| Section 1The active components in Hu-Lu-Ba-Wan improve insulin resistance via the ER-?/PI3K/Akt signaling pathway in HepG2 cellsObjectiveTo compare the effect of diosgenin(DSG),5-methoxypsoralen(5-MOP),diosgenin and 5-methoxypsoralen(DSG/5-MOP),which are contained in Hu-Lu-Ba-Wan,on insulin resistance in HepG2 cells and to explore their related molecular mechanism.MethodsTo establish the cellular model with insulin resistance,HepG2 cells were incubated in culture medium containing 10-6mol/L insulin for 36 hours.The corresponding intervention was administrated according to different groups:DSG(10-5mol/L)group,5-MOP(10-6 mol/L)group,DSG/5-MOP(10-5mol/L DSG and 10-6mol/L 5-MOP)group,?-Estradiol(10-6 mol/L)group.In addition,we set up a control group and a model group.The distribution and expression of estrogen receptor-a(ERa)were observed by Immunofluorescence laser scanning confocal microscope.Intracellular glycogen synthesis and cell supernatant glucose consumption were examined by enzyme method.Western blot method was conducted to determine the phosphorylated protein/total protein expression level of ER??sarcoma(src),the p85 regulatory subunit of phosphatidylinositol 3-kinase p85(PI3Kp85),protein kinase B(Akt),glycogen synthase kinase-3(?(GSK-3[?).Immunofluorescence technique was applied to detect the expression of glucose transporter-4(GLUT-4).ResultsWith the technique of immunofluorescence laser scanning confocal microscope,it was observed that ERa was expressed in plasmalemma,cytoplasm,and nuclei.Compared with control group,the expression of ERa level in cellular model group was decreased.Compared with model group,the expressions of ERa in DSG group,5-MOP group,DSG/5-MOP group and ?-Estradiol group were increased.Compared with control group,intracellular glycogen synthesis and the cell supernatant glucose consumption in model group were significantly decreased(P<0.01,P<0.05).Compared with model group,intracellular glycogen synthesis and the cell supernatant glucose consumption were significantly increased in DSG group,5-MOP group,DSG/5-MOP group and ?-Estradiol group(P<0.01,P<0.05).Compared with the DSG group,the cell supernatant glucose consumption in DSG/5-MOP group was significantly decreased(P<0.05).Compared with control group,the expressions of p-ERa/ER,p-src/src,PI3Kp85/?-actin,p-Akt/Akt,p-GSK-3?/GSK-3 in model group were significantly decreased(P<0.01,P<0.05).Compared with model group,the expressions of p-ERa/ER,p-src/src,PI3Kp85/?-actin,p-Akt/Akt(not significant of p-Akt/Akt in ?-Estradiol group),p-GSK-3p/GSK-3 were significantly increased in DSG group,5-MOP group,DSG/5-MOP group and ?-Estradiol group(P<0.01,P<0.05).Compared with DSG group,the expression of p-src/src in DSG/5-MOP group was significantly increased(P<0.01).Compared with control group,the protein expression of GLUT-4 in model group was significantly decreased(P<0.01).Compared with model group,the protein expression of GLUT-4 in DSG group,5-MOP group,DSG/5-MOP group and ?-Estradiol group was significantly increased(P<0.01,P<0.05).Compared with 5-MOP group,the expression of GLUT-4 in DSG/5-MOP group was significantly decreased(P<0.05).ConclusionsDSG and 5-MOP,two active components in Hu-Lu-Ba-Wan,may improve insulin resistance by activating the ERa mediated PI3K/Akt signaling pathways.Section 2The active component in Hu-Lu-Ba-Wan improves lipid metabolism disorder via the AMPK/ACC/CPT-1A signaling pathway in L02 cellsObjectiveTo compare the effect of different concentrations of diosgenin(10-5mol/L?10-6mol/L?10-7mol/L)on lipid accumulation in L02 cells and to explore the related molecular mechanism.MethodsTo establish the model of nonalcoholic fatty liver,L02 cells were incubated in culture medium containing 25 mmol/L glucose and 0.25 mmol/L palmitic acid(PA)for 24 hours.The corresponding intervention was administrated according to different groups:DSG(10-7 mol/L)low dose group,DSG(10-6mol/L)middle dose group,DSG(10-5mol/L)high dose group,A-769662(AMPK activator,10-5mol/L)group.Besides,we set up a control group and a model group.Intracellular reactive oxygen species(ROS),mitochondrial membrane potential(MMP),cell supernatant glucose consumption were detected by fluorometric method.Intracellular triglyceride(TG)content,glutathion peroxidase(GSH-PX),superoxide dismutase(SOD),catalase(CAT),malonaldehyde(MDA),the cell supernatant glutamic-pyruvic transaminase(ALT),glutamic oxalacetic transaminase(AST),y-glutamyltranspeptidase(?-GT)were determined by enzyme method.Western blot method was conducted to detect the phosphorylated protein/total protein expression level of adenosine monophosphate activated protein kinase(AMPK)and acetyl-CoA carboxylase(ACC)as well as the protein expression level of fatty acid synthase(FAS),carnitine acyl transferase-1A(CPT-1A),sterol regulating element binding protein-1c(SREBP-lc)and cytoplasmic cytochrome c.Furthermore,AMPK inhibitor Compound C was administrated for 5 hours before corresponding intervention to detect the phosphorylated protein/total protein expression level of AMPK and ACC.ResultsCompared with control group,the ROS content in model group increased while decreased in DSG low dose group,DSG middle dose group,DSG high dose group and A-769662 group.Compared with control group,MMP and cell supernatant glucose consumption were significantly decreased in model group(P<0.01).Compared with model group,MMP and cell supernatant glucose consumption were significantly increased in DSG low dose group,DSG middle dose group,DSG high dose group and A-769662 group(P<0.01,P<0.05).Compared with control group,the contents of TG,ALT,AST,?-GT were significantly increased(P<0.01).Compared with model group,the contents of TG,ALT,AST,y-GT in DSG low dose group,DSG middle dose group,DSG high dose group and A-769662 group were significantly decreased(P<0.01,P<0.05).Compared with control group,the contents of GSH-PX,SOD,CAT were significantly decreased(P<0.01).Compared with model group,the contents of GSH-PX,SOD,CAT in DSG low dose group,DSG middle dose group,DSG high dose group and A-769662 group were significantly increased(P<0.01).Compared with control group,the contents of MDA was significantly increased(P<0.01).Compared with model group,the contents of MDA in DSG low dose group,DSG middle dose group,DSG high dose group and A-769662 group were significantly decreased(P<0.01).Compared with control group,the protein expressions of p-AMPK/AMPK,p-ACC/ACC,CPT-1A/?-actin in model group were significantly decreased(P<0.01,P<0.05).Compared with model group,the protein expressions of p-AMPK/AMPK,p-ACC/ACC,CPT-1 A/?-actin in DSG low dose group(not significant of CPT-1A/?-actin),DSG middle dose group,DSG high dose group and A-769662 group were significantly increased(P<0.01,P<0.05).Compared with control group,the protein expressions of FAS/?-actin,SREBP-1c/?-actin and the cytoplasm cyc/?-actin in model group were significantly increased(P<0.01).Compared with model group,the protein expressions of FAS/?-actin,SREBP-1c/?-actin and the cytoplasm cyc/(3-actin in DSG low dose group,DSG middle dose group,DSG high dose group and A-769662 group were significantly decreased(P<0.01,P<0.05).Compared with DSG group(10-5 mol/L),the protein expressions of p-AMPK/AMPK?p-ACC/ACC in Compound C/DSG group(10-5mol/L Compound C +10-5mol/L DSG)were significantly decreased(P<0.01).ConclusionsDSG,the active component in Hu-Lu-Ba-Wan,may reduce intracellular lipid accumulation in L02 cells.The possible mechanism may relate to its activation of AMPK/ACC/CPT-1A signaling pathways,increase in lipolysis,reduction in lipid synthesis and improvement of oxidative stress. |
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