Resveratrol treatment alleviates motor symptoms and cognitive decline on A53T-PD mouse model and Hydroxypropyl-β-cyclodextrin encapsulation increases its efficacy
It is well known that PD is characterized by dyskinesia, autonomic dysfunction and cognitive impairment[24], and motor disorders and cognitive decline commonly occur in theA53T-PD mouse models at 9–16 months of age[25]. To evaluate the protective effects of resveratrol administration on PD, the A53T mice and WT littermate control were treated with low dosages of RES (30 mg/kg, L-RES), high dosages of RES (90 mg/kg, H-RES), and RHSD (30 mg/kg), followed by a panel of behavioral tests and cognitive function tests. As illustrated in Fig. 1A, three behavioral tests were performed to assess the motor functions of mice from different groups, including the Rota-Rod test for the motor coordination of mice from different groups, the grip strength test for muscle force of limbs, and the pole test for motor balance. In addition, the Morris water maze test and open field test were performed to assess the cognitive function (Fig. 1A). As shown in Fig. 1C-E, the A53Tmice were showed significant motor deficits compared to the WT mice, including decreased scores of the pole test (Fig. 1C), reduced limb muscle strength (Fig. 1D), and shorter duration on the rod (Fig. 1E). In the Morris water maze test (Fig. 1F, G), longer latency (Fig. 1I), slower swimming (Fig. 1J), and less frequent visits to the target quadrant (Fig. 1K) of A53T mice compared with WT mice. In the open field test (Fig. 1H), the groups of mice exhibited comparable levels of locomotor activity (total distance traveled) (Fig. 1L). On the contrary, resveratrol treatment alleviated motor and cognitive deficits in the A53T-PD mouse model in a dose-dependent manner, and the efficacy of RHSD (30 mg/kg) was more significant than same dose RES, indicating that RHSD could induce a remarkable recovery from the motor ability and the spatial memory disorder of PD mice. However, no differences were observed in WT mice treated with or without RHSD. Furthermore, there was no significant difference in body weight between A53T-PD mice and wild type mouse, resveratrol treatment also unremarkably changed the weight of the mice (Fig. 1B).
Resveratrol treatment alleviates gastrointestinal dysfunctions and rebuilding the intestinal barrier of A53T-PD mice model
Recent research has linked central neuropathology in Parkinson’s disease to increased barrier permeability of the colon[26]. Given the common occurrence of dyssomnia, gastrointestinal symptoms, and intestinal barrier deterioration in PD patients, our study aimed to measure the impact of scent perception, gastrointestinal functions, and intestinal barrier on PD pathology. In our study, we found that PD patients took longer to find food compared to the wildtype (WT) group (Fig. 2A). However, this was significantly shortened by administration of L-RES, H-RES, and RHSD (Fig. 2A). Furthermore, we collected fecal pellets to measure the water content percentage and colon motility. A53T mice presented with markedly reduced fecal water content percentages (Fig. 2B) and pellet numbers (Fig. 2C), which were significantly elevated by RES administration in a dose-dependent manner. Notably, RHSD was more effective than L-RES (Fig. 2B, C). Additionally, the study revealed significant differences in fecal output frequencies between PD group mice and WT group mice (indicated by a 20-min fecal collection experiment) (Fig. 2D). Nevertheless, treatment with H-RES and RHSD significantly increased the fecal output at each observed time point. However, the effect of L-RES was observed solely in the last time point.
In addition to testing the functions and structures of intestinal barriers, an in vivo intestinal permeability assay was performed to measure the concentration of FITC-dextran in the serum. Higher concentration of FITC-dextran indicates heightened gut permeability. The PD group had increased concentration of FITC-dextran compared to the WT group, while L-RES, H-RES, and RHSD administration significantly reduced serum FD4 concentrations (Fig. 2E). TEM analysis was also conducted to detect changes in the structures of intestinal barriers. Results showed impaired epithelium with sparse microvilli, and decreased and discontinuous electron-dense material with wider paracellular gaps in the colon slides of A53T mice. After RHSD treatment, the epithelium was present with regularly organized microvilli and intact tight junctions (Fig. 2F). Immunofluorescence staining was used to measure tight junction protein expression in the colon. Results showed disrupted and decreased expression of ZO-1 and Occludin in the PD group. RHSD administration preserved ZO-1 and Occludin expression and integrity (Fig. 2G, H). To test whether the restoration of intestinal barriers could lead to decreased LPS levels, serum LPS levels were measured using ELISA. Results showed that LPS levels in the serum of the A53T group were elevated to more than three times those of the WT group. Conversely, L-RES, H-RES, and RHSD administration significantly reduced serum LPS levels (Fig. 2I). Consistently, the level of LPS in the feces of A53T mice was significantly elevated, while RHSD treatment reduced the abnormal LPS levels (Fig. 2J). These data suggest that RES preserves the intestinal barrier impairment in the A53T transgenic mouse model.
Abnormal NO pathways are associated with Parkinson's disease[27]. Given recent evidence that elevated NO mediates axonal degeneration and activates cyclooxygenases to provoke neuroinflammation. NO also instigates a down-regulated secretion of brain-derived neurotrophic factor (BDNF), which is essential for neuronal survival, development and differentiation, synaptogenesis, and learning and memory[28].The abnormal serum concentration of NO in the A53T transgenic mouse model was detected by ELISA analysis. As shown in Fig. 2K, the serum concentration of NO in the A53T group was significantly higher than those in the WT group (Fig. 2K). In contrast, resveratrol treatment reduced serum NO levels in the A53T mouse model in a dose-dependent manner, and the efficacy of RHSD was more significant than same dose RES, indicating that resveratrol could significantly restore the serum NO level of A53T mice and H-β-CD encapsulation increases its efficacy (Fig. 2K).
Resveratrol treatment reduced the accumulation of α-Syn and restored homeostasis in the colon of A53T-PD mice model
In recent years, there has been increasing recognition of a low-grade inflammatory state in the gut of Parkinson's disease (PD) patients. There is substantial evidence that prodromal lesions appear in the bowel in a significant fraction of PD patients up to 20 years before the onset of central nervous system (CNS) manifestations[29]. In this study, we used H&E staining to evaluate immune infiltration and epithelium impairment in the colon. The H&E histological scores were remarkably higher in the A53T mice than in the WT group, while treatment with high-dose resveratrol (H-RES) and resveratrol hyaluronic acid sodium diester salt (RHSD) reduced the scores significantly (Fig. 3A, D). These findings suggest that H-RES and RHSD treatment significantly attenuate PD-related histological characteristics and inflammation in the colon of A53T transgenic mice. It has been debated whether the submucosal detection of α-Syn+ Lewy neurites in colon biopsy samples is specific for PD[30–32]. Human α-Syn overexpressing transgenic mice exhibit insoluble α-Syn aggregates within enteric neurons, which precedes onset of subcortical changes[33]. To determine the inhibiting effects of resveratrol administration on α-Syn aggregation in A53T transgenic mice, the expression of α-Syn and Ser129-phosphorylated α-synuclein (S-129 α-Syn) in the colonic mucosa and muscular layers were measured by immunohistochemistry staining. Results showed increased expression of α-Syn (Fig. 3B, E) and S-129 α-Syn (Fig. 3C, F) in the PD group, whereas RHSD administration significantly decreased the expression of α-Syn and phosphorylated α-Syn. It has been demonstrated that mice have a "monocyte waterfall" from circulation to the intestine to maintain the macrophage pool in the gut in a CCR2-dependent manner[34]. These monocytes terminally become mature resident Ly6Clow/− CX3CR1hi MHC-IIhi macrophages that express IL-10 and maintain intestinal homeostasis in the resting intestine [34, 35]. Additionally, compelling evidence has shown that enteric glia are important homeostatic cells of the intestine. Bidirectional communication between enteric glia and immune cells contributes to gastrointestinal immune homeostasis [36]. However, it was not reported whether intestinal macrophages and enteric glia are disordered by insoluble α-Syn aggregates. Therefore, we used GFAP and CX3CR1 to label enteric glia and intestinal resident macrophages respectively. Global scanning imaging was performed by immunofluorescence detection to observe the co-localization and expression levels of α-Syn with the glial cells and macrophages. As shown in Fig. 3G-J, the large accumulation of α-Syn in the colon of A53T mice was significantly inhibited by RHSD treatment (Fig. 3G, H). The number of GFAP+ cells had no significant difference between the groups (Fig. 3G, J). The number of CX3CR1+ cells was remarkably higher in the A53T mice group than in the WT group, while RHSD administration reduced the number of CX3CR1+ cells significantly (Fig. 3G, I). In addition, we also observed that α-Syn co-localized with GFAP in the colon mucosa and co-localized with CX3CR1 in the submucosa (Fig. 3G). These findings suggest that RHSD restored intestinal homeostasis disrupted by the accumulation of insoluble α-Syn in A53T transgenic mice.
RHSD administration attenuates PD-associated histological features in the substantia nigra and striatum of the A53T transgenic mice
To further investigate how RHSD treatment protects dopaminergic neurons, we conducted immunofluorescence staining of the substantia nigra (SN) and striatum regions to detect the number of dopaminergic neurons and the activation of glial cells. We used tyrosine hydroxylase (TH) as the marker for dopaminergic neurons, GFAP for astrocytes, and Iba-1 for microglia. The number of dopaminergic neurons in the SN and striatum of the RHSD treatment group increased significantly compared to the A53T group (Fig. 4A, B, Fig. S1A, B). The number of Iba-1+ cells showed a significant reduction in both the SN and striatum regions of the RHSD administration group compared to the A53T group (Fig. 4A, D, Fig. S1A, D). However, RHSD administration did not significantly reduce the number of GFAP + cells in either the SN or striatum regions of the A53T group (Fig. 4A, C, Fig. S1A, C). These results suggest that RHSD treatment reversed PD-associated neuroinflammation primarily by inhibiting microglia activation. Another important hallmark of PD, α-Syn and S-129 α-Syn expression, was also measured using immunohistochemistry staining in the SN and striatum regions. The results showed that the expression of α-Syn and phosphorylated α-Syn was significantly increased in the PD group, whereas RHSD administration significantly decreased the expression of both proteins (Fig. 4E, F, Fig. S1E, F).
RHSD reverses gut dysbiosis in A53T transgenic mice
In recent years, accumulating reports have found that intestinal microbiota dysbiosis plays a vital role in the occurrence and development of Parkinson's disease [35, 36][37, 38], and this feature also exists in animal models of PD. Alterations and oscillations of the gut microbiota precede the onset of neuropathy and motor dysfunction, as observed in the A53T mouse model [39].To explore how RHSD administration protects the A53T transgenic mouse model by modulating microbiome community structures, we carried out metagenomic sequencing on fecal samples from mice in the WT, A53T, and RHSD treatment groups. NMDS decrease-dimension analysis showed that the fecal microbiota of A53T transgenic mice was different from that of WT mice based on the abundance of each taxonomic hierarchy, while the material similarity of the RHSD group was similar to that of the WT group. The analysis of NMDS based on the phylum and species level is presented in Fig. 5A and Fig. 5B (Stress < 0.2, NMDS analysis is reliable). Bray-Curtis PcoA showed clear separation among groups, consistent with the correlation heatmap (Fig. S2A and S2B). To further identify the critical bacteria that contribute to the development of PD, we used Metastats and LEfSe analysis to look for different species between groups at the phylum level (Fig. S2D and S2E) and species level (Fig. 5C). The histogram of LDA value distribution and evolutionary branching plots show biomarkers with statistical differences between groups (Fig. 5D and 5E), as well as the differential abundances of gut microbes at various taxon levels among all three groups (Fig. 5E). Lactobacillus murinus and Lactobacillus reuteri are the intestinal probiotics that were significantly increased by RHSD (Fig. 6A). To find the specific bacterial species that might mediate the metabolic benefits of RHSD, we analyzed metagenomics data at the species level. A total of 1,289,948 genes were annotated in the WT group, while only 1,184,096 genes were annotated in A53T mice. Different from the reduced species number found in the A53T group, RHSD supplementation increased the gene number to 1,391,317 (Fig. S2C). Additionally, a total of 174 species were aberrant in A53T mice and reversed by RHSD treatment, determined with the double criteria of both fold change > 2 (or < 0.5) and p < 0.01. The differential species were mainly from the Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria phyla. Among them, 44 species were reduced in the PD group and reversed by RHSD treatment, mostly from the Proteobacteria and Bacteroidetes phyla, while the other 130 species showed opposite changes, mostly from the Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria phyla (Fig. S3). Moreover, we removed unclassified species, and selected those with stricter double criteria of both fold change > 2 (or < 0.05) and p < 0.001 in the RHSD treatment group, as shown in Table S1. These data suggest that RHSD plays an essential role in regulating gut microbiota composition in A53T transgenic mice.
We further performed a functional analysis of KEGG pathway enrichment to interpret the functions of the significantly altered bacteria. A total of 35 KEGG pathways at level 2 were identified (Fig. S2F), and 19 KEGG pathways at level 3 were identified (Fig. 6B). The altered flora mainly belonged to genetic information processing and metabolism pathways in A53T mice and were significantly reversed by RHSD treatment, including the ribosome, protein export, purine metabolism, methane metabolism, carbon fixation in photosynthetic organisms, lysine biosynthesis, and drug metabolism-cytochrome P450.
RHSD reverses metabolomic changes inA53Ttransgenicmice
Several studies have identified compositional and metabolic alterations in the Parkinson's microbiota have been strongly linked to the development and progression of the disease[40, 41].To elucidate the effect of RHSD on gut microbial metabolism in A53T mice, Untargeted metabolic profiling using LC/MS in positive and negative mode was performed on fecal samples. The unsupervised PCA, which was performed to visualize the general differences among samples, showed clear separation among groups in both positive and negative modes (Fig.S4A, B). Differential metabolites in RHSD treatment group were shown on the volcano plot compared with the A53T group in both positive and negative modes (Fig. 7A, B). The differential metabolites were displayed in Venn diagrams that were significantly altered in two between-group comparisons (Fig. 7C for positive modes and Fig. 7D for negative modes). A total of 103 and 35 different metabolites were selected with the criteria of either VIP > 1 (multivariate statistical analysis), p < 0.05 (univariate statistics) and fold change ≥ 2 or ≤ 0.5 in positive and negative modes (Fig. 7C, D). Most of the different metabolites are related to amino acids and their metabolites (28 and 6), Benzene and substituted derivatives (24 and 5), and Aldehyde, Ketones, Esters (11 and 4) in positive and negative mode. Top 20 metabolites with fold difference showed in TopFcBarChart of both positive and negative modes (Fig. 7E, F). Similar results were also shown in differential metabolite radar maps (Fig. S4C, D). Among them, 11 metabolites were significantly decreased and one metabolite was significantly increased in A53T mice, which were reversed by RHSD administration in positive modes (Fig. 7G). In addition, 3 metabolites were significantly altered in A53T mice and reversed by RHSD administration in the negative mode (Fig. 7H). The compounds corresponding to the index are given in Table S2.
Furtherly, we next carried out the KEGG function annotation and enrichment analysis on the all of differential metabolites. Differential metabolite clustering heatmap of KEGG pathways covered 2 classes of metabolites including Hormones and hormone related compounds, Alkaloids, and Benzene and substituted derivatives in positive modes (Fig. 7I). Among them, estriol (MEDN1407) and desmethylcitalopram (MW0006739) were enriched through differential enrichment analysis (Fig. 7I). Arginine and proline metabolism, Steroid hormone biosynthesis, Drug metabolism-cytochrome P450, and Ovarian steroidogenesis were enriched significantly P-value (Fig. S4E, F). It is interesting to note that the metabolism of terpenoids and polyketides, amino acid metabolism and drug metabolism-cytochrome P450were also identified based on altered bacterial function (Fig. S2F), the steroid hormone biosynthesis seemed no correlation with bacterial function (Fig. 6B). However, we have also noted that terpenoids regulate estrogen synthesis via aromatase CYP19, a key step rate-limiting enzyme and a member of the cytochrome P450 superfamily[42]. Additionally, desmethylcitalopram is a routine treatment for Generalized anxiety disorder[43]. These findings suggested that RHSD was effective in reversing endogenous α-Syn induced dysregulated metabolism, which may be attributed to its ability to regulate gut microbiota composition and metabolism.
RES Modulates Gut Microbiome Metabolites in A53T Transgenic Mice
Spearman's correlation analysis was used to perform the correlation analysis between the 30 hormone-related compounds (Table S3) and biomarker species, as well as the 46 other metabolites (Table S4) and biomarker species. Principal Component Analysis (PCA) of the metabolome and microbiome directly showed that the PDR group was closer to the WT group in metabolome among sample groups (Fig. 8B), and the analysis of microbiome at the species level was consistent (Fig. 8A). The upregulated metabolites by RHSD negatively correlated with the biomarkers of A53T transgenic mice, and positively correlated with the biomarkers of RHSD administration group mice. On the contrary, the majority of other metabolites downregulated by RHSD were positively correlated with the biomarkers of A53T transgenic mice, and negatively correlated with the biomarkers of RHSD administration group mice (Fig. 8C, D).
Furthermore, to better interpret the correlations of these significantly altered bacteria and metabolites, Spearman's correlation analysis was performed between the significantly difference bacterial species and the19 metabolites with fold difference which reversed by RHSD administration (Table S4). The results showed that the high abundance of microflora in A53T mice feces was positively correlated with the high content of Lithospermoside, 5-(Diphenylphosphinyl) pentanoic acid, Sulfadimethoxine, and Vinblastine (Fig. S5A). We noted that the high abundance of bacteria in the feces of A53T mice was closely related to inflammation and other pathological phenomena. For example, Arcobacter butzleri is an emerging enteric pathogen increasingly identified in intestinal infectious diseases and is likely under-reported in neurodegenerative diseases [44, 45]. Interestingly, the 19 metabolites were not significantly correlated with Lactobacillus_murinus and Lactobacillus_reuteri, but were significantly correlated with Lactobacillus_vaccinostercus, Lactobacillus_rapi, and Lactobacillus_shenzhenensis (Fig. S5A). In addition, the level of Secnidazole, Leu-Gly-Val, Ciclopirox, and Estriol was positively correlated with Lactobacillus_vaccinostercus (Fig. S5B). It has been shown that Lactobacillus spp. in the gut microbiota exert anti-inflammatory effects by repairing the damaged gut barrier, suppressing pro-inflammatory factors in the lymphatic circulation, and improving the ratio of regulatory versus pathogenic T cells[46].Our study highlights the important role of bacteria and metabolites in resveratrol-associated beneficial effects in A53T transgenic mice.
RHSD affects transcriptomics changes inA53T transgenic mice
Using Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) as an indicator to measure the level of transcript or gene expression, this study aimed to screen for differential genes. The screening conditions for differential genes were |log2Fold Change|>=1 and FDR < 0.05. The results showed that 217 differential genes were screened, of which 126 were expressed up-regulated and 91 were expressed down-regulated in the volcano map (Fig. 9A).
To explore the differences in the expression of estrogen receptor in the midbrain of A53T transgenic mice, we determined the expression of Gpr30 and Esr1. The results showed that estrogen receptors were up-regulated by RHSD in A53T transgenic mice, but no significant difference (Fig. 9B). In total, 698 genes were differentially regulated (FC ± 1.5, false discovery rate [FDR]-corrected p < 0.05) by RHSD in three comparison groups (Fig. 9C). There were 11 genes that were aberrant in A53T mice and reversed by RHSD treatment (Fig. 9D). Among them, 8 genes were expressed up-regulated and 3 genes were expressed down-regulated in A53T transgenic mice, while RHSD reversed (Fig. 9E).
Pathway significant enrichment analysis was performed using the pathway in KEGG database as a unit, and a hypergeometric test was applied to find out the pathway significantly enriched in differentially expressed genes compared with the whole genome background (Fig. 9F). It is interesting to note that the pathway of olfactory transduction, calcium signaling pathway, dopaminergic synapse, and serotonergic synapse were enriched by RHSD treatment (Fig. 9G).
Integrated analysis of microbial differential metabolites and host transcript profiles
The first step was to perform a correlation analysis between differential microbial metabolites and host genes detected between the A53T and A53TR groups. The Spearman correlation coefficients for differential metabolites and genes were calculated using the R software. Based on the calculated values of |cor|>0.8 and p-value < 0.05, the Cytoscape software was used to plot the heatmap and network of correlation for differential metabolites and genes (Fig. 10A, B). One of the primary genes positively correlated with Lithospermoside and negatively correlated with Leu-Gly-Val was Ferredoxin reductase (Fdxr) (Fig. 10A, C). Fdxr is located in the mitochondrial inner membrane and is orthologous to human FDXR. A previous study found that Fdxr mutant mouse brain tissues had increased astrocytes, which could potentially be associated with neurodegeneration [47]. The data from this study further sheds light on the pathogenic mechanism of FDXR-mediated central neuropathy and suggests an avenue for mechanistic studies that could ultimately aid in treatment. Anoctamin 2 (Ano2) was positively correlated with Leu-Ser-Asn and Secnidazole (Fig. 10A, C). Ano2 is orthologous to human ANO2 and is expressed in various bodily regions[48]. Our findings suggest that resveratrol could potentially restore olfactory function by regulating the metabolism of intestinal microbes in A53T transgenic mice. Additionally, dopamine receptors D1 (Drd1) were positively correlated with Rivastigmine (Fig. 10A, D). Drd1 is orthologous to human DRD1 and regulates spines in striatal direct-pathway and indirect-pathway neurons, with knockout Drd1 mice (Drd1−/−) commonly used as models of PD[49]. In conclusion, microbial metabolites were found to be correlated with host transcription regulation that is associated with Parkinson's disease progression.