| OBJECTIVE Mitochondrial oxidative damage may be an important factor leading to development failure and restricted clinical application of many drugs. Exogenous antioxidants can significantly improve drug-induced mitochondrial oxidative damage. Resveratrol(Rev) is a promising natural antioxidant, and it has wide range of biological functions. This study focused on investigate the ability of resveratrol to ameliorate troglitazone-mediated mitochondrial damage and subsequent apoptosis of human hepatoma Hepa RG cells, and a preliminary exploration of its occurrence mechanism to provide data support for improving the hepatotoxicity induced by exogenous drugs.METHODS The experimental groups were divided into five groups: control group( RPMI 1640 only with 0.1% DMSO), Tro(50μM), Tro(50μM) +Rev(15μM), Tro(50μM)+Rev(7.5μM) and Tro(50μM)+Rev(3.75μM) group. MTT assay was performed to detect the viability of resveratrol-treated, troglitazone-treated and 50 μM troglitazone interaction with different concentrations of resveratrol-treated Hepa RG cells. Each group Hepa RG cells were treated 48 h, after that, the content of ROS were detected by loading DCFH-DA probe, meanwhile the levels of lipid peroxidation is detected by the content of malondialdehyde, which is producted by lipid peroxidation. The level of ATP synthesis, the activity of mitochondrial respiratory chain complexes Ⅰ-Ⅳand total antioxidant capacity of intracellular were detected by using multifunctional microplate reader. the changes of mitochondrial membrane potential was detected by JC-1 staining and the cell apotosis was detected by Annexin VFITC/PI staining. And changes in mitochondrial ultrastructure were observed by electron microscopy. the activity of hydrogenper-oxidase( catalase, CAT), glutathione peroxidase(GSH-px) and superoxide dismutase(SOD) were inspected. Western blotting was used to detect SIRT1, SIRT3, p53, Bcl-2, and caspase-3 protein levels to preliminary explorat the mechanism of action.RESULTS The consequence based on the results of MTT assay and the literature data. We the choice of 50 μM Tro-treated group and 50 μM troglitazone interaction with three concentrations of resveratrol-treated Hepa RG cells for 48 h. Compared with the control group, 50 μM troglitazone group of reactive oxygen species(ROS) levels were increased to 2.03±0.04 times.(P<0.01), the production of MDA also increased from 3.85±0.60 μmol/mg to 15.22±3.24 μmol/mg(P<0.01). And accompanied by decreased of ATP synthesis(P<0.05), the mitochondrial respiratory chain complexes Ⅰ, Ⅱ, Ⅲ and Ⅳ activity also corresponding decrease(49.15±1.97)%,(65.67±4.18)%,(64.54±3.97)% and(57.55±3.4)%(P<0.05), mitochondrial membrane potential and total antioxidant capacity dropped significantly, apoptosis and necrosis rate were correspondingly increased(P<0.05), from mitochondrial ultrastructure, we also observed serious swelling of mitochondria and vacuolization, crest fracture or even disappear. And after adding resveratrol, the levels of ROS and lipid peroxidation were dropped significantly, in which, the 15 μM resveratrol intervention action group, ROS levels decreased to 1.26±0.17 times of the normal control group, malondialdehyde content is much lower than the normal control group(P<0.05). The production of ATP were increased, but also slightly lower than the normal control group. The activity of mitochondrial respiratory chain complexes Ⅰ, Ⅱ, Ⅲ and Ⅳ activity were appropriate increased to( 103.27±3.61)%,( 100.07±3.87)%,( 93.36±3.23)%,( 89.56±2.75)%,( P<0.05), those were close to the normal control group. The mitochondrial membrane potential and total antioxidant capacity were increased, the cells apoptosis has also been improved, mitochondrial ultrastructure gradually become normal, we can see clear outline and whole structure and morphology which were close to the control group, and almost all of above were exhibited concentration gradient. After further exploration of the mechanism, we find 50 μM troglitazone lead antioxidant enzymes(catalase, glutathione peroxidase, superoxide dismutase) activity were significantly decreased, which resulted ROS can not be timely elimination.Western Bolt results showed that resveratrol can reduced apoptotic protein expressions of p53 and caspase-3, those were consistent with the result of cell apoptosis. After the intervention effect of resveratrol, those above antioxidants activity rebounded sharply( SOD 37.35±1.52 units/mg, CAT 799.39±51.62 units/mg, GSH-px 1054.54±33.65 m U/mg)(P<0.01), and it also can upregulated the protein expression of the deacetylases SIRT1 and SIRT3 as well as the expression of the anti-apoptotic protein Bcl-2, whereas the expression of the apoptotic proteins p53 and caspase-3 were down-regulated to improve troglitazone-induced apoptosis.CONCLUSION Troglitazone can damage the mitochondrial respiratory chain, mitochondrial membrane potential, normal mitochondrial structure, and reduce ATP synthesis, total antioxidant capacity, some antioxidant activity, it also can increased apoptotic proteins, etc., eventually leading to Hepa RG apoptotic or death.While resveratrol can protect Hepa RG cells from damage, may be take the following two aspects: on the one hand, it can be activating the mitochondrial respiratory chain complexes, reducing the generation of ROS, inhibiting lipid peroxidation, increase ATP production, and at the same time activate some antioxidant enzymes, eliminate deposition of ROS, to maintain a balance in vivo oxidant / antioxidant and other aspects to regulate stability of mitochondrial structure and function. On the other hand, it also can be raised deacetylase SIRT1 and SIRT3, deacetylation p53, inhibiting cell apoptosis, and at the same time acting on mitochondrial membrane MPTP hole, so that decreased the expression of pro-apoptotic protein caspasse 3 and promoting anti-apoptotic protein bcl-2, inhibition of apoptosis happen, and ultimately to protect the role of the liver. |
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