Therapeutic Effect Of Stevioside On Hyperlipidemic Fatty Liver And Its Mechanisms In Rats

Objective:To investigate the therapeutic effect of stevioside on hyperlipidemic fatty liver and its possible mechanisms.Methods:In vivo experiment,SD rats were randomly divided into 5 groups,control group,model(fatty liver)group,stevioside 75 and 150 mg/kg groups,and fenofibrate 20 mg/kg group.A rat model with fatty liver was induced by feeding a high-fat diet.After 6 weeks of high-fat diet,the medicine-treated rats were then treated with stevioside or fenofibrate for an additional 6 weeks.Finally,all rats were sacrificed,the contents of serum and hepatic total cholesterol(TC),triglyceride(TG),and free fatty acid(FFA)as well as hepatic tumor necrosis factor??(TNF-?),interleukin-6(IL-6),and interleukin-8(IL-8)were measured,the hepatic weight index was calculated,and the hepatic morphological changes were observed under a light microscope.The expressions of hepatic peroxisome proliferator-activated receptor(PPAR)?/?,sterol regulatory element binding protein(SREBP)-1c,fatty acid synthase(FAS),diacylglycerol acyltransferase(DGAT),carnitine palmitoyl transferase(CPT)-IA,nuclear factor kappa B(NF-?B)p65,and inhibitor of kappa B-alpha(I?B-?)proteins were measured by Western Blot method.In vitro experiment,BRL hepatocytes were cultured in RPMI 1640 medium and oleic acid was used to induce the hepatocellular steatosis.After simultaneous treatment of oleic acid-stimulated hepatocytes with 100-400?M stevioside for 36 h,the contents of intracellular TG and FFA were determined,and the distribution of lipid droplets in hepatocytes were observed by Oil Red staining.After hepatocellular PPAR? gene silence by siRNA technology,we further observed whether the lipid-lowering effect of stevioside was affected,and the expressions of PPARa downstream proteins including SREBP-1c,FAS,DGAT,and CPT-1A in hepatocytes were determined by Western Blot method.On the other hand,the inflammatory response of fatty liver was mimicked and induced by oleic acid plus lipopolysaccharide(LPS).The level of supernatant TNF-? was measured after treatment with stevioside for 12 h or pretreatment with PPAR? inhibitor GW9662,and the expressions of hepatocellular PPAR?,NF-?B p65,and I?B-? proteins were also determined by Western Blot method.Results:After administration of stevioside 75-150 mg/kg for 6 weeks,the serum TC,TG,and FFA levels were decreased(P<0.01),the hepatic TC,TG,FFA,TNF-?,IL-6,and IL-8 contents and hepatic weight coefficient were also lowerd(P<0.05 orP<0.01).The hepatic histological evaluation showed that the degree of hepatic steatosis in stevioside-treated groups was significantly alleviated(P<0.01).Stevioside treatment could significantly increase the expressions of hepatic PPAR?/?,CPT-1A,and I?B-?proteins(P<0.05 or P<0.01),and decrease the expressions of hepatic SREBP-1c,FAS,DGAT,and NF-?B p65 proteins(P<0.05 or P<0.01).In vitro,the intracellular TG and FFA levels were significantly reduced after treatment with 100-400 ?M stevioside(P<0.05 or P<0.01).The results of Oil Red staining also showed that the intracellular lipid droplets in the stevioside-treated groups were significantly decreased(P<0.05 or P<0.01).When PPARa gene was silenced,the lipid-lowering effect of stevioside and its reversing effect on SREBP-1c,FAS,DGAT,and CPT-1A protein expressions were abolished.Stevioside also decreased the NF-?B p65 protein expression and increased the I?B-? protein expression in hepatocytes stimulated by oleic acid plus LPS(P<0.05 or P<0.01),and decreased the cultured supernatant TNF-?level(P<0.05 or P<0.01).However,these effects of stevioside were cancelled after pretreatment with GW9662.Conclusion:Stevioside had a better therapeutic effect on high-fat diet-induced fatty liver,and its mechanisms might be related to the activation of PPAR?/? and subsequent regulation of the protein expressions of its genes involved in lipid metabolism and inflammation.