Alamandine/MrgD Pathway Modulates Gut-Bone Marrow Axis in Aging

Figures

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  Figure 1.. Reversal of the increased intestinal permeability in aging by Alamandine. A and B. Shown were representative western blots of MrgD, MasR and β-actin proteins in the colon tissue lysates from young and old mice. The expression of MasR was increased in the Old colons of both males and females (P < 0.05, n = 6) while that of MrgD was decreased in the male (*P < 0.05, n = 6) but unchanged in females compared to the Young. C. Dose-dependent effect of Alamandine (Ala) on the intestinal permeability in the Old mice tested by FITC-dextran permeability. Permeability was higher at all three time points tested in the Old group compared to the Young (***P < 0.001, n = 4). At the dose of 1 μg/Kg/min for four weeks, Ala reversed the increased permeability in the Old mice (P < 0.001, n = 4) compared to the untreated group. The effect at lower doses, 0.01 and 0.1 μg/Kg/min for four weeks, was not significant (n = 4). D and E. Ala treatment reversed the increased permeability in both male and female Old mice (***P < 0.001, n = 7). Effect of Ala in the Old group was not observed when concurrently administered with D-Pro7-Ang-(1–7) (n = 5). Two-way ANOVA followed by Bonferroni’s multiple comparison…

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  Figure 2.. Alamandine restored epithelial barrier integrity in the aging colon. A. Shown were representative light microscopic images of Hematoxylin–Eosin (H&E) staining of colon sections. Histological changes depicted disruption of epithelial continuity with atrophied crypts in the Old mice, which was reversed by Ala treatment (n = 5). B - D. Shown were representative light microscopic images of Alcian Blue PAS staining of colon sections. The number of Goblet cells per villus was decreased in the Old group compared to the Young (*P < 0.05, n = 15 villi/5 mice) that was increased by Ala (* P < 0.05, n = 15 villi/5 mice). Mucus layer was either thinner or absent in the Old colons compared to the Young (*** P < 0.001, n = 6) that was increased by Ala (**P < 0.001, n = 6). Data sets were analyzed by Kruskall–Wallis test followed by Dunn’s test. E. Shown were representative fluorescence images of claudin 1 and occludin immunohistochemistry with nuclear counter stain DAPI in colon sections derived from the experimental groups, Young, Old and Ala treated Old mice. Decreased expression of claudin 1 and occludin in the colons of Old mice were reversed by Ala (n = 6 mice).

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  Figure 3.. Alamandine restored intestinal stem cell layer in the aging colon. A. Shown were representative fluorescence images of MrgD and Lgr5 immunohistochemistry with nuclear counter stain DAPI in colon sections of Young mice showing evidence for the colocalization of MrgD with Lgr5. B. Shown were representative fluorescence images of Lgr5 and Olfm4 with nuclear counter stain DAPI in colon sections derived from the experimental groups, Young, Old and Alamandine (Ala) treated Old mice. Decreased Olfm4 and Lgr5 dual positive structures in the Old group was restored by Ala-treatment. C. Shown were representative light microscopic image and fluorescent images of colon organoid derive from Young mice with MrgD and Lgr5 immunohistochemistry showing evidence for colocalization of MrgD with Lgr5. D and E. Shown were representative light microscopic images of colon organoids derived from the experimental groups, Young, Old and Ala treated Old mice. The number and the diameter of organoids were lower in the Old group (***P < 0.001) that were increased by Ala-treatment (**P < 0.001). F. Shown were representative western blots of Wnt3a in the colon lysates derived from three experimental groups,…

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  Figure 4.. Alamandine restored Wnt3a levels in the colonic organoids derived from Old mice via MrgD/Gαs/AC/CREB/β-catenin pathway. A and B. Shown were representative western blots of Wnt3a or claudin proteins with β-actin as an internal control in the lysates of colon organoids treated with different concentrations of Ang-(1–7) or Ala with or without A779 or D-Pro7-Ang-(1–7). C - D. The organoids derived from the Old mice showed lower levels of Wnt3a and claudin1 compared to that of Young mice (**P < 0.01). Ang-(1–7) (100 nM) and Ala (1 nM) increased Wnt3a and claudin1 that did not achieve statistical significance. Increase in the Wnt3a or claudin1 by Ala at 10 and 100 nM was significantly higher than the untreated Old-colon organoids (**P < 0.01) and similar to that observed in the Young. Effect of Ala (10 nM) on Wnt3a in the Old-colon organoids was not affected by the simultaneous treatment with A779 (1 μM) but decreased by D-Pro7-Ang-(1–7) (1 μM) (*P < 0.05). A779 or D-Pro7-Ang-(1–7) (1 μM) did not show any increase in the Wnt3a levels of Old-colon organoids. E. Shown were representative western blots of Wnt3a and and β-actin proteins in the colon organoids derived from the Young or Old…

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  Figure 5.. Restructuring of dysbiotic gut microbiota in the aging by Alamandine. A. Histograms showing a decrease in the richness (Chao1 index) of the microbiota in the Old group compared with the Young, which was restored by Ala. B. Beta diversity as tested by principal coordinate analysis by using Brey–Curtis was significantly different among three groups depicting differences in microbial composition (PERMANOVA analysis). C. Correlation of microbial families with functional measures of gut permeability carried out by Spearman’s rank correlations (*P < 0.05, **P < 0.01 and ***P < 0.001). D and E. Gut microbial metabolites, acetate and butyrate were higher in cecum of Old mice treated with Ala (*P < 0.05) compared with the untreated. In the serum, the propionate levels were higher in the Old compared to the Young that was decreased by Ala-treatment. Kruskal-Wallis test followed by Dunn’s test. F. Shown were heat map and comparison of the concentrations of bile acid metabolites in the cecum. The levels of GDCA and TCDCA were higher or lower, respectively, in the Old groups compared to the Young (*P < 0.05, n = 10). The levels of GDCA was decreased and that of TUDCA, TCDCA, TCA and GCDCA…

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  Figure 6.. Aging-associated gut wall inflammation is ameliorated by Alamandine. A. Shown were representative fluorescence images of CD80 and CX3CR1 immunohistochemistry with nuclear counter stain DAPI in colon sections from different treatment groups. B and C. Increased number of CD80 cells in the old mice colon were decreased while the number of CX3CR1 cells were increased by Ala treatment in old mice (*P < 0.05, **P < 0.001 vs old untreated, n = 6). D. The tissue concentrations of cytokines such as IL-13, MCP-5, MIP-1a, CXCL4 CXCL9, CXCL13, CCL24 and CCL17 were higher in colons from the Old mice compared to the Young, which were decreased by Ala. Effect of Ala on CXCL13, CCL24 and CCL17 was not significant. CXCL1 was higher in the Old colons compared to the Young but was not significant and it was significantly decreased by Ala. *P < 0.05, **P < 0.001, Kruskal-Wallis test followed by Dunn’s test was used for statistical significance.

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  Figure 7.. Aging-associated increase in the myelopoiesis in the bone marrow was decreased by Alamandine. A. Shown were representative flow cytometry dot plots of CD45+Ly6G−CD11b+Ly6C+CD115+ monocytes, CD45+Ly6G−CD11b+Ly6C+F4/80+CD80+CCR2+ Mϕ (M1) and CD45+Ly6G−CD11b+Ly6C+F4/80+CD206+CX3CR1+ Mϕ (M2) in the bone marrow from three experimental groups of mice. B - E. Monocytes and M1 Mϕs were higher in the Old group compared to the Young (***P < 0.001 and **P < 0.01, respectively) that were decreased by Ala-treatment (*P < 0.05) though the decrease was not significant in M1 Mϕs. M2 Mϕs were decreased in the Old group (***P < 0.001) that resulted in the higher ratio of M1/M2 Mϕs. Ala-treatment increased the number of M2 Mϕs and decreased the ratio (*P < 0.05). F. Shown were representative light microscopy images of colonies derived from the bone marrow cells undergoing CFU-GM assay. G. Shown were representative flow cytometry dot plots of monocytes, M1 Mϕ and M2 Mϕ in the colonies derived from three experimental groups of mice. H – K. Monocytes and M1 Mϕs were higher in the colonies derived from Old mice compared to the Young (***P < 0.001 and *P < 0.05, respectively). Monocytes were decreased…

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  Figure 8.. Pro-myelopoietic potential of bacterial metabolites in the aging was reversed by Alamandine. A. Shown were representative light microscopy images of colonies derived from the bone marrow cells treated with cecal supernatants (CS) from three different experimental groups and subjected to CFU-GM assay. B. Shown were representative flow cytometry dot plots of CD45+Ly6G−CD11b+Ly6C+CD115+ monocytes, CD45+Ly6G−CD11b+Ly6C+F4/80+CD80+CCR2+ Mϕ (M1) and CD45+Ly6G−CD11b+Ly6C+F4/80+CD206+CX3CR1+ Mϕ (M2) derived from CFU-GM colonies. C – F. Monocytes were higher in the Old-CS (O-CS) treated group compared to the Young-CS (Y-CS) (*P < 0.05) that were lower in the Old-Ala-CS (O-Ala-CS) treated group (**P < 0.01). M1 Mϕs showed increasing trend in the O-CS group but was not significant. M2 Mϕs were decreased in the O-CS group (*P < 0.05) that resulted in the higher ratio of M1/M2 Mϕs (*P < 0.05). O-Ala-CS treatment did not alter the number of M2 Mϕs but decreased the ratio (*P < 0.05). G. Shown were representative light microscopy images of Mϕs derived from the bone marrow cells treated with cecal supernatants (CS) from three different experimental groups and subjected to Mϕ polarization assay.…