The main component of polysaccharides from Hippophae rhamnoides L. improves HFD-induced neuronal damage by regulating tryptophan synthesis and metabolism

Abstract

Excessive neuroinflammation can lead to neuronal damage, resulting in cognitive impairment and an increased risk of neurodegenerative diseases. This research aimed to examine the impact and underlying mechanisms of SPa, the main component of polysaccharides from Hippophae rhamnoides L., on neuronal damage induced by a high-fat diet (HFD) in mice. Initially, SPa significantly enriched the microbial communities associated with tryptophan synthesis and metabolism, such as Pseudoflavonifractor, Muribaculum, and Oscillibacter. Specifically, SPa restored the decrease of 5-HT and indole derivatives and the increase of KYN, and promoted the production of IL-22 by activating the indole derivative ligand AHR to alleviate HFD-induced intestinal inflammation and barrier damage. At the same time, SPa effectively alleviated HFD-induced behavioral impairments by alleviating neuroinflammation via the AHR–NF-κB–NLRP3/Caspase-1–IL-1β/IL-18 signaling pathway and improving neuronal damage via the BDNF/TrkB pathway. Therefore, we conclude that SPa can ameliorate HFD-induced neuroinflammation and neuronal damage via the gut microbiota–tryptophan metabolism–AHR axis.

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Introduction

Neurodegenerative diseases affect approximately 50 million patients globally. By the year 2050, it is projected that the prevalence of neurodegenerative diseases will soar to approximately 115 million cases.¹ The onset and progression of neurodegenerative diseases are driven by a mix of factors, including aging, genetics, environment, oxidative stress, and inflammation. Neuronal damage in the brain regions can lead to deterioration of cognitive functions and result in neurodegenerative diseases in severe cases. Notably, obesity can induce neuroinflammation and neuronal damage, and increase the risk of developing neurodegenerative diseases.² Neuroinflammation represents a sophisticated reaction of the central nervous system to diverse stimuli and significantly contributes to the maintenance of brain homeostasis.³ Research has indicated that obesity resulting from the prolonged intake of a high-fat diet (HFD) exerts an impact on learning and memory in rodents and leads to the development of neuroinflammation. The development of this condition might be associated with the disruption of the blood–brain barrier, which allows peripherally derived pro-inflammatory cytokines to infiltrate the brain and induce inflammatory responses in the hippocampus. This subsequently leads to alterations in the expression of genes like BDNF and PSD-95. This has a substantial impact on the morphology of the brain and synaptic plasticity, ultimately leading to neuronal dysfunction in the brain.⁴ Moreover, recent investigations have highlighted that plant-derived polysaccharides are capable of modulating the gut microbiota composition and alleviating brain inflammation and neuronal damage via the microbiota–gut–brain axis.⁵ Thus, polysaccharides hold significant promise for the prevention and amelioration of neurodegenerative diseases.

Tryptophan (Trp), an essential amino acid, has a remarkable impact on the microbiota–gut–brain axis. Natural plant polysaccharides have the potential to alter the gut microbiota composition, thereby impacting Trp metabolism.⁶ The metabolites of Trp are crucial for the maturation of both the central and enteric nervous systems, and they are vital in regulating neuronal activity and systemic inflammatory cascades.⁷ Hence, the gut microbiota emerges as a pivotal target for polysaccharides to prevent and improve neurodegenerative diseases, with Trp and its metabolites playing important roles. Under inflammatory conditions, dysregulated Trp metabolism via the key enzymes TPH and IDO leads to reduced 5-HT synthesis and elevated KYN, contributing to neurotoxicity and impaired neuroplasticity.⁸ Additionally, in Trp metabolism, indole derivatives produced by the gut microbiota via the indole pathway can act as ligands for the aryl hydrocarbon receptor (AHR). These derivatives induce the expression of IL-22 in DCs and ILC3 to regulate epithelial cell integrity and immune responses.⁹ In the brain, the activated NLRP3 inflammasome triggers the cleavage of pro-IL-1β into active IL-1β, triggering inflammation and promoting the activation of microglia and the occurrence of neuroinflammation. Nevertheless, AHR has the ability to bind to the NLRP3 promoter, thereby suppressing the transcription and activation of the NLRP3 inflammasome.^10,11

Sea buckthorn (Hippophae rhamnoides L.), as a medicinal and edible plant, was first documented in the early 8th century in the Tibetan medical classic “The Moon King's Medical Diagnosis”. Sea buckthorn polysaccharides (SP), extracted from the fruits of sea buckthorn, have been reported to possess anti-inflammatory, immunomodulatory, antioxidant, antibacterial, anticancer, cholesterol-lowering, and preventive effects on cancer and cardiovascular diseases.¹² Our previous research showed that SP can activate brown adipose tissue, promoting thermogenesis and thereby inhibiting lipid accumulation and weight gain, and it could ameliorate HFD-induced cognitive dysfunction.^13,14 Additionally, we have successfully extracted and purified a major component of sea buckthorn polysaccharides with a relatively uniform molecular weight distribution, which we have named SPa.¹⁴ However, the precise bioactive constituents within SP, especially the main component SPa, as well as the roles of Trp and its metabolites in the prevention and improvement of neurodegenerative diseases are still not fully understood.

In this study, we investigated the impact of dietary SPa supplementation on impairments of the intestinal barrier and neuronal damage in HFD-fed mice via the gut microbiota–tryptophan metabolism–AHR axis. The fundamental outcomes of this study are as follows: (1) SPa enriched the microbial communities associated with tryptophan synthesis and metabolism; (2) SPa restored the decrease of 5-HT and indole derivatives and the increase of KYN in obese mice, and activated AHR through indole derivatives, promoting IL-22 production and enhancing intestinal barrier integrity and gut homeostasis; (3) SPa alleviated HFD-induced brain inflammation via the AHR–NF-κB–NLRP3/Caspase-1–IL-1β/IL-18 signaling pathway; and (4) SPa improved HFD-induced neuronal damage and synaptic dysfunction through the BDNF/TrkB signaling pathway.

Materials and methods

Preparation of SPa

SPa was extracted and separated according to the methods described by our earlier study.¹⁴ In brief, SPa was extracted from fresh sea buckthorn berries via water extraction and ethanol precipitation. The berries were dried at 60 °C, ground, defatted, and then sieved. The material was mixed with deionized water at a 1 : 25 solid-to-liquid ratio and heated at 80 °C for 1 hour. After centrifugation at 4 °C and 4000 rpm, the supernatant was concentrated by rotary evaporation, blended with anhydrous ethanol at four times its volume and maintained at 4 °C for 48 hours. The solution was then deproteinized, dialyzed, and freeze-dried to obtain the crude SP with a yield of approximately 6.1%. The crude SP was further purified using a DEAE-52 column and a Superdex-75 column with a refractive index detector, yielding the purified polysaccharide fraction SPa. Among them, SPa accounts for approximately 47.99% of SP. The constituent monosaccharides of SPa include rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), and galacturonic acid (GalA), the molar ratios of which were 2.1 : 44.6 : 19.7 : 28.2 : 5.3. Its basic structural information is provided in Tables S1 and S2.

Animal experimental design

Thirty-six 8-week-old male C57BL/6 mice were obtained from Xi'an Jiaotong University (Xi'an, China). Following a 1 week acclimatization period under standard conditions (12 hours light/dark cycle, 50% ± 15% humidity, 22 ± 2 °C), the mice were randomly assigned to three groups (n = 12 per group): (1) the RC group received a standard diet; (2) the HFD group was fed a 60% fat diet; and (3) the HFD + SPa group was given an HFD with 0.1% (w/w) SPa (Fig. 1A). The composition of the nutrients of the diet is shown in Table S3. After 17 weeks, fecal samples were collected, and mice were fasted for 12 hours. They were then anesthetized with 2% tribromoethanol and euthanized via cervical dislocation. Serum, brain, and intestinal tissues were harvested and stored at −80 °C for analysis. Animal procedures were approved by the ethics committees of Xi'an Jiaotong University and Northwest A&F University (approval number: SP2022006), adhering to the Guide for the Care and Use of Laboratory Animals (ISBN10: 0-309-15396-4).

Fig. 1 SPa alleviated HFD-induced weight gain and cognitive impairment. (A) Experimental plan. (B) Weight gain of mice over 18 weeks. (C) Percentage of time spent exploring the novel object in the novel object recognition test. (D) Percentage of time spent in the open arms in the elevated plus maze test. (E) Percentage of time spent in the open arms in the elevated plus maze test. (F) Total distance in the open field test. (G) Average speed in the open field test. (H) Trajectories of mice in the novel object recognition, elevated plus maze, and open field tests. Data are presented as mean ± SEM, n = 6–8. *p < 0.05, **p < 0.01 and ***p < 0.001.

Behavioral experiment

The elevated plus maze test, the open field test and the novel object recognition test follow the previous method;¹⁵ please refer to the SI for the specific methods.

Hematoxylin and eosin (H&E) staining

After fixation in 4% paraformaldehyde and embedding in paraffin, brain and colon tissues were sectioned using a paraffin microtome (Leica RM2255, Nuremberg, Germany), deparaffinized with xylene, and stained with hematoxylin and eosin, and mounted with neutral gum for microscopic examination using an optical microscope (Olympus BX51, Tokyo, Japan), and histological scoring was performed to assess tissue damage.¹⁶

Alcian blue staining

The colon tissues were subjected to fixation in 4% paraformaldehyde. After immersion in Alcian acid solution, the tissues were stained with Alcian blue staining solution and then counterstained with nuclear fast red staining solution. Sections were dehydrated using graded ethanol, cleared with xylene, and mounted with neutral gum for microscopic examination of morphological changes using an optical microscope (Olympus BX51, Tokyo, Japan), and semi-quantitative analysis was performed using ImageJ software.

Immunohistochemistry

Colon tissues were prepared as paraffin sections and dewaxed and dehydrated. After antigen retrieval, serum blocking, and incubation with primary antibodies at 4 °C overnight, the sections were washed and incubated with secondary antibodies the next day. Sections were dehydrated using graded ethanol, cleared with xylene, and mounted with neutral gum for microscopic examination of morphological changes using an optical microscope (Olympus BX51, Tokyo, Japan).

Transmission electron microscopy

Half of the brain tissue was immersed in 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, and dehydrated using ethanol. The tissues were then infiltrated and embedded to prepare ultrathin sections (80 nm). After double-staining with uranyl acetate and lead citrate, these sections were examined using an HT7700 transmission electron microscope.

Gut microbiota analysis

Metagenomic sequencing method was performed on mouse fecal samples to determine the gut microbiota, please refer to the SI for the specific methods.

Tryptophan content detection

50 mg of feces or 50 µL of serum was weighed and poured into an EP tube, followed by the addition of 400 µL of extraction solution (acetonitrile : methanol : water = 2 : 2 : 1). The samples were ground using a tissue grinder. Subsequently, the samples were stored in a −20 °C freezer for 1 hour. The supernatant was concentrated via centrifugation, and redispersed in 200 µL of a 10% methanol–water solution. After re-centrifugation, the supernatant was obtained for LC-MS/MS analysis. Please refer to the SI for details on the liquid chromatography and mass spectrometry conditions.

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA extracted from the tissue samples was utilized for first-strand complementary DNA (cDNA) synthesis through reverse transcription. Real-time quantitative PCR was performed and specific primers were designed using NCBI BLAST, as detailed in Table S4. mRNA expression levels were quantified relative to GAPDH.

Western blotting (WB)

Protein samples were prepared from the mouse liver and colon tissues and quantified. Subsequent SDS-PAGE gel electrophoresis was performed, followed by wet transfer. After blocking with 5% skimmed milk, primary antibody solutions were applied dropwise, including AHR (1 : 1000), NF-κB (1 : 1000), p-NF-κB (1 : 1000), and the internal reference β-actin (1 : 2000). Finally, the signals were captured and analyzed by using an imaging system (Champ Chemi 610 Plus, Beijing, China).

Data analysis

The sample size selected was determined based on previously published studies in this field.^17,18 The same batch of animal samples was used for pathological observation, gut microbiota and tryptophan metabolite analysis, and the assessment of relevant gene expression in the gut–brain axis. The exact sample size for each experimental group has been clearly indicated in the legends of all relevant figures. Data are presented as mean ± SEM. Statistical analyses and graph generation were conducted using SPSS 20.0 (IBM Corp., Armonk, NY, USA) and Origin 2021 (OriginLab, USA). The graphical abstract was created with Biorender.com. Metagenomic sequencing data were analyzed using the Wilcoxon rank-sum test or Kruskal–Wallis test. For animal experiments, the one-way ANOVA test and Tukey's multiple comparison analysis were employed to determine the differences among the groups, with significance levels indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

Results

SPa alleviated HFD-induced weight gain and cognitive impairment

Initially, we conducted an 18 week feeding experiment in mice (Fig. 1A). During the 18 week study, mice fed an HFD gained significantly more weight than those on a standard diet (RC) (p < 0.001). This excessive weight gain was notably diminished following SPa treatment (p < 0.001), indicating that SPa supplementation in the diet could inhibit HFD-induced weight gain (Fig. 1B). Food intake did not result in a marked variation among all the groups, indicating that SPa had no impact on the appetite of the mice. Compared with the RC group, the HFD and HFD + SPa groups exhibited greater energy intake, but no pronounced difference was noted between the two groups (Table S5).

Subsequently, the impact of SPa supplementation on working and spatial memory in mice was assessed via the novel object recognition test (Fig. 1C and H). The HFD group exhibited a significantly reduced frequency of exploring the novel object relative to the RC and HFD + SPa groups. This suggests that the HFD induced memory impairment, which was notably ameliorated by SPa supplementation (p < 0.01), suggesting that SPa positively affects working and spatial memory in mice.¹⁹ Additionally, we assessed the effects of SPa on anxiety-like behavior and exploratory behavior in mice using the elevated plus maze and open field tests (Fig. 1D–H). The frequency of exploring open arms in the elevated plus maze test and the average speed in the open field test were significantly reduced in the HFD group versus the RC group (p < 0.05). These parameters were significantly enhanced following SPa intervention, suggesting that SPa can effectively alleviate HFD-induced anxiety-like and exploratory behavioral changes in mice.¹⁵

SPa restored HFD-induced intestinal inflammation and intestinal barrier damage

To assess the mitigating effects of SPa on intestinal inflammation and barrier disruption, histopathological and biochemical analyses were performed on the mouse colon tissues. The H&E and Alcian blue staining images (Fig. 2A) revealed that the HFD caused distortion of colonic crypts and depletion of goblet cells in mice, with inflammatory infiltration in the lamina propria. However, dietary supplementation with SPa significantly improved colonic damage. Histological scoring of the HE-stained images and quantitative analysis of the mucin percentage in the Alcian blue-stained sections consistently revealed congruent results (Fig. 2B and C). Research has shown that IFN-γ is a key factor in inflammatory diseases, as it induces the synthesis of iNOS to produce NO, thereby exacerbating conditions such as infections, inflammation, and tumors. In addition, COX-2 is rapidly induced at sites of inflammation, forming prostaglandins that intensify pain and inflammation.²⁰ Our RT-qPCR results indicated that the mRNA levels of Cox-2, Inos, and Ifn-γ were significantly elevated in mice fed the HFD (p < 0.01), which were markedly ameliorated by dietary SPa supplementation (Fig. 2D–F). These findings suggest that SPa in the diet helps to improve HFD-induced intestinal inflammation in mice. Additionally, the HFD diminished the expression of tight junction proteins (Claudin-1, Occludin, and ZO-1) linked to intestinal barrier function,²¹ while SPa supplementation significantly increased the expression of those (p < 0.001) (Fig. 2G–J). These results indicate that SPa in the diet helps to reverse HFD-induced intestinal barrier damage in mice.

Fig. 2 SPa restored HFD-induced intestinal inflammation and intestinal barrier damage. (A) Representative images of H&E and Alcian blue staining of the mouse colon tissues (×200). (B) Histological damage score, (C) the mucin area ratio, and the relative mRNA expression of (D) Cox-2, (E) Ifn-γ, and (F) Inos in the mouse colon. (G) Representative immunohistochemical images of Claudin-1, Occludin, and ZO-1 proteins in the mouse colon (×200). Relative mRNA expression of (H) Claudin-1, (I) Occludin, and (J) Zo-1 in the mouse colon. Data are presented as mean ± SEM, n = 6–8. *p < 0.05, **p < 0.01 and ***p < 0.001.

SPa ameliorated HFD-induced dysbiosis of tryptophan-metabolizing bacteria

We subsequently examined how the HFD and SPa influenced the overall structure of the gut microbiota through metagenomic sequencing of the fecal samples from mice. Initially, the unweighted pair group method with arithmetic mean (UPGMA) findings highlighted significant variations among the three groups (Fig. 3A). Moreover, the observed index in the HFD group was markedly reduced compared with the RC group (p < 0.001), indicating a reduction in microbial community abundance. However, SPa supplementation greatly restored the loss of microbial abundance (Fig. 3B). Furthermore, we evaluated the relative abundance of fecal microbiota in the three groups at different taxonomic levels (Fig. 3C and D). Fecal microbiota analysis showed that the relative abundance of Bacteroidota, Verrucomicrobiota, Spirochaetota, and Deferribacterota was reduced in HFD-fed mice compared to the RC group, while the relative abundance of Actinomycetota, Uroviricota, Bacillota, and Chlamydiota was increased. Dietary SPa supplementation corrected these changes in microbial community structure. In addition, the HFD-fed mice exhibited markedly reduced relative abundance of Pseudoflavonifractor, Muribaculum, Duncaniella, and Oscillibacter compared with the RC and HFD + SPa groups at the genus level (p < 0.05). Conversely, the relative abundance of Eubacterium and Helicobacter was markedly higher (p < 0.05) (Fig. 3E). The LDA bar chart showed the significantly different species with an LDA score greater than 3.0, that is, the biological categories with statistical differences. The analysis results indicated that in the RC group, the relative abundance of Desulfovibrionaceae_bacterium_LT0009, Helicobacter_ganmani, Heminiphilus_faecis, Muribaculum_intestinale, and Paramuribaculum_intestinale was significantly increased. In the HFD group, Lachnospiraceae_bactenum_10_1, Granulimonas_faecalis, Faecalibaculum_rodentium, Acetatifactor_muris, Coriobacteriaceae_bacterium, and Acetatifactor_aquisqranensis have higher relative abundance. SPa intervention significantly increased the relative abundance of Lachnospiraceae_bacterium, Bacterium_1XD42_87, Bacterium_0_1XD8_71, Phocaeicola_vulgatus, Roseburia_zhanii, Firmicutes_bacterium_ASF500, and Petralouisia muris (Fig. 3F). In addition, to identify the microbial genes and associated functional pathways present in the feces of mice, we used metagenomics to generate metabolic functional profiles. As shown in Fig. 3G, we counted the number of unigenes annotated by KEGG and found that the number of unigenes for phenylalanine, tyrosine and tryptophan biosynthesis was 5614, while the numbers for tyrosine metabolism, tryptophan metabolism, and phenylalanine metabolism were 1806, 1165, and 2717, respectively. Moreover, we identified that in the KEGG database, the abundance of tryptophan synthase, aromatic amino acid lyase, dihydrolipoamide dehydrogenase, aldehyde dehydrogenase (NAD+), and tryptophanase microbial communities related to tryptophan metabolism was reduced by the HFD, but significantly improved by SPa (Fig. 3H–L and Table S6).

Fig. 3 SPa ameliorated HFD-induced dysbiosis of tryptophan-metabolizing bacteria. (A) Unweighted pair group method with arithmetic mean (UPGMA) of β-diversity. (B) Observed index representing the α-diversity of the gut microbiota. (C) Relative abundance of microbial phyla. (D) Relative abundance of microbial families. (E) Relative abundance of microbial genera. (F) LEfSe analysis of differential bacteria species. (G) Number of unigenes annotated by KEGG pathways. Average abundance of microbial communities related to (H) tryptophan synthase (trpB), (I) aromatic amino acid lyase (hutH and HAL), (J) dihydrolipoamide dehydrogenase (DLD, lpd and pdhD), (K) aldehyde dehydrogenase (NAD+) (ALDH), and (L) tryptophanase (tnaA) in the KEGG database. (M and N) Stamp analysis of differential strains involved in tryptophan metabolism. Data are presented as mean ± SEM, n = 3–5. *p < 0.05, **p < 0.01 and ***p < 0.001.

Finally, we analyzed the relative abundance of bacteria in the gut that have been reported to metabolize tryptophan. The relative abundance of Peptostreptococcus_anaerobius, Bacteroides_thetaiotaomicron, Bacteroides_ovatus, and Bacteroides_fragilis in the HFD group was significantly lower than that in the RC group. However, the relative abundance of Parabacteroides_distasonis, Bacteroides_fragilis, Bacteroides_thetaiotaomicron, Bacteroides_ovatus, Peptostreptococcus_anaerobius, and Clostridium_perfringens in the HFD + SPa group was significantly higher than that in the HFD group (Fig. 3M and N). The above results indicate that SPa can ameliorate HFD-induced gut microbiota dysbiosis, and the microbial communities associated with tryptophan metabolism pathways underwent significant changes following HFD and SPa interventions.

SPa improved HFD-induced tryptophan metabolism disorder

Tryptophan is a key amino acid in maintaining gut homeostasis and primarily affects the host's metabolism through three metabolic pathways: the indole, the kynurenine, and the serotonin pathways.²² As shown in Fig. 4A–E, in contrast to the trend of KYN, the levels of L-Trp, 5-HT, 5-HTP, and indole derivatives (indole, IPA, IAA, ILA and IA) were strikingly decreased in the HFD group (p < 0.05). However, these levels were restored following SPa intervention. Notably, IDO serves as the key rate-limiting enzyme in the kynurenine pathway, while the key enzyme in the serotonin pathway is TPH1.²³ Therefore, we measured the relative mRNA expression levels of TPH1 and IDO1. As shown in Fig. 4G and H, compared with the RC group, the mRNA expression of Tph1 was significantly downregulated in the HFD group, while the mRNA expression of Ido1 was markedly elevated (p < 0.01). Interestingly, the expression of these two key enzymes was significantly reversed following SPa intervention (p < 0.01). Additionally, in the indole pathway, tryptophan is metabolized into indole and AHR ligands (IPA, IAA and IA), which activate AHR to promote IL-22 production in ILCs and induce the expression of downstream AHR-related genes and a series of biological effects, the most common of which are CYP1A1 and CYP1A2.^23,24 In our study, feeding an HFD significantly reduced the protein expression levels of AHR in mice (p < 0.05), which was significantly improved following SPa intervention (p < 0.05) (Fig. 4I and J). Likewise, relative to the RC group, the HFD group displayed significantly lower relative mRNA expression levels of Il-22, Cyp1a1, and Cyp1a2 (p < 0.001), which were markedly increased following SPa (p < 0.001) (Fig. 4H, K and L). The above results indicate that SPa can regulate HFD-induced tryptophan metabolism disruption, and promote IL-22 production by activating AHR, thereby enhancing epithelial integrity and gut homeostasis.

Fig. 4 SPa improved HFD-induced tryptophan metabolism disorder. Concentration of (A) L-Trp, (B) KYN, (C) 5-HT, (D) 5-HTP, and (E) indole derivatives in the mouse feces. Relative mRNA expression of (F) Tph1, (G) Ido1, and (H) Il-22 in the mouse colon. (I) Representative western blotting analysis of AHR and β-actin in the mouse colon. (J) Relative protein expression of AHR in the mouse colon. Relative mRNA expression of (K) Cyp1a1 and (L) Cyp1a2 in the mouse colon. IPA: indole-3-propionic acid, IAA: indole-3-acetic acid, ILA: indole-3-lactic acid, IA: 3-indoleacrylic acid; data are presented as mean ± SEM, n = 3–8. *p < 0.05, **p < 0.01 and ***p < 0.001.

SPa restored HFD-induced neuroinflammation by regulating intestinal tryptophan metabolism

To evaluate whether tryptophan metabolism in the gut affects the distribution of tryptophan in the brain via the blood–brain barrier, we measured the levels of tryptophan in the serum and brain. As depicted in Fig. 5A–J, in line with the alterations in gut tryptophan levels, the concentrations of L-Trp, 5-HT, 5-HTP, and indole derivatives (indole, IPA, IAA, ILA and IA) in the serum and brain of mice in the HFD group were significantly lower than those in the RC group (p < 0.01). Conversely, the level of KYN was markedly elevated (p < 0.01). However, these changes were significantly reversed by SPa intervention. Similarly, we measured the changes in the key enzymes of the kynurenine pathway and serotonin pathway in the brain, IDO1 and TPH2, and found that SPa notably enhanced the reduced relative mRNA expression of Tph2 and attenuated the elevated relative mRNA expression of Ido1 in the HFD group (p < 0.001; Fig. 5K and L). Additionally, the relative protein expression levels of p-NFκB/NF-κB were elevated, whereas the relative protein expression level of AHR was reduced in the HFD group. These changes were markedly ameliorated by SPa intervention (p < 0.05; Fig. 5M and O). Moreover, the HFD-group mice exhibited significant upregulation of Nlrp3, Caspase-1, Il-1β, and Il-18 mRNA levels in the brain compared with the RC group (p < 0.01). In contrast, the HFD + SPa group revealed levels analogous to the RC group (Fig. 5P–S). Therefore, we conclude that SPa modulates HFD-induced neuroinflammation by regulating intestinal tryptophan metabolism.

Fig. 5 SPa restored HFD-induced neuroinflammation by regulating intestinal tryptophan metabolism. (A) L-Trp, (B) KYN, (C) indole derivatives, (D) 5-HTP, and (I) 5-HT content in the mouse serum. (E) L-Trp, (F) KYN, (G) indole derivatives, (H) 5-HTP, and (J) 5-HT content in the mouse brain. Relative mRNA expression of (K) Tph2 and (L) Ido1 in the mouse brain. (M) Representative western blotting analysis of NF-κB, p-NFκB, AHR, and β-actin in the mouse brain. Relative protein expression of (N) p-NFκB/NF-κB and (O) AHR in the mouse brain. Relative mRNA expression of (P) Nlrp3, (Q) Caspase-1, (R) Il-1β, and (S) Il-18 in the mouse brain. IPA: indole-3-propionic acid, IAA: indole-3-acetic acid, ILA: indole-3-lactic acid, IA: 3-indoleacrylic acid; data are presented as mean ± SEM, n = 3–8. *p < 0.05, **p < 0.01 and ***p < 0.001.

SPa ameliorated HFD-induced brain histopathological changes and neuronal damage

We then assessed the histopathological alterations of mice via H&E staining. As depicted in Fig. 6A, the hippocampus of mice in the RC group displayed tightly packed, conical-shaped neurons with distinct cytoplasm and nuclei, and no significant abnormalities were detected. In contrast, HFD-fed mice displayed marked neuronal degeneration and pronounced nuclear pyknosis in the cerebral cortex, with loosely arranged neurons and evident neuronal loss in the CA1 and DG regions of the hippocampus. Relative to HFD-fed mice, those in the HFD + SPa group exhibited significantly improved histopathological features, indicating that dietary SPa supplementation reduced the extent of degenerating neurons. Similarly, SPa markedly attenuated the elevated mRNA levels of Tnf-α, Il-6, and Ifn-γ in the brains of HFD-fed mice (p < 0.001; Fig. 6B, C and E). These findings substantiate that SPa can effectively mitigate histopathological alterations and inflammatory responses in the brains of mice fed an HFD.

Fig. 6 SPa ameliorated HFD-induced brain histopathological changes and neuronal damage. (A) Representative micrographs of the cerebral cortex, hippocampal CA1, and DG regions in the mice (H&E staining, ×200). Relative mRNA expression of (B) Tnf-α and (C) Il-6 in the mouse brain. (D) Ultrastructure of the hippocampal synapses. Relative mRNA expression of (E) Ifn-γ, (F) Psd-95, (G) Bdnf, and (H) Trkb in the mouse brain. Data are presented as mean ± SEM, n = 6–8. *p < 0.05, **p < 0.01 and ***p < 0.001.

Additionally, the maintenance of synaptic integrity is vital for the transmission of signaling molecules between neurons.³ We observed the morphology of the postsynaptic density structure using transmission electron microscopy. The HFD led to a remarkable reduction in the postsynaptic density area in the hippocampus of mice (a 63.2% reduction compared with the RC mice). However, SPa intervention significantly improved the postsynaptic density area (a 56.4% increase compared with the HFD mice) (Fig. 6D). In addition, we found that SPa supplementation significantly improved the decreased relative mRNA levels of Psd-95, Bdnf, and Trkb in the HFD-fed mice (p < 0.01; Fig. 6F–H). The aforementioned findings imply that SPa could potentially alleviate HFD-induced synaptic dysfunction and neuronal damage.

Correlation analysis between the brain tryptophan metabolite levels and other biochemical indicators

To elucidate the correlations among the brain tryptophan metabolites and gut microbiota, neuroinflammation, and neuronal damage in the experimental mice, we performed Pearson's correlation analysis on the aforementioned data. As shown in Fig. 7, the levels of 5-HTP, 5-HT, and indole derivatives (indole, IPA, IAA, ILA and IA) in the brain exhibited significant positive correlations with Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bifidobacterium pseudolongum, Parabacteroides distasonis, and Peptostreptococcus anaerobius, while showing significant negative correlations with Clostridium perfringens and Lachnospiraceae bacterium. Conversely, KYN showed significant positive correlations with Clostridium perfringens and Lachnospiraceae bacterium, but significant negative correlations with Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bifidobacterium pseudolongum, Parabacteroides distasonis, and Peptostreptococcus anaerobius. Furthermore, the levels of 5-HTP, 5-HT, and indole derivatives in the brain were significantly positively correlated with the gene expression of TPH2, AHR, PSD-95, BDNF, and TrkB, while showing significant negative correlations with the gene/protein expression of IDO1, p-NF-κB/NF-κB, NLRP3, Caspase-1, IL-1β, and IL-18. In contrast, KYN displayed an opposite correlation pattern. In summary, these results indicate that the ameliorative effects of SPa on HFD-induced neuroinflammation and neuronal damage are closely associated with the composition of the gut microbiota, the levels of tryptophan metabolites in the brain, the NLRP3 inflammasome, and synaptic integrity.

Fig. 7 Correlation analysis of brain tryptophan metabolites with gut microbiota and various biochemical indicators. The color of the boxes indicates the level of the relevant index. *p < 0.05 and **p < 0.01.

Discussion

In this study, the results showed that SPa intervention significantly alleviated HFD-induced weight gain, cognitive impairment, intestinal inflammation, gut barrier damage, and microbial metabolic disorders in mice. Furthermore, SPa markedly restored HFD-induced gut microbiota dysbiosis, reversed the decreased levels of L-Trp, 5-HTP, 5-HT, and indole derivatives in the fecal, serum, and brain tissues, and suppressed the elevated KYN levels. Additionally, SPa significantly ameliorated cerebral neuroinflammation and synaptic ultrastructural integrity. Collectively, these results reveal that SPa exerts definite protective effects against HFD-induced neuronal damage through regulation of the gut–brain axis function.

SPa, as the main component of SP, was found in our previous studies to have a M_w, M_n, and PDI of 9944 Da, 8251 Da, and 1.21, respectively. SPa is composed of Rha, Ara, Gal, Glc, and GalA in a molar ratio of 2.1 : 44.6 : 19.7 : 28.2 : 5.3.¹⁴ Obesity is an inflammatory state that can lead to the development of neuroinflammation, which promotes the production of peripherally derived pro-inflammatory cytokines that cross the blood–brain barrier and may ultimately lead to neuronal damage in the brain.⁴ Long-term consumption of a high-fat diet (HFD) is a known factor promoting cerebral inflammation and neuronal injury. Dietary supplementation with 0.1% (w/w) SPa has been shown to significantly ameliorate HFD-induced obesity, disruption of intestinal immune homeostasis, and hepatic inflammation.^14,25 However, the role and underlying mechanisms of SPa in preventing and mitigating HFD-induced neuronal damage in mice remain incompletely elucidated.

In our study, HFD-fed mice exhibited significant cognitive impairment in behavioral tests (Fig. 1C–H), accompanied by accumulation of inflammatory factors in the brain, leading to neuronal damage and compromised synaptic integrity (Fig. 6C–F). These pathological manifestations were markedly ameliorated by SPa intervention. These findings are consistent with previous studies showing that polysaccharides (Eucommiae cortex polysaccharides and Lycium barbarum polysaccharides) improve cognitive impairment in HFD-fed mice.^26,27 AHR is a ligand-activated transcription factor expressed in various cell types, including immune cells, microglia, and intestinal epithelial cells.²⁸ Notably, studies have shown that AHR, the ligand of an indole derivative, negatively regulates the activity of the NLRP3 inflammasome by inhibiting the activation of NF-κB. NLRP3 induces the activation of Caspase-1, converting inactive pro-IL-1β and pro-IL-18 into bioactive IL-1β and IL-18, thereby triggering neuroinflammation.^11,29,30 These mechanistic insights are consistent with our experimental findings. In this study, SPa intervention significantly alleviated the HFD-induced decrease in AHR expression levels and the increase in NF-κB, NLRP3, Caspase-1, and inflammatory factor (IL-1β and IL-18) expression levels (Fig. 5M–S). Additionally, we found that SPa significantly alleviated the reduced levels of BDNF and its downstream protein TrkB in the brains of HFD-fed mice and activated the major synaptic protein PSD-95.³¹

Tryptophan, an indispensable amino acid for protein synthesis, is a key player in the microbiota–gut–brain axis. Tryptophan metabolites generated by the gut microbiota are capable of crossing the blood–brain barrier and regulating neuronal activity in the brain.⁷ Detection of L-Trp and its metabolite levels in the mouse serum and brain tissues showed that SPa significantly ameliorated the HFD-induced decreases in 5-HTP, 5-HT, and indole derivatives, as well as the increase in KYN (Fig. 5E–J). Tryptophan metabolism is governed by two key enzymes: TPH and IDO. Under duress, the conversion of Trp to 5-HT and KYN becomes dysregulated. The excessive conversion of Trp to KYN is closely related to the systemic inflammation response and the overexpression of IDO1, consequently reduces TPH2 expression and compromises 5-HT synthesis.^8,23 SPa intervention normalized the elevated levels of IDO1, the rate-limiting enzyme in the KYN pathway, and restored the diminished levels of TPH2, the key enzyme in the 5-HT pathway, in mouse brain tissues (Fig. 5K and L). Moreover, the dysregulation of intestinal tryptophan metabolites plays a central role in the pathogenesis of many neurological and psychiatric disorders, with the gut microbiota directly or indirectly affecting tryptophan metabolism and the corresponding behavioral and cognitive changes.⁹ Zhang et al. found that Morinda officinalis oligosaccharides can favorably modulate the tryptophan-5-HTP-5-HT metabolic pathway via the gut microbiota.³² Since peripheral 5-HTP can cross the blood–brain barrier and rapidly metabolize into 5-HT, it increases brain 5-HT levels. This elevated 5-HT counteracts neuronal damage, thereby promoting neuroprotective effects.³² Our analysis of Trp and its metabolites in mouse fecal samples showed that the HFD induced marked disruption of intestinal tryptophan metabolism (Fig. 4A–E). These findings, consistent with the parallel trends observed in the tryptophan metabolite levels across the fecal, serum, and brain samples, provide further support for the conclusion that gut-derived tryptophan metabolites cross the blood–brain barrier to regulate neural activities in the brain. Furthermore, the integrity of the intestinal barrier effectively prevents systemic infections and inflammatory responses and maintains the body's internal homeostasis.³³ Indole derivatives (IA, IAA and IPA) can act as AHR ligands to activate AHR. AHR not only regulates downstream signals CYP1A1 and CYP1A2 but also promotes IL-22 expression, thereby improving the integrity of the intestinal barrier.²³ Moreover, it was observed that SPa restored the diminished levels of AHR, CYP1A1, CYP1A2, and IL-22 in the colon of HFD-fed mice. These findings are consistent with the conclusions of our study (Fig. 2 and 4H–L). In the context of obesity, elevated fecal KYN levels are associated with adverse metabolic profiles and intestinal inflammation, as increased IDO1 activity shifts tryptophan metabolism away from indole derivative production toward KYN generation.⁸ In our study, the HFD increased both KYN content and expression of its key enzyme IDO1 in the mouse intestine, along with significantly elevated levels of inflammatory factors (COX-2, iNOS and IFN-γ). However, SPa intervention substantially ameliorated these alterations (Fig. 2B–D and 4F and G). Our findings show that SPa effectively mitigates HFD-induced intestinal barrier damage and gut inflammation, with Trp and its metabolites playing crucial roles in this protective mechanism.

Studies have shown that HFD-induced cognitive decline may correlate with alterations in the abundance, diversity, and structure of the gut microbiota. Thus, alterations in this microbial community have been identified as crucial factors in neuroinflammation and the resulting cognitive impairments.⁵ Plant-derived polysaccharides can modulate tryptophan metabolism by reshaping the composition of the gut microbiota.^26,27 Our results showed that, at the genus level, SPa ameliorated the HFD-induced reduction in the relative abundance of Pseudoflavonifractor, Muribaculum, Duncaniella, and Oscillibacter in the mouse feces, all of which are involved in tryptophan metabolism.³⁴ At the species level, compared with RC mice, HFD-fed mice exhibited significantly decreased abundance of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus, Bifidobacterium pseudolongum, Parabacteroides distasonis, and Peptostreptococcus anaerobius, along with markedly increased levels of Clostridium perfringens and Lachnospiraceae bacterium. These findings indicated that SPa significantly alleviated HFD-induced gut microbiota dysbiosis (Fig. 5). Furthermore, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ovatus, and Parabacteroides distasonis are capable of metabolizing Trp to produce indole derivatives (IAA, IPA and ILA).^35–40Bacteroides thetaiotaomicron inhibits indole production in Escherichia coli through monosaccharide metabolites, thereby making more tryptophan available for utilization by Peptostreptococcus anaerobius, consequently enhancing ILA and IPA generation.⁶ Reportedly, Bifidobacterium pseudolongum metabolizes Trp to produce IAA and protects synapses from microglial phagocytosis in an AHR-dependent manner, thereby improving cognition.⁴¹ Conversely, Lachnospiraceae bacterium produces long-chain fatty acids and exacerbates diet-induced obesity by impairing intestinal integrity.⁴² Similarly, tryptophan synthase is involved in the biosynthesis pathway of tryptophan metabolism, aldehyde dehydrogenase (NAD+) oxidizes indole-3-aldehyde (IAAld) to IAA, and tryptophanase catalyzes the decomposition of L-tryptophan into indole.⁴³ In our study, the abundance of these enzyme-related bacterial communities was reduced by the HFD, while improved by SPa intervention. Furthermore, our results revealed that the reduced abundance of microbial communities associated with tryptophan synthase and aromatic amino acid lyase, both critical enzymes functioning at distinct stages of tryptophan synthesis and degradation, was restored following SPa intervention in the HFD group, as identified in the KEGG database (Fig. 3H–L).⁴³

In summary, the findings of this study indicate that SPa potentially ameliorates HFD-induced neuroinflammation, neuronal damage, and synaptic dysfunction by modulating the gut microbiota and tryptophan metabolism, thereby regulating the AHR–NF-κB–NLRP3/Caspase-1–IL-1β/IL-18 and BDNF/TrkB signaling pathways. These results suggest that SPa alleviates HFD-induced cerebral inflammation and neuronal injury via the microbiota–tryptophan–brain axis. However, further validation through fecal microbiota transplantation (FMT) experiments and studies employing AHR antagonists is required. Additionally, this study has certain limitations: whether the effects of SPa are dose-dependent remains to be established, and although SPa may improve neuronal damage by altering 5-HT and its key metabolic enzyme TPH2, as well as KYN and its key metabolic enzyme IDO1, the precise underlying mechanisms warrant further investigation.

Author contributions

Jing Peng and Lili Chang: drafted the manuscript, performed the experiments, and analyzed the data; Mingyou Yuan, Chendi Wang, Zishuo Zhao, Huizhen Yan, Xiangyu Han and Xuyang Qin: investigated and performed the experiments, and analyzed the data; Meng Zhang and Xiulian Li: method guidance; and Ying Lan: review. All authors read and approved the final manuscript.

Conflicts of interest

The authors declare no conflict of interest.

Abbreviations

SP	Sea buckthorn polysaccharides
SPa	The main component of sea buckthorn polysaccharides (SP)
RT-qPCR	Real-time quantitative polymerase chain reaction
IL	Interleukin
TNF-α	Tumor necrosis factor-α
KEGG	Kyoto Encyclopedia of Genes and Genomes
NF-κB	Nuclear factor-κB
HFD	High-fat diet
AHR	Aryl hydrocarbon receptor
Trp	Tryptophan
IPA	Indole-3-propionic acid
IAA	Indole-3-acetic acid
ILA	Indole-3-lactic acid
IA	3-Indoleacrylic acid
DCs	Dendritic cells
ILC3	Group 3 innate lymphocytes
5-HT	5-Hydroxytryptamine
KYN	Kynurenine
BDNF	Brain-derived neurotrophic factor
PSD-95	Postsynaptic density protein-95
NLRP3	NOD-, LRR- and pyrin domain-containing protein 3
Caspase-1	Cysteinyl aspartate specific proteinase 1
TrkB	Tropomyosin-related kinase B
IFN-γ	Interferon gamma
CYP1A1/CYP1A2	Cytochrome P450 1A1/1A2
LC-MS/MS	Liquid chromatography-tandem mass spectrometry
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase

Data availability

The data supporting this article are available from the corresponding author upon reasonable request. Supplementary information (SI) is available and includes: Basic structural information of SPa, Composition of nutrients of standard diet. Elevated Plus Maze, Open Field Test, Novel Object Recognition, Gut Microbiota Analysis, Tryptophan Content Detection, Primer Sequence Table. Daily food and energy intake of mice, and Metabolic microbiota of tryptophan metabolism related proteins. See DOI: https://doi.org/10.1039/d5fo04010h.

Acknowledgements

This work was financially supported by the Natural Science Foundation of China (32202041), the National Undergraduate Training Program for Innovation and Entrepreneurship (XN2025011073), and the Special Financial Grant from the China Postdoctoral Science Foundation (2023T160533).

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Footnote

† These authors contributed equally to this work.

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