Effect Of Soothing Liver And Invigorating Spleen Methods And Recipes On Expression Of UCP₂mRNA And Protein Of Hepatic Tissue In NAFLD Rats

Objective:To observe the therapeutic effects of soothing liver,invigorating spleen,integrated methods and their typical recipes on NAFLD rats,to explore the possible mechanism,and to provide experimental foundation for the clinical use of the soothing liver and invigorating spleen therapies treating NAFLD.Methods:76 male SD rats were randomly divided into two groups:high fat diet group(n=66,fed with high fat diet) and the normal group(n=10,fed with normal diet).At the end of 12th week,6 rats were randomly selected for hepatic histopathology examination.When the rat model of NAFLD was confirmed,the remaining 60 NAFLD rats were subdivided into 6 equal subgroups:soothing liver group,invigorating spleen group,integrated group,Sanqizhiganwan group,model control group and natural recovery group.Rats in the anterior 4 groups were fed with corresponding medications by perfusion while the remaining rats were fed with equal volume of distilled water. Meantime,rats in the model group were continuously fed with high fat diet while rats in other groups were fed with normal diet.At the end of 20th week,all rats were sacrificed.TG,TC,AST, ALT contents in the serum and TC,TG contents in the hepatic homogenate were detected by automatic biochemical analyzer.Plasma insulin concentration was measured by radioimmunoassay.The expression of UCP₂mRNA and UCP₂ proteins in liver tissue were respectively examined by reverse transcription polymerase chain reaction(RT-PCR) and immunohistochemistry respectively.Results:(1) Compared to normal group,contents of serum TC,TG,LDL-C in model control group were significantly increased(P＜0.01),while content of serum HDL-C was obviously decreased (P＜0.01).Compared to model control group,contents of serum TC,TG,LDL-C in treatment groups were significantly decreased(P＜0.01),especially in the soothing liver and integrated group(P＜0.01);Serum HDL-C was increased,especially in the soothing liver group, invigorating spleen group,integrated group and natural recovery group(P＜0.01).(2)Contents of serum AST,ALT in model control group were significantly increased(P＜0.01, compared to normal group),while they were decreased in the treatment groups,especially in the soothing liver,invigorating spleen,integrated and natural recovery group(P＜0.01,compared to model control group).However,no statistical significance was found between the treatment groups.(3) Contents of TC,TG in the hepatic homogenate in model control group were significantly increased(P＜0.01,compared to normal group),while TC content was decreased in invigorating spleen group.Compared to model group,TG content in the treatment groups were decreased (P＜0.05).(4) Light microscope observation revealed medium fatty degeneration in hepatocytes in model group:swelling of hepatocytes and different sizes of lipid droplets inside the cytoplasm. Inflammatory cells were seen in the portal area.No obvious fatty degeneration was seen in hepatic tissue of soothing liver,invigorating spleen and integrated group,especially in the soothing liver group.Liver tissue of natural recovery group and Sanqizhiganwan group showed mild fatty degeneration.(5) Result of RT-PCR showed that UCP₂ mRNA expression was little in hepatic tissue of normal group,high expression in model control group(P＜0.05,compared to normal group). Compared to model control group,UCP₂ expression in liver tissue of other groups was decreased, especially for the soothing liver,invigorating spleen and integrated group(P＜0.05) and the least expression is in the soothing group.(6) Immunohistochemistry showed no expression of UCP₂ protein in liver tissue of normal group,high expression in model control group.Compared to model control group,UCP₂ protein expression in hepatic tissue of other groups was dramatically decreased(P＜0.01).No statistical significance was found between invigorating spleen group and integrated group(P=0.14),while it was found between other two groups(P＜0.05).Conclusion:(1)Removal of the cause(high fat diet control) is significant in reversing NAFLD in the early stage.(2)Soothing liver,invigorating spleen methods and recipes showed good prevention function in NAFLD:improving lipid metabolism and reducing serum AST and ALT.(3) Disturbance of the spleen and liver function were the main pathogenesis of NAFLD in the early stage,and therapies of soothing liver and invigorating spleen are the main therapeutic methods.Soothing liver therapy has a relative better effect in the treatment of NAFLD in the early stage.(4) There was a high expression of UCP₂ mRNA and protein in liver tissue of model rats with NAFLD and decreased expression in the treatment groups,which suggest a close correlation between UCP₂ protein and occurrence and development of NAFLD.Soothing liver and invigorating spleen recipes can down-regulate the expression of UCP₂,which maybe the therapy target for Chinese herbs treating NAFLD.