| Objective:In this study,a model of antibiotic induced intestinal microflora disorder in mice was established to observe the effects of different doses of Tributyrin(TB)on intestinal microflora,fecal short-chain fatty acids,intestinal inflammation and intestinal mucosal barrier related indexes in mice,and to explore the repair effect of TB on intestinal microflora disorder.Method: Thirty-two male C57BL/6 mice were randomly selected,and 24 of them were given 0.2 m L ceftriaxone sodium solution(400 mg/m L)by intragastric administration,twice a day for 7 days,to establish the intestinal flora disorder model of mice.Modeling mice(ABx group)were randomly divided into three groups: model group(M),TB low-dose group(TL)and TB high-dose group(TH),with 8 mice in each group.The other 8 mice were used as control group(NC).TL and TH groups were given 0.3 g/kg.BW and 3g/kg.BW TB solution by intragastric administration,respectively,and NC and M groups were given the same amount of normal saline by intragastric administration for 11 consecutive days.During the intervention period,body weight,food intake,water intake and fecal characteristics of the 4 groups of mice were monitored.After the intervention,the mice were sacrificed,and the cecum weight and colon length of the 4 groups were measured.Hematoxylin-eosin staining(HE)and Alcian blue staining(AB)were used to observe the histopathological changes of colon.16 S r RNA sequencing method was used to determine and analyze the changes of fecal intestinal flora of mice before intervention and on the 7th and11 th day after intervention.The content of short-chain fatty acids in mice feces was detected by gas chromatogram-mass spectrometry(GC-MS)The levels of lipopolysaccharide(LPS)and zonulin in serum of mice were detected by enzyme-linked immunosorbent assay(ELISA).Real-time quantitative PCR(RT-PCR) was used to detect mRNA expression levels of TNF-α,IL-6,NLRP3,ASC,caspase-1,IL-1β,MUC2,ZO-1,Occludin,E-cadherin and β-catenin in colon tissue of mice.The expression levels of NLRP3,ASC,caspase-1,IL-1β,ZO-1,Occludin,E-cadherin andβ-catenin in colon tissue of mice were detected by Western blot.Results: Compared with NC group,cecal index of mice in M group increased(P<0.05)and increased colon length(P<0.05);Compared with M group,cecal index of TL group and TH group was significantly decreased(P<0.05),shortened colon length(P<0.05).The intestinal flora analysis results before TB intervention showed that compared with NC group,the intestinal flora α diversity index(Chao1)of antibiotic treated mice(ABx group)decreased significantly(P<0.05),the intestinal flora structure was significantly different from that of NC group(P<0.05).The intestinal flora analysis of mice on the 7th day of intervention showed that the intestinal flora structure of TL group and TH group was significantly different from that of M group(P<0.05),which was closer to NC group;Compared with M group,αdiversity in TL group and TH group increased,but the difference was not statistically significant.The species composition analysis showed that the relative abundance of Muribaculaceae,Bifidobacterium,Lachnoclostridium and Parasutterella in TL group was higher than that in M group.The relative abundance of Lachnoclostridium and Akkermansia in the TH group increased,while the relative abundance of Bacteroides and Enterococcus in the 2 groups decreased significantly(P<0.05).Intestinal flora analysis of mice on the 11 th day of intervention showed that Chao1,Shanon and Simpson indices of intestinal flora of mice in TL group were significantly higher than those in M group(P<0.05);The intestinal flora structure of the NC group was similar to that of the M group,and was obviously separated from the NC group.Compared with M group,the relative abundance of Muribaculaceae and Bifidobacterium in TL group was significantly increased(P<0.05),the relative abundance of Enterococcus was decreased significantly(P<0.05);Compared with M group,α diversity index of mice in TH group was increased,but there was no statistical difference.PCo A results showed that the intestinal flora structure of TH group overlapped with TL group,was similar to NC group,and was obviously separated from M group(P<0.05);Compared with M group,the relative abundance of Muribaculaceae in intestinal flora of mice in TH group was significantly increased(P<0.05),the relative abundance of Enterococcus was significantly decreased(P<0.05).The results of GC-MS analysis showed that the relative contents of acetic acid,propionic acid,isobutyric acid,butyric acid and valeric acid in feces of antibiotic mice(ABx group)were significantly decreased before TB intervention(P<0.05).On day 7 after TB intervention,compared with NC group,the relative contents of acetic acid,propionic acid and butyric acid in feces of group M were significantly decreased(P<0.05);Compared with group M,the relative contents of acetic acid and butyric acid in group TL were significantly increased(P<0.05).The contents of acetic acid,butyric acid and valerate in TL group were significantly higher than those in TH group(P<0.05).On day 11 after TB intervention,compared with group M,the relative contents of acetic acid,propionic acid and butyric acid in feces of TL group were significantly increased(P<0.05),there was no significant difference in the contents of short-chain fatty acids in TH group(P>0.05).ELISA results showed that compared with NC group,serum expressions of LPS and zonulin in M group were significantly increased(P<0.05);The expression of LPS and zonulin in TL and TH groups was significantly lower than that in M group(P<0.05),and TL group was significantly lower than TH group(P<0.05).RT-PCR and Western blot results showed that compared with NC group,mRNA and protein expressions of inflammation-related indicators,including TNF-α,IL-6,NLRP3,ASC,caspase-1 and IL-1β,were significantly increased in M group(P<0.05);Compared with the M group,mRNA and protein expressions of inflammation-related indicators were significantly decreased in the TL group(P<0.05),and TL group was lower than TH group(P<0.05).Results of mRNA and protein of intestinal barrier related indexes showed that compared with the NC group,mRNA expression of MUC2,mRNA and protein of ZO-1,Occludin,E-cadherin andβ-catenin in the M group significantly decreased(P<0.05),mRNA and protein expressions in TL group were significantly increased(P<0.05),while the TH group had no significant change.Conclusion: Moderate amount of triglyceride can improve the diversity of intestinal flora,increase the relative abundance of beneficial bacteria and reduce the relative abundance of harmful bacteria in mice.At the same time,it can improve the level of short-chain fatty acids,reduce intestinal inflammation,enhance intestinal mucosal barrier,and play a role in repairing the imbalance of intestinal flora caused by antibiotics in mice and restoring intestinal function. |
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