Apple polysaccharide improves age-matched cognitive impairment and intestinal aging through microbiota-gut-brain axis

Aging is a spontaneous and inevitable process in which the body's physiological and psychological adaptability to the environment gradually decreases and tends towards death¹. The results of China's seventh population census show that China's aging population is further deepening, and the world's aging population is also increasing². Population aging brings heavy pressure to the healthcare system. Aging is often accompanied by low immune function, skin relaxation, decline in memory and cognitive ability, metabolic disorder, and decline in stem cell differentiation, and is related to a variety of diseases, such as cancer, diabetes, autoimmune diseases. Aging is accompanied by the failure of various organ functions throughout the body³. Due to its immune and nutrient intake functions, the intestine is considered an important organ that regulates the body's ability to extend lifespan⁴. The father of probiotics, Mechinikov, said, " Aging begins with the intestines. Only when the intestines are healthy can the body be healthy. The toxins produced by the gut microbiota are the main cause of aging and disease in the body."⁵. The gut microbiota present in the intestine will experience dysbiosis with aging, resulting in a decrease in beneficial bacteria and metabolites, an increase in intestinal lipopolysaccharide and inflammation levels, damage to intestinal barrier function, and accelerated aging process⁶.

The gut microbiota refers to all microbial colonies that are designated to grow in the host's intestines, known as "hidden organs"⁷. Under normal circumstances, there are approximately 100 trillion bacteria in the adult human body, of which 80% exist in the intestines⁸. The gut microbiota contains approximately 3.3 million genes, which is 150 times the number of genes in humans⁸. Among them, Firmicutes and Bacteroidetes are the most important phyla, representing the bacterial core of the human microbial community⁹. Research has shown that as age increases, the community structure and function of gut microbiota change, including changes in microbial diversity and relative abundance¹⁰. In addition, the level of lipopolysaccharides (LPS) in the intestinal cavity increases, and the abundance of short chain fatty acids with anti-inflammatory effects decreases, inducing inflammatory reactions and accelerating intestinal aging¹¹. As a cell wall immune stimulatory component of Gram-negative bacteria, LPS can bind to toll like receptor 4 (TLR4) in their lipid A region, forming a complex that activates the nuclear factor-κB (NF-κB) signaling pathway, leading to the synthesis and secretion of inflammatory factor, which destroy the permeability of mucosa, reduce tight junction proteins and induce cell aging¹². However, a study suggests that altering the composition of gut microbiota and reversing probiotic levels can regulate barriers and counteract intestinal aging¹³. Therefore, it is a way to maintain intestinal homeostasis and delay intestinal aging by regulating the gut microbiota, improving intestinal mucosal barrier and reducing the level of pro-inflammatory cytokines¹³.

Although aging is an irreversible and inevitable process, the speed of aging is controllable, and traditional Chinese medicine molecules have unique theories and rich experience in delaying aging¹⁴. Epidemiological studies have shown that eating apples can reduce the risk of chronic diseases such as cancer, cardiovascular disease, asthma and diabetes¹⁵. In addition, basic research has shown that apple polysaccharides (AP) can reduce the risk of cardiovascular disease, cancer, and neurodegenerative diseases¹⁶. The results of in vitro and in vivo experimental studies indicate that AP can prevent the occurrence of chemically induced chronic colon cancer in mice and inhibit the migration and invasion of colorectal cancer cells ¹⁷. The gut microbiota of mammals largely relies on dietary polysaccharides as an energy source, and most polysaccharides also require the participation of gut microbiota in degradation¹⁸. The dietary polysaccharides that reach the colon have a significant impact on the ecology and balance of gut microbiota¹⁹. Research has shown that AP can regulate the composition of gut microbiota, alter the concentration of short chain fatty acids in the intestinal lumen, improve chronic inflammation, and reduce intestinal permeability in high-fat induced mice²⁰. However, there are currently no reports on the use of AP to delay intestinal aging. In this study, we evaluated the composition of the microbiota, intestinal permeability, and inflammatory response to detect the regulatory effect of AP on intestinal aging in 18 months mice, and further explored potential mechanisms.

In this study, the therapeutic effect of AP on delaying intestinal aging in mice was evaluated by administering it to mice that grew naturally to 18 months. We explored the levels of inflammation and oxidative stress, intestinal aging markers, intestinal barrier function and fecal microbial composition in mice, and confirmed that AP can delay intestinal aging.

Neuronal degeneration is the main pathological process in aging, which can lead to progressive memory loss and cognitive impairment. Previous studies have reported that aging mice exhibit statistically increased oxidative damage and poorer cognitive function in the Y-maze test²¹. Similarly, D-galactose induced simulated aging mice can exhibit neuronal damage accompanied by cognitive decline²². This study found that AP can reverse cognitive impairment in aging mice, and improve their spatial learning and passive memory performance. BDNF is one of the most abundant neurotrophic factors in the central nervous system, playing a crucial role in nourishing and protecting nerves, as well as enhancing synaptic plasticity²³. Synaptic related proteins, especially PSD95 and SYP, are markers of synaptic plasticity and play a crucial role in memory²⁴. Research has confirmed that compared to 5-month-old mice, the levels of PSD95 and SYP proteins in 22-month-old mice are significantly downregulated²⁵. This study found that the expression of BDNF, PSD95 and SYP in the brain tissue of aging mice decreased, further confirming the cognitive dysfunction of aging rats. AP intervention can effectively reverse the decline of BDNF, PSD95 and SYP during the aging process, indicating that AP improves cognitive dysfunction in aging rats by improving synaptic plasticity. One factor contributing to age-related cognitive decline is the increase in oxidative stress²⁶. Previous studies have found that supplementing with concentrated apple juice can improve oxidative damage and cognitive decline in food deficient aging mice²¹. The research results of Zhonghua Liu et al. revealed that theaflavins can improve cognitive impairment in aging mice induced by D-galactose by increasing their antioxidant capacity²⁷. Consistent with previous studies, our study found that AP maintained the redox balance in mice, exhibiting strong antioxidant effects, increasing CAT, SOD, T-AOC, GSH-px activity, and reducing MDA levels.

Most elderly people develop a mild pro-inflammatory state, which is associated with increased susceptibility to various age-related diseases, and pro-inflammatory cytokines IL-1β, IL-6 and TNF-α play a crucial role in the amplification cascade of inflammation²⁸. Research reports that exposure to D-galactose can increase TNF-α, IFN- γ, IL-6 and IL-1β levels of pro-inflammatory factors²⁹. In our study, aging mice showed an increase in serum pro-inflammatory cytokine levels, and AP administration for 3 months could reverse this phenomenon. TLR4/NF-κB is a classic inflammation related signaling pathway that plays an important role in mediating inflammation³⁰. Activation of TLR4 leads to NF-κB enters the nucleus from the cytoplasm, leading to the release of a large number of pro-inflammatory factors³¹. Upregulation of the TLR4/NF-κB inflammatory pathway in the mouse intestine can be observed in the SAMP6 mouse model, leading to an inflammatory response in the mouse intestine³². Mao et al. revealed that D-galactose-induced aging mice exhibit activation of the TLR4/NF-κB signaling pathway in intestinal tissue, and downregulation of TLR4 and NF-κB protein expression can repair aging induced intestinal barrier damage³³. In this study, we revealed that AP can downregulate the activation of the TLR4 signaling pathway in intestinal tissue, inhibit the release of pro-inflammatory factors, and reduce systemic and intestinal inflammatory responses in aging mice.

Chronic progressive inflammation and oxidative stress with age can disrupt the intestinal epithelial barrier and increase intestinal permeability³⁴. Previous studies have found that in elderly rats, the presence of goblet cells producing neutral mucin is lower, which leads to a weaker mucus layer and lower buffering capacity³⁵. D-galactose-induced mice exhibit increased intestinal permeability and higher levels of serum endotoxin were observed. The AB/PAS staining results intuitively demonstrated the protective effect of AP on the intestinal barrier. Occludin and ZO-1 were tight junction (TJ) proteins that play a crucial role in maintaining intestinal epithelial cell adhesion and barrier homeostasis³⁶. Previous studies have shown that with the increase of age, the body may undergo a series of changes, especially in the integrity of the intestine³⁷. Dawn M.E. Bowdish et al. found that the intestinal permeability and inflammatory level of aging mice increased³⁸. Tran et al. indicated that compared to young baboons, the expression of ZO-1 and occludin in colon tissue of elderly baboons was reduced, and permeability was significantly increased³⁹. Previous study has revealed that apple extract can inhibit TNF-α induced TJ dysfunction was associated with increased expression of TJ proteins ZO-1 and occludin⁴⁰. Li et al. reported that apple polyphenols can increase the protein expression of ZO-1 and Occludin, upregulate the protein levels of MUC-2 and TTF3, improve the integrity of the intestinal barrier and the function of goblet cells⁴¹. Similarly, we observed disruption of the intestinal mucosal barrier in aging mice, and administration of AP could reverse this result.

It has been confirmed that p21^waf1 (p21) is associated with important biological functions such as regulating cell cycle, DNA repair, and apoptosis, while P16^lnk4a (p16), as a regulator of cellular aging, can reduce the risk of age-related diseases⁵⁸. Due to the accumulation of p16 and p21 in various tissues during normal aging processes in rodents and humans, they are used as aging markers⁴². Previous study has observed an increase in p21 and p16 protein expression in liver and kidney tissues in D-galactose induced aging mouse model⁴³. Masternak et al. have shown that Dasatinib and Quercetin alleviate intestinal aging in mice by reducing the mRNA levels of p16 and p21 in intestinal tissue⁴⁴. Similarly, in this study, we observed that AP can reduce the expression levels of p16 and p21 caused by aging. However, the increased expression of these aging markers can also be caused by active macrophages⁴⁵. We further performed SA-β-Gal staining on mouse colon tissue, and the results showed that AP can inhibit β-galactosidase activity and delay intestinal aging. SA-β-Gal positive aging cells were observed in a 5-FU induced intestinal aging model⁴⁶. Research reports that when intestinal progenitor cells transform into absorbing or secreting cells and migrate towards the villous basement, cell proliferation stops as differentiated cells leave the crypt⁴⁷. In our study, we observed a decrease in the number of Ki67⁺progenitor cells in elderly mice compared to young mice, and AP treatment rescued these progenitor cells. Aging is closely related to apoptosis, the TUNEL staining and apoptosis protein expression showed that AP can inhibit apoptosis in intestinal tissue⁴⁸. In summary, these results indicate that AP can regulate intestinal anti-inflammatory and antioxidant capacity, repair intestinal mucosal barrier, inhibit cell apoptosis, and delay intestinal aging in mice.

The gut microbiota changes with age, affecting gut barrier function and regulating cognitive abilities through the gut brain axis⁴⁹. Francesco Marotta revealed that age-related changes in gut microbiota composition include a decrease in microbial diversity, a decrease in dominant species abundance, and an increase in sub dominant species abundance⁵⁰. In this study, we observed differences in gut microbiota diversity in aging mice, characterized by a decrease in α-diversity and isolation of β-diversity. Additionally, we also observed differences in species abundance. The genus Helicobacter contains over 35 species. Helicobacter pylori is the most important factor in human health, and it is associated with gastritis, precancerous gastric atrophy, and intestinal metaplasia⁵¹. In aging mice, we observed a high relative abundance of Helicobacter, and AP significantly reduced the abundance of this genus in the feces of aging mice. Bilopila belongs to the Proteobacteria and is an opportunistic pathogen, B. wadsworthia synergizes with a high-fat diet to promote higher inflammatory responses, intestinal barrier dysfunction, and abnormal bile acid metabolism, leading to higher glucose metabolism abnormalities and liver fat production⁵². Bilopila can reproduce and accumulate in large quantities under the dual conditions of ketogenic diet and intermittent hypoxia, thereby causing damage to hippocampal function and increasing the risk of cognitive impairment⁵³. Compared with young mice, Bilopila was significantly enriched in fecal bacteria of aging mice, and the administration of AP reversed this result.

Notably, the relative abundance of Bacteroides increased in aging mice after AP treatment (OA), and Lefse analysis found that Bacteroides vulgatus was enriched in the OA group. Bacteroides vulgatus is one of the main Bacteroides in the human gut, with various beneficial effects. Previous study has reported that Bacteroides vulgatus and Bacteroides dorei can reduce the production of lipopolysaccharide in intestinal microorganisms and inhibit atherosclerosis⁵⁴. Meanwhile, Bacteroides vulgatus can also reduce the expression of colon TNF-α, IL-1β and IL-6 induced by DSS and improve colitis in mice⁵⁵. In addition, we also observed an increase in the genus Lactobacillus in the OA group, including Lactobacillus faecis and Lactobacillus taiwanensis. Jie Yu et al. revealed that Lactobacillus faecis belongs to Lactobacillus with high SCFAs production⁵⁶. Previous studies have shown that the abundance of Lactobacillus taiwanensis was low in colorectal cancer and cervical tumors, indicating that Lactobacillus taiwanensis may have a protective effect in the occurrence and development of colorectal and cervical tumors⁵⁷. Yan Qing Li et al. reported that Lactobacillus taiwanensis has antibacterial activity and applications in regulating intestinal immunity⁵⁸. Parabacteroides goldsteinii showed high relative abundance in the OA group. Studies have shown that Parabacteroides goldsteinii has shown good prebiotic effects, including relieving chronic obstructive pulmonary disease, improving obesity and type 2 diabetes, and treating autism⁵⁹. Kristiansen et al. revealed a correlation between the levels of inflammatory cytokines and the abundance of specific bacterial taxa, including Lachnospiraceae bacterium 28–4 and Alistipes inops⁶⁰. We observed an increase in the relative abundance of Alistipes inos in aging mice, while its relative abundance decreased after AP administration. In addition, there were high relative abundances of Parasutterella and Pantoea, as well as Burkholderiales bacterium YL45 and Pantoea agglomerans in the fecal microbiota of control group mice (YC). Parasutterella is known to be a high consumer of L-cysteine, and L-cysteine is known to improve the blood sugar level of rodents, so Parasutterella is considered to be important in the occurrence and development of type 2 diabetes and obesity⁶¹. Pantoea is a rare pathogen of human infectious diseases. Pantoea agglomerans is a genus of Pantoea, which may be the cause of opportunistic infections in humans, mainly from wounds made of plant materials or as hospital acquired infections, and occurring in individuals with weakened immune function⁶².

In summary, AP can improve the antioxidant capacity of aging mice, inhibit intestinal inflammation through the TLR4/NF-κB signaling pathway, repair the intestinal mucosal barrier, regulate intestinal microbiota, and ultimately alleviate intestinal aging and improve age-related cognitive dysfunction. Meanwhile, it was found that administering AP to young mice can reduce the relative abundance of opportunistic pathogens. These results indicate that daily intake of apples is safe and effective in improving intestinal aging during the aging process.

This study was carried out in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All animal experiments complied with “the Guide for the Care and Use of Laboratory Animals”, and were approved by the Animal Experiment Ethics Committee of Nanchang University (NCULAE-20221031089). The methods of this study were informed to all the authors and were conducted in accordance with relevant guidelines and regulations.