Chitosan Attenuates Obesity By Modifying The Intestinal Microbiota And Increasing Serum Leptin Levels In Mice

ObjectivesObesity refers to a certain degree of obvious overweight and thicker fat layer and the obesity state is produced by abnormal or excessive accumulation of damaging fat in vivo.The metabolism of body is becoming disordered with the development of obesity,resulting physiological or pathological diseases such as metabolic syndrome.At present,the proportion of obesity and overweight is increasing rapidly at worldwide at all ages.The number of obesity human had tripled from 1975 to 2013,and childhood obesity has become one of the most serious public health challenges in the 21st century.So the improvement of obesity is imminent and has become the focus of more and more scholars.The traditional obesity control strategies are mostly physical exercise,diet control,surgery and oral medicine.However,with the development of microbiology and bioinformatics in recent years,studies have shown that obesity is closely related to gut microbiota.Meanwhlile,many scholars is trying to improve obesity by regulating gut microbiota with prebiotics,and the results showed that the regulation of prebiotics and probiotics on obesity has a more promising and anticipated future.Chitosan has been used in food and medical industry as an easy obtaining dietary fiber in nature which has been proved to have many biological functions such as lipid and glucose regulation,healing promoting and reducing body weight.However,the antiobesity mechanism of chitosan and its effect on gut microbiota were still not cleared.By building mice obesity model,our study aimed to test the antiobasity effect of chitosan and explored the effect of chitosan on metabolism and gut microbiota in obese mice.We hope to find more intestinal flora which is related to obesity or antiobesity and provide new ideas or experimental basis for the control of obesity and the regulation of gut microbiota by probiotics and prebiotics.MethodsThe mice were fed with low fat diet(named group L),low fat diet+chitosan(named group LC),high fat diet(named group H),or high fat diet+chitosan(named group HC).The antiobesity effect of chitosan was verified by monitoring the increasing body weight.The effect of different diet on weight gain was tested through the regular monitoring of food intake.The fasting glucose was tested by ACCU-CHEK Performa and the oral glucose tolerance was tested by the oral glucose tolerance test(OGTT).The serum lipid level was tested at QILU HOSPITAL OF SHANDONG UNIVERSITY.The serum leptin and insulin was tested using Elisa Kit through enzyme linked immunosorbent assay(Elisa).The mRNA levels of inflammatory genes at different intestinal segments were tested by quantitative real-time polymerase chain reaction(qRT-PCR).The adipocyte size was shown by hematoxylin and eosin(HE)staining and was calculated by ImageJ.The data between groups were compared by one-way ANOVA and the results were expressed as mean±standard deviation(SD)unless otherwise specified.Statistical probability of P<0.05 was considered significant.The V3-V4 region of the 16SrDNA of fecal samples was sequenced on HiSeq2500 by Novogen Company.The data were quality controlled by Usearch software to remove low quality sequences.Uparse algorithm is used to cluster the effective tags of 230 samples,and 97%of them are classified into 1602 operational taxonomic units(OTUs).The OTUs were annotated with the "Ribosomal Database Project" database.The alpha diversity,beta diversity,difference and correlation analysis of annotated data was analyzed by R software.The R package "ggplot2" was used for dilution curve;The α diversity was analyzed by the R package "ggplot2" and "ggsignif",theβ diversity was analyzed by the R package "ape" and "ggplot2",and the significance analysis was analyzied with Wilcoxon test.In the difference analysis,the R package"dplyr" was used for screen,"deducer" was used for test,"fdrci" was used for correcting and "pheatmap" was used for exhibition.Spearman correction was analyzed by the R package "Hmisc" and was shown using cytoscape software.Results1.Effects of chitosan on improving obesity:the body weights of high fat diet feeding mice were significantly higher than the other three groups and chitosan supplement decreased the increasing body weight caused by the high fat diet.But the effect of antiobesity was not showed in low fat diet feeding mice.Chitosan supplement decreased the food intake compared with the normal high fat diet feeding.Chitosan supplement reduced the high body fat rate and adipocyte hypertrophy caused by high fat diet.Compared with the normal high fat diet,the increasing body weight caused by per unit diet was reduced when chitosan was supplemented.2.Effects of chitosan on metabolize:chitosan increased the serum leptin level in the high fat diet groups and increased the oral glucose tolerance in the low fat diet groups.But the level of fasting glucose,serum lipid,serum insulin and insulin resistance level didn’t change significantly.Chitosan reduced the chronic inflammation caused by high fat diet consumption;however it increased the normal intestinal inflammation level when added into the low fat diet.3.Effects of chitosan on gut microbiota:the gut microbiota was influenced by the chitosan intervention as the feeding time increased.Chitosan supplementation increased the anti-obesity-related species,such as Coprobacillus cateniformis and Clostridium leptum,and significantly decreased obesity-related species such as Clostridium lactatifermentans and Clostridium cocleatum.Moreover,spearman correlation analysis showed that Coprobacillus cateniformis and Clostridium leptum were positively correlated with serum leptin,Ruminococcus bromii was positively related to serum insulin.Conclusions1.Chitosan reduce the increasing body weight caused by high fat diet and show good effects on impoving obesity and reducing body fat rate.2.Chitosan show good effects on improving obesity as a prebiotic.Moreover,many obesity or antiobesity related species and its’ related effects are found by the difference analysis and correlation analysis of gut microbiota.Therefore,these results provide broader ideas and evidence for the future research on the function of such bacteria and the improvement of obesity.