Figure 6.

Broad-spectrum-antibiotics abolished differences of gut microbiota between AL and DR mice. Two-month-old mice were fed with AL diet or 30% DR diet for 14 days before MTX administration and the diet regimen was continued afterward. Then, all mice were intraperitoneally injected with MTX for 2 days at a dose of 120mg/kg (d-1) and 60mg/kg (d0). Seven days prior to MTX administration, mice were fed by gavage for 5 days and then in drinking water for the following days with broad-spectrum antibiotics or saline. Fecal samples were randomly collected from mice after 14 days on AL or DR diet before MTX injection and on day 2 after MTX injection for 16S rRNA gene sequencing (n = 5–8 mice per group randomly picked from 2 independent experiments) and qPCR analysis (n = 5 mice per group randomly picked from 2 independent experiments). (A) Shannon index of indicated groups based on alpha diversity analysis. (B) Variation of intestinal flora structure of indicated groups along PC1 and PC2 of PCoA based on the Bray–Curtis distance. (C) Relative abundance of the Lactobacillus showed by 16S rRNA gene sequencing. (D,E) qPCR analysis of the amounts of total bacteria (D) and Lactobacillus (E) of indicated groups (n = 5 mice per group, randomly picked from 2 independent experiments). Results were displayed as mean±SD. n.s; nonsignificant; *: P < .05; **: P < .01; ***: P < .001; ****: P < .0001 by one-way ANOVA. AL: mice on AL diet; DR: mice on DR diet; AL+Abx: mice on AL diet with Abx administration before MTX injection; DR+Abx: mice on DR diet with Abx administration before MTX injection; AL+Abx+MTX: mice on AL diet with Abx administration day 2 after MTX injection; DR+Abx+MTX: mice on DR diet with Abx administration day 2 after MTX injection.