| The integrity of red blood cell is the structural basis to ensure the normal physiological functions of the body.The red blood cell membrane is rich in lipid and protein, under pathological conditions,the generation and removal of free radicals is not in balance,the excess of free radicals easily oxidize unsaturated fatty acids, induce lipid peroxidation chain reaction. MDA is an important product of lipid peroxidation, it has a strong biological toxicity, is easy to crosslink with phospholipids, protein denatured or inactivated,resulting in the destruction of the membrane structure and the loss of physiological function.The excess of free radicals can also break protein peptide, lead to protein intramolecular or intermolecular crosslinking, damage protein secondary, tertiary and quaternary structure, reduce protein folding,increase irregular curl. Eventually lead to red blood cell hemolysis, reduced antioxidant enzyme activities,changed the structure of membrane proteins, changed cell surface morphology, destruct the membrane skeleton structure. Cell aging is the basis of Organism aging, free radical theory of aging is Accepted. Therefore, searching, screening free radical scavenging, blocking the lipid peroxidation chain reaction, increased antioxidant enzyme activity of antioxidant drugs more and more attention by researchers.Main active ingredient of trollions is flavonoids, and content of orientoside is highest. The antioxidant activity of the flavonoid compound is typically through a hydroxyl group represented by the reductive associated with the number and position of the hydroxyl group, steric hindrance, and molecular size. We find orientin and luteolinon have similar structures, C-glycosides combined glucose of the A ring is the difference.Orientoside can effectively clear O2-·,· OH, DPPH, improve antioxidant enzyme activity of aging mise induced by D-galactose, decrease MDA content, improve the function of the structure of nerve cells in the hippocampus of aging mice, and reduce the content of lipofuscin. In this research, we explore antioxidant effects of orientin in the cellular level, eatablise erythrocyte oxidative damage model and erythrocyte natural aging model, explore the protective effects of orientin on human erythrocytes, and screen structure-activity relationship of the antioxidant, research directions for the synthesis of anti-aging drugs.Eatablished red blood cell oxidative damage model,2%red blood cell suspension was added to300mmol·L-1H2O2, adequate response to1.5h at37℃. It was divided into blank group, model group, orientoside group (10,20,40μg·mL-1), luteolin group (10,20,40μg·mL-1), VC(20Ｌg·mL-1). Red blood cell hemolysis rate, ROS, MDA, SOD, CAT, GSH-Px, Na+-K+-ATPs, Ca2+-Mg2+-ATPs were detected, membrane proteins were analysed by SDS-PAGE, the red blood cell surface morphology was observed by SEM, membrane skeleton structure was observed by TEM.Eatablished red blood cell natural aging model, red blood cell incubated at37℃for48h. It was divided into blank group, model group, orientoside group (10,20,40Ｌg·mL-1), luteolin group (10,20,40μg·mL-1), VC(20μg·mL-1). Red blood cell hemolysis rate, MDA, SOD, CAT, GSH-Px, Na+-K+-ATPs, Ca2+-Mg2+-ATPs, SA was detected, membrane proteins were analysed by SDS-PAGE, the red blood cell surface morphology was observed by SEM, membrane skeleton structure was observed by TEM.1Effects of hemolysis rate on oxidative damage and the natural aging of red blood cellHuman red blood cell was damaged by H2O2. The hemolysis rate of model group increased, compared with blank group, it has significant difference (P<0.01); After giving orientoside, luteolin and vitamin C, the hemolysis rate gradually reduced, and compared with model group, it has significant differences (P<0.01); Compared with luteolin group, the hemolysis rate of10,20μg·mL-1dose group of orientoside was higher, and it has significant differences (P<0.05, P<0.01), and40μg·mL-1dose group of orientoside has no difference; Compared with VC group, the hemolysis rate of10,20μg·mL-1dose group of orientoside and luteolin was higher, and it has significant differences (P<0.05, P<0.01),40μg·mL-1dose group of orientoside and luteolin has no difference.Human red blood cell were incubated for48hours, and the hemolysis rate of model group increased. After giving orientoside, luteolin and vitamin C, the hemolysis rate gradually reduced, and compared with model group, it has significant differences (P<0.05, P<0.01); Compared with luteolin group, the hemolysis rate of10,20μg·mL-1dose group of orientoside was higher, and it has significant differences (P<0.05, P<0.01), and40μg·mL-1dose group of orientoside has no difference; Compared with VC group, the hemolysis rate of10,20μg·mL-1dose group of orientoside and luteolin was higher, and it has significant differences (P<0.05, P<0.01),40μg·mL-1dose group of orientoside and luteolin has no difference.2Effects of orientin on ROS, MDA, antioxidant enzymes, ATP s of human RBC induced by H2O2and aging RBC2.1Effects of orientin on ROS, MDA, antioxidant enzymes, ATP s of human RBC induced by H2O2Human red blood cell was damaged by H2O2, the fluorescence intensity of ROS was higer, the content of MDA was increased, the activities of antioxidant enzyme and ATPs was lower, compared with blank group, they had significant differences (P<0.01); After giving orientoside, luteolin and vitamin C, every indicators was improved, and they had a positive dose-effect relationship. Compared with modle group, the fluorescence intensity of ROS and activities of SOD, CAT, Na+-K+-ATPs of10μg.mL-ldose group of orientoside had significant difference (P<0.05, P<0.01), other indicators had no significant difference, and every indicators of20,40μg·mL-1dose group of orientosid and10,20,40μg·mL-1dose group of luteolin had significant difference (P<0.05, P<0.01).Compared with luteolin group, the ability of improving the fluorescence intensity of ROS, the content of MDA, activities of SOD, GSH-Px, Ca2+-Mg2+-ATPs on10,20μg·mL-1dose group of orientoside was lower than the same dose group of luteolin (P<0.05, P<0.01), and the activities of CAT, Na+-K+-ATPs had no significant difference.Compared with VC group, the ability of improving the fluorescence intensity of ROS, the content of MDA, activities of SOD, GSH-Px, Ca2+-Mg2+-ATPs on10,20μg·mL-1dose group of orientoside and luteolin was lower than VC group (P<0.05, P<0.01), and the activities of CAT, Na+-K+-ATPs had no significant difference,40μg·mL-1dose group of orientoside and luteolin has no difference.2.2Effects on MDA, antioxidant enzymes, ATP s, SA of aging RBCHuman red blood cell were incubated for48hours, the content of MDA was increased, the content of SA was lower, the activities of antioxidant enzyme and ATPs was lower, compared with blank group, they had significant differences (P<0.01); After giving orientoside, luteolin and vitamin C, every indicators was improved, compared with modle group, they had significant difference (P<0.01), and also they had a positive dose-effect relationship.Compared with luteolin group, the improvement of every indicators on10,20μg·mL-1dose group of orientoside was lower than the same dose group of luteolin (P<0.05, P<0.01), and40μg·mL-1dose group of orientoside had no difference.Compared with VC group, the improvement of every indicators on10, 20μg·mL-1dose group of orientoside and luteolin was lower than VC group (P<0.05, P<0.01),40(μg·mL-1dose group of orientoside and luteolin has no difference.3SDS-PAGE results of RBC membraneHuman red blood cell was damaged by H2O2, spectrin, anchoring proteins, band3protein, band4.1a protein, band4.1b protein and actin were weaker or even disappeared, compared with blank group, they had significant difference. Afert giving orientoside, band3protein, band4.1a protein and actin had been restored, Afert giving luteolin, spectrin, band3protein, band4.1a protein and actin had been restored, and afert giving VC, spectrin, anchoring protein, band3protein, band4.1a protein, band4.1b protein and actin had been restored, and they had a positive dose-effect relationship. The protein recovery degree of orientoside group was weaker than the same dose group of luteolin, meanwhile,40μg·mL-1dose group of luteolin and20μg·mL-1dose group of VC recovered significantly.Human red blood cell were incubated for48hours, modle group showed that spectrin and anchoring protein had aggregation phenomenon, band4.1a protein increased, band4.1b protein decreased,4.1a/4.1b had increased, and compared with blank group, they had significant difference. After giving orientoside, luteolin and vitamin C, spectrin and anchoring protein resumed,4.1a/4.1b were lower, but there were no differences between every groups4SEM results of human RBC induced by H2O2and aging RBCThe results show that red blood cell induced by H2O2and aging red blood cell were normal with blank group, the red blood cell of modle group surface morphology changed, most of the red blood cells shrinking, surface extending many spines edge serrations,compared with blank, they had significant difference. After giving orientoside, luteolin and vitamin C, red blood cell had been restored, and they had a positive dose-effect relationship. 5TEM results of human RBC induced by H2O2and aging RBCThe red blood cell membrane skeleton structure of the model group were fuzzy, there was no network-like structure, the J point was not clear and SP4fractured, compared with the blank group there were significant differences. After giving orientoside, luteolin and vitamin C, the red cell membrane skeleton J point gradually restored, SP4gradually became complete, the hole in the membrane skeleton were gradually smaller and network skeleton structure gradually appeared, and they had a positive dose-effect relationship. High dose group of orientoside and luteolin and VC group had a clear J point and SP4became complete.To sum up, orientoside and luteolin could protect red blood cells which been damaged by H2O2and time, they could inhibit the hemolysis rate, decrease MDA content, increase activities of antioxidant enzymes and ATPs, increase the concentration of SA, restore cell surface morphology and membrane skeleton structure; the antioxidant capacity of low, middle dose group of orientoside was weaker than the same group of luteolin, and the antioxidant capacity of high dose group of orientoside was in keeping with VC. |
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