Colon specific delivery of miR-155 inhibitor alleviates estrogen deficiency related phenotype via microbiota remodeling

Abstract

Compelling data have indicated menopause-associated increase in cardiovascular disease in women, while the underlying mechanisms remain largely unknown. It is established that changes of intestinal microbiota affect cardiovascular function in the context of metabolic syndrome. We here aimed to explore the possible link between host intestinal function, microbiota, and cardiac function in the ovariectomy (OVX) mouse model. Mice were ovariectomized to induce estrogen-related metabolic syndrome and cardiovascular defect. Microbiota was analyzed by 16s rRNA sequencing. miRNA and mRNA candidates expression were tested by qPCR. Cardiac function was examined by echocardiography. Colon specific delivery of miRNA candidates was achieved by oral gavage of Eudragit S100 functionalized microspheres. In comparison with the sham-operated group, OVX mice showed compromised cardiac function, together with activated inflammation in the visceral adipose tissue and heart. Lactobacillus abundance was significantly decreased in the gut of OVX mice. Meanwhile, miR-155 was mostly upregulated in the intestinal epithelium and thus the feces over other candidates, which in turn decreased Lactobacillus abundance in the intestine when endocytosed. Oral delivery of miR-155 antagonist restored the protective microbiota and thus protected the cardiac function in the OVX mice. This study has established a possible regulatory axis of intestinal miRNAs-microbiota-estrogen deficiency related phenotype in the OVX model. Colon specific delivery of therapeutic miRNAs would possibly restore the microbiota toward protective phenotype in the context of metabolic syndrome.

Keywords: Microbiota; cardiac function; colon specific drug delivery; metabolic syndrome; miRNAs.

Figure 1.

Changes of microbiota in OVX mice model. (A) Principal Component Analysis on the relative abundance of bacterial phylum. Each point represents a sample, plotted by the second principal component on the Y-axis and the first principal component on the X-axis, which was colored by group. (B) The relative abundance of OTUs (%) in the fecal bacterial community. (C) The relative abundance of Lactobacillus in the fecal bacterial community. Data are expressed as mean ± SEM. **p < .01.

Figure 2.

Transplantation of the normal fecal microbiota alleviates the inflammation and cardiac dysfunction in OVX mice. (A) Schematic representation of the experimental procedure. Fecal microbiota transplantation was done every other day after the sham or OVX operation. Body weight (B) and fat mass percentage (C) change after fecal transplantation. n = 5 for each group. (D, E) The expression of Tnfα, Mip1, and Mcp1 in the visceral adipose tissue (D) and heart (E) in mice received indicated treatments, as detected by qPCR. qPCR was performed at least in triplicates. Data are expressed as mean ± SEM of three biological replicates. *, p < .05. Short axis EF (eject fraction) value (F) and E/A value (the ratio of early and late mitral diastolic peak velocity) (G) in mice received indicated treatments. Data are expressed as mean ± SEM of 5 mice per group. *p < .05, **p < .01, ***p < .001.

Figure 3.

Involvement of miRNAs in microbiota remodeling in OVX mice. (A) Heatmap of the most abundant miRNAs expressed in the colon epithelium in control and OVX group. The color represents the normalized mean –dCt relative to internal control U6 in each mouse, with three mice in each group. (B, C) Validation of miR-155 (B) and let-7g (C) expression in the colon epithelium of control and OVX mice. Data are expressed as mean ± SEM of at least 3 biological replicates per group. (D, E) Relative expression of miR-155 (D) and let-7g (E) in the feces from the cecum of both control and OVX mice. Data are expressed as mean ± SEM of at least three biological replicates per group. *p < .05, **p < .01, ***p < .001.

Figure 4.

Host miRNAs enter the bacterium and regulate bacterial growth. (A) E. coli was cultured in the presence of FITC labeled miRNAs for 4 h, followed by fixation and DAPI staining. Images were acquired by confocal microscopy with a 100× objective. Data presented were representative of three independent experiments. Scale bar = 20 µm. Transfer of miR-155 (B) and let-7g (C) in the E. coli after 4 h incubation with control or indicated miRNAs. qPCR was performed at least in triplicates. NC is the miRNA mimic that can’t be detected by primers of miR-155 and let-7g. Data are expressed as mean ± SEM of three biological replicates. (D) Lactobacillus gasseri (ATCC 33323) growth in medium added with miR-155, let-7g or negative controls for 12 h. Data are expressed as mean ± SEM of three biological replicates. ***p < .001. (E) Growth curve of Lactobacillus gasseri (ATCC 33323) growth in medium added with negative control miRNA mimic or miR-155 at indicated doses. Data are expressed as mean ± SEM. *p < .05, 10 nM miR-155 vs. PBS, 5 nM NC, or 10 nM NC at 9 h; 5 nM miR-155 vs. PBS, 5 nM NC, or 10 nM NC at 12 h. **p < .01, 10 nM miR-155 vs. PBS, 5 nM NC, or 10 nM NC at 12 h.

Figure 5.

Microsphere mediated colon specific delivery of miRNAs. (A) Schematic illustration of the procedure how Eudragit S100 functionalized microsphere manipulated. miRNAs are incorporated in the Chitosan solution before mixed with the Eudragit S100: Methanol/dichloromethane solution, followed by ultrasonication. Next, the water-in-oil emulsion is poured into the PVA solution to form the water-in-oil-in-water (W1/O/W2) double emulsion. Representative bright-field microscope (B) and electron microscope (C) image of the fabricated Eudragit functionalized microspheres. (D) Encapsulation efficiency of miR-155 (D) loaded in the microsphere as assayed by qPCR. Enrichment of miR-155 in the feces (E) and cecal bacterium (F) from the cecum after orally delivered microspheres loaded with control or indicated miRNAs, NC is the miRNA mimic with no similar sequence as miR-155. qPCR was performed at least in triplicates. Data are expressed as mean ± SEM of three biological replicates. *p < .05, **p < .01.

Figure 6.

Oral delivery of miR-155 antagonism protects the heart in OVX mice via microbiota remodeling. (A) Schematic representation of the experimental procedure. OVX mice were treated with negative control or anti-miR-155 twice a week for 8 weeks, together with or without antibiotics in drinking water (1 mg antibiotic per g body weight per day). n = 5 for each group. (B, C) The expression of Tnfα, Mip1, and Mcp1 in the adipose tissue (B) and heart (C) in mice received indicated treatments, as detected by qPCR. Data are expressed as mean ± SEM of three biological replicates. (D, E) CD11b + percentage (D) and M1 cell percentage (E) in mice treated as indicated. Data are expressed as mean ± SEM of 5 mice per group. (F, G) Short axis EF value (F) and E/A value (G) in mice received indicated treatments. Data are expressed as mean ± SEM of at least 5 mice per group. *p < .05, **p < .01.

Figure 7.

Schematic summary of the study. miR-155 is significantly upregulated in the intestinal epithelium and thus the feces of OVX mice, which in turn remodels the microbiota, especially reducing Lactobacillus abundance. Remodeled microbiota results in activated inflammation in both adipose tissue and the heart, contributing to the cardiac dysfunction. Oral delivery of miR-155 antagonism encapsulated in the Eudragit S100 functionalized microspheres restores the beneficial microbiota and thus protects the cardiac function.