| ?-Lipoic acid(?-LA) is a free radical scavenger and potent natural antioxidant. It can scavenge numerous free radicals such as reactive oxygen and nitrogen species; It can chelate metal ions; It can regenerate other antioxidants. ?-LA is a critical component of the antioxidant network because of it regenerating other antioxidants. It can work both in water and fat, which gives it an unusually broad spectrum of antioxidant action. Consequently, ?-LA has been termed as the ‘ideal' antioxidant.Cadmium(Cd) is a heavy toxic metal that is ubiquitously present in the environment. Because of carcinogenic, teratogenic and mutagenic effects of cadmium, cadmium is an important environmental hazardous substances. Cadmium is toxic and carcinogenic in various organs, such as kidney, liver, bone and testis. Some studies have demonstrated that cadmium- related diseases including carcinogenesis and hepatotoxicity are related to cadmium- induced oxidative damage.Glutathione(GSH) is a tripeptide,which has an important physiological functions. GSH is present in two forms, reduced glutathione(r GSH) and oxidized glutathione(GSSG). r GSH plays a major role in cellular defense against oxidative and nitrosative stress, maintenance of cellular homeostasis and protection of the biological macromolecules oxidative damage.Therefore, from the point of view of regenerating GR by ?-LA, the research of the effect of ?-LA protecting against the oxidative damage induced by cadmium and regenerating glutathione in HepG2 cells. It will provide important information on whether ?-LA can be used as a potential therapeutic agent against cadmium- induced oxidative damage diseases. Part? Protection of ?-LA on the oxidative damage induced bycadmium in HepG2 cellsObjective: To determine whether ?- LA protects against the cytotoxicity and oxidative stress induced by cadmium in HepG2 cells.Methods: The Hep G2 cells were divided into normal control group, 100?M ?-LA treated group, 25?M cadmium chloride treated group, 10?M ?-LA and 25?M cadmium chloride co-treated group, 50?M ?-LA and 25?M cadmium chloride co-treated group, 100?M ?-LA and 25?M cadmium chloride co-treated group. Cells were treated with 100?M ?-LA and 25?M cadmium chloride for 16 h respectively. For the co-treatment groups, cells were pre-treated with ?-LA(10, 50 and 100?M) for 8h followed by treatment with 25?M cadmium for 16 h in the presence of ?-LA. Cell viability was measured by MTT assay, the levels of was measured by DCFH-DA method.Results: Compared with untreated cells, the viability of the cells treated with25?M cadmium alone was significantly decreased(p < 0.01), The level of ROS in the HepG2 cells treated with 25?M cadmium was significantly increased(p < 0.01); Compared with cells treated with 25?M cadmium alone, the viability of the cells treated with ?-LA(50 and 100?M) and 25?M cadmium was significantly elevated(p < 0.01), the level of ROS in the HepG2 cells treated with ?- LA(10, 50 and 100?M) and 25?M cadmium was significantly reduced(p < 0.01 or p < 0.05).Conclusion: Our results show that that ?-LA can antagonize oxidative stress and alleviate the cytotoxicity induced by cadmium in HepG2 cells.Part? The mechanism of the regeneration of rGSH by ?-LAObjective: To investigate how ?-LA replenishes rGSH through glutathione reductase in HepG2 cells treated with cadmium.Methods: As was mentioned above, the cells were treated with cadmium and ?-LA for 16 h. The intracellular concentrations of rGSH and GSSG were measured with the rGSH and GSSG assay kit, GR activity was measured by DTNB method, the mRNA levels of GR was measured by RT-PCR, the protein of GR was measured by western blotting.Results: Compared with untreated cells, the r GSH contents and the ratio of rGSH/GSSG in Hep G2 cells treated with 25?M cadmium were significantly decreased(p < 0.01). GR activity, the protein and mRNA levels of GR in Hep G2 cells treated with 25?M cadmium were also significantly decreased(p < 0.01 or p < 0.05); Compared with cells treated with 25?M cadmium alone, the r GSH contents and the ratio of rGSH/GSSG in the Hep G2 cells treated with ?-LA(50 and 100?M) and 25?M cadmium were significantly increased(p < 0.01), GR activity, the protein and mRNA levels of GR in the HepG2 cells treated with ?-LA(50 and 100?M) and cadmium were also significantly elevated(p < 0.01 or p < 0.05).Conclusion: These results suggested that the regeneration of rGSH by ?-LA was mediated by induction of gene and protein expressions of GR, and the increase of GR activity.Part? Effects of Nrf2/ARE signal pathway on regeneration ofglutathioneObjective: To explore the effect of Nrf2/ARE signal pathway on regeneration of glutathione.Methods: As was mentioned above, the cells were treated with cadmium and ?-LA for 16 h. The protein levels of p-Nrf2 and Nrf2 were measured by western blotting. Immunocytochemical staining was used to observe the influence of ?-LA on nuclear translocation of Nrf2 in cadmium-treated HepG2 cells.Results: Compared with untreated cells, the ratio of p-Nrf2/Nrf2 in Hep G2 cells treated with 25?M cadmium was significantly increased(p < 0.01). Compared with the cells treated with cadmium alone, the ratio of p-Nrf2/Nrf2 in the cells co-treated with ?-LA(10, 50 and 100?M) and cadmium was significantly elevated(p < 0.01).Nrf2 was immunocytochemical stained. There was no nuclear staining of Nrf2 in untreated cells, and Nrf2 was only in the cytoplasm. Very little nuclear Nrf2 staining was found in the cells treated with 25?M cadmium and treated with 100?M ?- LA respectively. The Nrf2 staining in the cells co-treated with 25?M cadmium and ?- LA(10 and 50?M) was present both in the cytoplasm and in the nucleus. In the 25?M cadmium and 100?M ?-LA co-treated cells, most of the Nrf2 staining was found in the nucleus.Conclusion: Nrf2/ARE signal pathway may play an important role on the regeneration of r GSH by ?-LA. We also found that ?-LA induced phosphorylation of Nrf2 and dose-dependently upregulated the nuclear translocation of Nrf2. It indicated that ?-LA regenerated r GSH through up-regulation of Nrf2 signaling pathway and induction of Nrf2 nuclear translocation. But the effects of Nrf2/ARE signal pathway on the regeneration of r GSH need to verify through Nrf2 inhibitor and Nrf2 knockout animals. |
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