| Objective: To study the effect of oxymatrine on hepatic lipid deposition induced by high fat and high fructose environment in vivo and in vitro.To explore the intervention effect of oxymatrine through upregulating miR-182 expression,and to explore its potential mechanism.Methods:1.In vivo experiment: Wistar rats at the age of 7 weeks were randomly divided into 2 groups: control group(NC)12,and high-fat high-fructose group(HFDHFr)24.After 4 weeks,the HFDHFr group was randomly divided into the HFDHFr group and the high-fat and high-fructose group with oxymatrine(HFDHFr+OMT),(12 rats in each group).Rats in HFDHFr+OMT group were given oxymatrine 80 mg/kg/d with 0.5% sodium carboxymethyl cellulose(CMC-Na)as the carrier,and rats in NC group and HFDHFr group were given only the carrier.At 4 weeks of drug intervention,subperitoneal glucose tolerance test(IPGTT)was performed to detect blood glucose at each time point and the area under the glucose curve were calculated.Fasting blood glucose was detected 4 weeks after drug intervention,Serum and liver tissue samples were collected.Fasting insulin level was detected by ELISA.Triglyceride content in liver tissue samples was detected.The histological changes of liver were observed by HE staining and oil red O staining.Total RNA and miRNA were extracted from liver tissues of rats,m RNA and protein expressions of sterol element binding protein 1c(SREBP-1c),acetyl-coenzyme A carboxylase(ACC),fatty acid synthase(FAS)and carnatine palml-transferase 1A(CPT-1A)in liver tissues were determined by real-time fluorescence quantitative PCR(RT-q PCR).Expressions of miR-182,miR-183-5p and miR-96-5p in liver tissues were detected by RT-q PCR.2.In vitro experiment: HepG2 cells were treated with different concentrations of oxymatrine: 0.1 mg/m L,0.2 mg/m L,0.4 mg/m L and 0.8 mg/m L.Cell survival rate was calculated after 0,12,24,36 and 48 h,respectively.HepG2 cells were divided into control group(CON),palmitic acid intervention group(PA)and oxymatrine intervention group(PA+OMT,OMT concentration of 0.2 mg/m L).Lipid deposition in cells was observed by oil red O staining.Mi RNA and total RNA were extracted from HepG2 cells.The expressions of miR-182,miR-183-5p and miR-96-5p in HepG2 cells were detected by RT-q PCR.m RNA and protein expressions of SREBP-1c,ACC,FAS and CPT-1A were detected by RT-q PCR and Western Blotting.In addition,HepG2 cells cultured with palmitic acid were transfected with miR-182 inhibitor,then oxymatrine intervention was administered.The protein expressions of SREBP-1c,ACC,FAS and CPT-1A were detected by Western blot.Results:In vivo experiment:1.Comparison of fasting blood glucose,fasting insulin and IPGTT results in each group: there were statistical differences in fasting blood glucose and fasting insulin in three groups(P<0.01).The blood glucose level of HFDHFr group was significantly higher than that of the NC group at 0 min,15 min,30 min,60 min and 120 min,and the area under the blood glucose curve(AUC)was significantly increased(P<0.01).The blood glucose of HFDHFr+OMT group was significantly lower than that of HFDHFr group at 0 min,15 min,30 min,60 min and 120 min;and the AUC of blood glucose was significantly reduced.(P<0.05).2.Effects of oxymatrine on lipid deposition in liver of rat fed with high fat and high fructose diet.2.1 Comparison of liver TG content in each group: Compared with NC group,the liver TG content in the HFDHFr group was significantly increased(P <0.01),liver TG content in the HFDHFr + OMT group was significantly reduced compared with HFDHFr group(P <0.01).2.2 comparison of liver histopathological test resultsIn H&E staining,the structure of liver cells in NC group was complete,and no obvious lipid droplet vacuoles were observed.In HFDHFr group,liver lobule structure was disordered,the liver cells were swollen,and a large number of lipid droplet vacuoles were observed in the cytoplasm.In HFDHFr+OMT group,the morphology of the liver cells was improved and the amount of lipid droplet was reduced.Oil red O staining: red lipid droplets were less in NC group;large amount of red lipid droplets were formed in HFDHFr group,whereas significantly decreased in HFDHFr+OMT group.3.Comparison of the m RNA and protein levels of SREBP-1c,ACC,FAS and CPT-1A in the liver of each group: compared with the NC group,m RNA and protein expressions of SREBP-1c,ACC and FAS in HFDHFr group increased(P<0.01),and m RNA and protein expressions of CPT-1A decreased(P<0.01).Compared with HFDHFr group,m RNA and protein expressions of SREBP-1c,ACC and FAS in HFDHFr+OMT group decreased(P<0.01),while m RNA and protein expressions of CPT-1A increased(P<0.01).4.Gene sequencing results showed that the expressions of miR-182,miR-183-5p and miR-96-5p in HFDHFr group were down-regulated compared with NC group.5.The expression of miRNA in the liver of each group: compared with the NC group,the expressions of miR-182,miR-183-5p and miR-96-5p in HFDHFr group were significantly decreased(P<0.01).Compared with HFD group,the expressions of miR-182,miR-183-5p and miR-96-5p in HFDHFr+OMT group were significantly increased(P<0.01).In vitro experiment:1.Cytotoxic effect of oxymatrineOxymatrine concentration of 0.2 mg/m L or less did not inhibit cell viability in 24 hours or shorter time.2.Effects of oxymatrine on lipid deposition of HepG2 cells cultured with palmitate2.1 Changes of TG content in HepG2 cells: compared with CON group,the TG content in HepG2 cells increased significantly after PA intervention(P<0.01).Compared with the PA group,the TG content of PA+OMT group decreased significantly(P<0.01).2.2 Oil red O staining results: no significant lipid droplets were observed in the Con group,a large number of lipid droplets were observed in the PA group,and significantly decreased in the PA+OMT group.3.Effects of oxymatrine intervention on m RNA and protein levels of SREBP-1c,ACC,FAS and CPT-1A in HepG2 cells cultured with palmitic acid: Compared with CON group,m RNA and protein levels of SREBP-1c,ACC,FAS in PA group increased(P<0.01),while m RNA and protein expression of CPT-1A decreased(P<0.01).Compared with PA group,m RNA and protein expression of SREBP-1c,ACC,and FAS in PA + OMT group decreased(P<0.01),and m RNA and protein expression of CPT-1A increased(P<0.01).4.Effect of oxymatrine on miRNA expression in HepG2 cells: Compared with the CON group,the expression of miR-182 in the PA intervention group was reduced(P<0.01);compared with the PA group,the expression of miR-182 in the PA+OMT group was increased.(P <0.01).The expressions of miR-183-5p and miR-96-5p in each group were not statistically significant(P>0.05).5.Effect of oxymatrine on protein expression of SREBP-1c,ACC,FAS,and CPT-1A after transfection of miR-182 inhibitor in HepG2 cells: Compared with CON group,protein expression of SREBP-1c,ACC,and FAS in PA group increased(P<0.01),protein expression of CPT-1A decreased(P<0.01);compared with PA group,protein expressions of SREBP-1c,ACC,and FAS in PA + OMT group decreased(P<0.01).Protein expression of CPT-1A increased(P<0.01).After transfection with miR-182 inhibitor,compared with PA+OMT group,protein expression of SREBP-1c,ACC and FAS increased in PA+OMT+miR-182 group(P<0.01),while CPT-1A protein expression decreased(P<0.01).6.HepG2 cells transfected with miR-182 mimic,the effects of oxymatrine and miR-182 mimic on the protein expression of SREBP-1c,ACC,FAS and CPT-1A,respectively:Compared with CON group,protein expression of SREBP-1c,ACC,FAS in PA group increased(P<0.01),protein expression of CPT-1A decreased(P<0.01);after transfection with miR-182 mimic,compared with PA group,protein expression of SREBP-1c,ACC,FAS protein in PA + miR-182 mimic group decreased(P<0.01),protein expression of CPT-1A increased(P<0.01);compared with PA group,protein expressions of SREBP-1c,ACC,and FAS decreased in PA + OMT group(P<0.01),while CPT-1A protein expression increased(P<0.01).Conclusions1.High-fat and high-fructose diet can induce the de novo lipogenesis in rat liver,and inhibit fatty acid ?-oxidation,while oxymatrine can inhibit lipogenesis and increase fatty acid ?-oxidation in fatty liver by high-fat-high-fructose feeding.2.PA can induce the activation of de novo lipogenesis pathway in HepG2 cells and inhibit CPT-1A expression which is the key enzymes fatty acid ?-oxidation,while oxymatrine can inhibit de novo lipogenesis related enzymes and increase CPT-1A expression.3.The mechanism by which oxymatrine improves PA-induced lipid deposition in HepG2 cells is related to its up-regulation of miR-182 expression,which inhibits de novo lipogenesis and increases fatty acid ?-oxidation.4.miR-182 overexpression is similar to the effect of oxymatrine on lipid deposition in hepatocytes,which can be used as a target for subsequent research... |
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