Effects of Different Oils Combined with Lycium Barbarum Extract on Chemical-Induced Liver Inflammation and Fibrosis in Rats

According to the statistical report by the Ministry of Health and Welfare, chronic liver diseases including cirrhosis have become the 7th leading cause of death in Taiwan, and 4,975 patients died from these diseases during 2012. Approximately 2.5-3 million persons carry hepatitis B surface antigen in Taiwan. Hepatitis B infection was strongly associated with the development of liver cirrhosis and hepatocellular carcinoma. Therefore, the prevention and therapy of hepatitis become one of the most important research issues in Taiwan. Our previous study showed that combined Lycium barbarum and Rehmannia glutinosa extracts significantly improved liver function index and alleviated liver inflammation and fibrosis in rats with CCl4-induced liver damage. Lycium chinense is in the family Lycium, Solanaceae, and Solanales, and its fruit goji or wolfberry, particularly Lycium barbarum L., has been widely used for traditional Chinese medicine. The active components of Lycium barbarum L. including polysaccharides, zeaxanthine, betaine, cerebrosides, taurine, scopoletin, pyrrole derivatives, and trace amounts of zinc, iron, and copper, have been reported to facilitate blood flow, lower blood glucose, and protect against fatty liver and atherosclerosis, and have beneficial effects on the functions of eyes, liver, and kidney. Additionally, olive oil rich in monounsaturated fatty acids can protect against non-alcoholic steatohepatitis and liver fibrosis, which exerts anti-inflammatory, antioxidant, immunomodulatory, and liver regeneration-mediated actions. The active components of olive oil include oleic acid, polyphenolic compounds (such as hydroxytyrosol), squalene, lignans, and other minor compounds. This 3-year proposal will be conducted for the following objectives: the 1st year: to prepare water-soluble and -insoluble Lycium barbarum extracts primarily containing polysaccharides and carotenoids, respectively, analyze the compositions of different Lycium barbarum extracts, and determine the most potential Lycium barbarum extract with anti-fibrotic effects in activated hepatic stellate cells (HSCs), the 2nd year: to investigate different dietary oils (olive oil vs. soybean oil) combined with Lycium barbarum extract (the most potential extract from the in vitro study in the first year) on liver inflammation and fibrosis induced by CCl4 in rats, and the 3rd year: to study the possible mechanisms of active components of dietary oil and/or Lycium barbarum extract per se in regulating inflammation in macrophages via mediating signal transduction of inflammatory response, and regulating liver fibrosis in activated HSCs via mediating signal transduction of hepatic fibrogenesis and apoptosis. In the first year, the fruit of Lycium barbarum will be extracted by hot water and organic solvents, respectively, and total carbohydrate, crude protein, crude fat, water, ash, monosaccharides, total polyphenols, and carotenoids will be analyzed in water-soluble and -insoluble Lycium barbarum extracts. The effects of Lycium barbarum extracts (0-1,000 g/mL) on cell viability, cell toxicity, and anti-fibrotic activity in rat activated HSC-T6 cells by H2O2 will be investigated, and the most potential Lycium barbarum extract will be determined and used for the following studies. In the second year, male Sprague-Dawley rats will be randomly divided into 7 groups: control, CCl4+5% soybean oil, CCl4+5% olive oil, CCl4+2.5% soybean oil+2.5% olive oil, CCl4+5% soybean oil+Lycium barbarum extract (0.1%, w/w; 50 mg/kg bw), CCl4+5% olive oil+Lycium barbarum extract (0.1%, w/w; 50 mg/kg bw), and CCl4+2.5% soybean oil+2.5% olive oil+Lycium barbarum extract (0.1%, w/w; 50 mg/kg bw) groups. Rats will be given the treatments one week before CCl4 injection. Carbon tetrachloride will be injected intraperitoneally once a week for 7 weeks. After 8-week treatments, liver will be excised for histopathological semi-quantitation of cell necrosis, fat accumulation, inflammation, and fibrosis. Blood samples will be collected for the indicators of hepatic necrosis. Inflammatory markers in plasma and liver, fibrosis regulators in liver, apoptosis in hepatocytes, and protein expression of apoptotic regulators in liver will be measured. In the third year, the effects of oleic acid (0-200 M), linoleic acid (0-200 M), and/or Lycium barbarum extract (0-1,000 g/mL) on cell viability and cell toxicity in rat HSC-T6 and mouse RAW 264.7 cells will be determined. After the treatments, protein and mRNA expression of inflammatory regulators for signal transduction in RAW 264.7 cells after stimulation by lipopolysaccharide will be investigated. Apoptosis, protein and mRNA expression of fibrogenic and apoptotic regulators for signal transduction in HSC-T6 cells will be measured after activation by H2O2. The results of this proposal provide such information in the potentially preventive or therapeutic role of olive oil and Lycium barbarum extract for protection against the development of liver inflammation and fibrosis in the future clinical trial.