CUMS and DEX treatments induce depression-like behaviours in female mice
We evaluated behaviours of mice with SPT, OFT, EPM, TST and FST to determine whether CUMS and DEX evoked depression-like or anxious behaviours. Figure 2 showed that mice in both CUMS and DEX groups displayed a significant decrease in the total travel distance, movement speed, frequency of rearing and crossing in open arena, as well as the time spent in open arms of the EPM and sucrose preference compared with the mice in the CON group (p<0.01). Mice exposed to CUMS or DEX had a significant increase of immobility time in TST and FST relative to mice in the CON group (p<0.01). While there was no significant difference in body weight between groups at the beginning of experiments (figure 2B), the body weight of mice (p<0.01) was significantly reduced and increased, respectively, relative to that of mice in the CON group (p<0.01). Collectively, these data suggest that we have succeeded in establishing CUMS and or DEX induced models of depression.
Figure 2CUMS and DEX treatments alter body weight and behavioural performances of mice. (A)Automated tracking in the large open field and the elevated plus-maze test. (B) Body weight of each mouse in each group was measured at week 1 and the last day of CUMS or DEX treatment and graphed. The weight of mice subjected to CUMS was less than the CON group and the DEX group gained more weight than the CON group after 5 weeks. Total distance, moving speed, frequency of rearing and crossing of mice in OPT (C–F) and time spent in open arms of EPM (G) were significantly decreased. (H)The sucrose preference test decreased notably in the CUMS and DEX group. The time of FST immobility (I) and TST immobility (J) were significantly increased. Data are shown as mean(SD) (n≥3 in each group). *p<0.05; **p<0.01, versus control group. CON, control; CUMS, chronic unexpected mild stress; DEX, dexamethasone; EPM, elevated plus maze; FST, forced swimming test; OFT, open field test; TST, tail suspension test.
Differences in the gut microbiota diversity in two mouse models of depression
To determine whether gut microbiota was altered in CUMS and or DEX treated mice, we collected faeces for analysing bacterial diversity and composition. The dilution curve in online supplemental figure S1 showed that the sequencing depth covered all species in the samples. The α diversity indices including chao1, observed species, shannon and simpson were used as indicators of species richness and uniformity in community ecology. As shown in online supplemental figure S2, there was a significant difference in the abundance and diversity between the CON group and CUMS group (p<0.05), whereas there was no significant difference between the CON group and the DEX group (p>0.05). These data suggest that CUMS but not DEX alters the abundance and richness of intestinal flora. β diversity analysis was used for comparing the differences in species diversity of a pair of intergroup samples. Anosim similarity revealed the differences between intergroups and intragroups. The differences of intergroups were significantly greater than those of intragroups (online supplemental figure S3, R=0.892, p=0.007; R=0.524, p=0.01). To further compare the species diversity, PCoA was conducted (online supplemental figure S4).11 12 As shown in figure 3, the relative abundance in the CUMS group was higher than that in the CON group, but the relative abundance of the DEX group was similar to that in the CON group. These results indicate that gut microbiota of mice subjected to CUMS are significantly different from the CON group, whereas the compositions of intestinal flora between DEX and CON groups are similar.
Figure 3Differential composition of gut microbiota in two depression models. (A) Comparison of the abundance of microbial phylum and genus in CON, CUMS and DEX groups . LDA score plot and cladogram plot from LEfSE analysis of the gut microbiota composition in CON, CUMS and DEX groups at baseline and end point. (B) Microbial taxa shown have an LDA score higher than 2 . CON, control; CUMS, chronic unexpected mild stress; DEX, dexamethasone; LDA, linear discriminant analysis; LEfSE, linear discriminant analysis effect size.
Figure 4Expression levels of proteins related to inflammation were changed in colon tissues and peripheral blood. (A) Western blot analyses of lysates prepared from colon tissues of mice in each group with indicated antibodies. The blots shown are representative of at least three independent studies. Bar graphs were quantitative data of western blot analyses and showed altered expressions of occludin, claudin-1 and IL-1β in the colon of mice treated with CUMS or with DEX. (B) Levels of CORT and IL-1β in peripheral bloods of mice in each group were measured and graphed. Data were shown as mean(SD). In both figure parts A and B, *p<0.05; **p<0.01; n.s., no significance versus control group. CON, control; CORT, corticosterone; CUMS, chronic unexpected mild stress; DEX, dexamethasone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-1β, interleukin-1β.
The species abundance and difference of gut microbiota in CUMS and DEX group
According to the results of species annotation, the corresponding histogram of each sample was analysed at phylum, class, order, family and genus classification levels respectively, so as to intuitively discern the abundance and proportion of species in different classification levels of each sample. At the phylum level, the abundance of Bacteroidetes (72.86 (2.43)%) and of Firmicutes (24.81 (4.24)%) in the CUMS group was higher and lower, respectively, when compared with the CON group (43.25 (14.17)%). There was no significant difference in the abundances of Bacteroidetes and Firmicutes between the DEX and CON groups. At the genus level, the abundances of Bacteroides (p<0.01) and Parabacteroides (p<0.01) were decreased significantly in the DEX group relative to that in the CON group. The abundance of Parabacteroides (p<0.01) as well as Lactobacillus and Staphylococcus (p<0.05) was decreased in the CUMS group. Escherichia/Shigella was found in the DEX group but not the CUMS or the CON group. Moreover, the relative abundances of Alloprevotella, Desulfovibrio, Parasutterella and Flavonifractor were significantly increased in the CUMS group relative to their corresponding abundances in the CON group. LDA effect size (LEfSe) was used to evaluate the impact of the abundance of each component and to identify the communities or species that are different between groups. Compared with the control group, mice in the CUMS group had an increase of the genera in their intestinal microflora, which included:Prevotellaceae, Alloprevotella, Oscillibacter, Desulfovibrionaceae, Mucispirillum, Olsenella, Allobaculum, Candidatus Saccharibacteria and Saccharibacteria genera incertae sedis, and a decrease of Bacilli, Lachnospiracea incertae sedis, Neisseriaceae, Neisseriales, Neisseria, Eubacteriaceae, Eubacterium, Butyricicoccus, Staphylococcus, Bacillales and Staphylococcaceae; Compared with the control group, mice in the DEX group had an increase of Enterobacteriaceae, Escherichia Shigella, Gammaproteobacteria, Enterobacteriales, Anaerofustis and Erysipelotrichaceae_incertae_sedis and a decrease of Clostridium XVIII. Relative to those in the CUMS group, Prevotellaceae, Alloprevotella, Ruminococcaceae, Oscillibacter, Desulfovibrionaceae, Deltaproteobacteria, Desulfovibrionales, Desulfovibrio, Deferribacteres, Deferribacteraceae, Deferribacterales, Burkholderiales, Mucispirillum, Olsenella, Allobaculum, Sutterellaceae, Candidatus Saccharibacteria and Saccharibacteria genera incertae sedis were significantly decreased, whereas Eubacteriaceae, Eubacterium, Enterobacteriaceae, Escherichia/Shigella, Gammaproteobacteria, Streptococcus, Enterobacteriales, Anaerofustis, Erysipelotrichaceae_incertae_sedis, Streptococcaceae, Peptostreptococcaceae, Lachnospiracea_incertae_sedis, Clostridium XI and Firmicutes were increased in the DEX group.
The intestinal barrier integrity and proinflammatory cytokine in the colon of depression mouse models
The intestinal epithelial barrier is the first line of defence of the host. Serious inflammation or many other intestinal diseases are associated with the integrity damage of the intestinal epithelial barrier. Accumulating evidence suggests that patients with chronic colon inflammation frequently have serious depression and anxiety morbidities.13 Therefore, we evaluated the colon epithelial barrier integrity by examining expression levels of claudin-1 and occludin, which are protein markers of the tight junction. While levels of claudin-1 were comparable in colon tissues of mice between CON and DEX groups, levels of occludin were significantly decreased in colon tissues of mice in the DEX group relative to those in colon lysates of mice in the CON group (p<0.05). Levels of IL-1β, which reflects inflammation, were significantly increased in colon tissues of mice in the CUMS group (figure 4A, p<0.01). These results suggest that DEX influences the colon permeability, whereas CUMS evokes inflammation in the gut.
CUMS and DEX groups mainly affected glia cells instead of neuron in the hippocampus
The hippocampus has been regarded as the main target in the formation of depression.14 Compared with those of the CON group, levels of GFAP and Nestin in the hippocampus of mice in both CUMS and DEX groups were significantly decreased (p-values <0.05). No significant difference in levels of neuron-specific neuronal nuclei (NeuN) among the three groups was noted (p>0.05). However, we found that levels of synapsin-1 and P2Y12 were decreased in the hippocampi of mice in both CUMS and DEX groups when compared with those in the CON group (figure 5, p<0.05). Compared with their expression in the control group, NLRP3 and IL-1β were upregulated in the CUMS group, but only IL-1β was found to increase in the hippocampus of mice in the DEX group (p<0.05). Collectively, these results suggest that the activation of glial cells occurs in the brain of CUMS or DEX induced mouse model of depression.
Figure 5Differential protein expressions of GFAP, P2Y12, Nestin, NeuN, Caspase-1, IL-1β, NLRP3 and Synapsin-1 in the hippocampus of two depression models. (A) Lysates were prepared from hippocampal tissues of mice in each group and analysed by SDS-PAGE and western blot with indicated antibodies. (B–I) Shown are data from one animal in each group. Data are shown as mean (SD) (n=3 in each group). *p<0.05; **p<0.01, versus control group. CON, control; CUMS, chronic unexpected mild stress; DEX, dexamethasone; GFAP, glial fibrillary acidic protein; IL-1β, interleukin-1β; NeuN, neuronal nuclei; NLRP3, nucleotide-binding domain-like receptor protein3; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.