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. 2020 Jun 22;10:10065. doi: 10.1038/s41598-020-66786-0

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The microbial suppression decreases aortic miR-204 and improves endothelial function in db/db mice. (a) The fecal microbiota load in the db/db and db/db-204^−/− mice following antibiotics treatment (A) and microbiota recolonization (F). n = 5–10. (b) ISH showing aortic miR-204 following antibiotics-induced microbial suppression and recolonization (blue, magnification ×40, Scale bar; 20 µm). (c) Quantification of miR-204 expression in ‘b’. “n(N)” for ‘c’ where ‘n’ represents the number of images and ‘N’ represents the number of mice. db/db: n = 8(2), db/db-A: n = 8(2), db/db-AF: n = 8(2). (d,e) The acetylcholine mediated vascular relaxation of the aortic rings isolated from db/db and db/db-204^−/− mice after microbial suppression (A) and recolonization (F). The experimental replicate is shown as “n(N)” where ‘n’ represents the number of aortic rings and ‘N’ represents the number of mice. db/db: n = 21(7), db/db-A: n = 9(5), db/db-AF: n = 12(5), db/db-204^−/−: n = 12(4), db/db-204^−/–-A: n = 4(3), db/db-204^−/–-AF: n = 14(6). (f) Immunostaining of aortic sections showing upregulation of endothelial apelin with antibiotics treatment and reversal with recolonization (magnification ×63, Scale bar; 10 µm). (g) Quantification of endothelial apelin in ‘f’. db/db: n = 9(2), db/db-A: n = 6(2), db/db-AF: n = 6(2). (h) Immunostaining of aortic sections showing upregulation of endothelial APJ with antibiotics treatment and reversal with recolonization (magnification ×63, Scale bar; 10 µm). (i) Quantification of endothelial APJ in ‘h’. db/db: n = 8(2), db/db-A: n = 8(2), db/db-AF: n = 8(2). The experimental replicate for ‘g’ and ‘i’ is shown as “n(N)” where ‘n’ represents the number of images and ‘N’ represents the number of mice. *p < 0.05, and ***p < 0.001 vs. indicated group. The data is shown as mean, and the error bar represents s.e.m. A one-way analysis of variance (ANOVA) followed by Tukey’s test was performed. The significance of difference between two curves was analyzed by using global non-linear regression. Ach, acetylcholine; PE, phenylephrine; vWF, von-Willebrand factor.