Delphinidin ameliorates learning and memory deficits in APP/PS1 mice

Following the experimental design in Fig. 1A, APP/PS1 mice were randomly divided into two groups (n = 8): APP/PS1 mice orally administered equal amounts of PBS (APP/PS1), and APP/PS1 mice orally administered 15 mg/kg/day delphinidin in PBS (APP/PS1 + Dp) for 8 weeks. C57BL/6 J mice (7-month-old) were administered equal amounts of PBS (WT) for 8 weeks as previously described [39]. Body weight was measured once a week. The experimental design is shown in Fig. 1A. Subsequently, the MWM and Novel Object Recognition NOR tests were conducted to assess spatial learning and memory, evaluating delphinidin’s effect on spatial learning and memory ability. During the spatial acquisition training period of MWM, the APP/PS1 mice presented a higher escape latency compared to the WT mice. The escape latency of the delphinidin-treated APP/PS1 mice was significantly decreased compared to the vehicle-treated APP/PS1 mice (Fig. 1B). During the probe trial, vehicle-treated with APP/PS1 mice exhibited reduced platform crossing frequency and spent significantly less time in the target quadrant compared to WT mice. However, delphinidin-treated APP/PS1 mice demonstrated an increased frequency of platform crossings and longer duration in the target quadrant compared to vehicle-treated APP/PS1 mice (Fig. 1C-E).

In the novel object recognition test, wild-type mice exhibited a preference for the novel object, whereas vehicle-treated APP/PS1 mice showed no significant preference between the familiar and novel objects. Following treatment with delphinidin, APP/PS1 mice spent significantly more time exploring the new objects (Fig. 1F-G). These results suggest that delphinidin treatment may ameliorate cognitive deficits and enhance learning and memory abilities in APP/PS1 mice.

Transcriptome sequencing analysis reveals that delphinidin exhibits an anti-aging signature in the hippocampus of APP/PS1 mice

To further investigate how delphinidin ameliorates cognitive deficits in APP/PS1 mice, we performed bulk RNA sequencing on the brain of mice from three different groups. The Venn diagram was created to identify differentially expressed genes (DEGs) shared between WT mice versus APP/PS1 mice and APP/PS1 mice versus delphinidin-treated APP/PS1 mice (Supplementary Fig. 2). Our analysis revealed the gene expression profiles of APP/PS1 mice differed significantly from those of WT mice. The heatmap illustrates that the DEGs profile of delphinidin-treated APP/PS1 mice closely resembles that of WT mice, indicating that delphinidin treatment may effectively restore gene expression patterns disrupted by the pathological change in APP/PS1 mice (Fig. 2A). As depicted in the volcano plot, the transcriptomic analysis revealed 870 DEGs in the brains of APP/PS1 versus WT mice, with 581 genes showing elevated expression and 289 genes displaying reduced expression (Fig. 2B).

Fig. 2

Effects of delphinidin on the gene expression profiles in APP/PS1 mice. A A heatmap illustrating hierarchical clustering of co-regulated differentially expressed genes (DEGs) across three distinct experimental groups. B, C Volcano plotting displaying DEGs between WT versus APP/PS1 mice and APP/PS1 versus delphinidin-treated APP/PS1 mice. D The heatmap showing DEGs associated with cell senescence. E, F The diagram showed the top 20 significantly down-regulated (E) and up-regulated (F) KEGG pathways of DEGs between APP/PS1 and delphinidin-treated APP/PS1 mice. G, H KEGG and GO term of gene set enrichment analysis between APP/PS1 and delphinidin-treated APP/PS1 mice. n = 4 mice. Dp: delphinidin; WT: wildtype

Our findings revealed that the transcriptomic comparison between delphinidin-treated APP/PS1 mice and vehicle-treated APP/PS1 mice identified 103 upregulated genes and 242 downregulated genes (Fig. 2C). Notably, among these genes, we discovered a principal cluster of genes that was downregulated in delphinidin-treated APP/PS1 mice compared to vehicle-treated APP/PS1 mice (Fig. 2D). This suggests that delphinidin treatment induces changes in the differential gene expression profile of APP/PS1 mice, which appear to exhibit an anti-aging signature [42, 43]. Further analysis showed that delphinidin treatment significantly downregulated genes associated with senescent microglia in APP/PS1 mice, including TREM2, ApoE, Cst7, Itgax, Tyrobp, Ccl4, Lyz2 and Cd68. These results suggest that delphinidin has the potential to resist aging-related molecular changes in AD mice, likely by reversing the cellular senescence of microglia.

Next, DEGs between delphinidin-treated APP/PS1 mice and vehicle-treated APP/PS1 mice submitted to KEGG pathway enrichment analysis. In the top 20 significantly down-regulated KEGG pathways, delphinidin treatment significantly reduced the pathways associated with redox reaction in APP/PS1 mice (Fig. 2E). Additionally, gene set enrichment analysis (GSEA) revealed that most DEGs related to reactive oxygen species, myeloid cell activation involved in the immune response, and myeloid leukocyte mediated immunity were downregulated in delphinidin-treated APP/PS1 mice compared to vehicle-treated APP/PS1 mice (Fig. 2G). The KEGG pathway analysis revealed an enrichment of synapse-related transcriptional upregulation in delphinidin-treated APP/PS1 mice compared to vehicle-treated APP/PS1 mice (Fig. 2E). Additionally, GSEA indicated that DEGs associated with dendrite morphogenesis, regulation of synaptic vesicle cycle, nerve growth factor and long-term potentiation were upregulated in the delphinidin-treated group (Fig. 2H). The SAUL_SEN_MAYO gene set, a newly identified signature of aging-related genes, is primarily composed of SASP factors [44]. Our research initially demonstrated an upregulation of the SAUL_SEN_MAYO gene set in APP/PS1 mice. Treatment with delphinidin downregulated the genes within the SAUL_SEN_MAYO gene set. These findings suggest that delphinidin may play a potential role in mitigating aging-related SASP changes in APP/PS1 mice. Among the top 20 significantly enriched KEGG pathways, our analysis highlighted the cAMP signaling pathway as particularly prominent in delphinidin-treated APP/PS1 mice compared to vehicle-treated APP/PS1 mice, suggesting its potential role in the observed biological effects. Considering that cAMP is a crucial precursor in AMP biosynthesis, which subsequently influences the activation energy of the AMPK signaling pathway. To summarize, the functional analysis of DEGs indicated that delphinidin may alleviate cognitive impairment by reducing oxidative stress, modulating the immune response, inhibiting cell senescence, and upregulating synapse-related process, while also enhancing AMPK signaling pathway.

Delphinidin prevents synapse loss and reduces Aβ plaques in APP/PS1 mice

It is well-established that the loss of synaptic function is associated with cognitive impairment. Our RNA sequencing enrichment analysis results suggest that delphinidin treatment promotes synaptogenesis and long-term potentiation in APP/PS1 mice (Fig. 2H). To further investigate the effects on synaptic plasticity, we assessed hippocampal synaptic integrity by analyzing postsynaptic PSD-95 and presynaptic Synaptophysin via immunofluorescence and western blot. Consistent with our quantitative data, visual inspection of these new confocal images reveals a sparser structure and reduced density of both PSD-95 and Synaptophysin puncta in the hippocampal CA1 region of vehicle-treated APP/PS1 mice compared to WT mice. Importantly, the images from the delphinidin-treated APP/PS1 mice clearly show increased density and a more robust appearance of both PSD-95 and Synaptophysin puncta in the CA1 region, indicating improved synaptic structures (Fig. 3A-B). Supporting this, Western blots confirmed delphinidin mitigated the overall hippocampal loss of PSD-95 and Synaptophysin protein in APP/PS1 mice (Fig. 3C-E). Additionally, Immunofluorescence staining for the dendritic marker Map2 revealed structural deficits in APP/PS1 mice that were significantly improved by delphinidin treatment (Supplementary Fig. 3A-B). These findings indicate an enhancement of synaptic plasticity in APP/PS1 mice following delphinidin treatment.

Fig. 3

Delphinidin prevents synaptic loss and reduces Aβ plaques in APP/PS1 mice. A Representative PSD-95 and Synaptophysin immunostaining in the CA1. Scale bar = 50 μm. B Quantitative intensity analysis of PSD-95 and Synaptophysin immunostaining in the hippocampus. C Western blot analysis of PSD-95 and Synaptophysin in the hippocampus of mice. D, E Quantification of PSD-95/GAPDH (D) and Synaptophysin/GAPDH (E) in C. F Representative immunohistochemistry (IHC) staining of Aβ plaques in the brain. Scale bar = 200 μm. G, H Quantitative analysis of Aβ IHC staining number (G) and area (H) in the cortex and hippocampus. I Representative Th-S positive Aβ plaques in the brain. Scale bar = 500 μm. J Quantitative intensity analysis of Th-S positive staining in the brain. K Western blot analysis of Aβ and GAPDH in the hippocampus of mice. L Quantification of Aβ/GAPDH in I. n = 4 mice. Data were presented as mean ± SD. ###p < 0.0005, #### p < 0.0001 versus WT mice treated with vehicle, * p < 0.05, ** p < 0.005 versus APP/PS1 mice treated with vehicle. Dp: delphinidin; WT: wildtype

Extracellular accumulation of Aβ is a well-established pathological marker in APP/PS1 mice. To investigate whether delphinidin treatment can reduce Aβ plaque formation in the brain of APP/PS1 mice, we conducted immunohistochemistry and thioflavin S (Th-S) staining assays. Our findings revealed the presence of senile plaques in both the cortex and hippocampus of APP/PS1 mice. Notably, delphinidin treatment significantly decreased both the area and number of Aβ plaques in the cortex and hippocampus of APP/PS1 mice (Fig. 3F-H). Correspondingly, delphinidin treatment effectively decreased the intensity of Th-S staining in APP/PS1 mice (Fig. 3I-J). The hippocampal Aβ protein level was decreased in delphinidin-treated APP/PS1 mice compared with that in vehicle-treated APP/PS1 mice (Fig. 3K-L).

Investigating a potential mechanism for delphinidin’s effects on Aβ, we assessed microglial phagocytosis in vitro. Primary microglia treated with delphinidin showed significantly increased uptake of Cy3-labeled Aβ₄₂, measured by flow cytometry and immunofluorescence (Supplementary Fig. 4A-D). This suggests delphinidin promotes Aβ clearance by enhancing microglial phagocytic activity, explaining the reduced in vivo Aβ plaque load. These results suggest that delphinidin treatment significantly alleviates Aβ pathological alterations. Collectively, our findings indicate that delphinidin may delay or prevent neurodegenerative changes in APP/PS1 mice.

Delphinidin suppresses microglia activation, neuroinflammation and oxidative stress while upregulating the AMPK/SIRT1 pathway in APP/PS1 mice

Neuroinflammation and oxidative stress play key roles in the pathogenesis of Aβ pathology. Given the important role of glial cells in neuroinflammation, we assessed reactive astrocytes and microglia by GFAP and Iba-1 immunostaining, respectively. Delphinidin treatment significantly decreased reactive microglia in both the cortex and hippocampus of the APP/PS1 mice (Fig. 4A-B). Immunohistochemistry results showed prominent GFAP activation in the cortex of APP/PS1 mice but no significant effect of delphinidin treatment when quantifying astrocyte area (Fig. 4C). To further investigate delphinidin’s impact on astrocytes and microglia, we performed Western Blot analyses on protein lysates from both hippocampal and cortical tissues. Consistent with reactive astrogliosis, we confirmed that GFAP protein levels were significantly increased in both the cortex and hippocampus of APP/PS1 mice compared to WT mice. Interestingly, our results revealed that delphinidin treatment significantly reduced the elevated GFAP protein levels specifically in the hippocampus and cortex of APP/PS1 mice. In the cortex, delphinidin showed a slight decreasing trend in GFAP protein levels, but this did not reach statistical significance (Fig. 4D-F). Western blot analysis showed the increased levels of IBA1 in the hippocampus and cortex of APP/PS1 mice compared to those of the WT mice. Delphinidin treatment reduced protein level of IBA1 in the hippocampus and cortex of APP/PS1 mice (Fig. 4D-F).

Fig. 4

Delphinidin suppresses microglia activation in APP/PS1 mice. A Representative IHC staining of IBA1 and GFAP in the brains of APP/PS1 mice treated with delphinidin or vehicle and their WT littermates treated with vehicle. Scale bar = 50 μm. B Quantitative analysis of IBA1 immunostaining area in A. C Quantitative analysis of GFAP immunostaining area in A. D Western blot analysis of GFAP, IBA1 and GAPDH in the cortex of mice. E Western blot analysis of GFAP, IBA1 and GAPDH in the hippocampus of mice. F Quantification of GFAP/GAPDH and IBA1/GAPDH in D and E. n = 4 mice. Data were presented as mean ± SD. # p < 0.05, #### p < 0.0001 versus vehicle-treated WT mice, * p < 0.05, ** p < 0.005, *** p < 0.0005 versus vehicle-treated APP/PS1 mice, ns, not significant. Dp: delphinidin; WT: wildtype

To further explore the levels of inflammatory cytokines, we found that protein levels of IL-1β and IL-6 in the hippocampus of the APP/PS1 mice were higher than those of WT mice, whereas delphinidin markedly decreased the levels of these proinflammatory cytokines in the APP/PS1 mice (Fig. 5A-C). In addition, delphinidin administration reduced the mRNA levels of inflammatory mediators IL-1β, IL-6 and TNF-α in both the cortex and hippocampus of APP/PS1 mice (Fig. 5D-E). Similar to the brain, the levels of proinflammatory cytokines and chemokines CXCL1, CCL2, CCL3, IL-1β, IL-6, and TNF-α were significantly increased in the serum of APP/PS1 mice. These levels decreased following treatment with delphinidin (Fig. 5F).

Fig. 5

Delphinidin reduces pro-inflammatory cytokine production and oxidative stress while upregulating the AMPK/SIRT1 pathway in APP/PS1 mice. A Western blot analysis of IL-1β, IL-6 and GAPDH in the hippocampus of mice. (n = 4 mice). B, C Quantification of IL-1β/GAPDH (B) and IL-6/GAPDH (C) in A. D The mRNA expression of IL-1β, IL-6 and TNF-α in the hippocampus were detected by qRT-PCR. (n = 4 mice). E The mRNA expression of IL-1β, IL-6, and TNF-α in the cortex were detected by qRT-PCR. (n = 5 mice). F Proinflammatory cytokines or chemokines CXCL1, CCL2, CCL3, IL-1β, IL6 and TNF-α levels were detected by cytokine array in the serum of APP/PS1 mice treated with delphinidin or vehicle and their WT littermates treated with vehicle. (n = 3 mice). G The level of MDA in the cortex and hippocampus of mice. (n = 8 mice for cortex, n = 4 mice for hippocampus). H The level of T-SOD activity in the cortex and hippocampus of mice. (n = 8 mice for cortex, n = 4 mice for hippocampus). I Western blot analysis of p-AMPK, AMPK, SIRT1 and GAPDH in the hippocampus of mice. (n = 4 mice). J, K Quantification of p-AMPK/AMPK (J) and SIRT1/GAPDH (K) in I. Data were presented as mean ± SD. # p < 0.05, ## p < 0.005, ### p < 0.0005, #### p < 0.0001 versus vehicle-treated WT mice, * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 versus vehicle-treated APP/PS1 mice. Dp: delphinidin; WT: wildtype

Increased levels of MDA and decreased activity of SOD are associated with Aβ plaque aggregation and the progression of AD. To investigate the potential impact of delphinidin on MDA levels and T-SOD activity, we measured MDA levels and T-SOD activity in both the cortex and hippocampus of the mice. The MDA content was significantly higher in the cortex and hippocampus of APP/PS1 mice compared to that in WT mice, as shown in Fig. 5G, indicating that APP/PS1 mice exhibit elevated levels of free radicals and cellular damage. Delphinidin treatment significantly reduced the MDA levels in the cortex and hippocampus of APP/PS1 mice. Furthermore, T-SOD activity was lower in the cortex and hippocampus of APP/PS1 mice compared to WT mice, while delphinidin treatment significantly increased T-SOD activity in APP/PS1 mice (Fig. 5H).

Since our RNA sequencing data indicated enrichment of the cAMP signaling pathway, and given that cAMP is a crucial precursor in AMP biosynthesis, which in turn influences the activation energy of the AMPK signaling pathway, we employed the western blotting analysis to determine whether delphinidin affects the AMPK signaling pathway. A significant decrease in the p-AMPK/AMPK ratio was observed in the hippocampus of APP/PS1 mice compared to WT mice (Fig. 5I-J). However, treatment with delphinidin markedly increased the p-AMPK/AMPK ratio in the APP/PS1 mice. Given the close relationship between SIRT1 and AMPK, we further investigated the expression levels of SIRT1. The protein expression of SIRT1 was decreased in the hippocampus of APP/PS1 mice compared to WT mice, while delphinidin treatment significantly enhanced the levels of this protein in APP/PS1 mice (Fig. 5I and K). To determine the cellular localization, we performed immunofluorescence co-staining. Significant co-localization of SIRT1 with the microglial marker IBA1 was observed in both primary microglia and BV2 microglial cells. This provides direct cellular evidence that SIRT1 activation by delphinidin takes place in microglia (Supplementary Fig. 6 A).

Delphinidin prevents microglial senescence in APP/PS1 mice

Previous studies have strongly indicated that inflammatory responses are a hallmark of cellular senescence, and recent findings suggest that cell senescence occurs in AD. To further investigate whether delphinidin influences cellular senescence, we examined senescence phenotypes using SA-β-gal staining. There was more SA-β-gal staining in both the hippocampus and cortex of APP/PS1 mice compared to WT mice. Notably, delphinidin treatment reduced SA-β-gal staining in the hippocampus and cortex of APP/PS1 mice (Fig. 6A-B). Western blot experiments showed that the protein level of senescence markers p16INK4a and p21cip1 in the hippocampus of APP/PS1 mice were significantly higher than those in WT mice. However, this increase was reversed by treatment delphinidin (Fig. 6C-D). Furthermore, the mRNA levels of p16INK4a and p21cip1 were also elevated in the hippocampus of APP/PS1 mice, but decreased following treatment with delphinidin (Fig. 6E and H).

Fig. 6

Delphinidin prevents microglial senescence in APP/PS1 mice. A Representative SA-β-gal staining in the brains of APP/PS1 mice treated with delphinidin or vehicle and their WT littermates treated with vehicle. Scale bar = 100 μm. B Quantification of SA-β-gal staining densities in A. (n = 4 mice). C Western blot analysis of p16INK4a, p21cip1 and GAPDH in the hippocampus of mice. (n = 4 mice). D Quantification of p21cip1/GAPDH in C. (n = 4 mice). E The mRNA expression of p16INK4a and p21cip1 in the hippocampus were detected by qRT-PCR. (n = 4 mice). F Gene set enrichment analysis of SenMayo gene set between WT versus APP/PS1 mice and APP/PS1 versus delphinidin-treated APP/PS1 mice. G Quantification of p16INK4a/GAPDH in C. (n = 4 mice). H The mRNA expression of p16INK4a and p21cip1 in the cortex were detected by qRT-PCR. (n = 5 mice). I Immunofluorescence of IBA1 (green) with p16INK4a (red) in the hippocampus and cortex of APP/PS1 mice treated with delphinidin or vehicle. Scale bar = 20 μm. J, K Quantification the proportion of p16INK4a-positive cells among IBA+ cells in the hippocampus (J) and cortex (K) of APP/PS1 mice treated with delphinidin or vehicle. (n = 4 mice). Data were presented as mean ± SD. # p < 0.05, ## p < 0.005, ### p < 0.0005, #### p < 0.0001 versus vehicle-treated WT mice, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 versus vehicle-treated APP/PS1 mice, ns, not significant. Dp: delphinidin; WT: wildtype

As mentioned earlier, the SASP as a marker of cellular senescence and involves the production of pro-inflammatory cytokines. The levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were significantly reduced following delphinidin treatment (Fig. 5D and E). Following treatment delphinidin, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were significantly reduced in APP/PS1 mice. The SAUL_SEN_MAYO gene set, a newly identified list of senescence-associated genes, is primarily composed of SASP factors [44]. Our research initially demonstrated that delphinidin treatment downregulated SAUL_SEN_MAYO genes in APP/PS1 mice (Fig. 6F). These results further confirm that delphinidin play a potential role in mitigating cellular senescence in AD mice.

Senescent microglia-mediated neuroinflammatory responses are believed to exacerbate neuronal damage, thereby driving the progression of age-related neurodegeneration in AD. Based on the RNA sequencing data revealed that delphinidin treatment down-regulated several genes associated with senescent microglia, including Apoe, Cst7, Tyrobp, Cd68, Itgax and Trem2 (Fig. 2C-D). To explore whether delphinidin inhibit microglia senescence in the brains of APP/PS1 mice, we co-stained for the cellular senescence marker p16INK4a alongside the microglia marker IBA1. Following delphinidin intervention, we observed a statistically significant reduction in the proportion of p16INK4a-positive cells among IBA+ cells in both the hippocampal and cortical regions of APP/PS1 mice, indicative of a mitigated senescent signature in brain (Fig. 6I-K).

To evaluate neuronal contribution and delphinidin treatment specificity, p16INK4a+/NeuN+ co-localization was performed. A minor increase in p16INK4a+ neurons was observed in APP/PS1 mice relative to WT, but delphinidin treatment showed only a minor trend towards reducing these p16INK4a-positive neurons (Supplementary Fig. 3C-D). Overall, our transcriptomic and immunofluorescence results indicate that delphinidin primarily alleviates microglial senescence, with minimal impact on neuronal senescence. Our data indicate for the first time that delphinidin exhibits anti-aging properties and alleviates cellular senescence in the brain of AD mice.

Delphinidin decreases microglial senescence in BV2 microglia cells

To investigate the effect of delphinidin on microglial senescence in vitro, we treated BV2 microglia cells with varying concentrations of delphinidin. Initially, we assessed the impact of different concentrations of delphinidin on BV2 microglia cells viability to determine a suitable concentration with minimal effects on cell viability (Supplementary Fig. 5).

Since Aβ induces microglial activation, triggering an inflammatory response and accelerating microglial senescence [45]. We next evaluated the effect of delphinidin on Aβ₄₂-induced microglial senescence in vitro, following the experimental setup detailed schematically in Supplementary Fig. 7. Our results demonstrated a significant increase in SA-β-Gal positive cells following Aβ42 treatment, indicating the induction of cellular senescence (Fig. 7A-B). Conversely, delphinidin treatment attenuated the Aβ42-induced increase in SA-β-Gal positive cells. Furthermore, delphinidin treatment significantly reduced the protein and mRNA levels of p16INK4a and p21Cip1 induced by Aβ42 (Fig. 7C-F). Our data indicate that delphinidin eliminates senescent cells induced by Aβ42. We further investigated whether delphinidin treatment could reduce the inflammatory response in BV2 microglia cells induced by Aβ42. The results indicated that Aβ42 increased the mRNA levels of IL-1β, IL-6, and TNF-α inflammatory cytokines in BV2 microglia cells, while delphinidin treatment significantly reduced the levels of these cytokines (Fig. 7F). We further investigated delphinidin’s effect on senescence induced by different amyloid-beta oligomers, specifically using Aβ₂₅₋₃₅ in BV2 microglia. Delphinidin treatment effectively counteracted Aβ₂₅₋₃₅-induced senescence, significantly reduced SA-β-Gal positive cells, attenuating p16 and p21 mRNA expression (Supplementary Fig. 8A-C). This demonstrates that delphinidin’s protective anti-senescence effects extend beyond Aβ₄₂ oligomers.

Fig. 7

Delphinidin ameliorates microglial senescence induced by the Aβ42 oligomers in BV2 microglia cells. A Representative SA-β-gal staining in BV2 microglia cells. Black arrows point to representative SA-β-gal staining positive cells. Scale bar = 20 μm. B Quantification of SA-β-gal staining positive cells in A. C Western blot analysis of p21cip1, p16INK4a and GAPDH in BV2 microglia cells. D, E Quantification of p21cip1/GAPDH (D) and p16INK4a/GAPDH (E) in C. F The mRNA expression of p16INK4a, p21cip1, IL-1β, IL-6, and TNF-α in BV2 microglia cells were detected by qRT-PCR. n = 4. Data are presented as mean ± SD. # p < 0.05, ## p < 0.005, ### p < 0.0005, #### p < 0.0001 versus vehicle-treated cells, * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 versus Aβ42 oligomers-treated cells. Dp: delphinidin

Delphinidin decreases excessive ROS production while upregulating the AMPK/SIRT1 pathway in BV2 microglia cells

Excessive ROS production, as senescence marker, was significantly upregulated in BV2 cells treated with Aβ42, as assessed by flow cytometry and microscopy. Notably, delphinidin treatment attenuated the production of ROS induced by Aβ42 (Fig. 8A-D). Similarly, delphinidin treatment markedly reduced the intracellular levels of ROS induced by Aβ₂₅₋₃₅ (Supplementary Fig. 8D-G). Given delphinidin's potent antioxidant properties, it may alleviate cellular senescence by decreasing ROS production.

Fig. 8

Delphinidin decreases excessive ROS production while enhancing the AMPK/SIRT1 pathway induced by the Aβ42 oligomers in BV2 microglia cells. A, B Assessment of ROS production in BV2 microglia cells via flow cytometry following loading with the ROS indicator DCFH-DA. C Representative image of BV2 microglia cells loaded with DCFH-DA (green) and Hoechst (blue). Scale bar = 50 μm. D Quantification the proportion of DCFH-DA positive cells in C. E Western blot analysis of p-AMPK, AMPK, SIRT1 and GAPDH in BV2 microglia cells. F, G Quantification of p-AMPK/AMPK (F) and SIRT1/GAPDH (G) in E. n = 4. Data are presented as mean ± SD. ### p < 0.0005, #### p < 0.0001 versus vehicle-treated cells, * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 versus Aβ42 oligomers-treated cells. ROS, reactive oxygen species; Dp: delphinidin

We further evaluated the effect of delphinidin on the AMPK and SIRT1 in BV2 cells induced by Aβ42. A significant decrease in the p-AMPK/AMPK ratio and SIRT1 expression was observed in BV2 cells treated with Aβ42. However, treatment with delphinidin markedly increased the p-AMPK/AMPK ratio and SIRT1 expression in the Aβ42 induced BV2 cells (Fig. 8E-G).

Delphinidin inhibits microglial senescence induced by Aβ42 through the AMPK/SIRT1 pathway

To investigate whether delphinidin protects against Aβ42-induced microglial senescence by modulating the AMPK pathway, we conducted in vitro experiments co-administering delphinidin with the AMPK inhibitor Compound C. As anticipated, the results showed that Compound C effectively inhibited AMPK activation and suppressed SIRT1 expression (Fig. 9A-C).

Fig. 9

Delphinidin inhibits microglial senescence induced by Aβ42 through the AMPK/SIRT1 pathway. A Western blot analysis of p-AMPK, AMPK, SIRT1 and GAPDH in BV2 microglia cells. B, C Quantification of p-AMPK/AMPK (B) and SIRT1/GAPDH (C) in A. D Representative SA-β-gal staining in BV2 microglia cells. Scale bar = 20 μm. E Quantification of SA-β-gal staining positive cells in D. F, G Assessment of ROS production in BV2 microglia cells via flow cytometry following loading with the ROS indicator DCFH-DA. H Western blot analysis of p21cip1, p16INK4a and GAPDH in BV2 microglia cells. (I-J) Quantification of p21cip1/GAPDH (I) and p16INK4a/GAPDH (J) in C. K The mRNA expression of p16INK4a, p21cip1, IL-1β, IL-6, and TNF-α in BV2 microglia cells were detected by qRT-PCR. n = 4. Data are presented as mean ± SD. # p < 0.05, ## p < 0.005, ### p < 0.0005, #### p < 0.0001 versus Aβ42 oligomers and delphinidin treated cells, * p < 0.05, ** p < 0.005, **** p < 0.0001 versus Aβ42 oligomers-treated cells. ROS, reactive oxygen species; Dp: delphinidin

SA-β-gal staining revealed that Compound C inhibited the ability of delphinidin to reverse the reduction in the proportion of SA-β-gal-positive cells induced by Aβ42 (Fig. 9D-E). Flow cytometry analysis demonstrated that, in the presence of Compound C, delphinidin was unable to mitigate the ROS production induced by Aβ42 (Fig. 9F-G). Similarly, Compound C inhibited the ability of delphinidin to reverse the protein expression and mRNA level of P21CIP1 and P16INK4a triggered by Aβ42 (Fig. 9H-K). Similarly, Compound C inhibited the ability of delphinidin to reverse the mRNA levels of IL-1β, IL-6, and TNF-α inflammatory cytokines induced by Aβ42 (Fig. 9K).

In conclusion, these findings suggest that delphinidin exerts its protective effects against Aβ42-induced microglial senescence through targeted regulation of the AMPK/SIRT1 pathway.

Delphinidin directly binds to SIRT1

Recent research suggested that SIRT1 regulating metabolism and cellular senescence through activates AMPK [38]. Molecular docking simulation was performed to investigate the binding mode of delphinidin within the active site of the SIRT1 protein using AutoDock Vina 1.1.2 and PLIP. The molecular docking results indicated that hydrogen bonds were formed at the PHE-273, ILE-347, ASP-348, and VAL-412 residues between delphinidin and SIRT1 (Fig. 10A). The theoretical binding affinity of A to SIRT1 was calculated to be − 9.1 kcal/mol (mean value). This indicates a strong binding between delphinidin and SIRT1, which may potentially influence the structural function and biological activity of SIRT1.

Fig. 10

Direct interaction between SIRT1 and delphinidin. A The docking mode of delphinidin with SIRT1 protein. B Root Mean Square Deviation (RMSD) analysis showing the binding of delphinidin with SIRT1 protein. C Gyration radius (Rg) analysis showing the binding of delphinidin with SIRT1 protein. D The number of hydrogen bonds formed between delphinidin and SIRT1 protein. E Root Mean Square Fluctuation (RMSF) analysis showing the binding of delphinidin with SIRT1 protein

To further validate the binding affinity and stability between delphinidin and SIRT1, we performed molecular dynamics simulations on the docking results. The Root Mean Square Deviation (RMSD) values of the backbone atoms stabilized at approximately 0.30–0.42 nm after 20 ns, indicating that the system had reached a well-equilibrated state. The RMSD of the delphinidin stabilized at around 0.25–0.45 nm, suggesting that it could stably bind to the SIRT1's hydrophobic pocket (Fig. 10B). Furthermore, the radius of gyration (Rg) of the SIRT1 protein was analyzed during the simulation, and it was found to remain stable at around 2.0–2.2 nm, indicating that the protein-small molecule complex was relatively stable throughout the simulation (Fig. 10C). The number of hydrogen bonds formed directly between SIRT1 protein and delphinidin during the simulation was also analyzed. It was found that 3–6 stable hydrogen bonds were formed between SIRT1 protein and delphinidin, which played a crucial role in their stable binding (Fig. 10D). Additionally, the root-mean-square fluctuation (RMSF) of the SIRT1 protein backbone was analyzed, revealing that the regions spanning residues 184–196, 207–219, 273–305, 372–375, 395–405, and 414–425 exhibited higher flexibility, while the remaining regions showed relatively rigid structures (Fig. 10E).

These findings suggest that the complex formed between delphinidin and SIRT1 is remarkably stable and exhibits strong binding affinity. The results further demonstrate that delphinidin treatment significantly enhances the thermal stability of SIRT1. Overall, it is likely that delphinidin exerts its protective effects by specifically targeting and binding to SIRT1, thereby enhancing its stability and function.

 Delphinidin also mitigates microglial senescence in naturally aged mice

Glial activation, characterized by astrocyte and microglial activation, is a hallmark of brain aging. Immunohistochemical staining revealed a significant increase in IBA1 area in both the hippocampus and cortex of aged mice compared to young mice, but delphinidin treatment significantly reduced IBA1 area in both the hippocampus and cortex of aged mice (Fig. 11 A-B). Delphinidin treatment had no discernible effect on the GFAP area of young mice and aged mice (Fig. 11C).

Fig. 11

Delphinidin also reduces microglial senescence in aged mice. A Representative IHC staining of GFAP and IBA1 in the brains of young mice treated with delphinidin or vehicle and aged mice treated with delphinidin or vehicle. Scale bar = 50 μm. B Quantitative analysis of GFAP immunostaining area in A. C Quantitative analysis of IBA1 immunostaining area in A. D, E The mRNA expression of p16INK4a and p21cip1 in the hippocampus (D) and cortex (E) were detected by qRT-PCR. F Western blot analysis of p16INK4a, p21cip1 and GAPDH in the hippocampus of mice. (n = 4 mice). G, H Quantification of p21cip1/GAPDH (G) and p16INK4a/GAPDH (H) in F. I Immunofluorescence of IBA1 (green) with p16INK4a(red) in the hippocampus and cortex of aged mice treated with delphinidin or vehicle. Scale bar = 20 μm. J Quantification the proportion of p16INK4a-positive cells among IBA+ cells in the hippocampus and cortex of aged mice treated with delphinidin or vehicle. n = 4 mice. Data are presented as mean ± SD. # p < 0.05, ### p < 0.0005, #### p < 0.0001 versus vehicle-treated young mice, * p < 0.05, ** p < 0.005, *** p < 0.0005 versus vehicle-treated aged mice, ns, not significant. Dp: delphinidin

To further investigate cellular senescence, we examined the expression of senescence markers p16INK4a and p21Cip1 in young and aged mice. Notably, our findings revealed that the mRNA levels of p16INK4a and p21Cip1 were significantly upregulated in both the hippocampus and cortex of aged mice, but were notably reduced following treatment with delphinidin (Fig. 11D-E). Similarly, delphinidin treatment also decreased the protein levels of p16INK4a and p21Cip1 in the hippocampus of aged mice (Fig. 11F-H). We evaluated microglial senescence by co-staining p16INK4a with the microglia marker IBA1. Following treatment with delphinidin, a statistically significant reduction in the proportion of p16INK4a-positive cells among IBA+ cells was observed in both the hippocampal and cortical regions of aged mice (Fig. 11I-J). These findings indicate for the first time that delphinidin mitigates microglia senescence in the brain of aged mice.

Delphinidin suppresses neuroinflammation, oxidative stress while upregulating the AMPK/SIRT1 pathway in naturally aged mice

Furthermore, we investigated the effect of delphinidin on AMPK/SIRT1 signaling pathway in young and aged mice. A significant decrease in the p-AMPK/AMPK ratio and SIRT1 expression was observed in the brains of aged mice compared to those in young mice. However, treatment with delphinidin notably restored both the p-AMPK/AMPK ratio and SIRT1 expression in the brains of aged mice (Fig. 12A-C). The protein levels of IL-1β and IL-6, as well as the mRNA levels of IL-1β, IL-6, and TNF-α inflammatory cytokines, were significantly increased in the brains of aged mice compared to those of young mice. Delphinidin treatment significantly decreased the protein levels of IL-1β and IL-6 inflammatory cytokines in the brains of aged mice (Fig. 12D-F). Similarly, delphinidin treatment reduced the mRNA levels of IL-1β, IL-6, and TNF-α in aged mice (Fig. 12G-H).

Fig. 12