubator for 30 min, and the OD value of each well was measured by enzyme labeling at 450 nm, and 6 replicate wells were set up in each group, and the experiments were repeated for 3 times.
1.3 Intracellular reactive oxygen species detection
Intracellular reactive oxygen species (ROS) were detected by DCFH-DA fluorescent probe assay, and ROS expression was detected by DCFH-DA Reactive Oxygen Species Fluorescent Detection Kit (purchased from Suzhou Biyuntian Biotechnology Co., Ltd.) in the four groups of cells, and the experiments were repeated for three times.
1. 4 Detection of Caspase-3, Bcl-2, Bax, phosphorylated-Akt, Akt in Cells
The expression of Caspase-3, Bcl-2, Bax, phosphorylated-Akt and Akt in the cells was detected by Western blot, and the proteins of four groups of cells with different treatments were extracted, and the protein concentration was determined by BCA method. The protein concentration was determined by BCA method. The protein samples of each group were separated by electrophoresis, and the protein marker was added at the same time, the membrane was softened and sealed by skimmed milk powder, the primary antibody and secondary antibody were added sequentially, the exposure was performed, the film was scanned, and the bands were analyzed in grayscale by Image J software.
1.5 Statistical analysis
SPSS13.0 software was used to analyze the data, and the experiments were repeated three times, and the measurement data were expressed as (x- ± s), and different analysis methods were selected according to the characteristics of the data, and the data were inferred. The difference was considered statistically significant at P < 0.05.
2 Results
2.1 Effect of coenzyme Q10 on RPE-19 activity in human retinal pigment epithelial cells
The activity of the H2 O2-treated cells was significantly lower than that of the normal control group (P < 0.05). The cell activity of the coenzyme Q10 pretreated cells was significantly higher than that of the H2 O2-treated cells in the model group, and the differences between the 1 μmol/L coenzyme Q10 group and the 10 μmol/L coenzyme Q10 group and the oxidative stress model group were statistically significant (P < 0.05). The differences were statistically significant (P<0.05) when compared with the oxidative stress model group.
Table 1 Effects of coenzyme Q10 on RPE-19 activity in human retinal pigment epithelial cells.
Impact (x- ± s)
groups | OD value |
normal control group | 0. 985 ± 0. 023 |
Oxidative stress model group (H O2 2 ) | 0. 356 ± 0. 012 ∗ |
1 μmol/L Coenzyme Q10 group | 0. 496 ± 0. 022 ∗# |
10 μmol/L Coenzyme Q10 group | 0. 586 ± 0. 032 ∗# |
∗P < 0. 05, compared with normal control group; #P < 0. 05, compared with oxidative stress model group.
2.2 Effect of coenzyme Q10 on RPE-19 reactive oxygen species in human retinal pigment epithelial cells
The levels of intracellular reactive oxygen species were significantly higher in the H2 O2-treated cells than in the normal control group (P < 0.05). The levels of intracellular reactive oxygen species in the cells pretreated with coenzyme Q10 were significantly lower than those in the model group treated with H2 O2 alone, and the differences between the 1 μmol/L coenzyme Q10 group and the 10 μmol/L coenzyme Q10 group and the oxidative stress model group were statistically significant (P ＜ 0.05). The differences were statistically significant (P < 0.05) when compared with the oxidative stress model group.
Table 2 Effect of coenzyme Q10 on RPE-19 reactive oxygen species in human retinal pigment epithelial cells
groups | ROS value |
normal control group | 556.36 ±66.78 |
Oxidative stress model group (H O2 2 ) | 879.12 ±75.45 ∗ |
1 μmol/L Coenzyme Q10 group | 716.23 ±67.42 ∗# |
10 μmol/L Coenzyme Q10 group | 625.76 ±45.63 ∗# |
∗P < 0. 05, compared with normal control group; #P < 0. 05, compared with oxidative stress model group.
2. 3 Effect of coenzyme Q10 on the expression of Caspase-3, Bcl-2, Bax, phosphorylated-Akt, and Akt in human retinal pigment epithelial cells
3.
Coenzyme Q10 inhibited the expression of caspase-3 and Bax in human retinal pigment epithelial cells induced by H2 O2, and promoted the expression of Bcl-2, which can inhibit apoptosis. Compared with the oxidative stress model group, the expression of Caspase-3 and Bax was lower than that of the oxidative stress model group in the 1 μmol/L coenzyme Q10 group and the expression of Bcl-2 was higher than that of the oxidative stress model group in the 10 μmol/L coenzyme Q10 group.H2 O2 also induced the phosphorylation of Akt, and the phosphorylated-Akt was more than 1 μmol/L coenzyme Q10 group and the phosphorylated-Akt was more than 10 μmol/L coenzyme Q10 group in the 10 μmol/L coenzyme Q10 group. H2 O2 also induced the phosphorylation of Akt, and the expression of phosphorylated Akt in the 1 μmol/L coenzyme Q10 group and the 10 μmol/L coenzyme Q10 group was lower than that in the oxidative stress model. See Figure 1.
Fig. 1 Effect of coenzyme Q10 on the expression of Caspase-3, Bcl-2, Bax, phosphorylated-Akt, and Akt in human retinal pigment epithelial cells . *P < 0.05.
3 Discussion
Along with the aging of the society, the trend of age-related eye diseases is high. Age-related macular degeneration, glaucoma, cataract and other eye diseases cause serious visual impairment, and in severe cases, may lead to permanent blindness[6-8] . One of the most important factors causing these eye diseases is oxidative stress, and preventing and treating oxidative stress is of great significance in controlling the occurrence and treatment of these eye diseases.
Coenzyme Q10 is an antioxidant that is naturally present in the body and has been widely used in the prevention and treatment of many diseases[9 -11] . The content of coenzyme Q10 in various tissues and organs of the body decreases with age, and therefore its physiological capacity decreases accordingly, which is related to the occurrence of many chronic diseases. Exogenous supplementation of coenzyme Q10 can increase the level of coenzyme Q10 in the body, which can help it to play an antioxidant role in the body and prevent the occurrence of some chronic diseases[12 -13] .
There are few studies on the role of coenzyme Q10 in the occurrence and development of ocular diseases. In this study, we applied coenzyme Q10 to pre-treat human retinal pigment epithelial cells, and then induced oxidative damage with H2 O2, to investigate the protective effect of coenzyme Q10 against oxidative stress injury in human RPE cells and the mechanism of oxidative stress injury. The results showed that coenzyme Q10 was able to enhance the activity of H2 O2-treated retinal pigment epithelial cells and reduce the level of intracellular reactive oxygen species induced by H2 O2. A number of ophthalmic diseases are closely related to oxidative stress in retinal epithelial cells, such as senile cataract and diabetic retinopathy, etc. The anti-epithelial oxidative effect of coenzyme Q10 suggests that it has a potential value for clinical application.
Meanwhile, the present study showed that coenzyme Q10 inhibited the expression of Caspase-3 and Bax and promoted the expression of Bcl-2 in human retinal pigment epithelial cells. Coenzyme Q10 inhibits apoptosis in human retinal pigment epithelial cells in vitro, which may be one of the mechanisms of its efficacy. Akt is an important signal that regulates apoptosis, and coenzyme Q10 significantly inhibited the phosphorylation of Akt in human retinal pigment epithelial cells, suggesting that the possible mechanism of coenzyme Q is to inhibit Akt signaling.
In conclusion, the present study demonstrated the protective mechanism of coenzyme Q10 against oxidative stress injury in human RPE cells by in vitro study. This may be due to oxidative damage, inhibition of apoptosis-related factors (e.g., caspase-3, Bax, etc.), promotion of apoptosis-suppressing factors (e.g., Bcl-2, etc.), and inhibition of Akt phosphorylation.
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