Diabetes Metab J.  2024 May;48(3):390-404. 10.4093/dmj.2023.0109.

Supplementation of Clostridium butyricum Alleviates Vascular Inflammation in Diabetic Mice

• Zhou T
• Qiu S
• Zhang L
• Li Y
• Zhang J
• Shen D
• Zhao P
• Yuan L
• Zhao L
• Duan Y
• Xing C

• KMID: 2555771
• DOI: http://doi.org/10.4093/dmj.2023.0109

Abstract

Background
Gut microbiota is closely related to the occurrence and development of diabetes and affects the prognosis of diabetic complications, and the underlying mechanisms are only partially understood. We aimed to explore the possible link between the gut microbiota and vascular inflammation of diabetic mice.
Methods
The db/db diabetic and wild-type (WT) mice were used in this study. We profiled gut microbiota and examined the and vascular function in both db/db group and WT group. Gut microbiota was analyzed by 16s rRNA sequencing. Vascular function was examined by ultrasonographic hemodynamics and histological staining. Clostridium butyricum (CB) was orally administered to diabetic mice by intragastric gavage every 2 days for 2 consecutive months. Reactive oxygen species (ROS) and expression of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were detected by fluorescence microscopy. The mRNA expression of inflammatory cytokines was tested by quantitative polymerase chain reaction.
Results
Compared with WT mice, CB abundance was significantly decreased in the gut of db/db mice, together with compromised vascular function and activated inflammation in the arterial tissue. Meanwhile, ROS in the vascular tissue of db/db mice was also significantly increased. Oral administration of CB restored the protective microbiota, and protected the vascular function in the db/db mice via activating the Nrf2/HO-1 pathway.
Conclusion
This study identified the potential link between decreased CB abundance in gut microbiota and vascular inflammation in diabetes. Therapeutic delivery of CB by gut transplantation alleviates the vascular lesions of diabetes mellitus by activating the Nrf2/HO-1 pathway.

Keyword

Diabetes mellitus; Gastrointestinal microbiome; NF-E2-related factor 2; Vascular diseases

Figure

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  Fig. 1. The animal experiment procedure and vascular function change in the diabetic mice. (A) Schematic of experimental design. (B) Representative images of pulse wave velocity (PWV) measurement. PWV was determined at the ascending aorta and abdominal aorta, with the real-time electrocardiogram. (C) The PWV of the wild-type (WT) and db/db mice. (D) Hematoxylin/eosin (HE) and Masson’s trichrome staining of blood vessels. Quantitative analysis of wall thickness (E) and collagen volume fraction (%) (F). (G) Fluorescence microscope images of the dihydroethidium (DHE) staining of the reactive oxygen species (red) in blood vessels of WT and db/db mice. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). All images at 80× magnification. (H) Quantification of the DHE fluorescence intensity by the software Image ProPlus 6.0 (Media cybernetics) in (D). Data were presented as mean±standard error of the mean (n=6 mice/group). A t-test was used for two independent groups. aP<0.01, bP<0.001.

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  Fig. 2. Gut microbiota change in diabetic mice. (A) Alpha diversity was evaluated by Shannon, Chao1, and Faith-phylogenetic diversity (PD) indexes in gut flora. (B-E) Abundance of the main altered at classes (B), orders (C), families (D), and genera (E) levels in two groups, which displaying the decrease of Clostridia in db/db group. (F) The abundance of operational taxonomic units of Clostridia in the fecal bacterial community at classes, orders, families, and genera levels respectively in wild-type (WT) and db/db groups. Data were presented as mean±standard error of the mean (n=6 mice/group). Analysis of variance followed by the indicated post hoc test was performed to determine the significance among the three groups. A t-test was used for two independent groups. aP<0.05, bP<0.01, cP<0.001.

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  Fig. 3. Supplementation of Clostridium butyricum (CB) alleviates vascular phenotype, inflammation and oxidative stress in diabetic mice. (A, B) Schematic representation of the experimental procedure. (C) Weight change of every group. (D) Blood glucose change of every group. (E) The pulse wave velocity (PWV) of the wild-type (WT), diabetic (DB)-phosphate-buffered saline (PBS) and DB-CB group at ascending aorta to abdominal aorta. (F) Ultrasound representative images of PWV. (G) Vascular change as revealed by Hematoxylin/eosin (HE) and Masson’s trichrome staining. (H) Wall thickness, (I) collagen volume fraction (%) of the arteries of mice receiving CB treatment for 60 days. (J) Fluorescence microscope images of the dihydroethidium (DHE) staining of the reactive oxygen species (red) in arterial tissue of three groups mice. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). All images at 80× magnification. (K) Quantification of DHE fluorescence intensity by the software Image ProPlus 6.0 (Media cybernetics). (L, M) Real-time quantitative reverse transcription analysis of the mRNA expression of the inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in arterial tissue of three groups. Data were presented as mean±standard error of the mean (n=6 mice/group). Analysis of variance followed by the indicated post hoc test was performed to determine the significance among the three groups. aP<0.05, bP<0.01, cP<0.001.

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  Fig. 4. Clostridium butyricum (CB) supplementation increases the metabolite butyrate in diabetic mice. (A) Alpha diversity was evaluated by Shannon, Chao1 and Faith-phylogenetic diversity (PD) indexes in gut flora. (B) Beta diversity of gut microbiota in three group. (C) Venn diagram illustrates the overlap of operational taxonomic units in gut microbiota among three groups. (D) Linear discriminant analysis (LDA) effect size (LEfSe) identifies the taxa with the greatest differences in the abundance among three groups. Only the taxa with meeting a significant LDA threshold value of >2 were shown. Comparisons of the relative abundance of Clostridia in the fecal bacterial community at (E) bacterial order (orange), and (F) family (grey) levels. (G) The level of butyrate in the fecal samples. Data were presented as mean±standard error of the mean (n=6 mice/group). Analysis of variance followed by the indicated post hoc test was performed to determine the significance among the three groups. WT, wild-type; DB, diabetic; PBS, phosphate-buffered saline; ns, no significance; SCFA, short-chain fatty acid. aP<0.05, bP<0.01.

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  Fig. 5. Oral administration of Clostridium butyricum (CB) increases the expression levels of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in arteries of diabetic mice. (A, C) Immunofluorescence analysis using an antibody against Nrf2 and HO-1 on vascular histological sections prepared from animals orally administered CB. Images were acquired at 80× magnification (red=Nrf2 and HO-1; blue=4’,6-diamidino-2-phenylindole [DAPI]). (B, D) Quantification of the Nrf2 and HO-1 fluorescence intensity by the software Image ProPlus 6.0 (Media cybernetics). (E) The protein level of Nrf2 and HO-1 in arterial tissue of three groups mice was determined by Western blotting. Representative data of at least three independent experiments. (F, G) Quantification of relative density of the Nrf2 and HO-1 Western blotting bands by the software ImageJ v1.8.0. Data were presented as mean±standard error of the mean (n=6 mice/group). Analysis of variance followed by the indicated post hoc test was performed to determine the significance among the three groups. WT, wild-type; DB, diabetic; PBS, phosphate-buffered saline; KD, kilodalton; ns, no significance. aP<0.01, bP<0.001.

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  Fig. 6. Sodium butyrate (SB) inhibits high glucose (HG)-induced cellular inflammation and oxidative stress in multiple cells. (A, B, E, F) The expression level of the inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) mRNA was analyzed by quantitative polymerase chain reaction (qPCR), in HG-induced human umbilical vein endothelial cell (HUVEC) and RAW264.7 cells respectively. (C, D, G-J) The mRNA expression level of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was analyzed by qPCR, in HG-induced all groups. Data were presented as mean±standard error of the mean of three biological replicates. Analysis of variance followed by the indicated post hoc test was performed to determine the significance among the three groups. NG, normal glucose; ns, no significance; VSMC, vascular smooth muscle cell. aP<0.05, bP<0.01, cP<0.001.

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