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. Author manuscript; available in PMC: 2022 Feb 21.
Published in final edited form as: Sci Transl Med. 2021 Sep 22;13(612):eabg3459. doi: 10.1126/scitranslmed.abg3459

Fig. 5. Identification of a molecule, J11, able to induce BDNF release from enteric glia, improve motility, and reduce NEC in mice and human tissue ex vivo.

Fig. 5.

(A) The structural formula of “J11”, a molecular “hit” from a drug screen seeking molecules that induce BDNF from enteric glia. (B) Determination of BDNF concentration by ELISA from the enteric glia cell line EGC/PK060399egfr in which cells were treated with J11 (20μM) for the indicated time (n≥2 wells/time point) (C) Determination of BDNF concentration by ELISA in small intestinal lysates from wildtype or BdnfΔDTR mice at p11 after treatment with J11 (n≥7 mouse ileal tissues/group). (D) qRT-PCR expression of Tnf-α in ileal tissue of premature mice treated with LPS (5mg/kg) and J11 (20mg/kg) as indicated (n≥7 mouse ileal tissues/group). (E) FITC-dextran motility assay in wildtype or BdnfΔDTR mice at p11 treated with LPS (5mg/kg) and J11 (20mg/kg) as indicated (n>7 mice/group). (F to L) wildtype or BdnfΔDTR mice were induced to develop NEC in the presence or absence of J11(20mg/kg); shown at the end of the four-day model are (F to I) representative H&E images, (J) qRT-PCR expression in the intestinal epithelium of Tnf-α (n≥3 Bdnf deficient mice and n≥8 wildtype mice/group) (K) NEC severity scores (n n=6 histological sections scored/group). (L) FITC-dextran motility assay (n≥3 Bdnf deficient mice and n≥8 wildtype mice/group). (M, N) human intestinal tissue was obtained from patients undergoing surgery for NEC, were treated for 5 hours with either saline (“ctrl”) or J11 (50μg/ml) ex vivo in 10% fetal bovine containing Dulbecco’s modified growth media and assessed for (M) release of BDNF in the condition media by ELISA (n≥3 specimens/group) and (N) qRT-PCR expression of TNF-α in the tissue (n≥3 specimens/group). Each dot in dot graphs represents data from individual mice. Statistical significance was determined by one-way ANOVA, followed by Tukey’s multiple comparisons tests using GraphPad prism or unpaired t-test 9 software. *p<0·05, **p<0·01, ***p<0·001. Scale bars: 100μm. Mice experiments were repeated two-three times with at least 3–4 mice per group in each experiment. For knockout mutant mice experiments, the littermates of wild-type genotypes were used and underwent identical treatments with diphtheria toxin. Data of 4–5 de-identified resected bowl specimens at the time of stoma closure (healed NEC).