| ObjectiveIn this study,bacteria related to lipid metabolism were screened from the intestines of obese mice fed with high-fat diet,analyzed the effects of their metabolites on the adipogenesis of 3T3-L1 cells,in order to identify bacterial metabolites related to lipid metabolism and provide reference for studying their relationship with obesity.Methods1.C57 male mice(8 weeks old)were divided into a high fat group(given a60% high fat diet)and a control group(given a maintenance feed).After 10 weeks,mice in the high fat group were considered obese if their weight exceeded20% of the average body weight of the control group,and the white adipose tissue mass of the abdomen of the two groups of mice was measured.2.Use lipid metabolism enriched culture medium and screening solid culture medium(with addition of triglycerides)to conduct preliminary bacterial screening on the colon and cecal contents of obese mice.3.Apply the culture supernatant of the primary screening bacteria to induce differentiation of 3T3-L1 cells for rescreening.16 S r DNA identification,screening of solid medium and detection of lipase activity by p-nitrophenol method were carried out for bacteria promoting 3T3-L1 cell fat differentiation.4.The protein in the culture supernatant was purified by precipitation of ammonium sulfate saturated solution,dialysis and ultrafiltration with ultrafiltration tube,and the protein content was determined by Bradford protein concentration determination kit.According to the detection results of polyacrylamide gel electrophoresis,the obvious differential protein bands were detected by mass spectrometry.5.The culture supernatant was precipitated by alcohol precipitation method,and the protein was removed by Sevag method(chloroform:n-butanol=4:1).The content of polysaccharide was determined by phenol-sulfuric acid method.6.Using the CCK8 detection kit to detect the cytotoxicity of the crude purified protein and polysaccharide from the culture supernatant of the rescreened bacteria.The crude purified products of protein and polysaccharide were acted on 3T3-L1 cells to induce differentiation,and oil red O staining and lipid quantification were performed.7.Extracting DNA from the bacteria obtained from the re-screening and conducting genome re-sequence by Bioengineering(Shanghai)Co.,Ltd.,comparing the effective data of the sample to the reference genome,analyzing the genotypic differences between each sample and the reference genome,and conducting differential analysis and comparison of the variant genes between different groups.Results1.After 10 weeks,the amount of white adipose tissue in the abdomen of high-fat obese mice was significantly higher than that of the control group mice.2.12 bacterial strains were screened from the colon and cecal contents of high-fat obese mice.The supernatant of the above 12 bacterial cultures was applied to induce lipid formation in 3T3-L1 cells.The results of oil red O staining and lipid content determination showed that the supernatants of bacteria 2,6,9,11,and 12 could promote lipid formation in 3T3-L1 cells.Among them,bacteria12 and 2 had the most significant promoting effect.Therefore,these two strains were selected for the next experiment.3.The 16 S r DNA test results showed that these two strains had the highest homology with Staphylococcus aureus and were different strains.Therefore,the12 th strain was named Staphylococcus aureus 2(SA-12),and the 2nd strain was named Staphylococcus aureus 2(SA-2).4.Lipase measurement results: the diameter of the transparent ring produced by tributylglycerol ester solid culture medium SA-2: 1.05 cm,SA-12:1.30cm;Lipase activity SA-2: 56.73U/m L,SA-12: 118.70U/m L;The activity of SA-12 lipase was higher than that of SA-2.5.Lipase measurement results: the diameter of the transparent ring produced by the screening medium SA-2: 1.05 cm,SA-12: 1.30cm;Lipase activity SA-2: 56.73U/m L,SA-12: 118.70U/m L;The activity of SA-12 lipase is higher than that of SA-2.6.The crude protein purification results showed that the amount of SA-2protein was 1.26 mg/m L,and the amount of SA-12 protein was 1.92mg/m L.The crude purification results of polysaccharides showed that the content of SA-2polysaccharides was 351.5mg and SA-12 polysaccharides was 226.5mg.7.According to the CCK8 detection results,the crude protein purified products of the two strains were added to 3T3-L1 cells at a final concentration of50ng/m L,and the crude polysaccharide purified products were added to 3T3-L1 cells at a final concentration of 0.5 μ g/μ L.The results of oil red O staining and lipid quantification were consistent,indicating that compared with the triple inducer group,the experimental group added SA-12 protein crude purification product had an increase in lipid content,with no statistically significant difference(P>0.05).The addition of SA-2 protein crude purification product,polysaccharide crude purification product,and SA-12 polysaccharide crude purification product all had an increase in lipid content,with a statistically significant difference(P<0.05),And there was no statistically significant difference between these three different added products(P>0.05).8.Mass spectrometry detection of protein bands in the crude purified product of SA-12 protein that differ significantly from SA-2 revealed lip2.Compared with the standard strain ATCC25923,SA-12 showed specific non conserved mutations at amino acids 12,50,58,261,270,271,272,357,390.9.The resequencing results showed that both strains of bacteria were Staphylococcus aureus.Compared with the standard strain ATCC25923,SA-2had 14 mutation sites,SA-12 had 32580 mutation sites,and the two strains shared 3 mutation sites.Comparison with the KEGG database showed differences in lipid metabolism between the two strains of bacteria.Conclusions1.Two strains of Staphylococcus aureus SA-2 and SA-12 were screened from the intestinal contents of mice,and their culture supernatants can promote adipogenic induction of 3T3-L1 cells.2.The crude purified products of SA-2 protein and polysaccharide,as well as SA-12 polysaccharide,can promote the induction of adipogenesis in 3T3-L1 cells. |
PDF Full Text Request