| As a serious global issue on public health, people pay great attention on alcohol abuse and alcohol dependence, especially focus on teenager's drinking. Liver is the primary metabolism organ that the alcohol absorbs behind, is also the main target organ that the alcohol attached. Various mechanisms involved in the liver damnification, but oxidative damage is the common change in the initial stage of these diseases. It is the main performance that high generation of reactive oxygen species (ROS), imbalance of antioxidant system, lipid metabolic disorder, as well as biomacromolecule denaturation and generation of bulky adducts. All of this perfomence leads to the disorder of cell construction and function.Curcumin is a kind of natural antioxidant, belonging to polyphenol compound and it is almost innoxious to human beings. It shows a very strong anti-oxidative activity not only in food system, but also in endogenesis system. Recently, it was attracted particular interest as it is lead to exhibit the development of oxidize-related diseases. Its extensive biological activity includes anti-inflammation, anti-oxidation, anti-mutagenesis, reducing cholesterol, anti-artherosclerosis and anti- tumor (the DNA damage in anti-tumor process) etc. Curcumin has the power to clear free radicals, increase the free radicals scavengers and induce antioxidase activity besides the natural anti-oxidative activity for itself.Curcumin is the natural inducer of heme oxygenase-1 (HO-1). Among various genes encoding for proteins in possession of antioxidant characteristics, heme oxygenase-1 (HO-1) has been attracted interest for its modulattion by stress conditions and its antioxidant capacity. As a potential preferred target gene for preventing oxidative stress, increasing evidences support that induction of HO-1 represents an adaptive response or enhanced resistance to various oxidative stresses and diseases. Motterlini has confirmed that the HO-1 in endotheliocyte and nuerocyte can be induced by caffeic acid phenethyl ester (CAPE) and curcumin. Therefore, we can suppose that the antioxidative effects of these natural materials probably have related to activate HO-1 pathway. The recent research also confirms that curcumin help endotheliocyte to resist oxidative stress via inducing HO-1 expression under hypoxia environment.The HO-1 gene is regulated at transcription level. It is definite that certain antioxidant-response elements (ARE) lie at the upstream of promoter of HO-1 encoding gene. The discovered conjugated protein of ARE contains Nrf2 and MafK. The former can be activated by antioxidant and the latter is contrapositive. The activity of Nrf2 is usually inhibited via combination with chaperonin Keap1, which exists in cytoplasm. But, once inhibition disappears along with the accumulation of eletrophilic group or compounds that can modify mercaptan group, Nrf2 translocate to cytoblast and effect to ARE.According to above analysis, we hypothesize that curcumin induce HO-1 express via Nrf2/ARE signal pathway against ethanol-induced heptocyte damage. ObjectiveThis research plans to confirm the antioxidation of curcumin against ethanol-induced heptocyte damage and observe the molecular mechanism of curcumin inducing HO-1 and Nrf2 with hepatocyte as research object.MethodsIn the present study, we use different density ethanol to stimulate hepatocyte, and assemble cell at different time. Then we determine the activity of LDH, AST which released from hepatocyte, and determine the cellular MDA, SOD, GSH activity in order to observe the time-dose-response effects and establish the hepatocyte oxidative damage model. At the second part of this study, we use curcumin to pretreat hepatocyte before ethanol intake, and observe the time-dose-response defend effects of LDH, AST, MDA, SOD, and GSH in order to confirm curcumin can defend against ethanol-induced hepatocyte damage. For confirming the HO-1/Nrf2 pathway plays a important role in curcumin antioxidant, we use hemin (classic inducer of HO-1) and ZnPPIX (classic inhibitor) to stimulate hepatocyte, and comparing the LDH, AST, MDA, SOD, GSH lever with the result of curcumin stimulation, as well as abstract microsome via gradient centrifugation to determine the activity of HO-1 and protein expression. On this foundation, using western blot method to study the change of Nrf2 protein expression after curcumin stimulation with different MAPK signal pathway inhibitors in order to observe the molecular mechanism involved in relationship of curcumin and HO-1/Nrf2 pathway.Result(1) It is positive correlation between ethnaol stimulation dose and time and degree of damage. The vigor of hepatocyte degrade and degree of lipid peroxidation augment as well as the antioxidase degrade along with the increase of ethanol stimulate dose and time, it is significant difference at 100 mmol/L or 8 h comparing with normal hepatocyte.(2) Curcumin can protect hepatocyte against ethanol-induced damage. Pretreatment of curcumin can reduced GSH consume and inactivity of antioxidase and lipid peroxidant, which are provoked by ethanol. At the other hand, Curcumin can increase the cell metabolic activity and balance the antioxidative system to protect hepatocyte against oxidative damage.(3) Curcumin can induce HO-1 activity and protein express to educe antioxidation. 15 umol/L and 6 h is the befitting dose and time for curcumin to induce HO-1 activity. HO-1 activity is at the top when using 15μmol/L curcumin pretreated hepatocyte before ethanol stimulation. (4) ERK signal pathway inhibitor PD980059, P38 signal pathway inhibitor SB203580 and JNK signal pathway inhibitor SP600125 can not inhibit curcumin to induce the express of Nrf2 protein; but PI3K signal pathway inhibitor Wortmannin and LY294002 can inhibit curcumin to induce the express of Nrf2 protein. ConclusionCurcumin can protect hepatocyte against ethanol-induced oxidation damage. It is the main performance that increasing cell activity, reducing cell damage, raising antioxidase level and mitigating the degree lipid peroxidation as well as induce HO-1 activity and protein expression.HO-1 plays an important role in curcumin's antioxidation, its mechanism has the relationship with Nrf2 pathway, PI3K is the primary pathway leads to Nrf2 transfer to nucleolus. |
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