Marine Microalgae, Spirulina maxima-Derived Modified Pectin and Modified Pectin Nanoparticles Modulate the Gut Microbiota and Trigger Immune Responses in Mice

Abstract

This study evaluated the modulation of gut microbiota, immune responses, and gut morphometry in C57BL/6 mice, upon oral administration of S. maxima-derived modified pectin (SmP, 7.5 mg/mL) and pectin nanoparticles (SmPNPs; 7.5 mg/mL). Metagenomics analysis was conducted using fecal samples, and mice duodenum and jejunum were used for analyzing the immune response and gut morphometry, respectively. The results of metagenomics analysis revealed that the abundance of Bacteroidetes in the gut increased in response to both modified SmP and SmPNPs (75%) as compared with that in the control group (66%), while that of Firmicutes decreased in (20%) as compared with that in the control group (30%). The mRNA levels of mucin, antimicrobial peptide, and antiviral and gut permeability-related genes in the duodenum were significantly (p < 0.05) upregulated (> 2-fold) upon modified SmP and SmPNPs feeding. Protein level of intestinal alkaline phosphatase was increased (1.9-fold) in the duodenum of modified SmPNPs feeding, evidenced by significantly increased goblet cell density (0.5 ± 0.03 cells/1000 µm²) and villi height (352 ± 10 µm). Our results suggest that both modified SmP and SmPNPs have the potential to modulate gut microbial community, enhance the expression of immune related genes, and improve gut morphology.

Keywords: Bacteroidetes; Firmicutes; Spirulina maxima; intestinal alkaline phosphatase; metagenomics; modified SmP; mucin.

Figure 1

Growth performance and blood glucose level of mice during 4 weeks of modified SmP and SmPNPs treatment. (A) Body weight (g); (B) Body weight gain percentage (%); (C) Blood glucose levels (mg/dL). 4* denoted the fasting glucose level at the end of the experiment (4th week); (means ± standard deviation, n = 4 per replicate, 3 replicates per group).

Figure 2

Metagenomics analysis of mouse fecal matter. (A) Sequencing reads, Operational Taxonomic Units (OTUs) count and diversity indices; (B) Venn diagram of observed OTUs; (C) Rarefaction curves of control, modified SmP and SmPNPs treated mice (n = 4 per replicate, 3 replicates per each group).

Figure 3

Diet-specific changes of taxonomic composition of fecal microbial community of control, modified SmP, and modified SmPNPs treated mice. (A) Comparison of relative abundance of metagenomics based gut microbial phyla; (B) Comparison of relative abundance of metagenomics based gut microbial families; (C) Principal component analysis (PCA) of relative abundance of gut microbiota families. The percentage variance of PC1 and PC2 are represented in x and y axis, respectively, (Software version: R 3.6.1, packages. Vegan). (n = 4 per replicate, 3 replicates per each group).

Figure 4

Transcriptional responses of immune related genes in modified SmP and SmPNPs supplemented mice. (A) Relative mRNA expression in control, modified SmP, and modified SmPNPs treatment groups. Relative expression-fold was presented as mean ± standard error. Asterisk mark is used to indicate the significant difference between the pectin treatments and control (3 replicates/group); (B) Comparison of relative expression fold between the treatment groups by color schematic representation. Basal expression level was considered as 1.0-fold; upregulated and down regulated expression were considered as >1.0- and <1.0-folds, respectively.

Figure 5

Immunoblot analysis of duodenum intestine alkaline phosphatase expression (IAP) of mice fed with modified SmP and SmPNPs supplemented diet and the control. (A) IAP expression. β-actin was used to confirm equal loading of proteins; (B) Quantitative analyses of IAP expression in modified SmP and SmPNPs, which normalized to β-actin and compared with the control. Data are expressed as the mean ± standard error (n = 3).

Figure 6

Histological analysis of jejunum morphometry of mice fed with modified SmP and SmPNPs supplemented diet as compared with control. (A) Light micrographs of Alcian blue and periodic acid=Schiff (AB-PAS) stained histological sections; (B) Comparison of goblet cell density; (C) Comparison of villi height in control, modified SmP, and modified SmPNPs treated mouse gut. a, villus goblet cells; b, crypt goblet cells; c, paneth cells. * Significantly (p < 0.05) higher goblet cell density and villi height as compared with the control; # significantly (p < 0.05) higher villi height in modified SmPNPs supplemented group as compared with modified SmP group.