Frailty is a geriatric syndrome associated with both locomotor and cognitive decline, typically linked to chronic systemic inflammation, i.e., inflammaging. In the current study, we investigated the effect of a two-month oral supplementation with standardized extracts of H. erinaceus, containing a known amount of Erinacine A, Hericenone C, Hericenone D, and L-ergothioneine, on locomotor frailty and cerebellum of aged mice. Locomotor performances were monitored comparing healthy aging and frail mice. Cerebellar volume and cytoarchitecture, together with inflammatory and oxidative stress pathways, were assessed focusing on senescent frail animals. H. erinaceus partially recovered the aged-related decline of locomotor performances. Histopathological analyses paralleled by immunocytochemical evaluation of specific molecules strengthened the neuroprotective role of H. erinaceus able to ameliorate cerebellar alterations, i.e., milder volume reduction, slighter molecular layer thickness decrease and minor percentage of shrunken Purkinje neurons, also diminishing inflammation and oxidative stress in frail mice while increasing a key longevity regulator and a neuroprotective molecule. Thus, our present findings demonstrated the efficacy of a non-pharmacological approach, based on the dietary supplementation using H. erinaceus extract, which represent a promising adjuvant therapy to be associated with conventional geriatric treatments.
1. Introduction
Aging is a universal process characterized by a gradual decline in physical and cognitive functions. As age increases, a variety of changes occur, including brain atrophy, oxidative stress, and reduced antioxidant mechanisms, contributing to impairment of cognitive and locomotor performances [1].
The term frailty was proposed to describe a multisystemic impairment scenario, which negatively affects the health of an elderly individual, contributing to aggravate a clinical condition that is often already compromised. Recently, the World Health Organization (WHO) recognized frailty as an increasingly widespread syndrome that should be prevented, reversed, or at least mitigated to improve the quality of life in the elderly [2].
Frailty is a complex geriatric syndrome characterized by age-associated declines in physiologic reserve and functions through multiorgan systems, leading to enhanced vulnerability for adverse health outcomes [3,4]. Compelling evidence linked frailty to both immunosenescence and chronic systemic inflammation, the so-called inflammaging [5,6,7]. This latter mechanism is a typical biomarker of accelerated aging, being also a risk factor for cardiovascular diseases, cancer, dementia, cognitive decline, and physical disabilities [7,8]. In addition, inflammaging increases susceptibility to stress-related molecules [6,7].
Several works demonstrated that locomotor frailty predicts cognitive impairment and dementia during aging [9,10,11,12], and, in particular, the locomotor decline seems to be a risk factor of future cognitive deterioration [13,14]. In this scenario, the prevention, detection and the reversion of physical frailty is imperative, aimed at avoiding neurodegenerative diseases pathogenesis and cognitive impairment outcomes.
An imbalance in the REDOX system is critical not only in aging but even more in frail subjects. The accumulation of reactive oxygen and nitrogen species (RONS) during aging induces cellular damages and contributes to the onset of age-associated tissue impairment. Based on this evidence, the oxidative stress theory of aging was formulated and, over the years, the role of oxidative stress in the worsening of age-related diseases has been consolidated [15]. A redox imbalance is particularly critical in frail subjects during aging. Furthermore, oxidative stress induces the secretion of proinflammatory molecules and chemokines that promote protein degradation and contribute to cellular degeneration [16].
The brain is very sensitive to oxidative stress-induced damage, and the overproduction of free radicals during aging is suggested to be responsible for age-associated brain structural corrosion and functional decline [1]. Experimental and clinical evidence supported that age-related brain atrophy could worsen locomotor and cognitive performances [1]. Specifically, aged-derived increase of oxidative stress leads to cellular damages in the cerebellum, e.g., gray matter volume reduction and atrophy [17].
Furthermore, the morphological signs observed in cerebellum have been correlated with slow walking speed and reduction in both physical and social behaviors suggesting that frailty could promote neuronal degeneration in elderly patients [12,18]. Notably, cerebellar Purkinje neurons are vulnerable to aging, displaying considerable alterations in both morphology and function, e.g., cell number reduction and decrease in synapse density [19].
Hericium erinaceus is an edible and medicinal mushroom and it seems to stand out as an exceptional health-promoting species. Available generally in North temperate latitudes (including Italy), H. erinaceus can be identified by its long spines, for its appearance on hardwoods and its tendency to grow with a single tuft of dangling spines. Given its shape, it is also known as Lion’s mane and Monkey Head Mushroom [20,21]. H. erinaceus is able to regulate cytokines and mitogen-activated protein kinases expressions, and transcription factors at the molecular level: H. erinaceus performs medicinal activities at tissue, organ, and organism levels. Indeed, H. erinaceus synthesizes at least 70 different bioactive metabolites, such as β-glucans, erinacines, hericenones, alkaloids, sterols, and volatile aroma compounds [21]. Thanks to these biological compounds, H. erinaceus exerts many health-promoting properties [21], such as antibiotic [22,23], anticancer [24,25], antioxidant [26,27], antifatigue [28], antisenescence [14,29], neuroprotective [30,31,32], antidepressive, and antianxiety [33,34] activities.
Furthermore, both in H. erinaceus fruiting body and mycelium ergothioneine is found [35]. L-ergothioneine is a water-soluble thiol that can only be absorbed from the diet because animals and plants cannot synthesize this compound, produced solely by bacteria and mushrooms. L-ergothioneine displays antioxidant and cytoprotective capabilities [36,37]. An increasing number of scientific articles demonstrated the potential of L-ergothioneine as therapy for several diseases, such as preeclampsia [38,39], neurodegenerative [40,41,42,43], cardiovascular [44,45], and endothelial-muscular [46,47,48] pathologies.
Based on this knowledge, H. erinaceus appears an excellent candidate to prepare novel mushroom-based pharmaceuticals/medicines and functional foods [20]. Nevertheless, the standardization of medicinal mushrooms-derived dietary supplements is still in development, since proper standards and protocols have yet to be identified [49].
Our previous findings demonstrated the neuroprotective action and nootropic effect of H. erinaceus in adult wild-type mice. Specifically, dietary supplementation with H. erinaceus was effective at (i) increasing hippocampal neurotransmission, locomotor performances and recognition memory in wild-type mice [50,51], and (ii) improving recognition memory in frail mice during aging, also inducing hippocampal and cerebellar neurogenesis [14].
In the current study, we investigated the effect of a two-month oral supplementation with standardized extracts of H. erinaceus (He1), containing a known amount of Erinacine A, Hericenone C, Hericenone D, and L-ergothioneine, on locomotor frailty and cerebellum of aged mice. We decided to supplement only frail mice, then compare them to healthy aged animals to explore the potential occurrence of a recovery process, in which H. erinaceus-supplemented frail mice could have reverted, at least in part, to healthy aging.
Specifically, we monitored the locomotor performances comparing animals belonging to the two experimental groups (healthy aged vs. frail mice), and, further, we evaluated the cerebellar volume and cytoarchitecture, together with inflammatory and oxidative stress pathways, jointly with a neuroprotective molecule and a key longevity regulator, focusing on senescent frail animals.
2. Results
2.1. Array of Metabolites in Hericium erinaceus Extract
Italian Hericium erinaceus (He1) was collected in Siena province (Tuscany, Italy) and identified on the macro- and micro-morphological characteristics of the species. Culturing and extraction procedures are meticulously reported in Materials and Methods section (see Section 4.3).
Using HPLC-UV-ESI/MS, and by comparison with standard solutions, the presence and amount of different metabolites, i.e., Erinacine A, Hericenone C, Hericenone D was identified and quantified [14,52]. Specifically, He1 extract contained 150 µg/g Erinacine A, 500 µg/g Hericenone C, and about 20 µg/g Hericenone D.
In addition, the amount of ergothioneine (ET) in mycelium and sporophore extracts of He1 was measured. The ET calibration curve was constructed with concentrations ranging from 10 to 350 µg/mL (see Section 4). Linear least-square regression analysis for the calibration curve showed correlation coefficient of 0.9925 with respect to the peak area (Figure 1, Panel A, top right insert), demonstrating a good linear relationship in the different ranges tested.
