The Study On The Regulating Effect Of Chitosan Oligosaccharide On Glucolipid Metabolism Disorder And Its Possible Mechanism

Objective: Obesity associated with diabetes,nonalcoholic fatty liver disease(NAFLD),cardiovascular disease(CVD),metabolic syndrome and cancer has become a major global health challenge.Thus,it is urgently necessary to improve an alternative and efficient therapeutic strategy to curb the increasing incidence of obesity.In recent years,natural compounds have received extensive attention,providing a new direction for the improvement of obesity.Chitosan oligosaccharides(COS)are a kind of short-chain,low-molecular-weight multifunctional oligosaccharides and produced from chitosan,a polysaccharide constituent of crustaceans such as shrimps,crabs,lobsters,and prawns,by enzymatic degradation or acidic hydrolysis.In addition,studies have shown that COS could improve dyslipidemia and suppress NAFLD.Regretfully,the protect effect and the underlying molecular mechanism of COS on lipid metabolism disorder have not yet been reported.Because of the critical role of liver in the lipid metabolism,we used palmitic acid(PA)-induced HepG2 cells and high-fat diet(HFD)-induced mice as experimental models to examine the effect of COS on glucolipid metabolism.Compared to chitosan,COS have been shown to have higher solubility,lower viscosity,and higher absorption rate.Therefore,we hypothesized that COS may be directly absorbed into the blood to reach the target organs,and then to improve lipid metabolism disorder.So far,several natural products have been proven to reverse glucose and lipid metabolism disorders by upregulating the expression of nuclear receptor peroxisome proliferator-activated receptor gamma(PPARγ).PPARγ is an important member of the nuclear receptor super family of transcription factors and plays a vital role in regulating metabolic disorders,including obesity,insulin resistance,and CVD.Due to the key role of PPARγ in the regulation of lipid metabolism,we aimed to explore the effect of COS on PPARγ in palmitic acid(PA)-induced HepG2 cells and high-fat diet(HFD)-induced mice.The intestinal microbiome has recently been implicated in multiple metabolic diseases.And increasing evidence has indicated the important effect of changes of composition and function in intestinal microbiome on lipid metabolism disorder.It has also found that chitosan can improve the weight gain of piglets on a high-fat diet by regulating the intestinal microbiome.And dietary supplementation with COS can alter the intestinal flora and intestinal metabolites in weaned Huanjiang mini-piglets.Therefore,the aim of this study was to further explore the effect of lipid metabolism disorder and COS on the intestinal flora by analyzing the intestinal flora of mice treated with different diet,and then to investigate the role of intestinal microbiome in the regulation of COS on lipid metabolism disorder.Methods:In this study,we used palmitic acid(PA)-induced HepG2 cells and high-fat diet(HFD)-fed C57BL/6J mice as experimental models in vitro and in vivo,to respectively explore the preventive effects of COS on glucolipid metabolism disorder and the underlying molecular mechanism.Cell experiments: HepG2 cells were maintained in Minimum essential medium(MEM).After reaching subconfluence,cells were incubated with 0,25,50,100 μg/mL COS for 12 h and then exposed to 100 μM PA diluted in culture medium for 24 h.Then cells were stained with 0.5% oil red O solution to observe the lipid accumulation in cells.The mRNA expressions of inflammatory cytokines,such as IL-6,MCP-1,TNF-α and fatty acid synthesis-related regulators,such as SCD-1,ACC-1,PKC1-α were detected by RT-PCR.And RT-PCRand western blot was respectively used to detect the mRNA and protein level of PPARγ.Animal experiments: We randomly divided 20 mouse,which are male C57BL/6J wild type,into four groups: CD group,HFD group,CD + COS(1 mg/m L in drinking water,about 200 mg/kg/d)group and HFD + COS group.After the treatment for five months,the body weight and fasting glucose level of each mouse were detected and intraperitoneal glucose tolerance test(IGTT)was conducted.Then the mice were sacrificed and liver tissues,stool samples were collected.Liver tissues of each group(n=5)were stained with 0.5% oil red O solution to observe the lipid accumulation in cells.The mRNA expressions of inflammatory cytokines,such as IL-6,MCP-1,TNF-α and fatty acid synthesis-related regulators,such as SCD-1,ACC-1,PKC1-α were detected by RT-PCR.The transcription and translation of PPARγ were detected by RT-PCR and western blot respectively.16 S rDNA microbial community analysis was used to analyze the dissimilarity of gut microbiota in phyla level and at family level of different groups of mice.Results: In present study,both HepG2 cells exposed to PA and mice chronically subjected to HFD exhibited evident lipid droplet accumulation,suggesting the formation of dyslipidemia.On the contrary,the imbalance of lipid metabolism in both HepG2 cells and liver tissues of HFD-fed mice was reversed by COS treatment.Especially,COS displayed significantly inhibitory effect on the glucose intolerance in mice with HFD.Our results showed that COS significantly down-regulated the over expression of proinflammatory cytokine(IL-6,MCP-1,TNF-α)and acid metabolism-related regulators(SCD-1,ACC-1,PKC1-α)in PA-induced HepG2 cells as well as in liver tissues of HFD-fed mice at m RNA level.Our study showed that PA and HFD induced a significant decrease in the expression of PPARγ at both mRNA and protein levels in Hep G2 cells and liver tissues of HFD-fed mice,which was almost totally reversed by COS treatment.The results of 16 S rDNA microbial community analysis showed that as compared to CD group,at phylum level the increased Firmicutes and Proteobacteria and decreased Verrucomicrobia,Bacteroidetes and Actinobacteria was observed in HFD group,accompanied by the augmented ratio of Firmicutes to Bacteroidetes(F/B).In contrast,COS treatment reversed this change in the microflora and significantly reduced the ratio of F/B in HFD-fed mice.At family level,COS significantly increased the abundance of beneficial bacteria Akkermansia muciniphila,which can improve lipid metabolic disorders.Conclusion: 1.We proved that COS displayed strong improvement on lipid accumulation in PA induced HepG2 cells and weight gain and impaired glucose tolerance in mice induced by HFD,COS also ameliorated the overexpression of proinflammatory cytokine(IL-6,MCP-1,TNF-α)and fatty acid metabolism-related regulators(SCD-1,ACC-1,PKC1-α)in vitro and in vivo.2.COS reversed the decrease expression of PPARγ at both mRNA and protein levels in PA induced HepG2 cells and in HFD induced mice.3.HFD can destroy the structure and stability of gut microbiota,and COS reversed this anomalous variation,and promoted the growth of beneficial bacteria.