Effects Of Two-phase Metabolic Modification On Curcumin And 6-Gingerol In Protecting PC12 Cells From Oxidative Damage

Oxidative damage is one of the important causes of neurodegenerative diseases.In the nervous system,when?-amyloid aggregation,dopamine oxidation and cerebral ischemia occur,H₂O₂ is generated and converted into hydroxyl radicals,which causes damages to lipids,proteins and DNA,resulting in neuronal cell apoptosis.Curcumin and 6-gingerol,two natural phenolic compounds,are the main components of ginger.They are well-known for various biological activities such as anti-oxidation,anti-inflammatory and anti-tumor.However,after entering the human body,these phenolic compounds often undergo two-phase metabolic processes to generate a variety of metabolites.Therefore,it is of significance to investigate the biological activity of these metabolites in order to reveal the preventive and therapeutic mechanism of dietary polyphenols on various diseases.In the present study,curcumin,6-gingerol and its major metabolic modifications?tetrahydrocurcumin,curcumin-?-D-glucuronide,6-shogaol,6-gingerol-4?-O-?-glucuronide?were examined for protective effects against oxidative damage in rat adrenal pheochromocytoma PC12 cells exposed to H₂O₂.The main contents and obtained results are summarized as follows:?1?Protective effects of curcumin,6-gingerol and its metabolites on PC12 cells agaist H₂O_2-induced oxidative damage.In H₂O₂-induced PC12 cells,the effects of curcumin,6-gingerol and their metabolites on cell survival rate,lactate dehydrogenase?LDH?release,intracellular reactive oxygen species?ROS?and malondialdehyde?MDA?content were investigated.The results showed that curcumin,6-gingerol and its metabolites all exhibited some degrees of protective effects on PC12 cells,which improved cell survival rate,reduced LDH release,and suppressed intracellular ROS and MDA levels.Except for 6-shogaol,these metabolic modifications exhibited weaker protective effects on H₂O₂-induced PC12 cells than their parent compounds.?2?Effects of curcumin,6-gingerol and its metabolites on antioxidant enzyme activity in H₂O₂-induced PC12 cells.Intracellular superoxide dismutase?SOD?activity was determined by the commercial kits,and the mRNA and protein expression of heme oxygenase 1?HO-1?and NAD?P?H quinone dehydrogenase 1?NOQ1?were measured by fluorescent quantitative PCR and Western Blot,respectively.The results showed that,compared with H₂O₂-treated group,the groups treated with curcumin,6-gingerol and its metabolites had higher SOD activity,and two-phase detoxifying enzymes?HO-1,NQO-1?mRNA and protein levels in PC12 cells.However,with the exception of 6-shogaol,the inducing effect of these metabolites on the antioxidant enzyme activity was significantly?P<0.05?weaker than that of their parent compounds.?3?Regulation mechanism of curcumin,6-gingerol and its metabolites on Nrf2-Keap1signaling pathway in PC12 cells exposed to H₂O₂.The experimental results show that curcumin and 6-gingerol promoted the expression of Nrf2 mRNA and proteins in PC12 cells,reduced the expression of Keap1 protein,and promoted the transfer of Nrf2 from the cytoplasm to the nucleus,thereby activating Nrf2 as well as downstream HO-1 and NQO-1.The protective effects of these metabolites exhibited a time-dependent manner.However,the two-phase metabolic modification has a certain effect on the activation of Nrf2 by curcumin and 6-gingerol.Among them,6-shogaol enhanced the activation of Nrf2 in PC12 cells.By contrast,the activation of Nrf2 by the other three metabolites was weakened.These data suggest that the two-phase metabolites affect the Nrf2 activation,leading to changes of its antioxidant activity of their parent compounds.After the expression of Nrf2 in PC12 cells was silenced by siRNA transfection,it was found that the protective effect of active ingredients on PC12 cells almost disappeared,indicating that Nrf2 might be a main target for them to exert anti-oxidative activity.