Protective Effect Of Curcumin On Liver Injury Induced By Endoplasmic Reticulum Stress In Mice

It is well accepted that liver disease has a development model of acute, chronic, liver fibrosis, cirrhosis and liver cancer. The diseases of liver system have become a major illness in clinical areas, including drug-induced liver disease, alcoholic liver disease, chemical-induced liver disease and so on. According to World Health Organization (WHO) statistics, the mortality of liver disease has risen to the fifth cause of death in the world. Therefore, the research on the pathogenesis and drug therapy of various liver diseases is increasingly important. Because of a wide range of pathogenic factors, endoplasmic reticulum (ER) stress-induced diseases have become the research hotspot in recent years. And also it’s an important pathogenic factor of many diseases including liver, so to establish a reliable animal modal of ER-induced liver injury and explore its pathogenesis have important significance to the prevention and treatment of liver injury.Curcumin (curcumin) has the functions of antioxidant, anti-tumor, anti-inflammatory, and free radical scavenge, and play an important role in the prevention and treatment of neurodegenerative diseases, cardiovascular, pulmonary and other chronic inflammatory diseases. Recent studies have demonstrated that curcumin has a fairly good protective effect on liver fibrosis and non-alcoholic fatty liver. However, it has not been reported whether curcumin has a protective effect on ER stress-induced liver injury.To study the protective effect of curcumin on ER stress-induced liver injury and its possible mechanism, we intended to establish a model of ER stress-induced liver injury by oral administration of tunicamycin. Research content includes following three aspects:1. Endoplasmic reticulum stress-induced liver injury modelTo establish an ER stress liver injury model induced by tunicamycin and observe pathological changes, biochemical markers, liver index GRP78 levels and liver index in mice. Biopsy results showed that normal hepatocytes are arranged radially around central vein, hepatic lobule structure is integrity, normal size hepatocytes, no degeneration and necrosis, no inflammatory cell infiltration in interstitial. There were significant liver tissue lesions in model group:swelling of the liver cells in most lobular, puff of cytoplasm, and some were ballooning, some have visible changes of apoptosis and necrosis. Biochemical results showed that compared with normal group, the levels of ALT and AST were significantly increased in model group (P<0.01). Liver index showed that compared with normal group, there are no significant changes in body weight of mice and the levels of liver index are significantly increased (P<0.01). Variance of the three indicators demonstrated that tunicamycin successfully induced liver injury. Glucose-regulated protein 78/immunoglobulin heavy chain binding protein (GRP78/Bip) is the chaperone of endoplasmic reticulum, and its up-regulation is a marker of endoplasmic reticulum stress (ERS). In this study, the mRNA and protein levels of GRP78/Bip were detected by qPCR and SP immunohistochemical techniques. qPCR results showed that compared with normal group, the ratio of GRP78/GAPHD in mice liver tissues increased significantly in model group(P<0.01). Immunohistochemical results showed that in normal group, there are no brown particles in the liver cytoplasm and the staining reaction is negative, whereas a lot of brown particles appear in the model group, the color is depth, the volume is crude and dense and the staining reaction is strong positive. Semi-quantitative analysis showed that the positive rate is 2% in normal group and 98% in model group, which indicated that tunicamycin leads to liver injury by playing roles in the endoplasmic reticulum stress.2. To investigate the protective effect of curcumin on tunicamycin-induced liver injury Biopsy results showed that curcumin groups (75、150、300 mg/kg) can alleviate the lesion scope and extent of hepatocytes with a dose-dependent and the infiltration of inflammatory cells is reduced. Biochemical results showed that compare with model group, curcumin groups (75、150、300 mg/kg) reduced elevated levels of ALT in serum (P<0.01). Liver index showed that compare with model group, curcumin groups (75、 150、300 mg/kg) reduced elevated levels of AST in serum (P<0.01 or P<0.05). Variance of the three indicators demonstrated that curcumin has a protective effect on liver injury. Viewed from the statistical analysis of biochemical indicators and liver index as well as the degree of pathological changes, we find that the protective effect of curcumin on liver injury is dose-dependent.3. To explore the mechanisms of curcumin on tunicamycin-induced liver injury3.1 The effects of curcumin on the content of mice liver homogenate MDA, SOD and GSH in tunicamycin-induced liver injury.Values of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) in tissue are used to detect antioxidant activity index, in which the up-regulation of GSH, SOD indicated the enhanced oxidation resistance, and MDA as end products of lipid peroxidation indicated the degree of oxidation. The experimental results show that compared with normal group, MDA levels in liver homogenates increased significantly, while GSH, SOD levels were significantly reduced in model group which indicated that tunicamycin-induced liver injury is associated with the imbalance of oxidation and antioxidant in vivo. Compared with model group, curcumin groups (75、150、300 mg/kg) can reduce the elevated levels of MDA in liver homogenates (P<0.01) and curcumin groups (150、300 mg/kg) can increase the reduced values of GSH, SOD(P<0.05), which indicated that the protective effects of curcumin on liver may associate with the enhanced reaction of eliminating and inhibiting free radicals as well as avoiding excessive lipid peroxidation, and suggested that its ability of antioxidant is dose-dependent.3.2 The effects of curcumin on the apoptosis of mice hepatocytes in tunicamycin-induced liver injury.In this study, situ nick end labeling (TUNEL method) was used to detect the apoptosis of hepatocytes. Experimental results showed that in normal group, there are no dark brown particles in the liver cytoplasm and the staining reaction is negative, whereas a lot of dark brown particles appear in the model group, the color is depth, the volume is crude and dense. Curcumin groups (75、150、300 mg/kg) can alleviate the lesion scope and extent of hepatocytes, which indicated that curcumin may protect liver injury by inhibiting apoptosis.3.3 The effects of curcumin on the GRP78/Bip of liver index in tunicamycin-induced liver injury.qPCR results showed that compared with model group,curcumin groups (75、150、300 mg/kg) can reduce the elevated levels of GRP78/Bip in mice liver tissues (P<0.01或 P<0.05). Immunohistochemical results showed a lot of brown particles appear in the model group, the color is depth, the volume is crude and dense. Curcumin groups (75、 150、300 mg/kg) can alleviate the lesion scope and extent of hepatocytes.Semi quantitative showed that positive rate reduced significantly (85%、45%、10%),which indicated that curcumin may protect liver injury by reduce the elevated levels of GRP78 /Bip.In summary, the above results demonstrated that curcumin has a significant therapeutic effect on ER-stress liver injury induced by tunicamycin. The mechanism may be related to the regulation of antioxidant and inhibition of hepatocytes apoptosis.