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. 2020 Jan 26;12(1):1714401. doi: 10.1080/19490976.2020.1714401

Figure 2.

Figure 2.

DR significantly protected small intestine and ISCs from lethal doses of MTX. 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, mice were intraperitoneally injected with saline as control or MTX for 2 days at a dose of 120mg/kg (d-1) and 60mg/kg (d0). Mice were sacrificed at certain timepoints and small intestinal tissue was collected for further analysis. (A-C) Representative images of H&E staining of sections on day 3 after MTX administration (scale bar: 50μm). Square frames show the enlarged images of arrowhead pointed areas (scale bar: 20μm). (D) Villi height (30 crypts were counted for each mouse, n = 5 mice per group randomly picked from 2 independent experiments.) (E) Crypt number per millimeter (crypt number per 15 mm length was counted for each mouse, n = 5 mice per group randomly picked from 2 independent experiments.) and (F) cell number per crypt (30 crypts were counted for each mouse, n = 5 mice per group randomly picked from 2 independent experiments) at indicated timepoints after MTX administration. (G) Representative images of immunofluorescent staining of PCNA of jejunum on day 3 after MTX treatment (scale bar: 20μm). (H) Basal crypt PCNA-positive cell number per crypt counted from the whole small intestine at indicated timepoints after MTX administration (40 crypts were counted for each mouse, and n = 5 mice per group randomly picked from 2 independent experiments). (I) Representative images of cultured crypts on day 6 in culture derived from mice on day 3 after indicated treatment. Lower line: zoomed in vision of images in square frames of the upper line. Scale bars: 100 μm (upper); 50 μm (lower). (J) Seeding efficiency of cultured crypts on day 3 and number of viable organoids from cultured crypts on day 6 (n = 5 mice per group, combined 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 test. AL+Saline: mice on AL diet and received saline injection; DR+Saline: mice on DR diet and received saline injection; AL+MTX: mice on AL diet and received MTX injection; DR+MTX: mice on DR diet and received MTX injection; d3: day 3 after MTX injection; d6: day 6 after MTX injection.