Effect Of Quercetin/hemeoxygenase-1/bilirubin System On Ethanol-induced Injury Of Rat Hepatocytes

Background With the improvement of people's living standards and the changes of lifestyle, alcoholic liver disease (ALD) has increasingly become the main liver disease just ranking after infectious liver diseases. The prevention and treatment strategies, hence, have correspondingly been acknowledged as an important public health problem in the world. In the latest decades, oxidative damage and inflammatory stress have attracted much attention in the pathogenesis of ALD. Nowadays, more and more studies have shown that quercetin possesses effective antioxidation, anti-inflammation, anti-apoptosis, and other biological activities against various diseases, including ALD. The protective effect of quercetin on ALD is thought to be involved in the induction of heme oxygenase-1 (HO-1), which may strengthen the haptic antioxidant system.However, bilirubin, a product of heme metabolism via HO-1 catalysis, was regarded as a toxic product in the past decades. Recently, extensive studies have found that bilirubin appears to exert potent antioxidant and anti-inflammatory effects at normal physiological concentration. Nevertheless, there is still little evidence whether bilirubin itself participate or mediate in the beneficial effects of HO-1 to prevent ethanol-induced hepatocytes injury or not. Thus, the present study concerning the protection of bilirubin, will not only further explore the protective mechanism of quercetin / HO-1 on hepatocytes injury resulted from ethanol, but also provide much more information about biological role of bilirubin.Objective 1. To investigate the potential protective effect and dose-response of bilirubin originated from catalytic metabolites of HO-1 induced by quercetin against ethanol-induced oxidative damage and apoptosis in rat hepatocytes.2. To explore the underlying protection of bilirubin against oxidative damage and inflammatory stress in hepatocytes.Methods1. Primary hepatocytes were isolated from Sprague-Dawley rat using a two-step collagenase perfusion procedure and incubated with 200 mmol/L ethanol for 24 h to induce oxidative damage. Meanwhile, the ethanol-intoxicated hepatocytes were co-treated with quercetin (100μmol/L), hemin (20μmol/L) or different doses of bilirubin (2, 10, 25 or 100μmol/L), etc. The redox indices were detected by spectrophotometry, which included the leakage of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), the activity of superoxide dismutase (SOD), as well as the levels of malondialdehyde (MDA), glutathione (GSH), direct bilirubin, and reactive oxygen species (ROS). In addition, mitochondrial membrane potential and apoptosis rate were determine by flow cytometry2. Exogenous inflammatory factors, TNF-α(5 ng/ml) plus IL-6 (2 ng/ml), were introduced to the system of ethanol-incubated (200 mmol/L) rat hepatocytes to mimic ethanol-derived inflammatory and oxidative stress on the cells. Besides ethanol and inflammatory factors, bilirubin (25μmol/L) was also added into medium for 24 h incubation, and then redox parameters mentioned above were detected to evaluate the potential effect of bilirubin on rat hepatocytes with ethanol-induced oxidative damage and inflammatory stress.Results1. Quercetin inhibited the overproduction of MDA and ROS, decreased enzymatic leakage of AST and LDH, suppressed GSH depletion and SOD inactivation, increased the level of endogenous direct bilirubin, maintained mitochondria membrane potential and reduced apoptosis rate in heaptocytes induced by ethanol. The similary effect was observed when ethanol-treated hepatocytes were incubated with hemin. ZnPP-IX, an inhibitor of HO-1, abolished the protective effect of quercetin on ethanol-treated hepatocytes.2. At the mild concentration (25 or 10μmol/L), bilirubin manifested protective activity by decreasing in the production of MDA and ROS, down-regulating GSH depletion and SOD inactivity, and reducing the enzymes leakage from hepatocytes substantially derived from ethanol, which was similar as quercetin or hemin described above in a dose-dependent manner. Low dose of bilirubin (2μmol/L) failed to exhibit the protective effect. Notably, high concentration (100μmol/L) exerted cytotoxicity to some extent, but also aggravated the detrimental effect of alcohol.3. Quercetin maintained mitochondria membrane potential and reduced apoptosis rate in hepatocytes injury resulted from ethanol. The similar function was found when ethanol-treated hepatocytes were incubated with hemin. ZnPP-IX abolished the protective effect of quercetin on ethanol-treated hepatocytes. Exogenous bilirubin (25μmol/L) maintained mitochondria membrane potential and reduced apoptosis rate of ethanol-treated hepatocytes.4. Compared with the alcohol exposure alone, inflammatory factors and ethanol co-treatment aggravated the leakage of AST and LDH, GSH depletion, SOD inactivity, as well as the eruption of MDA and ROS in hepatocytes injury. Such detrimental effect was also substantially inhibited by exogenous bilirubin (25μmol/L).ConclusionsQuercetin prevented rat primary hepatocytes from ethanol-induced oxidative damage and inflammatory stress, inhibited ethanol-elicited mitochondria membrane damage and apoptosis of hepatocytes through inducing HO-1. Bilirubin originated from HO-1 catabolism might mediate the protective effect rather than toxic product.