Effect Of Lycium Ruthenicum Murr. On Nonalcoholic Steatohepatitis Induced By High Fat Diet In ApoE Null Mice

In recent years,the occurrence of nonalcoholic fatty liver disease?NAFLD?is on the rise as the living style and dietary strueture have been changed,and there is lack of effective therapeutic methods.Long term medical treatment not only aggravates the burden of patients,but also has toxic side effects.Therefore,it is of great significance to search for active ingredients without toxic side effects from traditional Chinese herbal medicines.Lycium ruthenicum Murr.as a traditional Chinese herbal medicine,has attracted extensive attention in recent years.Recent studies have shown that Lycium ruthenicum Murr.has a wide range of antiinflammatory,anti-aging and remarkable antioxidant effects.In this study,the flavonoids compounds were extracted from Lycium ruthenicum Murr.using ethanol as solvent,and the effect and potential mechanism of Lycium ruthenicum Murr.on nonalcoholic fatty liver disease were explored.The main contents are:1)Ultrasonic-assisted extraction was adopted to extract the flavonoids compounds from Lycium ruthenicum Murr.using 70% ethanol as solvent.Total flavonoids content was determined by spectrophotometer using rutin as a positive control.The anti-oxidant activity assays also be marsured by spectrophotometer.The study showed that the extraction rate of total flavonoids from Lycium ruthenicum Murr.was 2.84%,the crude extracts had strong DPPH and OH radical scavenging activity within certain concentration range.2)Fifty male ApoE null(ApoE^-/-)mice were randomly divided into five groups: normal control group?NC?with standard diet feeding,model group?MOD?fed a high-fat diet and received normal saline,simvastatin group?SIM?fed a high-fat diet and received simvastatin?5 mg/kg?,Lycium ruthenicum Murr.group?LRM?fed a high-fat diet and received Lycium ruthenicum Murr.abstracts?140 mg/kg?and combinined group?SIM+LRM?fed a high-fat diet and received simvastatin?5 mg/kg?plus Lycium ruthenicum Murr.abstracts?140 mg/kg?.Normal saline,simvastatin and Lycium ruthenicum Murr.abstracts were given by gastric gavage for 4 weeks.Then mice were sacrificed,liver and epididymal white adipse tissue weight were measured.Serum total cholesterol?TC?,triglyceride?TG?,low density lipoprotein cholesterol?LDL-C?,high density lipoprotein cholesterol?HDL-C?,aspartate aminotransferase?AST?,alanine aminotransferase?ALT?,the expression levels of inflammatory cytokines and adipokines were measured.The glutathione peroxidase?GSH-Px?activity and malondialdehyde?MDA?content in liver tissue were determined using standard biochemical methods with commercially available kits.H&E staining was used to observe the pathological changes of adipose tissue and liver tissue.The lipid accumulation of liver tissue was observed by oil red O staining.Expression of TNF-alpha and IL-6 mRNA in liver tissue were determined by RT--PCR.The activity of SAPK/JNK and P38 signaling pathway and the expression of cytochrome P450,superoxide dismutase1?SOD1?and Catalase1 in liver tissue were detected by western-blot,as well as the expression of PPAR-? and PPAR-?,which are related to fat metabolism.Result showed that:1)Compared with the NC group,the weight of white fat around the epididymis was increased significantly in mice of MOD group.H&E staining showed bigger volume of white adipose tissue with hyperlipidemia and increased AST levels in serum.H&E staining of liver tissue showed enlarged cell volume and liver cells were arranged irregularly,the oil red O straining also showed bigger.fat droplets in liver tissue.The MDA level in liver tissue was higer than that of the NC group.The expression of inflammatory factors were enhanced,the SAPK/JNK signaling pathway was activated and the phosphorylation levels of JNK1/2 was increased,the transcriptional level of PPAR-gamma mRNA was also upregulated.2)Compared with the MOD group,Lycium ruthenicum Murr.and simvastatin significantly improved hyperlipidemia and hepatic fat deposition induced by high fat diet.Serum lipid profiles and the levels of AST was decreased.H&E staining showed that the liver cells arranged closely,the number of fat vacuoles decreased significantly.The number of red fat droplets also decreased significantly in the oil red O staining of liver tissue.The transcription and expression of inflammatory factors decreased and the SAPK/JNK signaling pathway was inhibited.The expression of SOD1,Catalase1 and GSH-Px activity were significantly enhanced in liver tissue with MDA content decreasd significantly.The liver damage caused by high fat diet was improved remarkedly,and the combined effect is better than that of single reagent intervention.At the same time,the volume of white adipose cells in SIM+LRM group increased significantly,with the levels of PPAR-? and PPAR-? upregulated.Conclusion:1)The study showed that the crude extracts of Lycium ruthenicum Murr.has strong antioxidant activity in vitro,and the activity increases with the increase of concentration in a certain range.2)Lycium ruthenicum Murr.combined with simvastatin decreases serum lipd levels and improves liver lipd deposition,the combined effect is more significant than single reagent alone.The combination of Lycium ruthenicum Murr.and simvastatin increases daily food intake and body weight gain of ApoE^-/-mice,and increases the generation of white fat around the epididymis.3)Lycium ruthenicum Murr.combined with simvastatin depresses oxidative stress and inflammation responses of mice induced by high fat-diet,increases antioxidant levels via upregulating the GSH-Px activities and the expressin of enmyes such as SOD1 and Catalase1 in liver,down regulates expression of inflammatory factors,supresses the activity of SAPK/JNK signaling pathway,increases the utilization of FFA and distribution in the periphery.