| BackgroundNAFLD is a clinical syndrome without excess alcohol intake and has the pathological characteristics such as hepatic steatosis and lipid accumulation. The incidence of this disease is increasing with average25%-30%of the world’s population involvement, exceeding the incidence of hepatitis B, hepatitis C and alcoholic liver disease and become the most common liver disease. In recent years, with the improvement of people’s living standards and lifestyle changes. China’s prevalence rate showed an increasing trend. However, the pathogenesis of NAFLD is still unclear.Previous studies have shown that the pathogenesis of NAFLD is related to insulin resistance (IR) and studies in recent years have shown that cavl is related to IR[2-7] indicating that cavl may play a pivotal role in the metabolic diseases.There are several studies have shown that up-regulation of cavl enhance the ox-LDL absorption of HepG2cell markedly, down-regulation of cavl could inhibit HepG2uptake of long chain fatty3H oleic acid9]; implying the role of cavl in mediating lipid uptake may enhance the progression of NAFLD. However, whether cavl participates in the pathogenesis and progression of NAFLD is still unclear.This article established C57BL/6mouse model of NAFLD induced by high fat and cholesterol diet, to detect serum lipid concentration, cavl mRNA and protein levels in NAFLD livers of mice, as well as changes in the distribution by automated biochemical analysis, qPCR, Western blot, immunohistochemical examination, to explore the role of cavl in the formation of NAFLD.ObjectiveThe animal model of NAFLD was established by giving C57BL/6mice high fat and cholesterol diet, to observe changes of mouse cavl mRNA, protein as well as distribution in the steatotic hepatocytes, and then explore its role in the pathogenesis of fatty liver disease.Methods1The animal model of non-alcoholic fatty liver disease (treatment group) was established by giving C57BL/6mice with14-week high fat and cholesterol diet, which is based on normal diet (fat<4%, cholesterol<0.02%) by adding15%lard and1.25%cholesterol. And animal anatomy was performed to collect serum and liver for determination later.2Once the serum of all mice was collected, TG, TC, HDL-C and LDL-C between two groups were determined.3RNA was extracted from mouse liver tissue and reverse transcripted; real-time quantitative PCR (SYBR) was performed according to cavl primer to analyze cavl mRNA by2-△△Ct method.4Western Blot was performed after total protein extraction from mouse liver to observe mice hepatic cavl protein in the treatment group and the control group, further to confirm whether the changes are corresponding with the mRNA changes.5Mice livers were fixed in formalin, H&E staining was done to observe hepatic changes between the two groups, and NAS points were counted.6Then the immunohistochemistry was done to further determine the characteristics and changes of cavl distribution in hepatic tissue.Results1All mice developed non-alcoholic fatty liver disease after given high fat and cholesterol diet after14weeks.2Serum TC of NAFLD mice was3.771±0.804mmol/L and significantly higher than1.940±0.300mmol/L of control mice. Both serum HDL-C and LDL-C of NAFLD mice increased compared with the control (HDL-C:2.224±0.428vs.1.120±0.066, P<0.01; LDL-C:1.241±0.663vs.0.510±0.191, P<0.01). However, there were no significant differences of serum TG between two groups (0.719±0.383vs.0.710±0.487, P>0.05).3qPCR was performed to analyze hepatic cavl mRNA level, and its expression in the treatment group was significantly higher than the control (1.536±0.226vs.0.980±0.272, P<0.05).4Western Blot analysis confirmed that hepatic cavl protein was significantly higher in the treatment than the control (0.643±0.240vs.0.100±0.130, P<0.01), in accordance to mNRA changes.5The pathological results of treatment liver showed fatty liver. Five mice showed suspect NASH and seven mice NAFL.6The immunohistochemistry showed distribution of cavl increased (0.251±0.0380vs.0.137±0.0003, P<0.05), mainly located in the plasma membrane of liver, cytoplasm and the membrane of lipid droplets; Elevated hepatic cavl expression is more apparent with increasing severity of fatty liver.ConclusionThis study has successfully established mouse model of non-alcoholic fatty liver disease with high fat and cholesterol diet digestion, and serum TC, HDL-C, LDL-C markedly increased in NAFLD mice, cavl expression in NAFLD mice significantly increased, cavl is mainly distributed in the plasma membrane of liver, cytoplasm and the membrane of lipid droplets, and the intensity of cavl expression is related to the degree of fatty liver. All the results imply that cavl may play a role in the NAFLD induced by high fat and cholesterol diet. |
