Effect Of Anti-tuberculosis Drugs On Gut Microbiota And Improvement Effect Of Lactobacillus Casei Supplement

Purpose:Continuous high-dose application of anti-tuberculosis(TB)drugs challenges intestinal microorganism homeostasis and gastrointestinal tolerance.First-line anti-TB drugs are the main driving factor of intestinal microorganism changes.In this study,anti-TB drug experiment and Lactobacillus casei ATCC334 supplement experiment were carried out.The objective is to explore the effects of anti-TB drugs(rifampicin,pyrazinamide)on gut microbiota and its metabolic function,and further study the potential protective effect of probiotics on gut microbiota and intestinal barrier,so as to provide theoretical basis for improving the adverse reactions of gastrointestinal tract caused by anti-TB drugs through dietary supplementation.Methods:(1)Experiment 1:30 male Wistar rats(7 to 8 weeks old,230 to 250g)were randomly divided into 3 groups(10 rats per group):control group(CN),isoniazid+rifampicin model group(HR),and pyrazinamide model group(PZA).CN group,HR group and PZA group were given 0.5%sodium carboxymethyl cellulose(CMC),HR suspension and PZA suspension,respectively.The intervention time was 6 weeks,during which body weight and food intake were recorded.Feces were collected before intervention,2 weeks and 6weeks after intervention,and gut microbiota diversity was analyzed by high-throughput sequencing 16S r RNA.Fecal SCFA target analysis was performed by GC-Q-MS platform,and association analysis was performed.(2)Experiment 2:To investigate the preventive effect of Lactobacillus casei on intestinal barrier caused by isoniazid+rifampicin.Four groups(10 rats per group)were set:CN group,HR model group,HR+low-dose Lactobacillus casei ATCC334 group(LLC)and HR+high-dose Lactobacillus casei ATCC334 group(HLC).Each group was given gavage twice a day,with an interval of 2hours.During initial intragastry,0.9%normal saline was given to the control group,HR group and PZA group,while L.Casei ATCC334 solution was given to the LLC group and the HLC group at 1.0×10~9CFU/kg·d and 2.0×10~9CFU/kg·d,respectively.After 2 hours,0.5%CMC was given to the CN group and HR suspension was given to the other groups.The intervention time was 6 weeks,and stool collection,high-throughput sequencing and SCFA target analysis were the same(1).After the intervention,aorta blood was collected and serum was collected for preservation.The serum levels of LPS andβ-defensin-2 were determined by limulus lysate reagent and enzyme linked immunosorbent assay,respectively.The corresponding samples of ileum and colon were collected.The colon preserved by paraformaldehyde was stained with HE to observe the morphological structure,histopathological score and goblet cell count.The contents of mucin 2(MUC2),intestinal lysozyme,Interleukin-6(IL-6),Interleukin-10(IL-10),Interleukin-12P70(IL-12p70)and Tumor necrosis factor-α(TNF-α)were determined by ELISA.Spearman regression was used to analyze the correlation degree between intestinal barrier index and intestinal microorganism.Results:Results:(1)The experimental results of the effects of anti-TB drugs on intestinal microflora and SCFA in rats showed that after the intervention.Compared with the control group,Two weeks of isoniazid+rifampicin treatment resulted in a 34.5%decrease in the number of intestinal microflora OTU(P=0.038),The Chao 1 index and Shannon index were decreased by 33.4%(P=0.028)and 24.1%(P=0.016),respectively,and the relative abundance of Firmicutes was decreased by 34.1%(P=0.038).Two weeks of pyrazinamide treatment resulted in a 12.6%decrease in OTU(P=0.057),a 16.0%decrease in ACE index,a 15.4%decrease in Chao 1 index,and a 72.3%decrease in Shannon index(P<0.001),respectively,Simpson index increased by 84%(P<0.001).At 6 weeks,compared with the control group,OTUs in the HR group were decreased by 18.6%(P=1.0),and the difference was not statistically significant.The ACE index and Chao1 index were decreased by 19.4%(P=0.017)and 25.4%(P=0.028)respectively.At the end of treatment,isoniazid+rifampicin mainly caused changes in intestinal Firmicutes and some Bacteroidetes genera,including a 150-fold decrease in Lachnospiraceae＿NK4A136＿group(P=0.023).Ruminococcaceae-UCG-005 was increased by 7 times(P=0.023)and Bacteroidetes by 9times(P=0.007).For isoniazid+rifampicin,at 6 weeks,the levels of butyrate,valerate and caproic acid in the HR group were reduced by 73.0%(P=0.003),46.5%(P=0.009)and 95.6%(P=0.01),respectively,compared with the control group.The results of the correlation analysis between intestinal microbiota and SCFA at 6 weeks showed that Bacteroides,Lachnospiraceae＿NK4A136＿group and Marvinbryantia were related to the content of SCFA(butyric acid,valeric acid and hexanoic acid)in feces.The relative abundance of Lachnospiraceae＿NK4A136＿group was positively correlated with the contents of butyric acid(r=0.69,P=0.018),valeric acid(r=0.64,P=0.033)and hexanoic acid(r=0.68,P=0.022).The relative abundance of Bacteroides was negatively correlated with the contents of butyric acid(r=-0.83,P=0.002),valeric acid(r=-0.74,P=0.009)and hexanoic acid(r=-0.77,P=0.005).The relative abundance of Marvinbryantia was negatively correlated with the contents of butyric acid(r=-0.68,P=0.022),valeric acid(r=-0.69,P=0.018)and hexanoic acid(r=-0.79,P=0.004).(2)The effect of Lactobacillus casei supplementation with anti-TB drugs on intestinal flora changes and short-chain fatty acid levels.Effects of Lactobacillus casei supplementation with anti-TB drugs on intestinal microflora and SCFA levels.At 2 weeks,the number of OTU,diversity index,microorganism structure and SCFA level in the low-dose and high-dose Lactobacillus casei intervention groups were similar to those in the HR group,and there was no statistical difference.After 6 weeks of intervention,the rank sum test showed that the relative abundance of Lactobacillus in both the LLc and HLc groups was higher than that in HR,but there was no statistical difference.The Lachnospiraceae＿NK4A136＿group level of LLc group and HLc group was the same as that of HR group(P<0.02).In addition,Blautia was significantly increased in the HLc group compared to the control group(P<0.01).After 6 weeks of intervention,the levels of butyric acid,valerate and hexanoic acid in HR group were 14.9±10.5μg/g(P=0.003),1.51±0.88μg/g(P=0.009)and 2.39±2.13μg/g(P=0.01)lower than those in control group,respectively.Compared with the HR group,low-dose and high-dose L.casei ATCC334 intervention increased butyric acid levels by 4.35±0.88μg/g(P=1.0)and 2.78±2μg/g(P=1.0).The contents of total SCFA,butyric acid and valeric acid in low and high dose Lactobacillus casei intervention groups were still significantly lower than those in control group.(3)The results of intestinal mucosal structure and function analysis showed that compared with the normal group,isoniazid+rifampicin treatment decreased goblet cell proportion and mucin-2 content in colon(P>0.50).High dose Lactobacillus casei supplementation significantly increased goblet cell proportion and colonic mucin 2 levels(P>0.05).In addition,Lactobacillus casei intervention increased colonic secretory immunoglobulin A(s Ig A)and interleukin 10(IL-10)levels,respectively,compared with the control group(P>0.50).Levels ofβ-defensin-2,IL-10 and s Ig A in the high-dose Lactobacillus casei group were significantly higher than those in the HR group(P>0.50).Spearman correlation analysis showed that the level of MUC2 in colon was significantly positively correlated with the level of Cyanobacteria(r=0.41,P=0.05),Bacillus(r=0.48,P=0.03),Roseburia(r=0.50,P=0.04)and[Ruminococcus]＿Gauvreaui＿group(r=0.59,P>0.001).Conclusion:The combination of isoniazid and rifampicin can change the composition of intestinal flora and decrease the level of short-chain fatty acids.Supplementation of Lactobacillus casei can improve the diarrhea degree of rats,which may be related to the regulation of intestinal flora,short-chain fatty acids and mucin-2 and increase the level of immune factors.