In Biophysical Department, Basic Medical Faculty, Pyongyang Medical College,
Reactive oxygen, resulting in the oxygen from metabolic process in living organism, is a main factor in the generation of various diseases, and so oxidative stress is an important work in clarifying the mechanism of disease and developing the methods of diagnosis and treatment.
Oxidative stress is an indicator of acceleration of oxidative and antioxidative reaction in an organism, and so it becomes a biological reaction to show excessive accumulation of free radical, the lack of capacity to remove free radical and damage assessment of bonding structure and function in the organism.
Hypertonic glucose plays an important role among the factors causing oxidative stress, because glucose itself in hypertonic glucose solution is easily metabolized to free radical or malondialdehyde(MDA), decreases activation of enzymes such as superoxide dismutase(SOD) and glutation peroxidase(GSH-Px).
A-lipoic acid, and dihydrogen lipoic acid(DHLA) which is a reduced production of α-lipoic acid in organism are a strong substance to remove reactive oxygen species and free radicals. A-lipoic acid neutralizes free radicals including superoxide radical and hyperoxidative products and DHLA removes free radical and reactive oxygen species including singlet oxygen. Also α-lipoic acid has a role to protect the erythrocytes membrane from ionic radiation.
In our country the basic researches on oxidative stress have been applied to several animal models and patients suffering from diseases until now. But these can be influenced by many external factors in the research process, so influences of individual factors on them couldn't be assessed effectively.
Though experiments on cell models which are conducted recently in the world we can not only avoid lacks in vivo, but also study rather safely and complete the impossible studies in the organism. And this method has an advantage to analyze research results because the affective factors are rather single and there are less complex relationships than in the organism.
In the world hypertonic glucose is mostly used as an oxidative stress agent in several kinds of the cultured cells, but pamitic acid is used less than the glucose.
So we made oxidative stress model in HepG2 cell with the hypertonic glucose solution and palmitic acid and clarified the effects of lipoic acid on the antioxidative activity.
Oxidative stress cell model by adding palmitic acid was made by mixing 8mL of DMEM culture medium(2% FBS and 5mmol/L glucose) with palmitic acid of 175μL, 351μL, 526μL, 702μL and then culturing for 24h, 48h in 37℃. After that we evaluated oxidative stress in normal group(NG: glucose 50mmol/L) and experimental group(1G; 100μM palmitic acid, 2G; 200μM palmitic acid, 3G; 300μM palmitic acid, 4G; 400μM palmitic acid), and hypertonic glucose group.
Antioxidative ability of lipoic acid was evaluated by adding several concentration of lipoic acid to hypertonic glucose culture medium and then culturing for 48h.
The changes of oxidative substances and antioxidative enzymes in oxidative stress cell model of HepG2 cell by palmitic acid began to appear after 24h with more than 200μM of palmitic acid and they were changed according to concentration and action duration of palmitic acid (p<0.05), such as hypertonic glucose group.
The oxidative and antioxidative indicators that changed in culture during the period of 48h of HepG2 cells in 400μM of palmitic acid medium with unequal concentration of lipoic acid showed some improvements and the antioxidative reaction was more apparent according to concentration of lipoic acid. (p<0.05)
We are going to introduce this method to research of reactive oxygen species that are important factors in generation of several disease and to scientification of diagnosis and treatment of disease in future.
