Abstract
Aging-related neuroinflammation drives cognitive decline; however, the mechanisms by which gut microbiota-modulating bioactive compounds, such as rice bran peptide KF-8, mitigate this process remain unclear. Here, KF-8 was shown to ameliorate age-related traits in aged mice by reshaping gut microbiota, notably by stabilizing Akkermansia muciniphila (AKK), to suppress systemic inflammation and cognitive deficits. Specifically, antibiotic-treated mice receiving KF-8 exhibited neuroinflammation and declined cognition. KF-8 and AKK synergistically attenuated pro-inflammatory pathways, particularly TNF-α, in the blood and in the hippocampus. While TNF-α antibodies mirrored KF-8’s benefits, TNF-α recombinant protein negated KF-8’s protective effects. Combined KF-8 and AKK interventions aligned with TNF-α antibody outcomes, underscoring TNF-α’s pivotal role. Our findings reveal that KF-8 enhances healthy aging by modulating gut microbiota, sustaining AKK, and suppressing TNF-α-driven neuroinflammation, thereby rescuing cognitive function in aged mice.
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Introduction
Aging of the nervous system, skin, skeletal muscles, immune system, and cells remains a hot topic of research in the field of anti-aging for scientists1,2. Recent research on both animals and humans highlights the potential of the gut-X axis to affect health, which is particularly important for the elderly population3,4,5,6. A significant hallmark of aging is chronic inflammation7. This pro-inflammatory state increases the incidence of various age-related diseases, such as cardiovascular disease, osteoporosis, Alzheimer’s disease, and cancer8. Aging is accompanied by changes in innate immunity, sarcopenia, and cognitive function decline, and the gut microbiota in the elderly differs from that of young people9. The gut microbiota affects neuronal regeneration and mutation through the gut–brain axis and plays a key role in coordinating brain development and behavior10. Both the brain and the gastrointestinal tract are important sensory organs; immune cells in the brain and gut constantly evaluate environmental factors. Gut–brain communication is crucial in the regulation of inflammatory pain perception, inflammatory responses, and immune homeostasis11. Known as the “second brain”, gut microbiota has the ability to regulate immune cells’ maturation and function in the central nervous system (CNS)12,13. Microglia are the chief immune cells of the CNS, and gut microbiota can activate them so they migrate to the CNS to trigger or inhibit neuroinflammation14. During the aging process, microglia promote cognitive dysfunction, including altered brain plasticity and neurodegeneration, as the hippocampus microglia become overactive and unbalanced15. According to these studies, gut microbiota composition can affect neuroinflammation and lead to behavioral and cognitive dysfunction. Controlling the gut microbiota may prevent age-related systemic inflammation and cognitive decline.
A symbiotic relationship exists between diet and gut microbiota, with nutrients being one of the most effective regulators of microbial community composition and function. In addition, a host’s physiology could be significantly affected by gut microbes, influencing their absorption, metabolism, and storage of nutrients16. Emerging evidence suggests that older adults who consume certain nutrients, such as proteins, at recommended levels in their diet can improve their cognitive scores17. Moreover, hydrolysis products of proteins and peptides possess favorable functional activities; for instance, liraglutide and walnut peptides can effectively alleviate neuroinflammation and improve cognitive dysfunction in APP/PS1 mice18. Since food or food-derived peptides are digested and absorbed in the gut, food-derived peptides can significantly alter the gut microbiota, potentially counteracting cognitive decline with natural peptides19. In our previous research, rice bran peptides improved the lifespan of D-galactose-induced aging mice20. However, it remains unclear whether the improvement of neuroinflammation and the protective effect on cognitive function in aged mice by rice bran peptides are related to the gut microbiome.
This study aims to provide evidence that the mitigation of neuroinflammation associated with aging is linked to alterations in the gut microbiota induced by rice bran peptides. In addition, it seeks to identify the key pathways through which rice bran peptides enhance neuroinflammation and cognitive function. Here, the potential mechanisms by which gut microbiota and specific probiotics mediate the effects of rice bran peptides on neuroinflammation and cognitive function in aged mice were explored. The findings demonstrate that rice bran peptides can significantly counter brain inflammation and improve cognitive function, mainly by changing the composition of the gut microbiota, especially the abundance of AKK, a gut probiotic related to rice bran peptides, which can extend the healthy lifespan of aged mice by reducing systemic and hippocampal inflammatory levels.
Results
Rice bran peptides promote healthy aging in mice
Meaningful anti-aging therapy should be capable of extending the healthy lifespan or prolonging the duration of healthy living. To investigate the impact of rice bran peptides on the healthspan of mice, rice bran peptides were administered orally twice weekly at a dosage of 10 mg/kg (KF-8 10 mg/kg) and 30 mg/kg (KF-8 30 mg/kg) starting from 14 months of age, for a period of 10 months. Through functional and tissue analyses of deceased and surviving animals, including the health status of gross, skeletal, liver, kidney, skin, aorta, intestine, and other tissues, rice bran peptides were found to significantly improve the survival rate of mice (Fig. 1A). Histopathological analysis of the aorta, skin, liver, kidney, and intestine in aged mice showed noticeable improvements after intervention with 30 mg/kg rice bran peptides (Fig. 1B, C). Micro-CT analysis of bone structure revealed that, compared to the control group, intervention with rice bran peptides significantly increased bone mass (bone mass/total volume percentage) in aged mice (Fig. 1D, E). In summary, the inclusion of rice bran peptides activates the anti-aging mechanisms in mice, as their intervention promotes healthy aging in mice.
A Experimental design and survival curves. Administration of rice bran peptide at varying concentrations (10 mg/kg, 30 mg/kg) via gastric gavage to 14-month-old mice until they reached 24 months of age, twice weekly. The Control group received an equivalent volume of phosphate-buffered saline (PBS). Survival curves were plotted (n = 20). B Photographs of mouse appearance. Images of the external appearance of mice from the Control group and the rice bran peptide intervention groups after 10 months (n = 3). Scale bar, 1 cm. C Representative histopathological images of mouse tissues and organs (aorta, skin, liver, kidney, colon) stained with hematoxylin and eosin (H&E). Scale bars, 200 μm and 50 μm. D Representative micro-CT images of the mouse femur. On the left are the coronal and mid-sagittal views of the growth plate and trabecular bone. Scale bar, 1 mm. On the right are the longitudinal, tangential, and lateral sections of the mid-shaft scan. Scale bar, 2 mm. E Quantitative micro-CT analysis. Comparison of bone volume per tissue volume (BV/TV%) between the Control group and the rice bran peptide intervention groups in mice (n = 6). Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc tests for multiple group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significant difference between groups.
Rice bran peptides modulate multiple senescence traits to promote healthy aging in mice
To explore the mechanisms by which rice bran peptides promote healthy aging in mice, the effects of 30 mg/kg rice bran peptides on the senescence traits of mice during their middle to old age was further investigated, which corresponds to 14–16 months of age, equivalent to human ages of 47–56 years21.
After treatment and assessment of mice with rice bran peptides (Fig. 2A), the body weight of the mice was observed not to increase with age (Fig. 2B). Rice bran peptides significantly reduced age-related hair loss and graying, though body length remained unaffected (Fig. 2C). The Organ index was calculated as organ weight/body weight × 100%. This metric reflects the relative size of the organ compared to body weight and is an important parameter for assessing adaptive changes or functional status in various tissues and organs during aging following nutritional intervention22. Organ indices, except for the liver, showed no significant differences (Fig. 2D). The analysis of neuromuscular function in mice revealed that the grip strength of mice in the rice bran peptide intervention group increased in the wire suspension test (Fig. 1E), and the time taken to traverse a balance beam was significantly reduced (Fig. 2F), indicating that rice bran peptides increased muscle strength and balance in mice. Gastrointestinal (GI) transit time increases with age23, and treatment with rice bran peptides resulted in faster transit times when mice were given non-absorbable carmine red dye orally (Fig. 2G), which could contribute to weight loss in these mice.
A Experimental design. Rice bran peptide was administered via gastric gavage for 3 months (KF-8, 30 mg/kg) to mice aged from 14 to 16 months, twice weekly. The control group received an equivalent volume of phosphate-buffered saline (PBS). Body weight was recorded weekly, and behavioral tests were conducted at 16 months of age. Sample collection and assays were performed at 17 months of age (n = 8). B Weekly body weight changes in mice (n = 5). C Photographs and quantitative analysis of mouse appearance and body length (from nose to anus) (n = 5). D Organ index in mice, calculated as: organ index = (organ weight (g)/mouse body weight (g)) × 100. (n = 4). E grip strength in mice (n = 5). F Time taken by mice to traverse a balance beam (n = 5). G Gastrointestinal (GI) transit ability in mice, measured as the time for the excretion of non-absorbable carmine red dye after oral administration (n = 5). H Expression levels of P16, P21, P53, and IBA1 in mouse hippocampal tissue were detected by western blotting, and protein bands were quantitatively analyzed using ImageJ software (n = 6). I–N Levels of 8-OHdG, CRP, GM-CSF, RANTES, IL-17A, and TNF-α in mouse serum were detected by ELISA (n = 5). O, P Immunofluorescence representative images and quantitative analysis of the reduction in microglial cell numbers in the hippocampal region by rice bran peptide (n = 5). Q Upregulation of neuronal transcription factor C-Fos mRNA expression by rice bran peptide (n = 3). R, S Immunofluorescence representative images and quantitative analysis of the promotion of skin and intestinal stem cell numbers by rice bran peptide (n = 5). Statistical significance between groups was determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant.
Inflammation through senescence-associated secretory phenotype (SASP) is a leading cause of brain aging, neurodegeneration, and dementia24. The expression of SASP markers (cyclin-dependent kinase inhibitor 2A (P16), cyclin-dependent kinase inhibitor 1A (P21), and P53) proteins or genes in the hippocampus was examined, and it was found that mice treated with rice bran peptides had lower SASP marker abundance compared to the control group. SASP or inflammation may influence each other. Additionally, the levels of microglia in the mouse hippocampus were examined, and it was found that rice bran peptides significantly reduced the expression of ionized calcium binding adaptor molecule 1 (IBA1) but increased neuronal activity of Fos proto-oncogene (C-Fos) (Fig. 2H, O, P, Q), alleviating neuroinflammation in mice. This suggests that rice bran peptides can reduce chronic inflammation and cellular senescence in the brain. They are also expected to improve cognitive impairments caused by neuroinflammation.
In addition to DNA damage in various cell types, aging also affects intercellular communication, which is associated with increased levels of 8-hydroxy-2’-deoxyguanosine (8-OHdG) and pro-inflammatory factors25,26. Supplementation with rice bran peptides reduced the levels of 8-OHdG in mouse serum, indicating that rice bran peptides can inhibit DNA damage and improve the survival rate of mice (Fig. 2I). Additionally, mouse serum levels of C-reactive protein (CRP), granulocyte-macrophage colony-stimulating factor (GM-CSF), RANTES (regulated on activation, normal T cell expressed and secreted), interleukin-17A (IL-17A), and tumor necrosis factor-α (TNF-α) were elevated at the middle-age stage, but these cytokine levels were significantly reduced after intervention with rice bran peptides (Fig. 2J–N), suggesting that the pro-inflammatory state during aging can be improved with continuous intervention of rice bran peptides.
The ability of tissues to regenerate decreases with age progression, primarily attributed to defects in tissue-specific stem cells27. Skin tissue senescent cells increase with age, while stem cells decrease28. Among the human body’s fastest-renewing tissues is the intestinal epithelium, and the weakened regenerative function of intestinal stem cells can damage the integrity of the intestinal epithelium29. Wnt target gene eucaine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) is expressed in stem or progenitor cells30. Stem cells in the gut lining and hair follicles of middle-aged mice with and without rice bran peptide treatment were studied. The treatment boosted Lgr5-positive cells in both areas compared to the controls (Fig. 2R, S). Therefore, supplementation with rice bran peptides can enhance the regenerative capacity of certain tissues by increasing their stem cell count.
Dysbiosis of the gut microbiome is one of the latest hallmarks of aging. It is influenced by various factors such as host genetic diversity (ethnicity), dietary factors, lifestyle habits, and geographical environmental conditions during the aging process31. A 16S rRNA analysis was conducted on fecal samples from middle-aged mice treated with rice bran peptides, and the results were compared with those of young mice at the ages of 4 and 18 months. The α-diversity, as indicated by the Simpson and Shannon indices, showed significant differences between the microbiota of mice treated with rice bran peptides and those of old and middle-aged mice (Fig. 3A, B). Bray–Curtis analysis revealed an R value of 0.593, higher than 0, indicating greater intergroup differences than intragroup differences, with a P value of 0.008, indicating statistical significance (Fig. 3C). Principal component analysis (PCA) and Venn diagrams showing the overlap of operational taxonomic units (OTUs) among different groups indicated differences in the gut microbiota of young, old, and rice bran peptide-treated middle-aged mice (Fig. 3D, E). Based on the Kruskal–Wallis test analysis of intergroup differences at the genus level, mice treated with KF-8 had a significant increase in AKK, Allobaculum, Lachnospiraceae_UCG-001, and Eubacterium siraeum_group relative abundances. The microbial community structure in the gut microbiota of 4-month-old and 18-month-old mice was compared to study aging-related changes in the microbiota, with changes in the relative abundance of AKK, Lachnospiraceae_UCG-001 bacteria (Fig. 3F). These results suggest that rice bran peptides may alter the structure of the gut microbiota and make it more similar to a younger phenotype.
A, B Analysis of alpha diversity among groups using Simpson and Shannon indices. Assessment of alpha diversity in young (4 months old) and old (24 months old) mice, with control groups and KF-8 intervention groups (treated from 14 to 16 months of age with KF-8 at a dosage of 30 mg/kg, twice weekly) (n = 3). C, D Visualization of fecal sample bacterial community characteristics among groups at the species level using Bray–Curtis distance matrix and principal coordinate analysis (PCoA) (n = 3). E Venn diagram illustrating the overlap of operational taxonomic units (OTUs) among groups (n = 3). F Heatmap of the most abundant features of differential gut bacteria at the genus level (n = 3). G Representative path plot during the MWM test phase (n = 5). H In the acquisition phase, the escape latency of the rice bran peptide treatment group was significantly reduced (n = 5). I The latency to reach the platform position (n = 5). J Number of crossings of the platform position by mice (n = 5). K Percentage of time spent in the platform quadrant by mice (n = 5). L Representative path plot of mice in the NOR test (n = 5). M Increase in the NOR index in mice of the rice bran peptide intervention group (n = 5). N, O In the NOR test, the time and frequency of activity in the natural peptide treatment group mice were significantly increased (n = 5). P Significant increase in the number of touches to new objects by mice in the rice bran peptide intervention group (n = 5). Q Representative path plot of mice in the open field test (n = 5). R, S Rice bran peptide promotes mouse movement, with significant increases in movement distance and speed (n = 5). T Time spent by mice in the central area (n = 5). U Increase in the percentage of mouse motor activity due to rice bran peptide intervention (n = 5). Differential bacteria were selected based on LEfSe analysis and the Kruskal–Wallis test. P < 0.05 indicates statistical significance. Statistical significance between groups was determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant.
Reduced exploratory behavior and cognitive function are common age-induced behavioral changes32. The MWM and NOR tests were used to assess the learning and memory functions of mice. Compared to the control group, mice with continuous rice bran peptide intervention had a gradually decreasing latency to escape the platform over a 5-day learning phase, and this latency was significantly lower than that of the control group (Fig. 3H). Spatial exploration tests observed that the time taken by rice bran peptide-treated mice to first cross the platform was significantly lower than that of the control group mice, and both the number of platform crossings and the time percentage in the original platform quadrant (target quadrant) were higher than those of the control group (Fig. 3G, I–K). The NOR test showed the intervention group had a significantly improved recognition index, enhanced activity levels (frequency and duration), and notably increased novel object contacts (Fig. 3L–P). Concurrently, an open field test (OFT) was conducted to assess the exploratory behavior, anxiety levels, and adaptability of mice, and the results showed that the distance traveled, speed, and time spent in the central area of the rice bran peptide intervention group mice all significantly increased (Fig. 3Q–U). These results indicate that rice bran peptides significantly improved the exploratory behavior and cognitive functions of middle-aged mice, which aligned with our observations of the elimination of brain inflammation and subsequent improvement in cognition due to rice bran peptides.
The gut microbiota is a prerequisite for rice bran peptides to improve neuroinflammation and cognitive function
The aging characteristics of untreated or rice bran peptide-treated mice in the presence or absence of intestinal bacteria were measured to identify whether rice bran peptides promote healthy aging and cognitive function by improving gut microbiota. A 3-month treatment with rice bran peptides improved cognitive function and altered gut microbiota in middle-aged mice (Fig. 4A–F). Here, mice pre-treated with an antibiotic (Abx) for 7 days were considered germ-free and then subjected to gavage intervention with rice bran peptides or fecal transplantation from these peptide-treated mice for an additional 4 months (Supplementary Fig. 1A). Mice treated with Abx for 3 or 7 days showed that intestinal bacteria were eradicated on the 7th day, as indicated by bacterial plating and detection of the V3-V4 region RNA (Supplementary Fig. 1B, C). Mice’s body weight and organ indices showed no significant differences between groups (Supplementary Fig. 1E, F). According to the results of MWM and NOR tests, the learning ability and cognitive function of Abx-treated mice were no different from the control group, and Abx treatment hindered the improvement of spatial memory and cognitive ability in mice treated with rice bran peptides (Fig. 4A–J). However, during the 5-day learning phase of the MWM, compared to the control group, Abx group, and Abx+KF-8 group, the fecal microbiota transplantation (FMT) group mice showed a significant reduction in the latency to escape the platform (Fig. 4B). In the exploratory experiment, mice exhibited a notable decrease in positioning cruise time, with the highest frequency of platform quadrant crossings and the longest duration in the target quadrant, all showing significant differences (Fig. 4C–E). However, the exploration test did not show any difference in swimming speed among the four groups of mice (Supplementary Fig. 1D). As with MWM, the results of the NOR experiment were similar. In Abx-treated specific pathogen-free (SPF) mice, the role of rice bran peptides in improving memory function was eliminated, and this effect was restored after FMT in Abx-treated SPF mice (Fig. 4F–J). Similarly, the results of the open field test also showed that the fecal bacteria of mice treated with rice bran peptides could also play a role in enhancing the exploratory behavior of Abx mice, while the spontaneous behavior of Abx-treated mice with direct rice bran peptide intervention did not improve (Fig. 4K–O). These results indicate that the gut microbiota is necessary for the improvement of spatial learning and memory functions mediated by rice bran peptides. In addition, the levels of 8-OHdG and inflammatory factors in mouse serum were also detected (Fig. 4P, Q), and the results showed that rice bran peptides inhibit DNA damage and improve the pro-inflammatory state during aging, mainly by acting through the gut microbiota as the main medium. Further combined with the results of mouse grip strength tests and balance beam experiments, it was found that the muscle endurance and coordination of mice were significantly improved in the Abx+FMT(KF-8) group of mice, while the Abx+KF-8 group of mice did not show any significant improvement compared to the control group (Supplementary Fig. 1G, H). The above results indicate that a healthy gut microbiota is necessary for rice bran peptides to improve cognitive function and promote healthy aging in mice.
A Representative path plot during the Morris Water Maze (MWM) test phase (n = 5). B In the acquisition phase, the escape latency in the rice bran peptide treatment group was significantly reduced (n = 5). C In the rice bran peptide treatment group, the latency to reach the platform position was significantly reduced (n = 5). D The number of times mice crossed the platform position (n = 5). E The percentage of time mice spent in the platform quadrant (n = 5). F Representative path plot of mice in the NOR test (n = 5). G The NOR index increased in mice of the rice bran peptide intervention group (n = 5). H, I In the NOR test, the number of activities and the time spent by mice in the rice bran peptide treatment group were significantly increased (n = 5). J The number of times mice in the rice bran peptide intervention group touched new objects significantly increased (n = 3). K Representative path plot of mice in the open field test (n = 5). L, M Rice bran peptide promoted mouse movement, with significant increases in movement distance and speed (n = 5). N The time mice spent in the central area. (n = 5). O Rice bran peptide intervention increased the percentage of mouse motor activity (n = 5). P–U Levels of 8-OHdG, CRP, GM-CSF, RANTES, IL-17A, and TNF-α in mouse serum were detected by ELISA (n = 5). V, W Immunofluorescence representative images and quantitative analysis of the reduction in microglial cell numbers in the hippocampal region by rice bran peptide (n = 3). Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc tests for multiple group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns indicates no significant difference between groups.
Rice bran peptides improve cognitive function in mice by enriching AKK
Our goal was to determine the role of specific bacterial taxa in mediating the improvement of neuroinflammation by rice bran peptides. Differences in gut microbiota between the Control and Abx+FMT(KF-8) groups were identified through 16S rRNA sequencing analysis of fecal samples, using both α-diversity and β-diversity assessments (Fig. 5A–C). Principal component analysis (PCA) showed no significant overlap in the positions of the two groups, indicating a high degree of discrimination between the two groups’ microbiotas; however, the Venn diagram showed an overlap of operational taxonomic units (OTUs) among different groups (Fig. 5D, E). The heatmap in Fig. 5F, G, based on the Kruskal test analysis of intergroup differences at the genus-level, showed a significant increase in the relative abundance of AKK, Enterorhabdus, Lachnospiraceae_UCG_001, Mucispirillum, etc., in the fecal samples of FMT mice. Current research reports that the abundance of AKK decreases with age, and supplementation with AKK can increase the thickness of the intestinal mucus layer in aging mice, improve their systemic immune status, reduce the deposition of Aβ plaques in the brains of APP/PS1 mice, and thereby alleviate cognitive impairments and anxiety33.
A, B Analysis of alpha diversity among groups using Simpson and Shannon indices (Control group and Abx+FMT group) (n = 3). C, D Visualization of intergroup fecal sample bacterial community characteristics at the species level using the Bray–Curtis distance matrix and Principal Coordinate Analysis (PCoA) (n = 3). E Venn diagram illustrating the overlap of OTUs among groups (n = 3). F Heatmap of the most abundant features of differential gut bacteria at the genus level (n = 3). G Abundance of AKK in the feces of mice from both groups (n = 3). H Weekly body weight changes in mice. Control group mice were gavaged with PBS, AKK group mice were gavaged with 200 μL of a 2 × 109 CFU/mL AKK bacterial suspension, twice a day, and AKK + KF-8 group mice, in addition to being gavaged with the corresponding volume of AKK bacterial suspension, also underwent rice bran peptide intervention (30 mg/kg KF-8) (n = 8). I Organ index in mice (n = 4). J Grip strength in mice (n = 5). K Time taken by mice to traverse a balance beam (n = 5). L Representative path plot during the MWM test phase (n = 5). M, N The latency to reach the platform position (n = 5). O Number of times mice crossed the platform position (n = 5). P Percentage of time mice spent in the platform quadrant (n = 5). Q Representative path plot of mice in the NOR test. R Increase in the NOR index in mice of the rice bran peptide intervention group (n = 5). S, T The number of activities and the time spent by mice (n = 5). U The number of times mice touched new objects (n = 3). V Representative path plot of mice in the open field test (n = 5). W, X Movement distance and speed (n = 5). Y Time spent by mice in the central area (n = 5). Z The percentage of mouse motor activity (n = 5). Statistical significance between groups was determined by Student’s t test. One-way ANOVA followed by Bonferroni post hoc tests was used to compare multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant.
To explore whether AKK mediates the improvement of cognitive function in aging mice by rice bran peptides, 2 × 109 CFU/mL AKK was administered intragastrically to mice every 2 days, or co-administered KF-8 (30 mg/kg) for 3 months. Post-intervention behavioral assessment revealed significantly enhanced physical fitness and cognitive function in AKK-treated mice, with superior improvement observed in the AKK + KF-8 co-intervention group. While the co-intervention maintained normal organ indices, it effectively curbed age-related weight gain (Fig. 5H, I). Notably, both intervention regimens significantly boosted grip strength and balance capabilities (Fig. 5J, K). From the MWM test results, the latency of mice to reach the escape platform during the 5-day learning phase became progressively shorter and was much less than that of the control group (Fig. 5L, M). In the exploration test, they took significantly less time to first cross the platform position compared to the control group (Fig. 5N), and the number of times and time spent crossing the platform quadrant were greater than those observed in the control group (Fig. 5O, P). Following the intervention, the NOR test results showed a marked increase in the recognition index, activity times, duration, and the number of times the new object was touched (Fig. 5Q–U). Moreover, in the open field test, mice traveled a greater total distance, spent more time crossing the central area, and had a higher proportion of movement activity compared to the control group (Fig. 5V–Z). These results indicate that rice bran peptides mainly delay aging and improve the physical fitness and cognitive function of middle-aged mice by enriching AKK.
KF-8 and AKK downregulate inflammatory pathways and senescence marker levels in mice
The combination of KF-8 and AKK treatment was found to lead to a significant improvement in cognitive function in elderly mice (Fig. 5L–Z); however, the underlying mechanisms of action have not yet been elucidated. Therefore, the potential pathways involved were explored. The extent of cellular damage and inflammatory cytokine levels in the peripheral blood of mice were initially measured, and it was found that levels of 8-OHdG, CRP, RANTES, IL-17A, GM-CSF, and TNF-α were highest in the Control group and lowest in the AKK + KF-8 group, with significant differences between all groups (Fig. 6A–F). Several studies suggest that there is an association between peripheral inflammation and cognitive decline in non-demented adults. Consequently, the expression levels of inflammatory genes and proteins in the hippocampus of mice we examined, and the results showed that pro-inflammatory cytokines, such as interleukin-6 (IL-6), TNF-α, and interleukin-1β (IL-1β), were decreased in the AKK and KF-8 treatment group (Fig. 6G–I, N). Notably, throughout the aging process, the brain’s immune cells, known as microglia, are continuously activated34. In this study, the number of microglia (IBA1) in the hippocampus was highest and neurons (C-Fos) lowest in aged mice, but these levels were significantly reversed in mice treated with AKK and KF-8 (Fig. 6M, N). Senescent cells gathering in brain tissue result in aging and the progression of diseases associated with aging35. Furthermore, the gene and protein levels of senescence markers in the hippocampus were examined and, as expected, AKK and KF-8 significantly reduced the levels of P16, P21, and P53 genes and proteins (Fig. 6J, K, N).
A–F Levels of 8-OHdG, CRP, GM-CSF, RANTES, IL-17A, and TNF-α in mouse serum were detected by ELISA (n = 5). G–M AKK and rice bran peptide regulate the mRNA expression of hippocampal pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) and senescence-associated factors (P16, P21, P53) as well as the neuronal transcription factor C-Fos (n = 3). N Protein levels of TNF-α, IL-6, IBA1, P16, P21, and P53 in the hippocampus were detected by western blotting, and the protein bands were quantitatively analyzed by Image J software (n = 6). O Volcano plot results of DESeq2 analysis for differentially expressed genes (DEGs) in the hippocampus of AKK-VS-Control and AKK + KF-8-VS-Control mice (n = 3). P, Q Venn and heatmap comparisons of total transcriptome data (n = 3). R Pathway enrichment analysis of biological processes conducted using RNA sequencing. S, T Construction of PPI protein-protein interaction network diagrams with Cytoscape and the top five hub genes selected by the MCC plugin. U Levels of TNF in the hippocampus of mice from different groups (n = 3). DEGs were identified using DESeq2 (|log2 fold change | > 2, adjusted P value < 0.01. One-way ANOVA followed by Bonferroni post hoc tests was used to compare multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant.
The CNS and the peripheral immune system continuously and extensively communicate with each other through various pathways, participating in the regulation of behavior and many other key neural functions throughout life36,37. To this end, the transcriptome changes in the hippocampal tissue of mice treated with KF-8 and AKK were analyzed. Based on raw quantitative count data, differential expression analysis was performed using DESeq2 (|log2(FoldChange)| >0.5 & padj <0.05), and 2064 differential genes (Fig. 6O, left) and 986 differential genes (Fig. 6O, right) were screened out by comparing AKK and AKK + KF-8 with the Control group, respectively, with 177 overlapping differential genes (Fig. 6P, Q). A further analysis of the function of the 177 differential genes through the KABOS database revealed that they were significantly related to biological processes such as redox processes, regulation of cell population proliferation, angiogenesis, inflammatory response, extrinsic apoptotic signaling pathway, oxidative stress cellular response, positive regulation of neuroinflammatory response, and canonical Wnt signaling pathway in Gene Ontology analysis (Fig. 6R). In addition, 59 genes with interaction scores greater than 0.4 were further selected by importing the 177 differential genes into the STRING database (Fig. 6S). Using Cytoscape to construct a PPI network, and screening with the MCC plugin for the top 5 Hub genes, TNF (MCC score = 18) was ranked as the top hub gene based on MCC scoring. The complete hub gene list with MCC scores is provided in Supplementary Table S2. TNF scored the highest (Fig. 6T). TNF was markedly lower in the AKK group than in the Control group, with an additional reduction observed after AKK + KF-8 treatment (Fig. 6U), which is consistent with the gene and protein levels of TNF-α detected in the hippocampus (Fig. 6H, N). Moreover, the differential genes of AKK and AKK + KF-8 mice were also compared, and 359 differential genes were screened out, which were significantly related to the regulation of neuroinflammatory response, positive regulation of neuronal differentiation, apoptosis process, inflammatory response, long-term memory, and other biological processes (Supplementary Fig. 2A–D). Overall, these results indicate that KF-8 and AKK inhibit pro-inflammatory pathways in the blood and hippocampus, participate in and regulate aging and neurodegenerative diseases, especially the expression levels of TNF-α.
KF-8 improves neuroinflammation and cognitive function in aged mice by reducing TNF-α levels
Neuroinflammation, being part of systemic inflammation, raises the odds of cognitive impairment, neurological disorders, and neurodegenerative diseases36,37. Both peripheral blood TNF-α levels and hippocampal tissue TNF-α mRNA or protein levels increase during the aging process, and intervention with KF-8 and AKK reverses the upregulation of TNF-α in aged mice. Here, the impact of elevated peripheral TNF-α levels on systemic inflammation and aging in aged mice was verified. Through tail vein injection, aged mice were given recombinant TNF-α protein (TNF-αRP) and TNF-α monoclonal antibody (TNF-α antibody), with IgG used as a control. Mice body weight changes revealed that mice treated with KF-8 + AKK or TNF-α antibody did not gain significant weight due to aging, in contrast to the Control group (Fig. 7A). The levels of 8-OHdG and inflammatory factors in the peripheral blood of mice were significantly reduced after KF-8 + AKK intervention, although KF-8 + AKK could not completely reverse the damage to systemic inflammation caused by TNF-αRP in mice. However, the combination of KF-8 + AKK and TNF-α antibody had a more significant effect on improving systemic inflammation than the TNF-α antibody alone (Fig. 7B–G). Similarly, the levels of inflammatory proteins and aging-related proteins in the hippocampal tissue of mice were measured. Compared to the Control group, KF-8 + AKK significantly reduced these levels, however, this difference was eliminated by TNF-αRP treatment (Fig. 7H). Further comparison revealed that the combined use of KF-8 + AKK and TNF-α antibody had consistent effects with the use of KF-8 + AKK.
Control group mice were gavaged with PBS, while KF-8 + AKK group mice underwent concurrent 30 mg/kg KF-8 gavage intervention and 200 μL of 2 × 109 CFU/mL AKK bacterial suspension, twice a day, for a duration of 2 months (from 14 to 15 months of age). On the 8th week of intervention, mice were treated with tail vein injections of TNF-α recombinant protein (TNF-αRP) and TNF-α monoclonal antibody (TNF-α antibody), with IgG used as a control. Body weight was recorded weekly, behavioral tests were conducted at 15 months of age, and sample collection and assays were performed at 16 months of age (n = 8). A Weekly body weight changes in mice. B–G Levels of 8-OHdG, CRP, GM-CSF, RANTES, IL-17A, and TNF-α in mouse serum were detected by ELISA (n = 5). H Protein levels of TNF-α, IL-6, IL-1β, IBA1, P16, P21, and P53 in the hippocampus were detected by western blotting, and the protein bands were quantitatively analyzed by ImageJ software (n = 6). Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc tests for multiple group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant.
Excessive production of TNF-α may also damage neural transmission in brain structures that regulate cognitive function38,39. In the spatial learning and memory function test of the MWM, the escape latency of mice in the KF-8 + AKK group, TNF-α antibody, and KF-8 + AKK + TNF-α antibody treatment groups was significantly reduced during the 5-day training phase; however, there was no significant difference between the KF-8 + AKK + TNF-αRP-treated mice and the Control group (Supplementary Fig. 3A). During the probe trial, KF-8 + AKK co-treatment significantly shortened escape latency and increased platform quadrant crossings/duration versus controls – effects abolished by TNF-αRP (Supplementary Fig. 3B–D). Moreover, mice receiving TNF-α antibody plus KF-8 + AKK showed significantly elevated platform crossings relative to controls. It was also observed that in the NOR test, the KF-8 + AKK + TNF-α antibody treatment group (but not the KF-8 + AKK + TNF-αRP treatment group) had significantly more activity times and recognition indices for the new object than the Control group (Supplementary Fig. 3E–I). In the OFT test, mice in the KF-8 + AKK group, TNF-α antibody, and KF-8 + AKK + TNF-α antibody treatment groups were observed to have higher total movement distance, time moving in the central area, and activity time compared to the Control group and KF-8 + AKK + TNF-αRP mice (Supplementary Fig. 3J–M). In addition to tests of learning and cognitive abilities, the grip strength and balance of mice were also tested, and the results were consistent. These results indicate that KF-8 + AKK and TNF-α antibody reduce neuroinflammation and promote the protection of cognitive function, while TNF-αRP eliminates the protective effect of KF-8 + AKK on the cognitive function of aged mice.
Discussion
The gut microbiota plays a crucial role in the aging process of the human body. Gut bacteria collaborate with the host to regulate immunity, metabolism, and the nervous system through dynamic bidirectional communication along the “gut–brain axis”11. Studies have shown that aging in humans leads to changes in the gut microbiota and can also accelerate the development of immunosenescence and inflammation-related aging40. Notably, unhealthy lifestyles—particularly high-fat diets (HFD)—exacerbate systemic and neuroinflammation, accelerating aging processes. HFD promotes gut dysbiosis and bacterial translocation, activating peripheral TLR4/NF-κB signaling and central microglial hyperreactivity, thereby elevating TNF-α and IL-1β in the hippocampus41. Nutrient intake, especially proteins/peptides, when consumed in appropriate amounts beyond the daily diet, can effectively alleviate neuroinflammation and cognitive dysfunction17,18. Current research indicates that natural peptides have the potential to ameliorate drug-induced neuroinflammation and cognitive dysfunction in murine models by modulating the composition of the gut microbiota. While these studies can replicate the neuroinflammatory processes triggered by specific pathogens, they primarily concentrate on the structural composition of intestinal bacteria. Consequently, there is a lack of definitive evidence clarifying the mechanisms underlying neuroinflammation and age-related cognitive decline42,43,44. The uniqueness of our study lies in the use of aged mice over 14 months for FMT and specific bacterial community validation, and both were intervened with rice bran peptides and regular feed, reducing the interference of different diets and ages. The behavior of healthy aging in mice after rice bran peptide intervention was delineated across multiple aspects, including hair condition, physical fitness, microbial composition, key regulated bacterial taxa, learning and cognitive abilities, peripheral blood inflammation levels, and pathways and protein levels associated with neuroinflammation. Moreover, bacteria and bacterial-related functional markers regulated by rice bran peptides that can improve the healthspan of mice were identified and independently validated. In summary, this study focused on the regulation of the gut microbiota by rice bran peptides to improve systemic inflammation, providing preliminary insights into aging and longevity-related characteristics (including the microbiome, neuroinflammation, and cognitive function).
Previous studies have shown that natural peptides play a key role in the anti-aging process mainly through pathways such as oxidative stress, inflammation, apoptosis, and collagen synthesis45,46,47. Changes in the gut microbiota associated with age are closely related to the decline in cognitive ability, muscle mass and strength, bone loss, skin homeostasis, vascular aging, immunosenescence, and metabolic changes48. Rice bran peptides shape the gut microbiota of aged mice and improve various aging phenotypes, such as enhanced muscle endurance, coordination, exploratory behavior, cognitive ability, and gastrointestinal transit ability in mice, reduced cellular damage, systemic inflammation, and SASP marker levels, and positive effects on stem cells (skin and small intestine); more importantly, it was confirmed in Abx pre-treated aged mice that the gut microbiota is a necessary condition for rice bran peptides to improve neuroinflammation and cognitive function, just as metformin, which has been shown in previous studies to have weight loss, cardio protection, lipid-lowering, anti-tumor, and diabetes treatment effects, mainly improves cognitive function and extends lifespan in mice through the gut microbiota49,50.
AKK is a longevity bacterium that can achieve good anti-aging and life-extension effects through microbial regulation, such as balancing the gut microbiota, maintaining microbial diversity, anti-inflammation, metabolism assistance, and immune regulation. It was observed that rice bran peptides increased the α-diversity of gut bacteria in aged mice, while also significantly increasing the relative abundance of AKK, Eubacterium siraeum_group, Lachnospiraceae_FCS020_group, etc., which decrease with age. Interestingly, it was also found that AKK remained in high abundance in the feces of mice after 3 months of FMT following Abx treatment. This finding indicates that AKK, as a probiotic in the gut related to rice bran peptides, can improve inflammation-related aging in mice. This finding is consistent with many studies showing that the abundance of AKK increases in super-centenarians51, but is negatively correlated with overweight, obesity, untreated type 2 diabetes, or hypertensive patients52. Furthermore, the level of AKK is associated with reduced cognitive function and serum inflammatory marker levels53, which is consistent with our research results. Our findings reveal that the differential gene expression in mice subjected to a combined intervention of rice bran peptides and AKK, as opposed to intervention with AKK alone, remains significantly correlated with biological processes and signaling pathways, including the regulation of neuroinflammatory responses and neuroactive ligand-receptor interactions. These results suggest that rice bran peptides may synergistically enhance the proliferation and activity of AKK, thereby attenuating inflammation and improving cognitive function in aged mice. This finding aligns with the recently documented synergistic interaction between palmitic acid and AKK, which enhances colitis outcomes54. The study uncovers a novel positive feedback loop involving rice bran peptides, which promotes an increased abundance of beneficial gut bacteria, which in turn, contributes to a more favorable gut microbiota composition and a higher prevalence of selectively regulated gut bacteria, ultimately facilitating improved healthy aging and disease management. However, future studies need to investigate how rice bran peptides induce the enrichment of other gut microbes to beneficially affect chronic inflammation and healthy aging.
During the aging process, the impact of immune responses and neuroinflammation in the nervous system on the brain is multifaceted. In neuroinflammatory responses, the active pro-inflammatory state of microglia leads to the loss of neuroprotective functions, neuronal dysfunction, and the accumulation of brain tissue damage55,56. Our results show that AKK and KF-8 downregulate pro-inflammatory pathways in the nervous system and blood and reduce the levels of proteins associated with aging markers, including microglia marker protein IBA1 and pro-inflammatory factor TNF-α. These findings align with recent research, indicating that AKK enhances the concentration of short-chain fatty acids, including acetate and butyrate, within the body. In addition, it has been observed to downregulate the expression of pro-inflammatory cytokines, such as IBA1, TNF-α, and IL-6, in microglia, thereby ameliorating neuroinflammation and mitigating cognitive function43,44. Interestingly, it was found that KF-8 synergizes with AKK to significantly inhibit the systemic inflammatory biomarker TNF-α. A meta-analysis of several prospective studies has shown that TNF-α is a key pro-inflammatory molecule associated with the onset and potential progression of AD and other neurodegenerative diseases43,44. High levels of peripheral TNF-α have been identified as blood markers for frailty and sarcopenia in the elderly57. Excessive production of peripheral TNF-α leads to an increase in TNF-α concentration in the brain. In the brain, TNF-α is physiologically involved in regulating the function of the blood-brain barrier, synaptic plasticity and scaling, and neuronal function, which may be detrimental to neurogenesis58,59. Due to the limited ability of TNF-α antibodies to enter the brain, the practical value of TNF-α antibody-based therapies in improving neuroinflammation and cognitive function in neurodegenerative diseases is limited. In light of this limitation, some studies have suggested another approach to targeting TNF-α: to use small molecules to inhibit or regulate the biosynthesis of TNF-α38,60. Here, it was reported that small-molecule rice bran peptides may be an effective method for improving neuroinflammation and cognitive function in aged mice by regulating gut microbes (AKK) to reduce peripheral TNF-α production. In this study, TNF-α recombinant protein (TNF-αRP) and TNF-α antibodies were administered to mice, with behavioral assessments indicating that increased levels of TNF-α negated the enhancements in learning and memory attributed to rice bran peptide and AKK in aged mice. Notably, the administration of TNF-α antibody alone was less effective in mitigating neuroinflammation in elderly mice compared to the combined treatment with rice bran peptide, AKK, and TNF-α antibody. These findings corroborate previous research suggesting that small molecules play a regulatory role in TNF-α biosynthesis. With the progression of aging, in the early stages, microglia can be activated to release TNF-α by sensing the earliest neuronal stress, and during the neuroinflammatory process, infiltrating T cells release IFN-γ, which keeps microglia active and proliferative, producing and releasing large amounts of TNF-α61,62. It was found that rice bran peptides stably regulate AKK and inhibit the overactivity of microglia in the hippocampus. TNF-α acts as a core regulatory node in the synergistic anti-aging effects of KF-8 + AKK, and its inhibition sufficiently explains the systemic amelioration of the aging-associated inflammatory network. The latest studies highlight that AKK has been found to increase the concentration of short-chain fatty acids (such as acetate, butyrate) in the body, reducing the expression of pro-inflammatory cytokines in microglia, such as TNF-α, IL-6, improving neuroinflammation and cognitive aging63,64,65.
In summary, the potential mechanisms by which rice bran peptides regulate the gut microbiota and the impact of specific probiotics on neuroinflammation and cognitive function were explored. Our results demonstrate that rice bran peptides can significantly repair neuroinflammation and cognitive function in aged mice, with direct evidence supporting the role of rice bran peptides in mediating the gut microbiota’s effect on healthy lifespan. AKK is mediated by rice bran peptides and steadily regulates its abundance within the gut microbiota. It can regulate inflammation-related pathways in the host and improve neuroinflammation and cognitive function in aged mice by reducing the pro-inflammatory cytokine TNF-α systemically and in the hippocampus. Nevertheless, only male mice were examined here; future studies should evaluate sex-specific responses and consider age-dependent efficacy variations. Aged individuals exhibit altered gut permeability and microbiota composition, potentially modulating peptide absorption and anti-inflammatory effects66. Furthermore, the potential involvement of the blood-brain barrier (BBB) in mediating KF-8’s central effects warrants consideration. To further enhance the understanding of the synergistic effects of rice bran peptides and AKK on ameliorating neuroinflammation in aged mice, analyses from previous studies were integrated, exploring potential links between gut microbiota and metabolites. This comprehensive approach provides deeper insights into their potential mechanisms of action. Future investigations should comprehensively and explicitly verify the regulatory mechanisms by which gut microbiota metabolites influence key factors contributing to neuroinflammation. Although lifespans are increasing globally, years of healthy living are not keeping pace. Rice bran peptides could be developed as anti-aging nutritional supplements or functional foods to extend healthy lifespan, thereby reducing the burden on healthcare systems and the economy.
Methods
Mouse modeling and intervention
Male C57BL/6 mice (6–8 weeks old) were procured from Hunan SJA Laboratory Animal CO., Ltd. in Changsha, China. A temperature-controlled room was used for housing 3–5 mice per cage (22–25 g) with irradiated food and access to sterile water ad libitum, under a 12-h light and 12-h dark cycle, fresh HEPA-filtered air, and a temperature and humidity chamber were used to maintain them in a climate chamber with predefined pathogen-free conditions (21°C ± 1°C, 75% ± 10%) until they exceeded 14 months of age. The mice were then randomly assigned to groups, with 10 mice per group (except for two groups designated for survival curve analysis, containing 20 mice each). Rice bran peptides (30 mg/kg, molecular weight, 1002.06 Da; the amino acid sequence, KHNRGDEF, KF-8) were administered via oral gavage twice weekly to mice for 3 months20, after which they underwent behavioral testing or tissue collection (serum, skin, intestinal tissue, brain tissue (hippocampus), aorta, liver, kidney, heart, femur, and fecal samples), and body weight and food intake were recorded20. Mice (14–24 months old) were administered rice bran peptides (10 or 30 mg/kg) via twice-weekly oral gavage, and survival curves were generated. Through tail vein injection, aged mice were given recombinant TNF-α protein (TNF-αRP) and TNF-α monoclonal antibody (TNF-α antibody), with IgG used as a control. The animals involved in this study were treated in accordance with the guidelines set by the Ethics Committee of the Hunan Provincial Center for Laboratory Animals (Ethics Review No.: IACUC-2021(5)069) and the National Guidelines for Laboratory Animal Welfare (No. 119, Ministry of Science and Technology of the People’s Republic of China, 2006).
Morris water maze (MWM)
Spatial memory and learning functions related to the hippocampus were assessed using the MWM. Before the experiment, the water in the MWM was made opaque using a non-toxic, white, odorless, water-based pigment (titanium dioxide), and testing was conducted using a 5 + 1-day training paradigm. Mice were tested four times daily, with each trial placing the mouse at a random position at the north, south, southeast, or northwest, and given 60 s to locate the submerged platform. If the mouse failed to locate the platform independently, it was guided there and allowed to stay on it for 20 s to familiarize itself with the location. Each mouse had approximately a 20-min interval between trials. Mice were probe-tested on the 6th day. The platform was removed, allowing mice to swim in the pool for 60 s during this period. The swimming path of the mice was tracked and recorded. The tests were always conducted between 1:00 PM and 5:00 PM, and a computerized animal tracking system (EthoVision XT Base; Noldus 2020 Software, Nottingham, UK) was used for data collection, and blinded analysis of mouse data was performed.
Novel object recognition (NOR) test
The NOR test is based on the rodents’ exploratory preference for novel objects and reduced exploration of familiar ones. This rationality serves as a benchmark test for assessing animal memory function, which is contingent upon the integrity of the hippocampus. Each mouse undergoes three phases completed over 3 days, and the detection was conducted at the same time every day (09:00–11:00 AM): first, a 5-min familiarization with the test environment, then exposure to two identical objects (i.e., AA) in the same room for 5 min, and finally, placement in the same room and exposure to two different objects for 5 min (replacing one of the original objects with a different one, i.e., AB). The exploration behavior of the mouse towards the two objects was analyzed (with the nose being within one centimeter of the object). Mouse exploration and testing were recorded using a computerized animal tracking system (EthoVision XT Base; Noldus 2020 Software, Nottingham, UK), and blinded analysis of mouse data was performed. The equation can be expressed as: Discrimination Ratio = (Tnovel - Tfamiliar)/(Tnovel + Tfamiliar). Active time: Cumulative duration (s) of nose being ≤2 cm from the object during active sniffing/touching, quantified automatically by video-tracking software. Number of activities: Total discrete approaches to the object (inter-contact interval ≥1 s), manually validated by blinded observers. Data normalized as intergroup percentages, excluding immobility (velocity <1 cm/s) or passive contact.
Open field test (OFT)
For 10 min, mice were placed in an open field of dimensions 30 cm by 30 cm by 21 cm with controlled lighting (~200 lx) and a soundproof environment. In the open field environment, mice were observed with a video camera and activity monitor software (EthoVision XT Base; Noldus 2020 Software, Nottingham, UK) to identify exploratory and depression-like behaviors, and blinded analysis of mouse data was performed. 3 indicators were assessed to characterize exploratory behavior: total distance traveled (reflecting overall locomotor activity and exploratory drive), activity in the center zone (typically quantified as time spent in the center, where entry into this zone was considered exploratory behavior under low anxiety-like states), and average speed (reflecting the level of locomotor activity). For depression-like behavior or reduced locomotor activity, the percentage of active time was measured; this metric is defined as the percentage of the total test time during which the animal was not in a state of complete immobility—except for respiratory movements—lasting longer than 2 s, and a significant decrease in this percentage is often interpreted as an expression of depression-like behavior or behavioral despair.
Wire net holding test
An assessment of the mouse’s grip strength and muscular strength was performed by testing the mouse’s grasping ability. The testing procedure has been modified according to previously described protocols. Mice were allowed to hold on the top of a standard metal cage lid for a few seconds before it was inverted, and the holding time was recorded. Blind analysis was conducted on the mouse data. The experiment was repeated three times, and the average value was calculated. Results are reported as grip strength = (weight (g) × holding time (s))67.
Balance beam test
Mice are assessed using the balance beam test for their balance ability, muscle strength, and motor coordination. The balance beam typically measures 1 m in length with a diameter of 17 mm and is cylindrical in shape. One end of the balance beam was placed inside a black box, and the mouse was placed on the other end of the beam. The time and number of slips the mouse took between crossing the balance beam and entering the black box within 60 s were recorded. The experiment consisted of a 2-day training period and a 1-day testing period. The experiment was repeated thrice, the average value was calculated, and a blinded analysis of mouse data was performed.
Antibiotic (Abx) treatment and fecal transplantation
A mouse microbiota depletion model was established by using Abx. Ampicillin (1 g/L; Pfizer, New York, NY, USA), vancomycin (250 mg/L), neomycin (1 g/L), and metronidazole (1 g/L) from Sigma-Aldrich, St. Louis, MO, USA, were administered to mice in sterile water for a duration of 7 days, with the solution being replaced daily. The Abx treatment was discontinued after confirmation that the mouse microbiota had been disrupted or depleted. 12-month-old mice were treated with rice bran peptide (30 mg/kg, once daily) via gastric gavage for 2 weeks, and their feces were collected and suspended in pre-reduced PBS with a ratio of 10 mL/g weight, then filtered through a 70-μm sterile filter membrane. The suspension of intestinal bacteria or rice bran peptides (30 mg/kg) was administered to 14-month-old SPF mice pre-treated with Abx via gastric gavage (200 μL), twice weekly for a continuous period of 4 months, with body weight and food intake recorded. Control group mice were administered pre-reduced PBS.
Cultivation and treatment of AKK
Anaerobic conditions at 37 °C were used to cultivate AKK (ATCC BAA835) under anaerobic conditions on brain heart infusion (BHI) medium containing 0.05% porcine gastric mucin type III (Sigma-Aldrich, St. Louis, MO, USA)68. The bacterial cells were then collected by centrifugation and resuspended at a concentration of 2 × 109 CFU/mL in pre-reduced PBS. For a continuous period of 3 months, mice pre-treated with Abx were administered oral gavage once every 2 days with 200 μL of the bacterial suspension or with PBS as the vehicle control.
Tissue sample collection and plasma cytokine assay
Tribromoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) was used to deeply anesthetize mice and perform retro-orbital blood sampling. Plasma was obtained by centrifugation at 4 °C for 10 min and used to measure TNF-α, 8-OHdG, CRP, RANTES, IL-17A, and GM-CSF cytokines with ELISA kits (Shanghai Enzyme-Linked Immunosorbent Technology Co., Ltd., Shanghai, China) as directed by the manufacturer. Mouse tissue samples were collected, weighed, and either fixed in 4% polyformaldehyde or stored at −80 °C for further research.
Histological HE staining, immunofluorescence staining, and quantitative analysis
Tissues were sectioned into paraffin slices and HE stained. Slices of 5 µm were fixed in 10% formalin. For immunofluorescence staining of brain sections (10 µm), the experimental process was carried out in accordance with previous studies69. After blocking, all slides were incubated with anti-IBA1 or anti-Lgr5 overnight (1:500; Abcam, Cambridge, MA). Next, they were incubated for 1 h at room temperature with the appropriate secondary antibody Alexa594 (1:1000; Thermo Fisher Scientific, Waltham, MA). DAPI counterstained nuclei for 15 min, and images were analyzed using an Olympus IX73 microscope. Image-Pro Plus 6.0 software was used to measure integrated optical density to quantify fluorescence intensity.
Micro-CT analysis
The femurs of mice were scanned using Micro-CT (μCT-50, Scanco Medical, Switzerland) at a resolution of 14.8 μm per layer. Image reconstruction and analysis of the Micro-CT data were conducted using Mimics software (v.13.0, Materialise NV). The analysis of the trabecular bone mass was confined to 100 sections at the distal growth plate of the femur. The three-dimensional reconstruction of the femur was performed with reference to the morphology of the distal growth plate.
Fecal microbiota 16S rRNA gene sequencing
Gut microbiota samples from mice were analyzed using fresh feces. Fecal samples were collected on the terminal day of the intervention period (at 16 and 17 months of age, respectively) following a 6-h fast. Mice were individually housed in autoclaved cages under sterile conditions with ad libitum water access. Spontaneous defecation was allowed during a 2-h window (09:00–11:00 AM) within the light phase of the circadian cycle, and samples were immediately collected into sterile tubes and stored at −80 °C until analysis. A previous protocol was followed to extract bacterial DNA from samples using the TIANamp Fecal DNA Kit (Tiangen, Beijing, China)70. To construct and sequence libraries, amplicons targeting the V3-V4 hypervariable regions of 16S rRNA were prepared. The primers used were as follows: 341F: 5’-CTACGGGNGGCWGCAG-3’ and 805R: 5’-GACTACHVGGGTATCTAATCC-3’. In the Qiime 2 pipeline, raw data were denoised using the DADA2 algorithm, DNA sequences were clustered at 100% similarity, low-quality sequences were removed and corrected, and chimeras were identified. To obtain species-based abundance distributions (ASV_table) for each ASV sequence, species annotation was performed. Subsequently, the ASV_table was then used to analyze relative abundance, alpha diversity, beta diversity, and intergroup differences. Adonis and Anosim were used to analyze differences in species composition and community structure between groups using statistical analysis methods. Differential bacteria were selected based on LEfSe analysis and the Kruskal–Wallis (KW) test. In statistical terms, significance is determined by the P value, where P < 0.05 indicates statistical significance.
RNA sequencing (RNA-Seq)
Invitrogen TRIzol was used to extract total RNA from the mouse hippocampus. Following enrichment for mRNA, reverse transcription of this mixture into double-stranded cDNA, purification, and quality control on the Illumina sequencing platform, a library was constructed for sequencing. Raw data was processed using FASTQ software to remove adapters and low-quality data. HISAT2 (v2.0.4)/Bowtie2 (v2.2.5) were used to align the clean data with the reference genome sequence. Stringtie software was used to quantify gene expression levels, and DESeq2 was used to determine differentially expressed genes (DEGs) ( | log2 (Fold Change) | > 0.5, Padj value < 0.05). Pathway enrichment analysis was performed using the KABOS database. Using Cytoscape (v3.9.1) and the String database (interaction scores exceeding 0.4), a protein-protein interaction (PPI) network was constructed. Hub genes were identified via the CytoHubba plugin using the Maximal Clique Centrality (MCC) algorithm, which calculates node importance based on maximal cliques in the network topology. The top 10 highest-scoring genes by MCC score were defined as hub genes.
Quantitative real-time PCR (qPCR)
A TRIzol reagent (Invitrogen) was used to extract the total RNA from hippocampal tissue. The HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Cat# R312-01) was used to reverse transcribe the RNA. ABI-7500 quantitative PCR instrument with SYBR Green Master Mix (Vazyme, Cat# Q131-02) was used to examine gene expression. The cycling protocol was 30 seconds at 95 °C, 40 cycles of 15 s each at 95 °C and 60 °C were performed, followed by 20 s at 72 °C and a final 5 min at 72 °C. In all reactions, quantitative PCR was conducted in triplicate with internal normalization by GAPDH. The 2−ΔΔCt method was employed for data analysis. All primers are listed in Supplementary Table 1.
Western blot
Immunoblotting of hippocampal tissue was carried out according to a reported protocol70. For 30 min, tissue was lysed in RIPA buffer containing protease inhibitors. A protein extract was obtained through centrifugation at 4 °C 12,000×g for 10 min. BCA was used to determine protein concentration, and proteins were separated on a 12% SDS-PAGE gel, PVDF membrane transfer, blocking with skim milk, and incubated overnight with TNF-α, P16, P21, P53, and IBA1 antibodies. After washing with 1×TBST, secondary antibodies were incubated on the membranes. PVDF membranes incubated with ECL reagent were visualized using a chemiluminescence imaging system, with GAPDH serving as an internal control.
Data and statistical analysis
All data are expressed as mean ± SD with 95% confidence intervals (95% CI) reported for all group comparisons, with an unpaired Student’s t test used for analysis of statistical significance. Multiple comparisons were conducted using one-way or two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests. Detailed statistical analyses, including sample sizes (n) and P values, are provided in the figure legends, with a threshold of P < 0.05 indicating statistical significance. GraphPad Prism 9 software was used to visualize the data.
Data availability
The raw 16S rRNA gene sequencing data and the RNA-seq data have been deposited in the Sequence Read Archive-National Centre for Biotechnology Information (SRA-NCBI) under the accession numbers PRJNA1215949; PRJNA1217109, respectively. All other data are available at the website https://figshare.com/s/a735bb67a8c4c604b654 and/or the Supplementary Materials.
Code availability
To access the analysis code for the sequencing data, can contact the corresponding authors.
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Acknowledgements
This study was supported by the National Natural Science Foundation of China (32372349/32572588), National Key Research and Development Program of China (2022YFF1100203), Science and Technology Innovation Talent Project of Hunan Province (2022RC3056), Natural Science Foundation for Distinguished Young Scholars of Hunan Province (No. 2021JJ10078), Key Research and Development Program of Hunan Province (2024AQ2020). Ying Liang is the primary corresponding author for this paper.
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Y.L. conceived the studies; Y.L., J.M., and Q.L. designed the studies; J.M., S.W., J.W., Y.C., and M.C. performed the experiments; J.M. and Y.L. wrote the manuscript; J.M., S.W., and M.C. prepared the tables and figures. Y.L. and Q.L. revised the manuscript and offered financial assistance. Y.L. and Y.S.L. provided procedural advice and revised the manuscript. All authors read and approved the final manuscript.
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Mu, J., Lin, Q., Wang, S. et al. TNF-α modulation by rice bran peptides: implications for gut microbiota stability and cognitive health in aging. npj Biofilms Microbiomes 11, 195 (2025). https://doi.org/10.1038/s41522-025-00830-7
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DOI: https://doi.org/10.1038/s41522-025-00830-7
