Effects Of Sodium Butyrate Supplementation On Inflammation,Gut Microbiota And Short-chain Fatty Acids In Helicobacter Pylori-infected Mice

Objective:To investigate the effects of sodium butyrate（SB）on the viability and main virulences of Helicobacter pylori（HP）in vitro,and to investigate the effects of SB supplementation in vivo on inflammation,gut microbiota and fecal short-chain fatty acids（SCFAs）of HP infected mice,in order to more clearly understand the relationship between gut microbiota and their metabolites,and to find a certain theoretical basis for clinically effective treatment of HP infection-related diseases.Methods:1.Under micro-oxygen culture conditions,the growth of HP ATCC 43504 strain in different time（0,24,48,72h）and different concentration of SB（0,5,10,20 mM）was detected by OD₆₀₀ determination.Total RNA was extracted by Trizol method,and the effects of different concentration of SB on the main virulence factors CagA and VacA of HP were detected by quantitative reverse transcription polymerase chain reaction（qRT-PCR）.2.24 male specific pathogen free（SPF）C57BL/6 mice were purchased and randomly divided into 3groups,which were the control group（CN group,n=8）,the HP infection group（HP group,n=8）and SB supplementation group（HPSB group,n=8）.C57BL/6mice in HP group and HPSB group were gavaged with HP SS1 strain.The infection of C57BL/6 mice by SS1 strain was confirmed by rapid urease test（RUT）,stool antigen detection test,the hematoxylin-eosin stain（HE）.Mice in HPSB group were gavage supplemented with SB,and other groups were gavage with the same amount of phosphate buffer saline（PBS）.The qRT-PCR was used to detect the levels of CagA and VacA,the main virulence factors of HP,and the levels of Toll-like receptor 2（TLR2）and Toll-like receptor 4（TLR4）in the gastric tissues of mice in 3 groups.Gastric tissues and serum samples of the 3groups were collected.The levels of tumor necrosis factor-α（TNF-α）,lipopolysaccharides（LPS）and interleukin-8（IL-8）in the samples of mice were detected by Enzyme-linked immunosorbent assay（ELISA）.Several proteins expression levels of p-IκBα,p-NF-κBp65,IκBαand NF-κBp65 in the gastric tissues of different groups of mice were detected by western blot（WB）.HE staining was used to observe the inflammation in the gastric of different groups of mice.The feces of mice from different groups were collected,and the total DNA of bacteria in fecal samples were extracted.The V3-V4 region of 16S r RNA gene was high-throughput sequencing using the gene sequencing platform,and then the sequencing data was processed through bioinformatics analysis.The content of SCFAs in fecal samples was determined by gas chromatography-mass spectrometry（GC-MS）,and the correlation between potential gut microbiota,SCFAs and inflammatory parameters was further analyzed by Spar CC.Results:1.The in vitro experiment showed that compared with the control group,the growth activity of HP in each dose of SB group was significantly decreased（P<0.05）,and the growth activity of HP was also decreased with the extension of SB treatment time（P<0.05）.There were significant differences in HP growth activity among different SB dose groups（P<0.05）.qRT-PCR results showed that SB treatment reduced the m RNA levels of CagA and VacA,the main virulence factors of HP,in a dose-dependent manner（P<0.01）.2.The results of RUT,fecal antigen test and HE staining showed that mice in the HP group and HPSB group were successfully infected with HP.3.The qRT-PCR results showed that the m RNA levels of CagA and VacA in HPSB group were significantly decreased compared with those in HP group（P<0.001）.TLR2 and TLR4 m RNA levels were increased in the HP group compared with the CN group（P<0.001）,and decreased in the HPSB group compared with the HP group（P<0.01）.4.Western blot results showed that the protein expression of p-IκBαand p-NF-κBp65 in HP group were increased compared with those in CN group,and the protein expression of p-IκBαand p-NF-κBp65 in HPSB group were decreased compared with those in HP group.The results of HE staining showed that a large number of mononuclear and neutrophil infiltrates were found in the HP group compared with the CN group.Compared with the HP group,the number of monocytes and neutrophils in the HPSB group was reduced.ELISA results showed that the levels of IL-8,TNF-αand LPS in the gastric tissues and serum of HP mice were increased compared with those in the CN group（P<0.001）,and the levels of IL-8,TNF-αand LPS in the gastric tissues and serum of HPSB mice were decreased compared with those in the HP group（P<0.001）.5.16S r RNA results showed that compared with the CN group,the relative abundance and diversity of gut microbiota of mice in the HP group increased,and compared with the HP group,the relative abundance and diversity of some gut microbiota of mice in the HPSB group changed.6.GC-MS results showed that the total levels of SCFAs in feces of acetic acid（AA）,propionic acid（PA）and butyric acid（BA）were basically similar among the three groups.Compared with the CN group,the levels of isobutyric acid（IBA）,BA,valeric acid（VA）and hexanoic acid（HA）in feces of mice in HP group were significantly decreased（P<0.05）.No significant differences were observed compared with the HP group,although the levels of AA,PA,and isovaleric acid（IVA）were reduced.7.Clostridium was positively correlated with IL-8 and TNF-αlevels.A significant positive correlation has been observed between Psychobiosis and IVA levels,while a significant negative correlation has been observed between Desulfovibrio and AA,HA,BA and VA levels.Conclusion:1.SB can inhibit the growth of HP in a dose and time dependent manner,respectively.SB can reduce the expressions of CagA and VacA,the main virulence factors of HP in a dose dependent manner.2.SB can reduce the main virulence factors of CagA,VacA and LPS,decrease the expression of TLR2 and TLR4,inhibite the IκBα/NF-κB pathway,decrease the production of TNF-αand IL-8,and reduce the inflammatory response of HP-infected mice.3.The gut microbiota and fecal SCFAs of HP-infected mice are changed,and the supplementation of SB have significant changes in some gut microbiota and may have no significant changes fecal SCFAs of HP-infected mice.4.Part of gut microbiota is related to inflammation and SCFAs,SB may reduce inflammation through its relationship with gut microbiota of HP-infected mice,and the specific mechanism remains to be further studied.