Hericium erinaceus Extract Exerts Beneficial Effects on Gut-Neuroinflammaging-Cognitive Axis in Elderly Mice

Abstract

Ageing is a biological phenomenon that determines the impairment of cognitive performances, in particular, affecting memory. Inflammation and cellular senescence are known to be involved in the pathogenesis of cognitive decline. The gut microbiota-brain axis could exert a critical role in influencing brain homeostasis during ageing, modulating neuroinflammation, and possibly leading to inflammaging. Due to their anti-ageing properties, medicinal mushrooms can be utilised as a resource for developing pharmaceuticals and functional foods. Specifically, Hericium erinaceus (He), thanks to its bioactive metabolites, exerts numerous healthy beneficial effects, such as reinforcing the immune system, counteracting ageing, and improving cognitive performance. Our previous works demonstrated the capabilities of two months of He1 standardised extract oral supplementation in preventing cognitive decline in elderly frail mice. Herein, we showed that this treatment did not change the overall gut microbiome composition but significantly modified the relative abundance of genera specifically involved in cognition and inflammation. Parallelly, a significant decrease in crucial markers of inflammation and cellular senescence, i.e., CD45, GFAP, IL6, p62, and γH2AX, was demonstrated in the dentate gyrus and Cornus Ammonis hippocampal areas through immunohistochemical experiments. In summary, we suggested beneficial and anti-inflammatory properties of He1 in mouse hippocampus through the gut microbiome-brain axis modulation.

Keywords: Hericium erinaceus; ageing; frailty; gut microbiota; gut–brain axis; hippocampus; neuroinflammaging.

Figure 1

He1 treatment reverted cognitive frailty evaluated as the knowledge component of recognition memory during physiological ageing in mice. (a): Scatter plot of Cognitive FI Values measured at T1 in control (T1 CNTR, orange) and in pre-supplemented He1 mice (T1 pre-He1, blue). (b): Scatter plot of Cognitive FI Values measured at T1 in control and in pre-supplemented He1 animals (T1, orange and blue, respectively), and at T2 in control (T2 CNTR, orange) and in He1-supplemented mice (T2 He1, blue). P-value was calculated by unpaired Student’s t-test: p < 0.01 (**).

Figure 2

Gut microbiome composition. (a): Alpha-diversity distribution box plots estimated as Faith’s phylogenetic distance (PD) in control mice and He1-treated animals at T1 and T2 (T1 CNTR: blue; T1 pre-He1: light blue; T2 CNTR: pink; T2 He1: fuchsia). (b): Non-metric multidimensional scaling (NMDS). Colours in the bidimensional NMDS plot are used, as shown in the legend. The ordinate analysis is founded on the Bray–Curtis distance matrix. The graphical plot and the ellipses were generated by the ggplot2 R package implemented with the stat ellipse function. (c): Differential abundance (of selected genera that significantly changed) at T2 in He1 treated vs. control mice.

Figure 3

H&E staining revealing the well-preserved physiological hippocampal cytoarchitecture in non-supplemented controls (a–e) and He1-treated (f–j) elderly mice. (a,f): Low magnification micrograph shows the whole hippocampus, formed of Cornus Ammonis (CA; subdivided into CA1, CA2, CA3, and CA4) and dentate gyrus (DG). (b,g): Higher magnifications of the DG area displaying three distinct layers: ML, GL, and PL. (e,j): Higher magnifications of the CA1 region, showing the typical three-layered structure: outer polymorphic layer, i.e., Stratum oriens (SO); middle pyramidal cell layer, i.e., Stratum pyramidale (SP); inner molecular layer, i.e., Stratum radiatum (SR) in control and He1-supplemented animal, respectively. Light microscopy magnification: 4× (a,f); 20× (b–e,g–j). Histograms display the quantitative valuation of shrunken cell density in DG and CA subregions. p-values calculated by unpaired Student’s t-test. **** p < 0.0001.

Figure 4

PSR staining evaluation under light microscopy. Representative hippocampal specimens showing parenchyma and blood vessels from control (a–d) and He1-treated animals (e–h). Microscopy magnification: 20× (a–h). Panel A: Histograms showing OD measurements in DG and CA subregions. p-values calculated by unpaired Student’s t-test. p-values: ** p < 0.01; **** p < 0.0001.

Figure 5

DAB-immunostaining reaction for CD45 in hippocampal DG and CA subfields from control mice (a–e) and He1-treated mice (f–j). Magnification: 40× (a–j). Panel A: Histograms depicting CD45-immunopositive cell density assessed in hippocampal DG and CA subregions of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. ** p < 0.01; **** p < 0.0001. Panel B: Histograms display CD45-immunopositive cell OD measured in hippocampal DG and CA subregions of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. ** p < 0.01; **** p < 0.0001.

Figure 6

Double immunohistochemical detection of GFAP (green signal) and IL6 (red signal) by fluorescence microscopy in control animals (a–e) and He1-treated (f–j) mice. DNA counterstaining with Hoechst 33258 (blue fluorescence). Magnification: 60× (a–j).

Figure 7

Panel A: Histograms illustrating GFAP-immunopositive cell density determined in DG and CA subregions of control and He1-supplemented mice. p-values determined by unpaired Student’s t-test. **** p < 0.0001. Panel B: Histograms indicating GFAP-immunopositive cell OD measured in DG and CA subregions of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. * p < 0.05; ** p<0.01; **** p < 0.0001. Panel C: Histograms illustrating IL6-immunopositive cell density determined in DG and CA subregions of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. * p < 0.05; **** p < 0.0001. Panel D: Histograms illustrating IL6-immunopositive cell OD determined in DG and CA subregions of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. **** p < 0.0001. Panel E: Histograms showing the GFAP- and IL6- double-immunopositive cell density, determined in DG and CA subregions of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. *** p < 0.001; **** p < 0.0001.

Figure 8

Immunocytochemical detection of p62 (red signal) by fluorescence microscopy in control animals (a–e) and He1-treated (f–j) mice. DNA counterstaining with Hoechst 33258 (blue fluorescence). Magnification: 60× (a–j). Panel A: Histograms displaying p62-immunopositive cell density measured in DG and CA subregions of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. *** p < 0.001; **** p < 0.0001. Panel B: Histograms presenting p62-immunopositive cell OD determined in DG and CA subregions of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. ** p < 0.01; **** p < 0.0001.

Figure 9

Representative micrographs depicting immunohistochemical reaction for γH2AX in hippocampal DG and CA subfields and in cerebral cortex from controls (a–e,k) and He1-treated mice (f–j,l). Magnification: 40× (a–l); 100× (insert in k,l). Histograms illustrating γH2AX-immunopositive cell OD assessed in hippocampal DG and CA subregions and in cerebral cortex of control and He1-supplemented mice. p-values calculated by unpaired Student’s t-test. **** p < 0.0001.