| Objective:To investigate the inhibitory effect of chitin oligosaccharide(NACOS)on lipid metabolism and its molecular mechanism.Methods: In vitro experiment:(1)By means of MTT,PA(0,25,50,100 and 200 ?M)were used for 24 hours,respectively,using a concentration gradient.We detected the OD val ue of HepG2 cell proliferation and calculate the survival rate by microplate reader,and evaluated the safe concentration of PA on the HepG2 cells;The HepG2 cells were pretreated with NACOS(0,50,100 ?g/ml)for 12 hour and then treated with PA for about 24 hours.The survival rate of HepG2 cells was determined by MTT method,and the optimal concentration of NACOS was determined.(2)Red oil O staining: HepG2 cells were pretreated with NACOS(0,50,100 ?g/ml)for 12 hours,then stimulated with PA for 24 hours,the morphology of HepG2 cells was observed by inverted microscope to evaluate the effect of NACOS on lipid deposition induced by PA and NACOSA inhibits the optimal concentration of HepG2 cells.(3)By means of Quantitative Real-time PCR,we measured the gene expression of Peroxisome proliferator coactivator receptor-1?(PGC1?),Interleukin-1?(IL-1?),Acetyl coenzyme 1(ACC1)and Cytochrome oxidase subunit 5b(Cox5b),Medium chain acyl coenzyme A dehydrogenase(Mcad),to evaluate the optimum time of PA inhibition activatied HepG2 cells;HepG2 cells were pretreated with NACOS(0,50,100 ?g/ml)for 12 hours,then stimulated with PA for 24 hours,measured the gene expression of PGC1??IL-1??ACC1?Cox5b?Mcad,to evaluate the inhibitory effect of NACOS on lipid metabolism in HepG2 cells induced by PA.(4)By means of western blot,we measured the expression of phosphorylation protein in MAPKs and PI3K/Akt pathway,such as p38,ERK1/2 and Akt for different time by PA,to evaluate the optimum time of PA,HepG2 cells were pretreated with NACOS(0,50,100 ?g/ml)for 12 hours,then stimulated with PA for 0.5 hours,we measured the expression of protein in MAPKs and PI3K/Akt pathway,such as p-p38,p-ERK1/2 and p-Akt,to evaluate the inhibitory effect of NACOS on lipid metabolism in HepG2 cells induced by PA.In vivo experiment,the male C57BL/6 mice were randomly divided into 4 groups(n=5),namely the normal control(NCD)group,high fat diet(HFD)group,NACOS group,NACOS+HFD group,the experiment for a total of 20 weeks.After 20 weeks,the mice were sacrificed by anesthesia and the liver tissue was extracted for examination.The main detection methods were following:(1)In various physiological indexes,to investigate the changes of physiological indexes such as body weight,drinking water and diet after administration of NACOS in C57BL/6 mice.(2)By the method of Quantitative Real-time PCR,liver tissue was determined.The gene expression of Peroxisome proliferator coactivator receptor-1?(PGC1?),Interleukin-1?(IL-1?),Acetyl coenzyme 1(ACC1)and Cytochrome oxidase subunit 5b(Cox5b),Medium chain acyl coenzyme A dehydrogenase(Mcad)was measured.(3)By means of western blot,we measured the expression of phosphorylation protein in MAPKs and PI3K/Akt pathway,such as p38,ERK1/2 and Akt.Results : the results showed that the in vitro experiment:(1)MTT cell activity experiment,in the range of PA 25-200 ?M,the HepG2 cell activity was not significantly inhibited,PA showed no cytotoxicity.In the concentration of NACOS 100 ?g/ml can slightly increase the activity of HepG2,but the difference was not statistically significant,also showed no cytotoxic effect and NACOS and PA combined with drug treatment.(2)Red oil O staining showed red fat particles in cytoplasm.After PA treatment showed a significant accumulation trend.Compared with PA alone,the pretreatment of NACOS could significantly inhibit the formation of lipid droplets in HepG2 cells induced by PA.(3)RT-PCR,the experimental results show that the HepG2 cells were treated with PA 100 ?M 3-24 h,the expression of transcription such as Peroxisome proliferator coactivator receptor-1?(PGC1?),Interleukin-1?(IL-1?),Acetyl coenzyme 1(ACC1)and Cytochrome oxidase subunit5b(Cox5b),Medium chain acyl coenzyme A dehydrogenase(Mcad)showed different degrees of increase(P < 0.05 or 0.01),and reached the peak in 6 h.(3)Western blotting showed that HepG2 cells treated with PA 100 ?M after that,the stimulation of mitogen activated protein kinase(Mitogen activated protein kinases,MAPKs)and phosphatidylinositol 3-kinase/protein kinase B(Phosphoinositide 3-kinase,PI3K)p38,ERK1/2 pathway and Akt kinase activated rapidly,the phosphorylation level is a time-dependent increaseing in 0-1h.Among them,p-ERK1/2 and p-Akt peaked at 0.5 h(P<0.05,vs contro L group),while p-p38 reached the highest level at 1h(P<0.01,vs control group).In vivo experiment results show that :(1)Competing with the normal control group which is the basic feed(NCD)mice,the average weight of mice in High fat diet group(HFD)increased significantly(P<0.01).On the contrary,when the mice were fed with NACOS(1 mg/ml)at the same time,they could significantly inhibit the increase of body weight(P<0.05 or 0.01).High fat diet and NACOS had no significant difference between the intake of feed and water.(2)The RT-PCR experimental results show that the level of PGC1??ACC1 and Mcad in the livers of HFD mice is significantly higher than the NCD mice's(P<0.05 or 0.01),Interleukin-1(IL-1)also increased significantly(P<0.05),but the expression of the transcription of Cox5 b in HFD group had no significant effect(P>0.05).Compared with HFD group,the disorder of lipid metabolism in mice after NACOS treatment,the transcription level of liver lipid metabolism regulation factor such as PGC1?,Mcad and ACC1 have different degrees of inhibition,Interleukin-1 also decreased,Cox5 b expression did not change significantly.(3)Western blotting results showed that compared with the NCD group,the expression level in HFD mice's liver,it's p-p38 and p-Akt protein increased significantly(P<0.05),while p-ERK1/2 has no obvious difference.Compared with HFD group,NACOS treatment could significantly inhibit the activation of p-p38 and p-Akt induced by high fat diet(P < 0.01 or 0.05).NACOS can reduce the expression levels of p-ERK1/2 protein(P < 0.05)Conc Lusion:(1)NACOS did not show cytotoxicity when administered in combination with PA.(2)NACOS significantly inhibited the formation of lipid droplets in HepG2 cells induced by PA.(3)NACOS inhibits the disorder of lipid metabolism and the overexpression of related inflammatory factors in HepG2 cells induced by PA stimulation.(4)NACOS significantly inhibited the expression of p-p38,p-ERK1/2 and p-Akt.In vivo experiments show that:(1)High-fat mice were treated with NACOS(1 mg/ml),which could significantly increase the body weight of mice.There was no significant effect of high fat diet and/or NACOS on the intake and drinking water.(2)NACOS treatment can effectively reverse the lipid metabolism caused by high fat diet.(3)NACOS reduces the occurrence of inflammatory response by inhibiting the activation of the MAPKs pathway,thereby preventing the occurrence of lipid metabolism disorders. |
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