Longevity-Associated Core Gut Microbiota Mining and Effect of Mediated Probiotic Combinations on Aging Mice: Case Study of a Long-Lived Population in Guangxi, China

Abstract

With an ageing population, healthy longevity is becoming an important scientific concern. The longevity phenomenon is closely related to the intestinal microflora and is highly complicated; it is challenging to identify and define the core gut microbiota associated with longevity. Therefore, in this study, 16S rRNA sequencing data were obtained from a total of 135 faecal samples collected as part of the latest sampling and pre-collection initiative in the Guangxi longevity area, and weighted gene co-expression network analysis (WGCNA) was used to find a mediumpurple3 network module significantly associated with the Guangxi longevity phenomenon. Five core genera, namely, Alistipes, Bacteroides, Blautia, Lachnospiraceae NK4A136 group, and Lactobacillus, were identified via network analysis and random forest (RF) in this module. Two potential probiotic strains, Lactobacillus fermentum and Bacteroides fragilis, were further isolated and screened from the above five core genera, and then combined and used as an intervention in naturally ageing mice. The results show a change in the key longevity gut microbiota in mice toward a healthy longevity state after the intervention. In addition, the results show that the probiotic combination effectively ameliorated anxiety and necrosis of hippocampal neuronal cells in senescent mice, improving their antioxidant capacity and reducing their inflammation levels. In conclusion, this longer-term study provides a new approach to the search for longevity hub microbiota. These results may also provide an important theoretical reference for the healthification of the intestinal microflora in the general population, and even the remodelling of the structure of the longevity-state intestinal microflora.

Keywords: antioxidant; core gut microbiota; long-lived seniors; network analysis.

Figure 1

Characteristics of gut microbiota in Changshou District, Guangxi. (A–D) Comparison of α-diversity of intestinal microflora based on Chao1, Simpson, Shannon, and observed species index between LG and YG groups in Guangxi longevity area over 90 years of age. * indicates p < 0.05, and ** indicates p < 0.01 (Wilcoxon rank-sum test). (E) indicates principal component PCA analysis between LG longevity and YG groups. (F) OTU Venn diagram between longevity area (LA) and non-longevity area (NLA). (F) Ensemble plot showing the number of OTUs for each age group in the longevity area.

Figure 2

Construction of microecological co-expression modules. (A) Dendrograms and heatmaps of traits for 135 samples obtained from the longevity area. Colours indicate the proportion of traits, including longevity (age older 90 years) and sex. (B) Cluster dendrogram of OTUs. The branches above represent OTU clusters based on differences in topological overlap and the Colours of the assigned modules.

Figure 3

Identification of core network modules associated with longevity and visualisation. (A) Heatmap of trait correlations between modules. Each row corresponds to a trait module, and each column corresponds to a trait. The numbers on the left of the cells represent the correlation coefficient r-values, and the numbers on the right represent the p-values corresponding to each module. (B) Correlation between module membership (MM) and gene significance (GS) in the longevity key module is indicated. The GS scatter plot of longevity traits versus MM in the mediumpurple3 module is shown here. The size of the rectangle above and to the right of the image indicates the relative size of the number of genes at the same level. A larger rectangle indicates a larger relative size of the number of genes contained at that level. (C) Circular layout of the microecological co-expression network of the mediumpurple3 module. The size of the circles in the plot represents connectivity, and the darker colour of the connecting lines indicates higher weights. (D) Identification of core OTUs in the microecological co-expression network of the mediumpurple3 module using cytoHubba plugin. The size of the circles represents connectivity, and a darker node colour indicates a higher score for the maximum neighbour component (MNC), i.e., better centrality in the network module.

Figure 4

Random forests and differential gene expression identify key genera in the longevity core module. (A) Random forest prediction of iconic longevity taxa within the module and ranking of top 10 importance based on longevity phenomena, with taxa in bold indicating that they are also present in the top 10 results for network-centric colonies in the core module. On the left-hand side of the figure, the increase in mean-squared-error (IncMSE) ranking is indicated, and on the right-hand side the increase in node purity (IncNodePurity) is indicated, with higher values of %IncMSE as well as IncNodePurity indicating higher importance of the variable. (B–F) Results of qPCR relative expression tests for key gut microbiota, with both YG and LG groups using non-longevity area seniors as controls. (B) indicates Lachnospiraceae NK4A136 group, (C) indicates Alistipes, (D) indicates Bacteroides, (E) indicates Blautia, and (F) indicates Lactobacillus. * indicates p < 0.05 and ** indicates p < 0.01.

Figure 5

Results of the open-field experiment. (A–D) Parameters of the open field experiment. (A) indicates the number of times the mice entered the central area of the open field, (B) indicates the total distance the mice moved in the open field, (C) indicates the number of times the mice stood upright on their hind limbs during the open field experiment, (D) indicates the number of times the mice groomed during the open-field experiment; * indicates p < 0.05. (E–G) In the open field experiment, there are behavioural pathways of mice, with red dots in the diagram indicating the starting point and blue dots showing the endpoint. (E) denotes control group C, (F) denotes low-dose group L, and (G) denotes high-dose group H.

Figure 6

Effects of probiotic combinations on inflammation and oxidative stress in the brains of naturally aged mice. (A–C) Pathological sections (400×) of the CA1 region of the hippocampus of each group of mice, where (A) indicates control group C, (B) indicates low-dose intervention group L, and (C) indicates high-dose intervention group H. (D–F) Effect of the complex probiotics on antioxidant activity in the brains of naturally aged mice, showing the changes in MDA (D), T-SOD (E), T-AOC (F), and GSH-Px (G). (H,I) Changes in the effect of probiotic combinations on brain inflammation in naturally aged mice results show changes IL-6 (H) and CRP (I). * indicates p < 0.05; ** indicates p < 0.01.

Figure 7

Effect of probiotic combinations on the core Guangxi longevity gut microbiota. (A–E) In this study, the intestinal genera of mice at the beginning of the intervention were used as controls to demonstrate the changes in the core longevity genera in the intestine of each group of mice at the middle (3 weeks) and end (6 weeks) of the probiotic combination intervention. (A) denotes Alistipes, (B) denotes Bacteroides, (C) denotes Blautia, (D) denotes Lachnospiraceae NK4A136 group, and (E) denotes Lactobacillus. * indicates p < 0.05; ** indicates p < 0.01.