Protective Effects Of Curcumin On Chronic Alcohol-induced Liver Injury Of Mice And Its Mechanisms

Objective To investigate the in vivo protective effects of curcumin on chronic alcohol-induced liver injury of mice and to explore its underlying mechanisms.Methods (1) Balb/C mice were randomly divided into five groups with fifteen mice in each group, A:normal control group; B:curcumin (150 mg/kg, orally) control group; C:alcohol model group; D:alcohol+curcumin (75 mg/kg, orally) group. E:alcohol+ curcumin (150 mg/kg, orally) group. Mice in C, D and E groups were given 10 ml/(kg·bw·d) alcohol (56% degree) for two weeks. Then the alcohol dose increased to 12 ml/(kg-bw-d) for another two weeks. Thereafter, the alcohol dose increased to 15 ml/(kg·bw·d) for four consecutive weeks, totally eight weeks. Homologous curcumin were dissolved in olive oil and given to groups B, D and E 1 h prior to alcohol treatment. Changes of body weight were recorded weekly. Twelve hours after the last administration, animals were sacrificed by cervical dislocation under mild anesthesia. Blood samples were collected and centrifuged at 1,500 g/min at 4℃ for 10 min to obtain serum. Livers were totally excised from the mice and weighed to calculate the liver index (liver weight/body weight× 100%). Two pieces of tissues from the same liver lobe in each animal were fixed with formaldehyde for pathological examination. Serum levels of Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG), total cholesterol (TCH), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) were detected colorimetrically using commercially available kits.(2) Preparing 10% liver homogenate for determination of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities as well as the malondialdehyde (MDA) contents using the commercial assay kits.(3) Preparing hepatic mitochondria by differential centrifugation for examination of the mitochondrial antioxidant enzymes such as SOD and GSH-Px activities as well as MDA contents. Hepatic mitochondrial membrane potential (MMP) was determined by Rh123. Ca2+-induced membrane permeability transition pore (MPTP) opening was evaluated by spectrophotometric assay. Na+/k+-ATPase, Ca2+-ATPase and Ca2+Mg2+-ATPase activities in hepatic mitochondria were detected using commercial assay kits.(4) Using the western blot analysis, we detected the protein levels of peroxisome proliferator activated receptory coactivator-la (PGC-la), nuclear respiratory factor 1 (NRF1) and Mn-SOD, and nuclear erythroid 2-related factor 2 (Nrf2) protein levels in nuclei as well as the phosphorylation of ERK, p38 and JNK mitogen-activated protein kinase (MAPK). Glucose regulated protein 78 kD (GRP78) protein levels, phosphorylation of protein kinase R-like ER kinase (PERK) and inositol requiring 1α (IRE1α), indicative of endoplasmic reticulum stress (ERS), were also examined using the western blot analysis. Additionally, protein levels of nuclear transcription factor-KB (NF-κB) and phosphorylated levels of IκBα, indicative of inflammatory signaling, were evaluated.(5) mRNA levels of CuZn-SOD, GSH-Px, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1), the downstream anti-oxidative enzymes of Nrf2 were examined using quantitative real-time PCR.Results(1) Evaluation of protective effects of curcumin on chronic alcohol-induced liver injuryCompared to the normal control group, the body weight were decreased while the liver weight and liver index were significantly increased in alcohol model group (P< 0.05 or P< 0.01). Curcumin treatment significantly reduced the liver index in comparison with the alcohol model group (P< 0.05 or P< 0.01). HE staining results showed that in the normal control group liver sections had normal hepatic cell with preserved cytoplasm, distinct nucleus and central vein. Photomicrograph of a section from alcohol model mice liver showed loss of cellular boundaries, severe edema, microvesicular steatosis and hydropic degeneration of hepatocytes around the central and interlobular veins. In curcumin-treated groups, compared to the alcohol model group, disorder of hepatic cord arrangement has been restored, and edema and fatty degeneration of hepatocytes was significantly reduced. Compared to the normal control group, serum levels of ALT, AST, ALP, TG, TCH, LDL-C were significantly elevated while serum HDL-C levels were reduced in alcohol model group (P< 0.01), which were significantly improved after administration of curcumin at the doses of 75 mg/kg and150 mg/kg(P< 0.05 or P< 0.01).(2) Effects of curcumin on alcohol-induced hepatic oxidative stress and mitochondrial function in miceCompared to the normal control group, hepatic SOD and GSH-Px activities in alcohol model group were significantly reduced while MDA contents were significantly increased after ethanol exposure (P< 0.01). But curcumin administration at the doses of 75 mg/kg and 150 mg/kg robustly increased SOD and GSH-Px activities, and decreased MDA contents (P< 0.05 or P< 0.01). Meanwhile, alcohol exposure decreased the hepatic mitochondrial MMP and increased opening of the MPTP in comparision with the control group (P< 0.01), which were reversed by curcumin treatment (P< 0.05 or P< 0.01). Furthermore, compared to the normal control group, the activities of Na+/k+-ATPase, Ca2+-ATPase and Ca2+Mg2+-ATPase as well as SOD and GSH-Px in hepatic mitochondria were significantly decreased while MDA contents were increased in alcohol-exposed group, which were reversed by curcumin treatment(P< 0.05 or P< 0.01). Additionally, compared to the normal control group, the protein levels of PGC-la, NRF1 and Mn-SOD, closely related to mitochondria function, were significantly reduced in alcohol-exposed group, which were elevated by curcumin treatment (P< 0.05 or P< 0.01).(3) Effects of curcumin on Nrf2-mediated anti-oxidative signaling pathway in alcohol-induced liver injury in miceCompared to the normal control group, Nrf2 protein levels in nuclei as well as mRNA levels of Nrf2-mediated anti-oxidative gene such as CuZn-SOD, GSH-Px, HO-1 and NQO-1 were significantly decreased in alcohol-treated mice (P< 0.01), whereas curcumin treatment reversed these changes (P< 0.05 or P< 0.01). Furthermore, compared to the normal control group, chronic alcohol exposure decreased the phosphorylated levels of ERK, and p38. However, curcumin treatment at the doses of 75 mg/kg and 150 mg/kg significantly increased the phosphorylation of ERK and p38 (P< 0.05 or P< 0.01).(4) Effects of curcumin on ERS and inflammatory response in chronic alcohol-exposed miceCompared to the normal control group, protein levels of GRP78 as well as phosphorylated levels of PERK and IRE1α in alcohol-treated group were significantly increased (P< 0.01). However, curcumin treatment reversed these changes (P< 0.05 or P< 0.01). Meanwhile, alcohol exposure led to increase the NF-κB protein levels in nuclei as well as phosphorylated levels of IxBa when compared to the normal control group (P< 0.01). However, alcohol exposure led to enhance the hepatic levels of TNF-α, IL-1β and IL-6 (P< 0.01), which were attenuated by curcumin treatment (P< 0.05 or P< 0.01).ConclusionsThe present study showed that curcumin attenuated alcohol-induced liver injury through activation of ERK/p38/Nrf2-mediated antioxidant signaling pathways, thereby up-regulation of detoxifying gene expression such as CuZn-SOD, GSH-Px, NQO1 and HO-1, through improvement of mitochondrial function by upregulation of PGC-1α, NRF-1 and Mn-SOD protein expressions, as well as through attenuation of ER stress.