| In the modern large-scale and intensive farming mode,the pursuit of the growth rate of livestock and poultry leads to higher and higher body fat deposition,which leads to the imbalance of body fat metabolism.Many studies have revealed that intestinal flora is closely related to the development of chronic metabolic diseases.The imbalance of intestinal flora can cause obesity,hypertension,colitis and other diseases.Orosomucoid 2(ORM2)is an acute phase reactive protein secreted by liver,which plays an important role in regulating lipid metabolic diseases and immunity.The composition of intestinal flora was found to be influenced by host genotypes,and the mechanism of action between Orm2 and intestinal flora in obesity-related lipid metabolic diseases remains unclear.Therefore,based on the establishment of Orm2 knockout mice,this study aims to explore the effect of Orm2 deletion on intestinal flora of mice,clarify the important role of Orm2 gene in intestinal flora regulation of body fat metabolism and its influence on intestinal inflammation and dynamic balance of pathogenic bacteria under the condition of high fat diet,and further provide a new strategy for the prevention and treatment of metabolic disorders.This study mainly includes the following four parts:Experiment 1: Effect of Orm2 knockout on intestinal flora in mice.The 8-week-old male Orm2 knockout mice and wild-type C57BL/6N mice were fed a normal diet for 12 weeks.The weight of the mice was recorded during this period,the metabolic level of the mice was detected by insulin tolerance test(ITT),fresh feces were collected from the mice and the genome was extracted.At the end of the experiment,the adipose tissue of mice was collected and observed by HE staining.The results showed that the body weight of Orm2 knockout mice was significantly higher than that of wild-type mice(P < 0.001).The weight of white fat and brown fat was significantly higher than that of wildtype mice(P < 0.001).HE staining showed hypertrophy of adipocytes in Orm2 knockout mice.In addition,the sensitivity of Orm2 knockout mice to insulin was significantly decreased(P < 0.05).The results of 16 S r RNA gene sequencing in mouse feces showed that there was no significant change in α diversity of intestinal flora of Orm2 knockout mice,but there was significant difference in β diversity analysis of intestinal flora between the two groups of mice.Further analysis of bacterial community composition at phylum level showed that compared with wild-type mice,Orm2 gene knockout significantly improved the relative abundance of intestinal Firmicutes and the ratio of Firmicutes to Bacteroidetes was significantly increased(P < 0.05).In addition,Linear discriminant analysis Effect Size(LEf Se)showed that the species richness of mice in the two groups was different at different levels.Firmicutes,Ligilactobacillus,Ligilactobacillus-unclassified played a major role in the intestinal tracts of Orm2 knocked out mice.The wild-type mice were mainly Muribaculaceae.These results indicate that the intestinal flora composition of mice induced by Orm2 knockout changes under normal diet.Experiment 2: Effect of Orm2 knockout on intestinal flora in mice with high-fat diet.In experiment 2,we changed the dietary pattern of mice to explore the effect of high-fat diet on intestinal flora of Orm2 knockout mice.Twelve male wild-type C57BL/6N mice and six male Orm2 knockout mice were divided into 3 groups(n=6): control group(CON group);High-fat diet group(HFD group);Orm2 knockout mice in high-fat diet group(KO-HFD group).Both the control group and the high-fat diet group lasted for 16 weeks,during which the body weight of mice in each group was recorded,the metabolic level of mice was detected by ITT,fresh feces of mice were collected and the genome was extracted.At the end of the experiment,the adipose tissue of mice was collected,and the adipose tissue morphology was observed by HE staining.The results showed that compared with mice in HFD group,the body weight of mice in KO-HFD group increased significantly after high-fat diet(P < 0.0001),white fat weight increased but no significant difference,brown fat weight increased significantly(P < 0.05),HE staining showed hypertrophy of adipocytes.Then,KO-HFD group showed significant insulin resistance(P < 0.05).16 S r RNA sequencing results showed that compared with HFD group,the number of ASV in KO-HFD group was significantly decreased(P < 0.05),α diversity was significantly decreased(P < 0.05),PCo A analysis showed significant difference in β diversity among the three groups.In addition,from the microorganisms displayed in the stacking diagram,it was found that the abundance of Clostridium-unclassified and Clostridium in KO-HFD group was significantly lower than that in HFD group(P < 0.05);LEf Se analysis also showed that Clostridium was the dominant bacterial enriched in HFD group.These results indicate that Orm2 knockout exacerbates obesity in mice with high-fat diet and affects the composition and structure of intestinal flora,suggesting that Clostridium may play a key role in obesity response induced by high-fat diet.Experiment 3: Effects of Clostridium butyricum supplementation on obesity and intestinal flora in mice.Based on the research results of experiment 2,probiotic Clostridium butyricum(CB)is selected to supplement mice in this experiment.8-week-old male wild-type C57BL/6N mice and male Orm2 knockout mice were randomly divided into 4 groups(n=8): control group(WT group);Orm2 knockout group(KO group);Wild-type mice Clostridium butyricum supplement group(WT+CB group);Orm2 knockout mice Clostridium butyricum supplement group(KO+CB group);All four groups were on a high-fat diet for 14 weeks.During the experiment,the body weight and water intake of mice were recorded,glucose tolerance test(OGTT)was used to detect the metabolic level of mice,fresh feces were collected from mice and genome was extracted.At the end of the experiment,the adipose tissue of mice was collected,and the adipose tissue morphology was observed by HE staining.The results showed that the body weight of mice in WT+CB group was significantly lower than that in WT group(P < 0.05),body weight of KO group and KO+CB group tended to be the same;There was no significant difference in daily water intake among all groups.Compared with WT group,fat weight in WT+CB group decreased(P < 0.05),the adipose cell volume decreased,but the adipose tissue weight and cell volume had no significant change in KO+CB group.Then,OGTT results showed that compared with WT group,mice in WT+CB group were more sensitive to glucose(P = 0.07).In addition,16 S r RNA sequencing results showed that CB supplementation significantly improved the βdiversity of mice in WT group,but had no significant effect on the KO group.Further analysis of the relative abundance of microflora at phylum level showed that the composition of intestinal flora was more different between WT group and WT+CB group.These results indicate that the supplementation of Clostridium butyricum can alleviate the obesity of wildtype high-fat diet mice and improve the diversity of intestinal flora species.Clostridium butyricum had no significant effect on Orm2 knockout mice.Experiment 4:Manipulating the intestinal microbiome alleviates intestinal inflammation and obesity in Orm2 knockout mice.Since the supplementation of Clostridium butyricum alone did not significantly alleviate the obesity of Orm2 knockout mice,a co-housing experiment was designed to explore the effects of microbiome on different genotypes of mice through the exchange of intestinal flora between mice based on their coprophagy habits.The experiment was divided into four groups,wild-type mice(WT group);Orm2 knockout mice(KO group);An equal number of male wild-type C57BL/6N mice(CO-WT group)and male Orm2 knockout mice(CO-KO group)were placed in the same cage.All mice were fed a high-fat diet for 14 weeks.During the experiment,the body weight of mice was recorded,the metabolic level of mice was detected by OGTT,fresh feces of mice were collected and the genome was extracted.At the end of the experiment,fat and small intestine tissues of mice were collected,and the morphology of adipose tissue was observed by HE staining,and the expression of genes related to inflammation in small intestine tissue was detected by real-time quantitative PCR.The results showed that compared with the KO group,the body weight of mice in the COKO group decreased significantly at week 14(P < 0.01),the weight of white adipose tissue and brown adipose tissue was also significantly decreased(P < 0.05).HE staining showed that the adipocytes in the CO-KO group were reduced,but there was no significant change in the CO-WT group.OGTT showed that mice in CO-KO group were significantly more sensitive to glucose than those in KO group(P < 0.05).16 S r RNA sequencing results showed that the β diversity of intestinal flora in KO group was significantly increased in the co-cage.At the genus level,the difference of intestinal flora composition between KO group and CO-KO group was more obvious,among which,Desulfovibrionaceae,Desulfovibrionaceae,Robinsoniella and Anaerotruncus showed an increase in relative abundance in KO group and a significant decrease in CO-WT group(P < 0.05),indicating a positive regulatory relationship between pathogenic bacteria and obesity.KEGG enrichment analysis showed that the main biological pathways in KO group mice were related to bacterial chemotaxis,lipopolysaccharide biosynthesis and chemotaxis signaling pathway under high-fat diet.However,the relative abundance of these pathways in the CO-KO group was reduced after co-cage.Secondly,m RNA levels of inflammatory cytokines in small intestine tissues were detected,and the levels of IL-4,IL-6 and IFN-γ in CO-KO group were significantly lower than those in KO group(P < 0.05).Further correlation analysis showed a positive correlation between pathogenic bacteria and inflammatory factors.These results suggest that the obesity induced by high-fat diet in Orm2 knockout mice may be related to intestinal microbial ecological imbalance and intestinal inflammation.In conclusion,the present study explored the changes in intestinal flora composition of wild-type and Orm2-knockout mice under high-fat diet,indicating that Orm2 deficiency exacerbates intestinal flora disturbance and intestinal inflammation induced by high-fat diet.Simultaneously,it provides a new insight into the complex microbiota,which is not only given a single probiotic in the treatment strategy of obesity,and further provides a new reference basis for the involvement of Orm2 in the regulation of intestinal flora in lipid metabolism related diseases. |
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