Study On Synergistic Effects And Mechanisms Of Ginsenoside Against Oxidative Stress In Human Embryonic Kidney 293 Cells

Ginsenosides are the main pharmacological components of Panax ginseng root, which are thought to be primarily responsible for the suppressing effect on oxidative stress. The present study is aimed to investigate the antioxidant effects of ginsenosides against H2O2-induced oxidative stress in human embryonic kidney 293(HEK-293) cells, to find ginsenosides with high activity and extract method with high effectiveness on yield and activity. In this work, the in vitro synergistic effects and mechanisms of ginsenoside against oxidative stress in HEK-293 cells were studied.At first, the pulsed electric field(PEF) extraction was applied to extract the ginsenosides from Panax ginseng root. The influences of electric field intensity, frequency and solid-to-liquid ratio were individually investigated and optimized. The results indicated that the highest yield of the ginsenoside was 0.981% ± 0.012% by PEF using the conditions of 60 KV/cm electric field intensity, pulse duration 8 ?s, and solid-to-liquid ratio was 1 : 100. The yield of the ginsenosides extracted by PEF is higher than the other four methods, including solvent cold soak extraction(SCSE), heat reflux extraction(HRE), ultrasound-assisted extraction(UAE) and microwave-assisted extraction(MAE). DPPH radical scavenging activity and oxygen radical absorption capacity(ORAC) were applied to evaluate the antioxidant activities of the ginsenosides. The PEF extracts displayed the higher DPPH radical scavenging activity and stronger oxygen radical absorption capacity. HEK-293 cells were incubated with H2O2 to establish oxidative stress damage cell model, and 400 ?M of H2O2 was choosen as the best concentration for injuring. The cell viability and surface morphology of HEK-293 cells were studied by MTS assay and scanning electron microscopy(SEM), respectively. The HEK-293 cell model suggested that the protective effect of ginsenosides was enhanced by PEF. DCFH-DA fluorescent probe assay was used to measure the level of intracellular reactive oxygen species(ROS), and further proved that the antioxidant activiry was improved by PEF, which showed have no related to its proliferation effect. The intracellular antioxidant activities of ginsenosides were evaluated by cellular antioxidant activity(CAA) assay. The EC50 of PEF extract is 150.03 ± 4.03 ?g/m L, which hold 2.5 times as SCSE(60.22 ± 2.69 ?g/m L). CAA values indicated that ginsenosides extracted by PEF had stronger cellular antioxidant activity than SCSE ginsenosides extracts.Secondly, DPPH radical scavenging activity was applied to filtrate the ginsenoside F1 with best activity from fourteen kinds of ginsenosides, followed by F2 and Rh2. The activities of PPD-type ginsenosides were better than PPT-type. But the activities of all the ginsenosides were poorer compared to Trolox(25 ?M). Ginsenoside Rb3 was filtrated with best activity by ORAC, followed by F1 and other common saponins, including Rb1, Rg1, Rg2 and Rb2. The activities of Rg3 and Ro were the poorest. The results suggested that the secondary metabolites of ginsenosides show stronger activities. And the activities of ginsenosides were different from their degradations and configurations. On the basis of oxidative stress HEK-293 cell model, ginsenoside F2 was filtrated with best cytoprotection, followed by Rb1 and Rg1, which are higher than damage group by 28%, 21% and 18%, respectively. To investigate the synergistic antioxidant effects, ginsenoside F2 was combined with antioxidant peptides from egg white(WNWAD and WLKFI), water-soluble vitamin E(Trolox), and cyanidin-3-O-glucoside(C3G). The results suggested that F2 and C3 G synergistically protect HEK-293 cells against H2O2-induced oxidative stress.In addition, the synergistic antioxidant effects and underlying mechanisms of ginsenoside F2(F2) and C3 G against H2O2-induced oxidative stress in HEK-293 cells were confirmed and investigated. The MDA level and activities of antioxidant enzymes were measured by corresponding assay kits. DCFH-DA fluorescent probe assay was used to measure the level of intracellular reactive oxygen species(ROS). The results showed that the combined pretreatment of ginsenoside F2 and C3 G significantly inhibited the generation of MDA and intracellular ROS, and increased the activities of antioxidant enzymes compared with the single pretreatment. Furthermore, quantitative real-time PCR(q RT-PCR) and Western blot were used to detect the proterin and m RNA expressions of Nuclear factor erythroid-derived 2(NF-E2)-like 2(Nrf2) and Kelch-like ECH associated protein 1(Keap1). Co-administrating F2 and C3 G significantly enhanced the protein and m RNA expressions of Nrf2 and reduced the expressions of Keap1 in a concentration-dependent manner. These results demonstrated that ginsenoside F2 and C3 G synergistically protect HEK-293 cells against H2O2-induced oxidative stress through reducing intracellular ROS, as well as activating Nrf2/Keap1 signaling pathway and intrinsic antioxidant activities.Finally, combined protective effects of ginsenoside F2 and C3 G against apoptosis mediated by H2O2-induced oxidative stress and the underlying mechanisms were explored in HEK-293 cells. The protein expressions of cleaved caspase-3, Bcl-2, Bax, cleaved PARP, and the levels of caspases-6, caspases-9, lactate dehydrogenase(LDH), Mitochondria membrane potential(MMP) were measured as the apoptosis associated markers. The phosphorylation of nuclear factor kappa B(NF-?B) p65 was assessed by Western blot and q RT-PCR. When HEK-293 cells were stimulated with H2O2, individual F2 and C3 G pre-incubation increased MMP, suppressed the leakage of LDH, significantly decreased the activities of caspases-6 and caspases-9, down-regulated the protein expressions of cleaved caspase-3, Bax, cleaved PARP, and up-regulated the expression of Bcl-2, down-regulated the protein and m RNA expressions of NF-?B p65. The synergistic effects were present in the combination of F2 and C3 G. The underlying mechanism of F2 and C3 G in synergistic anti-apoptosis was in partly relevant to the regulation of mitochondria-mediated cell apoptotic pathway and NF-?B pathway.In summary, this study showed that the ginsenosides exhibit synergistic effects on oxidative stress. The underlying mechanisms of synergistic effects of ginsenosides against oxidative stress are partly by targeting oxidative stress and apoptosis related signal pathways, which provide some theoretical basis on application of ginseng in food.