Effects Of Eucommia Ulmoides Oliv And Gardenia Extract On Autophagy In Monosodium Glutamate-induced Obese Mice And Its Mechanism

Objective: 1.To observe the effects of eucommia leaf and gardenia extract on autophagy regulation related genes and proteins in obese mice induced by glutamate.2.To analysis of the bile acids in the bile of obese mice induced by glutamate by ultra-high performance liquid chromatography-tandem mass spectrometry.3.To study the effects of the effective components on the autophagy of Hep G2 cells from eucommia and gardenia extracts and to discuss the corresponding mechanism for obesity treatment.Methods: 1.Subcutaneous injection of sodium glutamate(4 g/kg/d)intervened growth in mice,and normal control group mice were subcutaneous injected saline(10 μl/g/d).After4 weeks,mice were divided into 9 groups: normal control group(Normal),model group(Model),positive control group(RES),high dose group of eucommia leaf extract(EEH),low dose group of eucommia leaf(EEL),high dose group of gardenia extract(GEH),low dose group of Gardenia extract(GEL),high dose group combination(EEH+GEH),and low dose group of combination(EEL+GEL),10 mice in each group.After establishing a stable model,the expression of m RNA and protein of autophagy genes in mice was detected by real-time fluorescence quantitative PCR and the method of Western Blot,respectively.2.Establishment of lc-ms/MS method was used to determine the concentration of bile acids in mouse bile.Bile after acetonitrile protein deposition of LC-MS/MS analysis,chromatographic column to SEG protecol using C18 ENDURO(250 mm * 4.6 mm * 5microns),mobile phase of 4 tendency/L ammonium acetate solution-containing 0.05%formic acid and acetonitrile,gradient elution,and the flow rate of 1ml/min,sample 10 mu L,using electrospray ion source(ESI),negative ion multiple reaction monitoring mode(MRM).3.When lipid accumulation model of Hep G2 was established by oleic acid,oil red O staining was observed by detecting the accumulation of lipid droplets in each group after 24 h and the protein expression of Beclin 1,LC3-II/I,p62,FXR,CREB was detecting by Western Blot analysis.Besides,detection of Mitotracker-LC3 B was used to observe the mitochondrial autophagy by immuno-fluorescence,which was used to detect the effects of the active components on autophagy.Results: 1.Compared with the control group,model groups have significantly lower lipid droplets in liver and in epididymal fat cells of mouse and more obvious effects were observed in group EEL+GEL、EEH+GEH and RES.2.Compared with the control group,the expression of m RNA(ULK1、LC3-I、LC3-II、Atg5、Atg7、Beclin-1and Lamp2a)was significantly increased in liver fat cells of mouse(p<0.05).However,the expression of m RNA(ULK1、LC3-I、LC3-II、Atg5、Atg7、Beclin1 and Lamp2a)was significantly decreased in epididymal fat cells of mouse,which was more obvious in the EEH+GEH group.Besides,the expression of pro-inflammatory factors(TGFα、COL1α、MMP13 、 RELMα)was decreased while some of inflammatory factors(MGL-1 、CD206、IL-10)was increased to different degrees.3.Compared with the control group,the expression of protein(RAB7、Beclin-1、Atg5、CREB、CRTC2、PPARα、TFEB、PGC1α、AMPK、ULK1and LC3II/I)was significantly increased and the expression of protein(P62、FXR and P-m TOR)was significantly decreased in liver fat cells of mouse(p<0.05).However,opposite change of protein expression was observed in epididymal fat cells,which was more obvious in the EEH+GEH group.4.Compared with the control group,the concentration of bile compound(DCA、GDCA、GUDCA)was significantly reduced and the concentration of bile compound(CDCA、UDCA、GCA、GCDCA)was significantly raised(all both p<0.05),while no effect on concentration of compound(LCA and TUDCA)was observed.5.Compared with the control group,mitochondrial autophagy was significantly inhibited in Hep G2 induced by oleic acid,while opposite change was observed in Hep G2 induced by drug compound(CRO,CGA and CRO+CGA),which was more obvious in the CRO+CGA group.Besides,the proteins expression of Beclin1,CREB and LC3II/I were significantly increased(p<0.05)and the proteins expression of FXR and P62 were significantly decreased(p<0.05).Conclusion: The m RNA expressions of autophagy related protein were increased in liver and in adipose tissue in MSG obese mice.However,the proteins expressions of autophagy were decreased in liver while the proteins express of autophagy were increased in adipose.The autophagies in liver and adipose were both improved to different levels and obvious anti-inflammatory was observed in the treatment groups.The concentration change of bile acid subcomponent and the adjustment characteristics by treatment group were clarified preliminarily.The mitochondrial autophagy was enhanced in Hep G2 by CRO and CGA extracted from eucommia and gardenia,which help understand the mechanism of glycolipid metabolism by eucommia and gardenia extracts preliminarily.