How does astaxanthin protect against reactive oxygen species-induced mitochondrial damage?

 Astaxanthin is widely found in nature, especially in shrimp, crab, fish, algae, yeast and bird feathers, and plays a role in color development. Astaxanthin is a kind of carotenoid, has reduced lipid peroxidation, scavenging free radicals, delay aging, inhibit tumorigenesis and improve immunity, prevention of cardiovascular disease and other physiological functions[1 ,2] . In recent years, astaxanthin has been widely used in medicine, and has received more and more attention [3].

 

1 Material

1.1 Instruments

Allegra 64R low-temperature high-speed centrifuge (Beckman); 722 visible spectrophotometer (Shanghai Spectrum Instrument Co., Ltd.); Wellscan MK 3 enzyme labeling instrument (Bio-Rad, USA); Adventurer electronic balance (Ohaus, USA).

 

1.2 Drugs and reagents

Astaxanthin (Sigma, purity 95%); 3% hydrogen peroxide (H2O2) and vitamin C (China Pharmaceutical Shanghai Chemical Company); calf serum albumin (BSA) (Roche); superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO) and ATPase kit (Nanjing Jianjian Bioengineering Institute); dimethyl sulfoxide (DMSO) (Sigma, purity ≥999%); other reagents were domestic analytical purity. (Nanjing Jianjian Institute of Biological Engineering); dimethyl sulfoxide (DMSO) (Sigma, purity ≥99.9%); other reagents were domestic analytical purity.

 

1.3 Laboratory animals

Clean grade Wistar rats, weighing 180~210 g, provided by Harbin Medical University, Certificate of Conformity No. P00101014.

 

2 Methodology

2.1 Isolation and Preparation of Mitochondria [4]

Differential gradient centrifugation for the preparation of mitochondria: rats were killed by decapitation, the livers were dissected quickly and weighed accurately, and 10 % of the tissue homogenate was prepared by adding cold physiological saline in the ratio of 1:10 (w/v), and then centrifuged at 1,500 r/min for 10 min in a low-temperature and low-speed centrifuge to discard the precipitate, and then centrifuged the supernatant at 10,000 r/min for 15 min, and then the precipitate was mitochondria, which was suspended in 1 mL of the homogenate. The mitochondria were suspended in 1 mL of homogenate, and the protein was quantified by the Caulmers Brilliant Blue method, and the protein concentration of the mitochondrial suspension was adjusted to 2 g/L. The above processes were operated at 4 ℃.

 

2.2 H2O2 damage to mitochondria

The experiments are shown in Table 1 , mitochondrial protein suspension was added with sodium chloride, H2O2, vitamin C and different amounts of astaxanthin to form a mixture, and the mitochondria were incubated in a 37 ℃ water bath [5] for 1h.

 

2.3 Measurement of biochemical indicators

Mitochondrial MDA, NO and GSH contents as well as SOD and ATPase activities were determined according to the kit instructions.

 

2.4 Statistical treatment of data

The data were statistically analyzed by SPSS15.0 software and expressed as group

Differences were analyzed by one-way ANOVA, and P < 0.05 was considered ± statistically significant.

 

3 Results

3.1 Effect of astaxanthin on mitochondrial MDA content

See Figure 1 . As shown in Figure 1, the MDA content in the mitochondria of the H2O2 damage group was significantly higher than that of the control group, and the difference was statistically significant (P<0.01). The high dose group of astaxanthin could significantly inhibit the increase of MDA content caused by reactive oxygen species, and there was no significant difference with the control group (P>0.05).

 

3.2 Effect of astaxanthin on mitochondrial NO content

See Figure 2 . As shown in Figure 2, the NO content in the mitochondria of the H2 O2 damage group was significantly higher than that of the control group, and the difference was statistically significant (P<0.01). The high dose group of astaxanthin could significantly inhibit the increase of NO content caused by reactive oxygen species, and there was no significant difference with the control group (P>0.05).

 

3.3 Effect of astaxanthin on mitochondrial GSH content

See Figure 3. As shown in Figure 3, the GSH content in mitochondria of the H2O2 damage group was significantly lower than that of the control group, and the difference was statistically significant (P<0.01). The high dose group of astaxanthin could obviously inhibit the decrease of GSH content caused by reactive oxygen species, and there was no significant difference with the control group (P>0.05).

 

3.4 Effect of astaxanthin on mitochondrial SOD content

See Figure 4. As shown in Figure 4, the mitochondrial SOD activity in the H2O2 damage group was significantly lower than that in the control group, and the difference was statistically significant (P<0.01). Astaxanthin high-dose group can obviously inhibit the reduction of SOD activity caused by reactive oxygen species, and there is no significant difference with the control group (P>0.05).

 

3.5 Effects of astaxanthin on mitochondrial ATPase activity

See Figure 5. As shown in Figure 5, the activities of Na+,K+-ATPase, Mg2+-ATPase and Ca2+-ATPase in the H2O2 injury group were significantly lower than those in the control group (P<0.01). The activities of three kinds of ATPase in the low, medium and high doses of astaxanthin were stronger than those in the H2O2 injury group, and the ATPase activities increased with the increase of the dose of astaxanthin.

 

4 Discussion

Reactive oxygen species are the most important free radicals in the human body. Excessive reactive oxygen species can cause oxidative damage to biological macromolecules such as proteins and nucleic acids, resulting in aging, tumors, diabetes, and autoimmune diseases[6] . Hydrogen peroxide is the most common reactive oxygen species.

 

Mitochondria are membrane organelles of eukaryotic cells that perform important functions such as synthesizing ATP, generating ROS, regulating cellular oxidation/reduction signals, controlling apoptosis and gene expression. It has been found that mitochondria have a cytosol-independent nitric oxide synthase (NOS) system, which generates NO that directly damages the mitochondria. H2O2 damage to mitochondria generates large amounts of NO, which attacks the unsaturated fatty acids on the mitochondrial membrane, resulting in an increase in MDA and a decrease in mitochondrial activity[7,8] . The possible mechanism of astaxanthin to reduce NO production is the inhibition of NOS activity. H2O2 decreases SOD activity, depletes GSH, and decreases ATPase activity in mitochondria, resulting in abnormal mitochondrial energy metabolism and affecting the normal growth of the cells[9, 10] . Mitochondria are also the main calcium reservoir of the cell, and H2O2 reduces the activities of mitochondrial Ca2+-ATPase and Mg2+-ATPase, decreasing the Ca2+ uptake by mitochondria, and then cytoplasmic Ca2+ overload occurs, which affects the metabolism of the cell and even leads to the death of the cell.The activity of Na+-K+-ATPase of the mitochondrial membrane is decreased by H2O2, which leads to the retention of mitochondrial Na+ , swelling and even rupture of the mitochondria. H2O2 reduces Na+-K+-ATPase activity in the mitochondrial membrane, causing mitochondrial Na+ retention and mitochondrial swelling and rupture[11] . The results of this study showed that astaxanthin can increase mitochondrial SOD activity, reduce GSH consumption, and effectively counteract the reduction of Ca2+-ATPase, Mg2+-ATPase, and Na+-K+-ATPase activities, which is of positive significance in protecting mitochondria from oxidative damage by reactive oxygen species.

 

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