Spatial regulation of NMN supplementation on brain lipid metabolism upon subacute and sub-chronic PM exposure in C57BL/6 mice

Abstract

Background

Atmospheric particulate matter (PM) exposure-induced neuroinflammation is critical in mediating nervous system impairment. However, effective intervention is yet to be developed.

Results

In this study, we examine the effect of β-nicotinamide mononucleotide (NMN) supplementation on nervous system damage upon PM exposure and the mechanism of spatial regulation of lipid metabolism. 120 C57BL/6 male mice were exposed to real ambient PM for 11 days (subacute) or 16 weeks (sub-chronic). NMN supplementation boosted the level of nicotinamide adenine dinucleotide (NAD⁺) in the mouse brain by 2.04 times. This augmentation effectively reduced neuroinflammation, as evidenced by a marked decrease in activated microglia levels across various brain regions, ranging from 29.29 to 85.96%. Whole brain lipidomics analysis revealed that NMN intervention resulted in an less increased levels of ceramide (Cer) and lysophospholipid in the brain following subacute PM exposure, and reversed triglyceride (TG) and glycerophospholipids (GP) following sub-chronic PM exposure, which conferred mice with anti-neuroinflammation response, improved immune function, and enhanced membrane stability. In addition, we demonstrated that the hippocampus and hypothalamus might be the most sensitive brain regions in response to PM exposure and NMN supplementation. Particularly, the alteration of TG (60:10, 56:2, 60:7), diacylglycerol (DG, 42:6), and lysophosphatidylcholine (LPC, 18:3) are the most profound, which correlated with the changes in functional annotation and perturbation of pathways including oxidative stress, inflammation, and membrane instability unveiled by spatial transcriptomic analysis.

Conclusions

This study demonstrates that NMN intervention effectively reduces neuroinflammation in the hippocampus and hypothalamus after PM exposure by modulating spatial lipid metabolism. Strategies targeting the improvement of lipid homeostasis may provide significant protection against brain injury associated with air pollutant exposure.

Background

Fine particulate matter, with an aerodynamic diameter of 2.5 microns (PM_2.5) or smaller, poses a serious threat to human health, significantly contributing to the morbidity and mortality of various diseases [1, 2]. In addition to non-communicable respiratory diseases, epidemiological studies have revealed a close correlation between atmospheric PM_2.5 exposure and impairment of the nervous system [3,4,5,6]. While several studies have identified potential interventions, such as vitamin supplementation or omega-3 intake leading to reduced systemic damage [7, 8] and the use of traditional Chinese herbs leading to mitigation of cognitive impairment [9], in response to PM exposure, the urgent need remains for the development of effective interventions.

Inflammation serves as a common link in the development and progression of neurological diseases [10]. Previous studies have demonstrated that exposure to particulate matter (PM) consistently induces neuroinflammation, which in turn leads to damage within the nervous system [11]. Both short- and long-term exposure to PM has been demonstrated to be associated with neurological impairment, including acute stroke and chronic neurodegenerative diseases [3, 12, 13]. Environmental stressors can induce alterations in lipid metabolism within the brain, thereby disrupting cellular homeostasis [14]. It has been reported that the components of PM can enter the bloodstream via alveoli, causing systemic damage and subsequently inducing brain damage through oxidative stress, inflammatory damage, apoptosis, etc [15,16,17,18]. Lipid metabolism reprogramming is pivotal for the maintenance of microenvironment stability and the function of nerve system. For instance, microglial lipid metabolism plays a significant role in regulating microglial activation and functions, including migration, phagocytosis, and inflammatory signaling. Even minor disruptions in microglial lipid processing have been linked to changes in brain function in neuroinflammatory diseases [19]. Previous studies have revealed how PM exposure disrupted brain lipid metabolism in the course of the development of neurodegenerative diseases through induction of neuroinflammation [20, 21]. This highlights the intimate connection between dysregulation of lipid metabolism and inflammation. In our previous research, we developed a whole-body PM exposure system in Shijiazhuang City, Hebei Province, China. This system replicated real-ambient conditions, providing continuous exposure for experimental animals, closely resembling natural human exposure scenarios [22]. This exposure system represents an optimal model for the study of PM-induced nervous system damage and the mechanisms underlying the regulation of lipid metabolism.

The brain is an organ rich in lipid content. Dysregulations in brain lipid metabolism play a critical role in maintenance of the structural composition, signal transduction, and energy metabolism of the nervous system [23]. Furthermore, lipidome alterations in brain have been shown to significantly impact bioenergy, oxidative stress, and inflammation, which may contribute to the development of numerous diseases, including ischemic stroke and neurodegenerative diseases [24, 25]. The brain exhibits a unique structural zoning that is attributable to spatial regulation of nervous system and determines the specificity of disease development, such as stroke [26], epilepsy [27], and neurodegenerative diseases [28]. Consequently, the examination of the spatial lipid metabolome offers insight into region-specific metabolic changes within brain and provides a comprehensive understanding of the underlying molecular mechanisms. Previous studies have revealed changes in lipid metabolism across various organs in response to PM exposure [21, 29]. However, the analysis of whole brain lipidome is unable to distinguish the specificity of metabolic changes in different brain regions due to the heterogeneity of brain tissue. It is noteworthy that significant alterations in lipid metabolism have been observed in the hippocampus of Alzheimer’s disease (AD) models [30], emphasizing the necessity of elucidating spatial regulation patterns in lipid metabolism. Mass spectrometry imaging (MSI) has proven to be a valuable tool in elucidating the spatial distribution of complex small molecules in tissue slices, particularly in studies related to lipid metabolism [31]. Desorption Electrospray Ionization-Mass Spectrometry Imaging (DESI-MSI) enables the identification of specific metabolites in terms of their spatial distribution in tissue sections without the need for molecular labeling [31, 32]. Previous studies have investigated the lipidome of both the entire brain and specific brain regions in animal models of different diseases, unveiling significant differences in lipid metabolites among regions in mice such as hippocampus, cortex, and other areas [20, 21]. MSI analysis revealed regional changes in phospholipids, sphingolipids, and neutral lipids associated with cerebral ischemia, with a particular focus on the cortex and striatum in mice [33]. Hence, our hypothesis postulates that exposure to PM triggers distinct alterations in lipid metabolism regulation in various brain regions, with some regions displaying greater vulnerability to PM exposure. The objective of this study is to analyze the spatial distribution of lipid metabolism in mice following real-world PM exposure and identify key lipid molecules that could be implicated in the damage to nervous system.

Nicotinamide adenine dinucleotide (NAD⁺) decreases with age and in the context of related diseases. It serves as a crucial metabolic redox coenzyme in eukaryotes and its content, which is attributable to regulation of a variety of biological endpoints, including neuroinflammation, oxidative stress, cell death, and DNA damage [34]. Nicotinamide mononucleotide (NMN) is the precursor in the course of NAD⁺ biosynthesis. It has been demonstrated to be able to compensate for the lack of NAD⁺ and attenuate cognitive impairment and inflammatory responses through improving energy metabolism in the nervous system [35,36,37]. Previous studies have shown that NMN supplementation improves the function of neurons and attenuates nervous system injury through reduction of inflammatory responses, oxidative stress, and cell death [38,39,40]. We have demonstrated that NMN alleviated chronic lung injury induced by sub-chronic PM exposure in mice by alteration in regulation of immune function [41]. Supplementation with NMN has been shown to elevate NAD⁺ levels in the hippocampus, cortex, and other brain regions [42, 43], indicating that NMN exerts diverse effects across different areas of the brain. Consequently, further investigation is required to elucidate the precise impact of NMN supplementation on the metabolic regulation of brain regions influenced by PM exposure.

To elucidate the spatial regulation of NMN supplementation on brain lipid metabolism in mice upon subacute or sub-chronic PM exposure, we performed lipidomics and transcriptomics analysis to characterize lipid metabolism and pinpoint the most sensitive brain regions. This study identifies key lipid molecules in distinct brain regions that undergo changes in response to both PM exposure and NMN intervention. By focusing on metabolic regulatory strategies, we elucidate the singular role of NMN supplementation in mitigating region-specific lipid disruptions. The findings offer profound insights into the regulation of spatial lipid metabolism and reveal the mechanism by which NMN intervenes in response to PM exposure. This positions NMN as a promising intervention for health issues stemming from atmospheric PM exposure.

Methods

Real-ambient PM exposure and animal experiments

Six-week-old male C57BL/6 mice were purchased from the Beijing Vital River Laboratories. The experimental design and real-time PM exposure systems are described in previous studies [41, 44]. Mice were housed in cages under a 12-h dark/light cycle with ad libitum access to food and water. The β-NMN powder, with a purity of 99.9%, was purchased from Hygieia Biotechnology Co., Ltd. (XJY01220618). Mice received β-NMN supplement that was dissolved in drinking water (freshly prepared every three days) at a concentration of 500 mg/kg two weeks prior to PM exposure and maintained throughout the experiment. Mice were kept in isolated ventilated cages (IVC) and exposed to PM in a real-ambient PM exposure system as detailed in previous study [22]. Fresh ambient air was imported into the IVC chamber, with air channels equipped with or without a three-layer HEPA filter in the control (termed as air-filtered control group, Con) and exposure groups (termed as PM exposure group, PM), respectively. Physical parameters in chambers were carefully monitored to ensure consistent temperature, humidity, ventilation frequency, air flow rate, and noise levels. The concentration of PM_2.5 in the chambers was monitored using an Aerosol Detector DUSTTRAKTM II (TSI Incorporated, Shoreview, MN, USA). The PM_2.5 concentration levels in both the exposure chamber and the ambient environment are monitored in real time. The exposure concentration within the chamber typically ranged from 60 to 70% of the atmospheric concentration, ensuring that the mice experienced real-time fluctuations and that the equipment operated effectively. Prior to the commencement of the experiment, random groups were formed based on the mice’s weight to minimize any bias resulting from group imbalances due to the mice’s physiological factors. Male mice were divided into four groups (Con-H₂O, Con-NMN, PM-H₂O and PM-NMN) and administered with either H₂O or NMN (500 mg/kg) with or without PM exposure (n = 15/per group, 5 mice/cage). The body weights were recorded weekly (Figure S1). The neurobehavioral tests were conducted four days before the mice were sacrificed. At the end of the experiments at two time points (11 days or 16 weeks), the mice were euthanized by injection of pentobarbital sodium and the biological samples were collected for subsequent analyses. To reduce variations in human judgment during the experiment, personnel were trained to follow standardized operating procedures, which might ensure consistency in the collected samples. Detailed guidelines on utilizing animal samples were provided in Table S1. All animal procedures were conducted in accordance with the guidelines estabilished by the Animal Care and Protection Committee of Sun Yat-sen University (No. 2016-09).

Neurobehavioral assessments

To evaluate both hyperactivity and anxious behavior, an open field test (OFT) was employed, utilizing a 30-cm × 30-cm × 25-cm square white box (n = 5 /per group). Each mouse was observed for a five-minute period within the box. The duration and distance traveled in the central area of the maze were recorded and analyzed to assess the degree of anxiety. The water maze analysis system consisted of a circular pool with an escape platform, equipped with cameras and computers (ANHUI ZHENGHUA, n = 5 /per group). The temperature was maintained at 22 ± 2 °C. The escape platform was positioned to submerge 0.5 cm below the surface of the water. The shortest distance from the escape platform wall is 20 cm. Prior to the commencement of the formal experiment, the mice were permitted to freely swim in the water for a period of two minutes to allow them to become accustomed to their surroundings. During the four-day training period, four consecutive training sessions of 60 s each were conducted consistently at set intervals every day. The escape latency was meticulously calculated and assessed based on the daily average training values. After the training phase, the platform was removed for conclusive testing, during which the number of platform area crossings and the time spent in the target quadrant were evaluated. Evaluation of mouse performance included the measurement of escape latency during navigation tests and the determination swimming time in different quadrants during the space exploration experiment. The performance of learning and spatial memory was quantified by the following three indexes: escape latency, platform area crossings, and time in target quadrant.

Histopathological analysis of mouse brain

Mouse brains were extracted and fixed in 4% paraformaldehyde followed by paraffin embedding. Tissue sections (5 μm) underwent immumohistochemical staining (n = 5/per group). Anti-ionized calcium binding adaptor molecule 1 (Iba1) primary antibodies were utilized at a 1:1000 concentration (Abcam; ab178846-40) for staining activated microglia. Histological examinations were performed under a light microscope (NanoZoomer^®S360, HAMAMATSU PHOTONICS) and scanned with NDP.iew (2.9.22 RUO). Quantitative analysis of Iba1⁺ cells was conducted on brain sections from 4 ~ 6 mice per group were used for, counting positive cells within a 200× fixed field area, with a minimum of 3 fields per mouse.

Cytokine analysis

Cytokines in plasma, including tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) were quantified using enzyme-linked immunosorbent assay (ELISA) assay kit (R&D Systems, MN, USA) (n = 5/per group). Absorbance at 450 nm wavelength in ELISA plates was measured via a microplate reader (BioRad, Model 550). Malondialdehyde (MDA) concentrations in mouse plasma were measured employing a lipid peroxidation MDA assay kit (Beyotime, Nantong, China), and absorbance was recorded at 532 nm using a microplate reader (BioRad, Model 550). Duplicate examinations were conducted for each sample following standard curves as the manufacturer’s specifications.

Transcriptomics analysis of hippocampus and hypothalamus

Freshly isolated mouse hippocampus and hypothalamus tissue were immediately snap-frozen for RNA sequencing using the BGISEQ-500 sequencing technology platform by MGISEQ2000 (BGI Genomics Co., Ltd, China) across four groups of mice (n = 3/per group). The BGI Optimal Dual-mode mRNA Library Prep Kit was used to construct the mRNA library. The initial filtration of raw data was conducted using the SOAPnuke (v1.5.2) program. The clean reads were then aligned to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was utilized to align the clean reads to the reference coding gene set (version: GRCm38), after which gene expression levels were calculated using RSEM (v1.2.12). Differentially expressed genes (DEGs) were identified based on a fold change greater than 1.5 and an adjusted P value (q value) less than 0.05, as determined by Bonferroni analysis (Table S2-S5). DEG comparisons were conducted with and without PM exposure (PM- H₂O vs. Con-H₂O) or with and without NMN intervention (PM-NMN vs. PM-H₂O). To unravel the biological implications of DEGs between groups, the canonical pathway analysis and the disease and biological function analysis were carried out using Ingenuity Pathway Analysis (IPA) software (Qiagen, Germany), with a significant threshold set at P < 0.05.

Lipidomics analysis

Approximately 20 mg of brain samples from four groups of mice (n = 5/per group) were homogenized in 300 µL of cold methanol and 1 mL of methyl tert-butyl ether (MTBE) was added, followed by 30 min of rotation at room temperature. Subsequently, 200 µL of distilled de-ionized water (DDI) was introduced to facilitate phase separation. After a 5 min shaking period and a 15 min centrifugation at 10,000×g at 4 °C, the upper MTBE layer was collected, dried and reconstituted in 200 µL of acetonitrile (ACN)/isopropanol (v/v, 7:3) for instrument analysis. A pooled quality control (QC) sample, combining equal aliquots from each sample, was inserted into the analysis sequence at regular intervals, following the analysis of eight samples. The data were collected via ultra-performance liquid chromatography coupled with a Q-Exactive Orbitrap Mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) with data-dependent MS/MS acquisition were processed using LipidSearch 4.0 software (Thermo Fisher Scientific, San Jose, CA, USA) for peak picking, identification, and alignment. The identification results were screened by the grade (A: Lipid of which fatty acid chains and class were identified completely; B: Lipid of which class and some fatty acid chains were identified; C: Lipid of which class or fatty acid was identified; D: Others). In the results, we have seamlessly integrated three levels of ABC lipid molecule identification for further analysis, ensuring a high standard of data quality. Detailed LC and MS parameters were set as described previously [45]. The peak intensity and identity information exported from LipidSearch software were used for multivariate statistical analysis. Lipids with a P less than 0.05 and a fold change greater than 1.5 were regarded as differential lipids. Comparative analysis was performed between groups to assess the effects of PM exposure (Con vs. PM) and NMN supplementation (H₂O vs. NMN) by MetaboAnalyst5.0 (https://www.metaboanalyst.ca/).

DESI-MSI analysis

Mouse brain tissues were excised, immediately snap frozen in liquid nitrogen, and stored at − 80 °C in Con-H₂O, PM-H₂O, Con-NMN, and PM-NMN groups (n = 5/per group). The frozen brain tissues were sliced (10 μm thick), thaw-mounted onto a big glass slide (128 mm × 85 mm), and dehydrated for 30 min in a vacuum chamber. A minimal optimal cutting temperature compound (OCT) compound was applied to affix the specimens during frozen section preparation. A 2D Omni Spray Stage (Prosolia, Indianapolis, IN) was used for MSI analysis with a spatial resolution of 100 μm. Instrument setting parameters in detail were described previously [33]. DESI-MSI was performed in positive and negative ion modes at m/z 100–1200 with a resolution of 20,000 using a Synapt G2-Si qTOF-MS (Waters, Wilmslow, UK). Optimal MS sensitivity was achieved through MS inlet cleaning and source tuning prior to imaging analysis. Additionally, any changes in sensitivity were evaluated both before and after the imaging analysis. The top 1000 peaks, ranked by descending peak intensity, were extracted from the entire m/z range at 20,000 mass resolution using High Definition imaging (HDI) 1.5 software (Waters) and no lock mass correction was applied. Spectra were normalized by TIC based on the base peak intensity, and ion images were reconstructed using the same software. The m/z peaks were identified by aligning with the laboratory’s custom lipid database, which was created based on the theoretical values of various lipid adduct ions. Identification results are detailed in Table S6.

To quantify lipid changes, differences were specified based on Log₂ |FC|, with the following delineations: 0 ~ 0.05 defined as no significant change (“/”), 0.05 ~ 0.10 defined as “+/-”, 0.10 ~ 0.20 defined as “++/--” and greater than 0.2 defined as “+++/---”, leading to identification of key brain regions and lipid species by quantifying the effects of PM exposure (PM-H₂O vs. Con-H₂O) and NMN supplementation (PM-NMN vs. PM-H₂O) (Table S7).

Statistical analysis

Data are presented as the mean ± SEM. All statistical analyses were performed with SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA). Student’s t-test was applied for comparisons between two groups, whereas one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test was used for comparisons between multiple experimental groups. Differences were considered significant at P < 0.05. The P-value summary of the variations among the effect indicators and the evaluation of statistical power (1-β) are presented in Table S8. PLS-DA results were obtained by MetaboAnalyst (https://www.metaboanalyst.ca/) for four groups (Con-H₂O, PM-H₂O, Con-NMN, and PM-NMN) at different time points of PM exposure. All images were analyzed by Prism 9 (GraphPad Software Inc., California, USA) and Mastergo (https://mastergo.com/).

Results

NMN supplementation alleviates nervous system impairments induced by subacute and sub-chronic PM exposure

The daily mean concentration of PM_2.5 during the exposure period (16 weeks) was previously reported using a whole-body PM inhalation system from November 27th, 2019 to March 26th, 2020 with a comprehensive component analysis [44]. Specifically, we estabilished two time points for sample collection and analysis. 120 C57BL/6 male mice were divided into four groups (Con-H₂O, PM-H₂O, Con-NMN, and PM-NMN groups) and exposed to real ambient PM for 11 days (subacute) or 16 weeks (sub-chronic). Control mice (Con group) were housed in chambers equipped with three layers of HEPA filters, which provided an excellent barrier to ambient fine PM (PM₁ = 0). As shown in Figure S2, T1 (at day 11 after PM exposure) and T2 (at 16 weeks after PM exposure) represented typical exposure modes of subacute and sub-chronic PM exposure. The daily mean concentrations of PM_2.5 in ambient air and exposure chamber were 124.38 µg/m³ and 72.71 µg/m³, respectively at T1, and 123.52 µg/m³ and 73.94 µg/m³, respectively at T2. Thus, the mice in the exposure chambers received a high concentration of PM exposure that was truly representative of human PM exposure in the most polluted regions of China.

As a precursor of NAD⁺, NMN supplementation was administered via drinking water at 500 mg/kg body weight per day two weeks prior to PM exposure and maintained throughout the experiments. NMN supplementation efficiently elevated NAD⁺ levels by 2.04-fold (Figure S3). We evaluated the effect of NMN on PM exposure-induced systemic damage (Fig. 1). Subacute PM exposure induced significant inflammatory reactions and oxidative stress, while sub-chronic exposure did not show statistically significant differences. NMN supplementation in mice reduced cytokine and oxidative stress indicators to levels comparable to the unexposed group (P > 0.05) after subacute exposure. This suggests that NMN can mitigate inflammation and oxidative stress caused by subacute PM exposure and may reduce chronic inflammation caused by prolonged exposure. To investigate the effect of NMN supplementation on neuroinflammation induced by subacute or sub-chronic PM exposure, we examined microglial activation in brain tissue slices labeled with anti-iba1 antibody. As shown in Fig. 2A & Figure S4, microglial activation was more pronounced upon sub-chronic PM exposure compared to subacute exposure. Notably, the number of activated microglia in hippocampus significantly increased following subacute PM exposure, with 1.25-fold higher than that of the control (n = 5/per group, P < 0.05), but was alleviated by 27.29% in PM-NMN group (Fig. 2B). Similarly, the number of activated microglia in the hippocampus, internal capsule, thalamus, and hypothalamus of sub-chronically exposed mice were increased by 1.34-, 2.02-, 1.28-, and 1.46-fold (n = 5 /per group, P < 0.05), respectively, but reduced by 29.83%, 85.96%, 30.24%, and 39.88% upon NMN supplementation, respectively (P < 0.05, Fig. 2B-E). These results indicate that the differential neuroinflammatory response of different brain regions induced by PM exposure and NMN supplementation might underlie the spatiotemporal regulation in sensitivity of neuroinflammation and the extent of neurological impairment. The levels of inflammation-related genes in key brain regions further supported these findings (see Figure S5). After sub-chronic exposure, several inflammation-related genes were upregulated in the hippocampus and hypothalamus. However, this upregulation was less pronounced in the PM-NMN group compared to the PM-H₂O group, suggesting that NMN supplementation may indeed have a mitigating effect on the inflammatory response induced by PM exposure. Next, we assessed the effects of NMN supplementation on neurobehavioral abnormalities linked to anxiety and learning and memory impairments. Results from the water maze test indicated a 1.67- or 1.83-fold increase in escape latency upon subacute or sub-chronic PM exposure, indicating a deficit in learning and memory performance (Fig. 3A). Similarly, sub-chronic PM exposure resulted in a 59.18% (P < 0.01) reduction in platform area crossings and a 35.47% (P < 0.05) reduction in quadrant time, both of which were significantly reversed by 39.09 ~ 63.72% (P < 0.05) in PM-NMN group (Fig. 3A-C). In the open field test, we found that the overall distance, route in the central squares, and time in central squares were decreased by 33.62%, 37.18%, and 42.10%, respectively in mice sub-chronically exposed to PM compared with those of the controls, indicating that negative emotion of anxiety might present in mice with sub-chronic PM exposure (P < 0.05) (Fig. 3D-F). Significantly, NMN effectively reduced anxiety-related indicators by 35.71 to 42.41%. Overall, NMN supplementation alleviated neuroinflammation induced by sub-chronic PM exposure, leading to an improvement in neurobehavioral functions.

Fig. 1

Effects of NMN supplementation on systemic responses upon subacute and sub-chronic PM exposure. Legends Systemic effects about the inflammatory and oxidative responses in mice upon subacute and sub-chronic PM exposure were examined by measuring the plasma levels of (A) IL-6, (B) TNF-α, and (C) MDA. The results were presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. Con-H₂O: air-filtered control group administered drinking water; Con-NMN: air-filtered control group supplemented with NMN; PM-H₂O: PM exposed group administered drinking water; PM-NMN: PM exposed group with NMN supplementation

Fig. 2

Effects of NMN supplementation on microglial activation across various mouse brain regions upon subacute and sub-chronic PM exposure. Legends (A) Representative images (20×) depict the impact of sub-chronic PM exposure on specified brain regions, with enlarged images illustrating typical microglial morphology in the lower right corner. Quantification of iba1-labeled cells is presented across multiple brain regions, including the (B) hippocampus, (C) thalamus, (D) hypothalamus, and (E) internal capsule. The results were presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

Impacts of NMN supplementation on neurobehavioral alterations in mice following subacute and sub-chronic PM exposure. Legends Neurobehavioral assessments were conducted following subacute and sub-chronic PM exposure using the tests of open field and the Morris maze. Various indices, including (A) escape latency, (B) platform area crossings, (C) time in target quadrant, (D) time in central squares, (E) route in the central squares, and (F) overall distance, were examined and analyzed. The results were presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001

Effects of NMN supplementation on lipid metabolism of mouse brain in response to subacute or sub-chronic PM exposure

Next, we investigated the effects of NMN supplementation on brain functions through regulation of lipid metabolism. We performed lipidomics analysis on homogenates of coronal brain tissue slices and detected lipids in 34 subclasses under positive and negative ion modes. The analysis revealed profound changes in lipid components, including ceramide (Cer), monoglycosylceramide (Cer G1), lysophosphatidylcholine (LPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and triglyceride (TG). Remarkably, distinct variations in lipid profiles were observed under various treatment conditions, as depicted in the partial least squares-discriminant analysis (PLS-DA) score map (Figure S6). NMN supplementation reduced the levels of DG and LPC, which are associated with subacute or sub-chronic PM-induced inflammation and increased various glycerophospholipids linked to membrane stability (Figure S7). Collectively, these results suggest the potential involvement of NMN supplementation in lipid metabolism regulation, thereby enhancing the ability to resist external stimuli such as neuroinflammation.

As shown in Fig. 4A, we summarized the specific lipid perturbations, of which the content of Cers and lysophospholipids increased, accompanied by a decrease in multiple glycerol phospholipids following subacute PM exposure. A similar reduction in glycerophospholipids and increase in TG levels appeared in mouse brain following sub-chronic PM exposure, indicating a decreased membrane stability. The effects of NMN supplementation on regulation of lipid metabolism were characterized by a significant decrease in content of Cer or TG levels in mouse brain following subacute or sub-chronic PM exposure. In addition, increased glycerophospholipids (PCs and PEs) after both subacute and sub-chronic PM exposure implicated a beneficial impact on membrane stability. In summary, NMN supplementation promoted membrane stabilization and alleviated neuroinflammation by rectifying lipid perturbations, which might confer mice with resistance to PM exposure-induced inflammation and stress.

Fig. 4

Effects of NMN supplementation on spatial brain lipid profiles in mice following subacute and sub-chronic PM exposure. Legends (A) The variations in lipid metabolic profiles in homogenized brain tissue following subacute (left) and sub-chronic (right) PM exposure are illustrated in heatmaps, displaying the alterations in the contents of key differential lipids. (B) Heatmaps were generated utilizing relative log₂FC value of DESI-MSI data from PM-H₂O/Con-H₂O and PM-NMN/PM-H₂O, encompassing TG, DG, SM, LPC, PI, PS, PE, and PG in indicated groups of mice. The trends of increase or decline were visually indicated by the colors red or green, respectively, with the depth of color representing the magnitude of the changes

The hippocampus and hypothalamus are critical brain regions upon PM exposure and NMN intervention

Next, we investigated the role of NMN intervention in spatial lipid regulation in response to PM exposure by DESI-MSI. As illustrated in Fig. 4B with schematic diagrams of coronal brain slices, we delineated 5 brain regions, cortex, thalamus, hypothalamus, internal capsule, and hippocampus with distinct colors based on whole brain lipidomics analysis. Through high-resolution mass spectrometry in positive and negative ion modes, we identified a total of 516 lipids, which were categorized into 16 types, including fatty acid (FA), monoglyceride (MG), diacylglycerol (DG), TG, SM, PC, phosphatidic acid (PA), PE, PG, PI, PS, LPC, lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lysophosphatidylinositol (LPI), and lipopolysaccharide (LPS) (Table S6). We analyzed the lipid profiles under the condition of PM exposure or NMN supplementation (Con-H₂O vs. PM-H₂O or PM-H₂O vs. PM-NMN). The quantitative lipid values were transformed into Log2FC in specific regions labeled with different colors as shown in Fig. 4B. We assigned the value of Log₂ |FC| to indicate the degree of altered lipid species and identified the key brain regions in response to PM exposure (Con-H₂O vs. PM-H₂O) and NMN supplementation (PM-H₂O vs. PM-NMN). As shown in Table S7, the difference was scaled as follows: 0 ~ 0.05 defined as no significant change (“/”), 0.05 ~ 0.10 defined as “+/-”, 0.10 ~ 0.20 defined as “++/--“, and greater than 0.2 defined as “+++/---“. These results showed that the increase in DG and TG content occurred in multiple brain regions and the decrease in SM appeared in all brain regions except cortex following subacute PM exposure. In contrast, sub-chronic PM exposure yielded diverse lipid alterations. Increased DG and LPC and decreased PI and PS appeared in all brain regions. In addition, decreased SM was observed in cortex, internal capsule, and thalamus, while decreased PA, PE, and PG were observed in hippocampus, cortex, and internal capsule. These results indicate a potential impact on membrane structure and functions with prolonged PM exposure based on the result of reduced glycerophospholipids. Interestingly, the changes of DG and SM displayed the same perturbations trends in both subacute and sub-chronic PM exposure groups, which could be recognized as the critical lipids responsive to PM exposure. These findings unveiled that both subacute and sub-chronic PM exposure led to perturbation of spatial lipid metabolism with different patterns, which were characterized by increased TG and DG and decreased glycerophospholipids. Significantly, NMN supplementation not only reversed dysregulation of TG, DG, and LPC induced by PM exposure, but also augmented various glycerol phospholipids, which might be linked to the enhanced neuroinflammation enhanced by PM exposure.

To pinpoint critical brain regions affected by PM exposure and influenced by NMN intervention, we tallied the number of altered lipid classes in different brain regions (Table S9). Both subacute and sub-chronic exposures resulted in extensive lipid remodeling in several brain regions, with NMN reversing this trend in multiple brain regions under both exposure durations. However, the effect of NMN on spatial lipid perturbations varied across brain regions between subacute and sub-chronic exposure scenarios. The lipid perturbations in hippocampus, thalamus, and hypothalamus following subacute PM exposure were relieved with more than four kinds of lipid change upon NMN supplementation, suggesting that these three regions might be the most sensitive to subacute PM exposure. In contrast, the hippocampus, hypothalamus, and internal capsule appeared to be the most sensitive regions in response to sub-chronic PM exposure. Notably, NMN supplementation reversed a trend of lipid perturbations with 4 ~ 8 lipid subtypes involved in these regions. These results reinforce the notion that NMN supplementation reverses the disturbances of spatial lipid metabolism induced by both subacute and sub-chronic PM exposure. Specifically, hippocampus and hypothalamus emerge as key brain regions where NMN supplementation combats PM-induced neuroinflammation and dysfunction.

The changes of spatial lipid metabolism are closely linked to pathway regulations and biological functions

To further identify the major lipid classes affected by PM exposure and NMN supplementation, we counted the number of differential lipids of the same lipid class in the same brain region, adhering to the limits of FC greater than 1.5 and P less than 0.05 (Fig. 5A). TG exhibited significant elevation in multiple brain regions under subacute PM exposure and could be reversed by NMN with the most affected region in hippocampus. Significantly, certain molecular species of triglycerides (TG), like TG (60:10) and TG (56:2), exhibited elevated levels in the hippocampus, whereas TG (60:10) and TG (60:7) were increased in the hypothalamus (Figure S8). NMN supplementation counteracted alterations in various lipids, including TG (60:10), TG (56:2), and TG (60:7) in hippocampus and hypothalamus upon subacute PM exposure. Profound changes in lipid metabolism were evident in hippocampus, hypothalamus, and internal capsule, implicating their high sensitivity to sub-chronic PM exposure. Notably, the increased DG and LPC in several brain regions during both subacute and sub-chronic PM exposure were related to mRNA levels of critical metabolic enzymes associated with reduced DG degradation and increased LPC synthesis, suggesting that the changes in DG and LPC levels might be involved in regulation of inflammatory activation under cumulative PM exposure. DGAT and DGK serve as pivotal enzymes in the degradation of diacylglycerol (DG), and their activities were notably increased after NMN supplementation, implying a reduction in DG accumulation. At the same time, phospholipase A2 (PLA2) plays a crucial role in synthesis of lysophosphatidylcholine (LPC). The marked rise in PLA2 levels following PM exposure suggests elevated LPC content, which subsequently declines following NMN supplementation, indicating the potential of NMN supplementation to reduce LPC levels. (Fig. 5B). Moreover, specific molecular changes, such as elevated DG (42:6) in internal capsule and LPC (18:3) in the hypothalamus, may denote region-specific proinflammatory effects (Fig. 5C). Multiple differential glycerophospholipids decreased in the hippocampus and hypothalamus under sub-chronic exposure but raised by 18.45 ~ 19.72% in PM-NMN group. Similar trends were observed, such as decreased PG (38:8) in internal capsule and hippocampus and reduced PE (44:12) and PI (34:1) in hippocampus (Fig. 5C, P < 0.05). Importantly, NMN intervention reversed these specific lipid changes to varying degrees. Given the crucial role of glycerophospholipids in maintaining a stable microenvironment and alleviating local inflammation, these observations indicate that the distinct lipid profile altered by NMN intervention might predispose mouse brains with resistance to PM exposure-induced inflammation through regulation of key lipid molecules at brain regions, particularly in hippocampus and hypothalamus.

Fig. 5

Visualizing and quantifying spatial regulation of specific lipid metabolites in mice following subacute and sub-chronic PM exposure. Legends (A) Quantification of differential lipids in different brain regions (hippocampus, hypothalamus, thalamus, and internal capsule) in the indicated groups of mice upon subacute or sub-chronic PM exposure. The trends of increase or decline were visually indicated by the colors red or green, respectively, with the depth of color representing the magnitude of the changes. (B) mRNA levels of metabolic enzymes related to DG and LPC synthesis and degradation in the hippocampus and hypothalamus from transcriptomic results. Differential lipids in critical brain regions are presented using relative log₂FC values obtained from DESI-MSI results for (a) PM-H₂O/Con-H₂O and (b) PM-NMN/PM-H₂O. (c) Representative images illustrating the indicated groups were visualized, showcasing specific lipid molecular species and the corresponding brain region along with the respective m/z value

To validate the effects of PM exposure and NMN supplementation on key brain regions, we isolated the hippocampus and hypothalamus from mouse brains and conducted transcriptome analysis following sub-chronic PM exposure. Comparative analysis revealed a parallel pattern of pathway regulations in hippocampus and hypothalamus (Table S10). Inflammatory pathways were activated in both two brain regions, which was consistent with the decline of multiple glycerides in the hippocampus and the increase of LPC in the hypothalamus. However, the regulatory mechanisms underlying NMN supplementation differed between hippocampus and hypothalamus (Fig. 6A). In particular, the inflammatory pathways including CREB Signaling in Neurons, S100 Family Signaling Pathway, FAK Signaling, and Phagosome Formation were inactivated in the hippocampus following NMN supplementation. In contrast, enrichment pathways in hypothalamus were primarily related to the regulation of adaptive immune responses including T cell receptor signaling, PKCθ signaling in T lymphocytes, Th1 pathway and Th2 pathway.

Fig. 6

Effects of NMN supplementation on the regulation of canonical pathway and disease and bio-functions in hippocampus and hypothalamus upon PM exposure. Legends Transcriptional results unveiled (A) regulation in canonical pathways and (B) the annotation of disease and biological functions in both hippocampus and hypothalamus, presented as relative values derived from PM-H₂O/Con-H₂O or PM-NMN/PM-H₂O comparison upon sub-chronic PM exposure. The length of the target signifies the significance of the P value, where a longer target corresponds to a smaller P value. The depth of the color indicates the magnitude of the Z-score, with darker shades of red representing higher Z-scores

Next, we analyzed disease annotation and biological function modules within both brain regions in response to PM exposure and NMN supplementation. As depicted in Fig. 6B, PM exposure triggered microglial activation and inflammatory responses in the hippocampus. The hypothalamus, on the other hand, is instrumental in regulating emotional behavior, lipid metabolism, as well as cell death. After NMN supplementation, remarkable effects on cell differentiation, cell migration and immune response were observed in the hippocampus. However, in the hypothalamus, the impact on cell viability and cell survival pathways were more pronounced. These results suggest that PM exposure may induce inflammation and cellular deterioration in both regions. The effect of NMN on the two brain regions was different, which may inhibit the inflammatory effect of the hippocampus and regulate the adaptive immune response of the hypothalamus.

Finally, we analyzed the correlation between the altered specific lipids in the hippocampus and hypothalamus and the functional consequences of neurological impairment (Fig. 7). Three specific phospholipids, PG (38:8), PI (34:1), and PE (44:12) in hippocampus exhibited a negative correlation with the expression of inflammatory genes (r < -0.96), but a positive correlation with the neurobehavioral deficits (r > 0.96). These results indicated a close relationship between these specific lipids in hippocampus and regulation of emotional and memory functions. In parallel, the specific lipid LPC (18:3) in the hypothalamus show a positive correlation with the degree of inflammatory activation (r = 0.969 ~ 0.988) and a negative correlation with neurobehavioral deficits in open field test (r = -0.995 ~ 0.982). This implies that alteration in LPC (18:3) may be involved in emotional regulation within hypothalamus. Token together, these findings unveil the potential of distinct lipid profiles in different brain regions in mediating inflammatory activation and regulation of neurobehavioral functions.

Fig. 7

Correlation analysis between critical lipids and neurological damage indicators in hippocampus and hypothalamus upon PM exposure. Legends (A) The correlation between the key lipids PG (38:8), PE (44:12), PI (34:1), activated microglia (iba1⁺), inflammatory genes (Casp1, Ccl27b, Cxcl5, Nlrc5, S100a8, S100a9, Tnfrsf11a, and Tnfrsf11b), and neurobehavioral functions indicators from open field tests (Time in central squares, Route in central squares, Overall distance) and water maze tests (Escape latency and Platform area crossings) in hippocampus. (B) The correlation between the key lipids LPC (18:3), activated microglia (iba1⁺), inflammatory genes (Casp1, Ccl27a, Ccl27b, Il18rap, S100a8, S100a9, Tnfrsf11a, and Tnfrsf11b), and neurobehavioral functions indicators from open field tests (Time in central squares, Route in central squares, Overall distance) and water maze tests (Escape latency and Platform area crossings) in hypothalamus. Circle size reflects the correlation coefficient magnitude, with red indicating positive correlations and blue representing negative correlations

Discussion

Perturbations in lipid metabolism induced by PM exposure potentially contribute to nervous system impairments through regulation of neuroinflammation. Yet, the efficacy of interventions against PM exposure mediated by lipid metabolism in brain remains elusive. In this study, we demonstrate that NMN supplementation effectively attenuates neuroinflammation and impaired neurobehavioral functions induced by real ambient PM exposure. We revealed distinct lipidome patterns related to subacute and sub-chronic PM exposure. Remarkably, we pinpointed hippocampus and hypothalamus as the most sensitive brain regions in response to PM exposure and NMN intervention using DESI-MSI analysis. Specifically, we identified that critical lipid molecules, TG (60:10, 56:2, and 60:7), DG (42:6), and LPC (18:3), could potentially be involved in mediating neuroinflammation that contributes to the PM exposure-related impairment of nerve system. Additionally, functional annotation from transcriptomic profile further strengthens the notion that NMN supplementation plays an important role in regulating spatial lipid metabolism. The discrepancies observed in lipid metabolism, neurofunctions, and pathway regulation between two brain regions might be attributable to their varying responses to PM exposure. These findings uncover the novel effects of NMN supplementation against PM-induced neuroinflammation and offer a promising avenue for population-based intervention against PM exposure.

A 10-day inhalation exposure period was classified as subacute exposure [46], while a 16-week inhalation exposure period was classified as sub-chronic exposure. Previously we reported that PM exposure induced the histopathologic changes of mild chronic brain injury upon 12-week PM exposure [22]. The systemic response increased significantly at the subacute time point, far exceeding the sub-chronic exposure time frame, suggesting that the mice were in a state of subacute reaction following high-dose PM exposure. The selection of NMN dosage (500 mg/kg) is derived from population-based clinical studies and species extrapolation. Previous research indicates that a daily supplementation of 250–1250 mg NMN (equivalent to 4.17–20.83 mg/kg/day for a 60 kg adult) is the current standard clinical dose [47,48,49] and a conversion of the equivalent dose from human to mouse was 231.67 ~ 1157.41 mg/kg [50, 51]. Furthermore, consistent with previous findings from a 90-day NMN drinking intervention in rodents, the intervention dose can successfully increase NAD⁺ levels in various organs of mice without eliciting any adverse health effects [52].

While previous studies have demonstrated the neurotoxicity of PM exposure, little is known about the sensitivity of brain regions and the regulation of corresponding spatial lipid metabolism. PM exposure has been linked to induction of neuroinflammation in specific brain regions, particularly the hippocampus [53]. Behavioral abnormalities associated with hippocampal inflammatory response have been reported previously [53,54,55]. In addition, perturbations of lipid metabolism in cortex have been observed in mice following PM exposure [56]. Enhanced inflammation has been reported in rat striatum upon PM exposure [57] and similar effects have been observed in the rat cerebellum when exposed to burning smoke [58]. Despite numerous experiments highlighting the spatial effects of PM exposure on brain, identifying the brain regions most sensitive to PM exposure remains a challenge. In this study, we demonstrate that the hippocampus and hypothalamus are the most critical brain regions responding to PM exposure-induced neuroinflammation as indicated by microglial activation. Confirmatory neurofunctional alterations further validate the sensitivity of these brain regions.

Previous studies have revealed that dysregulation of lipid metabolism might be involved in mediating PM exposure-induced injury to the nervous system [20, 21]. Increased glycerides and decreased glycerophospholipids have emerged as significant features of lipid metabolism related to PM exposure-induced neurotoxicity. In a mouse model of Alzheimer’s disease (AD), PM exposure led to reduced content of glycerophospholipids and increased content of DG in hippocampus. These changes were thought to be associated with increased energy requirements, membrane composition, oxidative stress, and cell signaling pathways that play an important role in the development of AD [20]. In addition to similar changes in glycerophospholipids and DG, we found a significant alteration in content of LPC in multiple brain regions. Taken together, we have identified that dysregulation of these molecules including glycerophospholipids, DG, and LPC, may be associated with neuroinflammatory responses induced by PM exposure.

Alterations in sphingolipid metabolism emerge as particularly sensitive indicators of nervous system damage, closely intertwined with brain inflammation. As the core of sphingolipid metabolism, Ceramides (Cer) are key lipotoxic players involved in metabolic disorders [59]. Previous studies have reported the negative impact of Cer in the brain of neurodegenerative disease, precipitating inflammatory response, oxidative stress, and cellular apoptosis [60, 61]. Lipidomics analysis of A549 cells treated with PM for 24 h revealed a notable accumulation of Cer in lipid metabolism disorder [62]. Similarly, the lipidomics analysis following subacute exposure unveiled a surge in Cer levels that may be closely related to acute systemic response as well as regulation of lipid homeostasis, marking it as a distinctive lipid signature upon PM exposure.

The application of MSI technology has been widely used to visualize the spatial distribution and quantify specific lipid metabolites. This study unveiled region-specific lipid responses during subacute and sub-chronic exposures. Particularly, significant changes in TG molecular forms (60:10, 56:2, and 60:7) were identified in hippocampus, hypothalamus and thalamus following subacute PM exposure. The accumulation of neutral lipids such as TG has been identified as an early biomarker in cellular models linked to α-syn-associated neurodegeneration [63], as well as in contexts such as acute traumatic brain injury and chronic stress in mice [64]. Elevated TG levels of lipid accumulation could increase levels of local inflammatory response and oxidative stress, which are associated with activation of microglia and astrocytes. TG accumulate predominantly within glial cells in the form of lipid droplets. Research has confirmed that exposure of neurons to conditioned medium with a high concentration of lipid droplet content increases tau phosphorylation and neuronal apoptosis [65], suggesting that increased lipid droplets in glial cells can lead to neuronal cytotoxicity. On the other hand, increased lipid droplet formation and the beta-oxidation of fatty acids in astrocytes are key mechanisms to remove lipid toxicity and thereby protect neurons. Importantly, during hippocampal inflammation, elevated levels of free fatty acids can trigger astrocyte activation. Following the inhibition of mitochondrial oxidative phosphorylation, lipid droplet accumulation persists, leading to increased oxidative stress through activation of neuronal fatty acid oxidation, ultimately resulting in neuronal damage and death [66]. Furthermore, an increase in TG associated with pathological injury can be observed in the brain tissue of both AD and Parkinson’s disease (PD) patients. It has been suggested that lipid droplets containing TG as the major component play a role in brain tissue lipid homeostasis, and it has been proposed that the onset of neurodegenerative diseases may be induced by protein-induced lipodystrophy [67]. In this study, we reveal that TG accumulation may be involved in subacute exposure-induced nervous system damage, possibly by affecting lipid droplet homeostasis to promote inflammation via microglial activation. The specific TG molecules screened likely reflect the specificity of brain regions that respond to environmental stress. This study concurrently illustrated that NMN supplementation increased the levels of specific TG molecules in critical brain regions. The consensus that NMN improves mitochondrial energy metabolism could be pivotal in mitigating TG lipid toxicity in the nervous system. Moreover, we noted significant increases in DG and LPC across multiple brain regions after subacute and sub-chronic PM exposure. Lipids exhibit high sensitivity to local microenvironmental conditions and serve as crucial second messengers within nervous system, dynamically responding to real-time conditions through on-demand production [68,69,70]. The plasma membrane, enriched in sphingomyelin and phospholipids, especially PC, is not only the major structural component of mammalian membrane, but also acts as a vital source of lipid second messengers. Lipid mediators play a critical role in brain inflammation induced by exposure to environmental pollutants [71]. In particular, the increase in DG was significantly more pronounced in various brain regions after sub-chronic exposure compared to subacute exposure, suggesting a cumulative effect over time. Furthermore, lipidomics focusing on DG has been validated in both the cortex and plasma of AD patients [72]. Local inflammation associated with PM exposure may activate phospholipase C, leading to PC hydrolysis and subsequent increases in DG and LPC, with concomitant decreases in PC levels, a scenario conducive to increases in inflammatory mediators DG and LPC [73]. It has been reported that LPS-treated microglia exhibit high levels of LPC and DG, with these changes potentially mitigated by anti-inflammatory agents [74]. This highlights the potential contributions of DG and LPC to neuronal dysfunction and their role as potential biomarkers of brain inflammation [75]. We specifically investigated DG (42:6) and LPC (18:3), which may play a significant role in regulating inflammation in specific brain regions. This study showed that NMN supplementation effectively regulated the activity of metabolic enzymes associated with DG and LPC, leading to a reduction in the production of inflammatory signaling molecules and maintaining membrane stability. This mechanism ultimately promotes improved nervous system function by supporting cellular homeostasis. However, more research is needed to fully understand the complex interactions between NMN and lipid metabolic enzymes in the synthesis of specific lipids. In recent decades, great efforts have been made to develop effective interventions to mitigate the adverse health effects of PM exposure. B vitamin supplementation has been shown to attenuate PM-induced inflammation and oxidative stress [7]. Supplement of polyunsaturated fatty acids ameliorated increased plasma oxidative stress levels in elderly individuals exposed to PM [8]. NMN is known to have anti-aging activity and biological activity related to NAD⁺ recovery through improving mitochondrial function, reducing inflammation and oxidative stress, and promoting DNA damage repair [37, 76]. NMN plays an important role in regulating mitochondrial fragmentation and reactive oxygen generation, and improving energy metabolism and oxidative stress, thus effectively alleviating acute ischemic brain injury in mice [39]. In this study, we found that NMN supplementation effectively alleviated lipid metabolism perturbations induced by PM exposure, mainly by relieving neuroinflammation. In addition to ameliorating PM-induced changes in critical inflammatory lipid mediators, NMN supplementation induced uniform changes in several other lipids across all brain regions. Specifically, the levels of LPI in five brain regions following subacute and sub-chronic PM exposure seemed to increase notably with NMN supplementation. The increase corresponded to a significant enhancement in microglial activation across all brain regions, suggesting a potential neuroprotective role of LPI in regulating microglial function. Previous reports indicate that LPI hinders microglial phagocytosis via G protein coupled receptor 55 (GPR55)-dependent pathway and suppresses NO production via GPR55-independent pathway, exhibiting an anti-inflammatory effect in cultured activated microglia [77]. GPR55 is widely expressed in the nervous system, and LPI is its endogenous ligand, which is involved in various brain functions such as social interaction, memory, and anxiety [78,79,80,81]. Additionally, GPR55 has been shown to influence barrier susceptibility by modulating T lymphocyte migration [82]. Under physiological conditions, T cell infiltration into the brain parenchyma is rare, but the reciprocal movement of T lymphocytes between tissue fluid and blood is possible. Research has substantiated that lymphocyte migration establishes a connection between the brain and immune system, underscoring neurons’ capacity to modulate immune responses through innervation of lymphatic organs in the context of central nervous system diseases [83]. The observed impact of NMN supplementation on lymphocytes in this study suggests its involvement in the regulation of immune integrity, potentially mediated by the interaction between GPR55 and LPI, which represents a key mechanistic pathway. Taken together, we demonstrate that NMN could offer neuroprotection by regulating inflammatory lipid mediators and immune homeostasis.

Differential lipid alterations were evident under subacute and sub-chronic exposure, revealing distinct regional responses. The hippocampus responds to inflammation and oxidative stress induced by acute stimuli, such as hypoxia and ischemic injury [84, 85]. Exposure to high concentrations of PM leads to significant inflammation of brain tissue [86]. Therefore, subacute PM exposure induced significant lipid changes in hippocampus, which reflected significantly increased pressure in the local microenvironment and induced activation of microglia. On the other hand, the internal capsule, which functions as a critical junction between brain regions, brainstem, and spinal cord, remains relatively elusive in relation to environmental exposure. In this study, we reveal that sub-chronic PM exposure caused pronounced lipid perturbations in the internal capsule, potentially associated with neurobehavioral abnormalities. These differences in sensitive brain regions between subacute and sub-chronic exposure imply varying biological consequences. Subacute exposure appears reliant on stressors such as hippocampal inflammation, whereas sub-chronic exposure may affect a wider range of brain tissues through the internal capsule.

However, there are some limitations to this study. Although we utilized mice to replicate potential population exposure scenarios to particulate matter through the respiratory system, it is important to interpret these findings with caution given the inherent variances between mouse and human biology. However, it is clear that these findings provide valuable insights that can guide adjustments in public health policies. For a comprehensive evaluation of response variation across brain regions, a more detailed exploration of the individual responses of different brain regions in conjunction with the metabolome of a specific brain region is required. It is critical to distinguish the distinct metabolic profiles of each brain region. While we have identified significant brain regions and key lipids that respond to different modes of PM exposure and NMN supplementation, unraveling the complex mechanisms linking altered lipids and neurological damage will require additional research.

Conclusion

In this study, we investigated alterations in lipid profiles across various brain regions following subacute and sub-chronic exposure to PM, highlighting the importance of the hippocampus and hypothalamus in PM exposure and NMN supplementation. Additionally, we identified essential lipids linked to these regions, such as TG, DG, and LPC. NMN has the potential to enhance immune function, regulate the metabolism of key lipids, and ultimately mitigate nervous system damage resulting from PM exposure by suppressing inflammatory pathways. These findings offer innovative approaches for dietary supplements, especially in safeguarding against nervous system impairments caused by environmental stressors.