Inhibition of CD38 and supplementation of nicotinamide riboside ameliorate lipopolysaccharide-induced neuroinammation in the hippocampus

Neuroinammation is initiated by the activation of the brain’s innate immune system in response to an inammatory challenge. Insucient control of neuroinammation leads to enhanced or prolonged pathology in neurological conditions, including multiple sclerosis, traumatic brain injury, and Alzheimer’s disease. Nicotinamide adenine dinucleotide (NAD + ) plays critical roles in cellular energy metabolism and calcium homeostasis. Our previous study demonstrated that the deletion of CD38, an enzyme that converts NAD + to calcium-mobilizing second messengers, increased NAD + levels in the brain and suppressed neuroinammation, glial activation, and demyelination in a cuprizone-induced demyelination model mouse. However, the direct effects of CD38 and NAD + on neuroinammation have not been claried. Here, we investigated the effect of CD38 inhibition and NAD + replacement in lipopolysaccharide (LPS)-induced neuroinammation in mice.


Abstract
Background Neuroin ammation is initiated by the activation of the brain's innate immune system in response to an in ammatory challenge. Insu cient control of neuroin ammation leads to enhanced or prolonged pathology in neurological conditions, including multiple sclerosis, traumatic brain injury, and Alzheimer's disease. Nicotinamide adenine dinucleotide (NAD + ) plays critical roles in cellular energy metabolism and calcium homeostasis. Our previous study demonstrated that the deletion of CD38, an enzyme that converts NAD + to calcium-mobilizing second messengers, increased NAD + levels in the brain and suppressed neuroin ammation, glial activation, and demyelination in a cuprizone-induced demyelination model mouse. However, the direct effects of CD38 and NAD + on neuroin ammation have not been clari ed. Here, we investigated the effect of CD38 inhibition and NAD + replacement in lipopolysaccharide (LPS)-induced neuroin ammation in mice.

Results
CD38 expression in the cortex and hippocampus increased after LPS administration. In ammatory responses and glial activation after LPS injection were signi cantly lower in CD38 KO mice than in WT mice. Pre-administration of apigenin or NR for 7 d increased NAD + levels in the brain and signi cantly suppressed the induction of cytokines and chemokines after LPS administration in mice. Moreover, LPSinduced glial activation and neurodegeneration were signi cantly suppressed under the same conditions.
In cell culture, LPS-induced in ammatory responses were suppressed by treatment of primary astrocytes or microglia with apigenin, NAD + , NR, or 78c. Finally, all these compounds suppressed the translocation of p65 to the nucleus by LPS in cultured microglia.

Conclusions
CD38-mediated neuroin ammation is linked to NAD + consumption, and CD38 inhibition and NR supplementation may be bene cial for preventing neuroin ammation in pathological conditions. Background Neuroin ammation is a biological response initiated by tissue injury or infection in the central nervous system (CNS) to eliminate pathogenic components and induce tissue remodeling. However, insu cient control of neuroin ammation leads to the progression of many neurological conditions such as multiple sclerosis, traumatic brain injury, and Alzheimer's disease [1][2][3]. Glial cells, including microglia and astrocytes, are involved in the immune response in the CNS and play important roles in the development of neuroin ammation. It is well documented that sustained in ammatory responses cause the release of harmful mediators such as cytokines and chemokines from activated glial cells, and further affect neuronal cells by triggering neurodegeneration [4][5][6][7]. Therefore, suppression of neuroin ammation may be an important therapeutic target in neurological diseases.
CD38 is a type II and type III transmembrane protein [8] that catalyzes the formation of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP + ) from nicotinamide adenine dinucleotide (NAD + ) and related metabolites [9] to mobilize calcium from intracellular stores [9][10][11][12][13][14][15]. CD38 has diverse functions. For example, it promotes the secretion of insulin from pancreatic beta cells and oxytocin from hypothalamic neurons, thereby promoting social behaviors that are de cient in mouse models of autism spectrum disorder (ASD) [16][17][18][19][20][21]. CD38 is also associated with glial cell functions. We previously demonstrated that under physiological conditions, CD38 regulates the maturation of astrocytes and differentiation of oligodendrocyte precursor cells using NAD + in the brain [22]. We also recently discovered that CD38 is crucially involved in the cuprizone-induced demyelination model in mice. CD38 expression was increased in the brain after cuprizone administration in a manner associated with the production of pro-in ammatory molecules, glial activation, and subsequent neurodegeneration.
Deletion of CD38 suppresses these phenotypes by increasing NAD + levels in the brain [23]. CD38 has also been reported to play a critical role in the pathology of experimental autoimmune encephalomyelitis, another model of demyelination in mice [24]. NAD + is synthesized in four gene-encoded biosynthetic pathways from tryptophan, nicotinic acid (NA), nicotinamide (NAM), and nicotinamide riboside (NR) [25,26]. The NR biosynthetic pathway is both unique and highly e cient in rodents and humans [27] and corresponds to a biosynthetic pathway that is transcriptionally induced in heart failure [28] and central brain injury [29]. Moreover, in an Alzheimer's disease model, NR improved both cognitive function and protected against neurodegeneration [30,31].
The NAD + level in the brain can also be increased by compounds that depress NAD + consumption. Thiazoloquin(az)olin(on)e, 78c, is a chemical that has a speci c inhibitory effect on CD38 activity (CD38i) and has been reported to arrest age-related NAD + decline [32,33]. Similarly, a natural avonoid apigenin (4'5,7-trihydroxy avone) [34][35][36], was reported to inhibit CD38 enzymatic activity and increase intracellular NAD + levels [37]. The effects of these compounds on neuroin ammation have been tested separately, but not together in the same model. In this study, we investigated the anti-in ammatory effects of inhibition of CD38 and the supplementation of NR on lipopolysaccharide (LPS)-induced neuroin ammation.

Animals and treatments
Wild-type (WT) and CD38 knockout (KO) male ICR mice (10-11 week old) were used for the experiments (body weight; 30-36g). CD38 KO mice were generated as described previously and backcrossed for more than eight times [38]. All mice were housed in 345 × 168 × 140 mm cages in a temperature-controlled room (24-25 °C) with 12-h light-dark cycles. Food and water were available ad libitum. LPS injection was performed as previously described [39]. In brief, 10 µg LPS dissolved in 5 m L of sterile PBS or control sterile PBS was injected into the right lateral cerebral ventricle (0.3 mm caudal to bregma and -1.0 mm from lateral to midline at a depth of 3.0 mm) using a microsyringe and stereotaxic coordinates. The animals were sacri ced at various time points as described in the Results section. Apigenin (40 mg/kg) or NR (400 mg/kg) was administered intraperitoneally once per day for 7 consecutive days, followed by LPS injection 6 h after the nal administration of apigenin or NR. Mice were anesthetized and sacri ced at the indicated times after LPS injection. To avoid the effect of injection-mediated brain damage, the contralateral (left) side of the cerebral cortex or hippocampus was analyzed in all experiments. All animal experiments were performed in accordance with the guidelines and approved by the Animal Care and Use Committee of Kanazawa University (AP-194042).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed as previously described [40]. In brief, total RNA was extracted from the cerebral cortex, hippocampus, or cultured cells using the FASTGene TM RNA Basic Kit. (FG-80250, Nippon Genetics Co., Ltd), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems, Warrington, UK). Individual cDNA sequences were ampli ed using the Thunderbird TM SYBR qPCR ® Mix (QPS-201, Toyobo Co., Ltd.) with speci c primers. To measure differential expression, the comparative Ct method was used for data analyses in MxPro 4.10 (Agilent Technologies Inc.). The primer sequences are listed in Additional le 4: Table S1.

Differential expression analysis of published microarray studies
Microarray expression pro les and associated platform data were downloaded from the Gene Expression Omnibus (GEO) database for three studies: GSE49329, GSE102482, and GSE122815 using the "GEOquery" Bioconductor package [41]. Speci c information regarding sample treatment and preparation can be found at the respective GEO accession numbers. Differentially expressed genes between control and LPS-treated cells or mice were identi ed using the GEO2R wrapper (https://www.ncbi.nlm.nih.gov/geo/geo2r/) around the Bioconductor package "limma" [42]. P-values were adjusted to correct for false positives using the Benjamini-Hochberg method. Plots of NAD-related genes were generated using GraphPad Prism v8.

Analysis of NAD + levels in brain tissues
After administration of apigenin or NR for 7 d, hippocampal tissues were harvested from WT mice, and NAD + levels were measured with a commercially available NAD + /NADH assay kit (E2ND-100, bioassay System, SFC, USA) according to the manufacturer's protocol. The absorbance at 570 nm for each sample was measured using a Multiskan GO Microplate Spectrophotometer (Thermo Fischer Scienti c, MA, USA).

Glial cell cultures
Astrocyte and microglial cultures were prepared from mixed glial cultures as previously described [23]. In brief, mixed glial cells were harvested from the cerebral cortices of WT neonatal mice (P1 to P3). After 14 d of cultivation, cells were collected and incubated with CD11b MicroBeads (130-093-634, microbeads conjugated to monoclonal anti-human/mouse CD11b antibody, Miltenyi Biotec, Bergisch Gladbach, Germany) and applied to a magnetic column tted into a MidiMACS TM cell separator (Miltenyi Biotec). The cells were separated into CD11b-positive and CD11b-negative fractions. The CD11b-positive fraction, which contained microglia, was used for the experiments 24 h after plating. The CD11b-negative fraction, which contained astrocytes, was plated and used for experiments after reaching con uence.

NF-kB nuclear translocation
Cultured microglia were plated in eight chamber slides and treated with CD38 apigenin, NAD + , and NR for 4 h followed by LPS stimulation for 1 h. Cells were then xed with 4% PFA containing 0.2% NP-40. Cells were processed for immunocytochemistry experiments with antibodies against Iba1 and p65 (8242, Cell Signaling Technology, Tokyo, Japan 1:200). Cell nuclei were visualized with DAPI (Sigma). Immunohistochemical labeling was visualized with alexa488-or Cy3-conjugated secondary antibodies, and images were obtained using a laser scanning confocal microscope EZ-C1. The nuclear uorescence intensity of p65 was determined by Integrated Density -(Area of selected cell × mean uorescence of background) using ImageJ software.

Statistical analysis
The experimental results are expressed as mean ± standard error of the mean (SEM), with the number of experiments indicated by "n." No statistical evaluations were performed to predetermine sample size, but our sample sizes were similar to those generally used in the eld. One-way ANOVA followed by the Tukey-Kramer test or two-way ANOVA followed by Scheffe's F test was used for the statistical analysis. P values < 0.05 were considered statistically signi cant.

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CD38 expression was increased after LPS injection.
We rst investigated the expression of CD38 in LPS-induced neuroin ammation (Fig. 1a). Consistent with our recent report in the cuprizone-induced demyelinating model [23], RT-qPCR analysis revealed that the expression of Cd38 mRNA gradually increased in both the cerebral cortex and hippocampus, and reached signi cance after 6 h (p < 0.01) and 12 h (p < 0.05), respectively, of intracerebroventricular (i.c.v.) injection of LPS. Furthermore, the level of Cd38 mRNA expression was higher in the hippocampus than in the cerebral cortex at any time point after LPS injection (Fig. 1b). Western blot analysis con rmed the gradual increase in CD38 protein expression after LPS injection. The expression level of CD38 protein was also higher in the hippocampus than in the cerebral cortex (Fig. 1c). Furthermore, analysis of microarray data also showed elevated expression levels of CD38 mRNA in the brain after LPS injection and in the primary microglia after LPS stimulation (Additional le 2: Fig. S1). Interestingly, we found that other NAD +consuming enzymes, PARPs (poly (ADP-ribose) polymerases), were also signi cantly increased by induction of neuroin ammation (Additional le 2: Fig. S1). These results suggest that CD38 and other NAD-consuming molecules may be involved in LPS-induced neuroin ammation.
LPS-induced neuroin ammation and glial activation are attenuated in CD38 KO mice.
To evaluate the effect of CD38 deletion on LPS-induced neuroin ammation and glial activation, we analyzed the expression of pro-in ammatory genes and glia-associated genes after LPS injection (Fig.  2a). RT-qPCR analysis revealed that the expression of genes such as Il1b, IIl6, Tnf, Nos2, Ccl2, and Ccl3 was robustly increased in WT mice 6 h after LPS injection (Fig. 2 b-g). In all cases except Il1b, mRNA expression reached a peak 6 h after LPS injection and gradually decreased thereafter (Fig. 2 c-g). In the case of Il1b, the high level of expression continued to 12 h after LPS injection (Fig. 2b). The expression of these genes was signi cantly lower in CD38 KO mice than in WT mice (Fig. 2 b-g). The expression of mRNAs for Gfap and Iba1, an astrocytic and microglial marker, respectively, gradually increased and reached signi cantly high levels at 12 h and 24 h, respectively, after LPS injection in WT mice. Expression of Gfap and Iba1 was signi cantly lower in CD38 KO mice (Fig. 3a). Western blot analysis con rmed the increase of GFAP and Iba1 protein after LPS injection, but the level was signi cantly lower in CD38 KO mice than in WT mice 24 h after LPS injection (Fig. 3b). Immunohistochemical analysis further revealed that the immunoreactivity of GFAP and Iba1 was clearly increased in both the CA1 and CA3 regions of the hippocampus 24 h after LPS injection, but the level was signi cantly lower in CD38 KO mice (Fig. 3c-d).
These data indicate that deletion of CD38 suppresses LPS-induced neuroin ammation and glial activation.
The phenotypes in CD38 KO mice described above and in our recent study [23] suggest that pharmacological inhibition of CD38 and/or boosted brain NAD + level may be capable of depressing pathological in ammation in neurological diseases. Therefore, we assessed the effect of apigenin, a natural avonoid that acts as a CD38 inhibitor [37], and NR, an NAD + precursor [26], on NAD + levels in the context of LPS-injected neuroin ammation (Fig. 4a). The NAD + levels in the hippocampus were signi cantly higher in apigenin-or NR-administered mice than in control mice, and the levels were similar in both conditions (Fig. 4b). WT mice were pre-administered with apigenin or NR for 7 d, and then injected with LPS 6 h after the nal administration of each compound. RT-qPCR analysis revealed that the induction of in ammatory genes such as Il1b, Il6, Tnf, Nos2, Ccl2, and Ccl3 was suppressed in compound-pre-administered mice than in control mice (Fig. 4c-h). Immunohistochemical analysis revealed enhanced levels of immunoreactivity for GFAP and Iba1 24 h after LPS injection, but levels were reduced in apigenin or NR pre-administered mice compared to the control group of mice (Fig. 5a-c).
Furthermore, we examined NAD + levels and neuroin ammation in CD38 KO mice administered NR or apigenin (Additional le 3: Fig. S2). CD38 KO mice showed signi cantly higher NAD + levels than WT mice in any group (Additional le 3: Fig. S2b). Consistent with CD38 as the target of apigenin, apigenin did not increase the elevated NAD + level in CD38 KO mice. Additionally, CD38 KO mice exhibited lower expression of proin ammatory genes as shown in Fig. 2, and apigenin or NR did not further decrease these gene expression programs (Additional le 3: Fig. S2c-h). These results suggest that the levels of CD38 and NAD + determine the state of neuroin ammation and the activation of both astrocytes and microglia in the brain.

NR and apigenin attenuated LPS-induced neurodegeneration
As LPS-induced neuroin ammation often leads to neurodegeneration [43][44][45][46], the effect of apigenin and NR on neuronal damage was evaluated after LPS injection. Immunohistochemistry for nonphosphorylated neuro lament H (SMI32), a marker of damaged axons, revealed that neurodegeneration occurred within 24 h after LPS injection in both the CA1 and CA3 regions of the hippocampus, and the level was signi cantly lower in apigenin or NR pre-administered mice (Fig. 6a, b). Consistent with these results, immunohistochemistry for MAP2 revealed that the intensity of normal axons and dendrites decreased after LPS injection, and this decrease was partially recovered by apigenin or NR preadministration (Additional le 4: Fig. S3a, b). These results suggest that administration of NR or apigenin attenuated not only LPS-induced neuroin ammation but also subsequent LPS-induced neurodegeneration.
NR, apigenin and 78c reduced in ammatory response in vitro.
Silencing of the cd38 gene and the addition of NAD + suppressed LPS-induced activation of astrocytes and microglia in vitro [22,23], the effects of NR, apigenin, and 78c, the latter a CD38-speci c inhibitor, on the in ammatory response were examined using cultured microglia and astrocytes. RT-qPCR analysis revealed that LPS strongly upregulated the expression of pro-in ammatory genes, such as Il1b, Il6, Tnf, Nos2, Ccl2, and Ccl3 in microglia and/or astrocytes. In microglia, the expression of Il1b and Il6 was signi cantly reduced by all compounds, and that of Tnf and Nos2 was signi cantly decreased by NAD + and NR, and apigenin, respectively (Fig. 7a-d). In addition, in astrocytes, all compounds showed a tendency to suppress the induction of in ammatory genes, although the expression of Il6 was signi cantly decreased by apigenin, and that of Tnf and Ccl3 was signi cantly reduced by NAD + after LPS injection. These results suggest that inhibition of CD38 and increased NAD + status directly suppress LPS-induced in ammatory responses, especially in microglia.
It is known that LPS binds to toll-like receptor 4, which activates the NF-κB cascade and consequently induces pro-in ammatory genes. To determine the relevance of CD38 inhibition and supplementation of NAD + in the NF-κB signaling pathway, the nuclear translocation of NF-κB was examined after LPS stimulation in cultured microglia. Immunocytochemical analysis revealed that the intensity of p65, a major component of NF-κB, in the nucleus was strongly increased 1 h after LPS stimulation. In contrast, it was signi cantly reduced by all the compounds (Fig. 8a, b). These results suggest that intracellular NAD + suppresses LPS-induced NF-κB activation in microglia.

Discussion
In the current study, we investigated the effect of CD38 inhibition and supplementation of NAD + on LPSinduced neuroin ammation in mice. CD38 expression was increased both in the hippocampus and in the cerebral cortex after LPS injection. Deletion of CD38 decreased LPS-induced neuroin ammation and glial activation Consistently, pre-administration of apigenin and NR increased NAD + concentration in the brain, and suppressed LPS-induced in ammatory response, glial activation, and neurodegeneration. In primary cultured glial cells, compounds such as 78c, apigenin, NAD + , and NR effectively suppressed the activation of NF-κB and the induction of cytokines and chemokines after LPS treatment. These results suggest that inhibition of CD38 and supplementation with NAD + precursors both depress neuroin ammation and have therapeutic potential for some neurological diseases.

The role of NAD + and CD38 in LPS-induced neuroin ammation
To date, several studies, including ours, have suggested the involvement of CD38 in neuroin ammation.
Deletion of CD38 in the APP.PS Alzheimer's disease model exhibited a signi cant reduction in Aβ plaque load and microglia/macrophage accumulation in the brain compared with APP.PS mice with CD38 intact [47]. Our previous study also revealed that CD38 KO mice ameliorated demyelination, glial activation, and neuroin ammation in cuprizone-induced demyelination [23]. To explore the direct effects of CD38 deletion or inhibition, and supplementation of NAD + on neuroin ammation, we employed a mouse model of LPS-injection into the lateral cerebral ventricle in the current study. Our results demonstrated that CD38 deletion or inhibition, and supplementation with NAD + suppressed neuroin ammation at earlier stages (within 6 h) after LPS administration in vivo ( Fig. 2 and 4) and in vitro ( Fig. 7 and 8), and these effects seem to be correlated with the increased level of NAD + in the brain (Fig. 4b) [23].
The biology of in ammation suggests that expression of in ammatory markers, including CD38, cytokines, and chemokines, constitutes a homeostatic attempt at controlling infection or environmental damage. However, uncontrolled in ammatory responses are clearly neurotoxic. The behavioral de cits of mice with CD38 deletion suggest that the formation of CD38-dependent second messengers, including cADPR [11] and NAADP [15], is required for oxytocin signaling, social intelligence, and parenting [17]. In the case of neuroin ammatory responses, we consider that the formation of CD38-dependent signals might induce such activities and/or elevated NAD might depress such activities.
Expression of glial markers such as GFAP and Iba1 ( Fig. 3 and 5), and those of neurodegeneration such as SMI32 (Fig. 6) were increased after LPS injection, and the levels were reduced in the conditions where CD38 was deleted or inhibited, and NAD + synthesis was supported by precursor supplementation. Since proin ammatory genes were elevated at earlier stages than glial markers after LPS administration, it is likely that the suppressive effects on glial activation are secondary to the anti-in ammatory effect of CD38 deletion or inhibition, and support of NAD + synthesis. However, we cannot rule out the possibility that CD38 directly regulates glial activation, since CD38 enhances GFAP expression during the postnatal development stages in vivo [22].
In contrast to the potential mechanisms by which CD38 and calcium release promote in ammatory gene expression, CD38 inhibition and NR supplementation both converge on elevated NAD + -mediated suppression of in ammatory responses. We recently showed that coronavirus infection induces transcriptional induction of a set of PARP-related genes that depleted cellular NAD + and that supplementation with NAD + precursors or pharmacological activation of NAD + synthesis can boost the activity of the highly transcribed PARP genes while providing partial protection against viral replication [48]. Here, we show that many of the same PARP genes are upregulated after LPS stimulation in vitro and in vivo (Additional le 1, Fig. S1). We therefore suggest that depressed activities of NAD-dependent enzymes (other than CD38) mediate in ammatory gene transcription. Three potential mediators are SIRT2, which reportedly prevents microglial activation by promoting NF-κB deacetylation [49], SIRT1, which potentially depresses NF-κB activity via deacetylation of p65/RelaA [50] and PARP10, which inhibit activation of NF-κB and downstream target genes in response to IL-1b and TNF-a in a manner that depends on its catalytic activity and poly-ubiquitin binding activities [51]. As glia-derived cytokines such as IFN-g and TNF-a synergistically promote neuronal degeneration with other toxic factors [52], the reduced levels of neuronal damage by NR or apigenin administration are likely attributed to decreased induction of pro-in ammatory molecules. Consistent with this proposed mechanism, three weeks of oral high-dose NR has been shown to depress the circulation of in ammatory cytokines in a small placebocontrolled trial of older men [53]. NAD + may also protect neurons directly because NAD + degradation has been directly linked to axonal degeneration. Sterile alpha and the TIR motif containing 1 (SARM1) initiates a local destruction program of axons through a process that involves the catastrophic depletion of axonal NAD + [54].
These results are not without caveats. For example, apigenin showed stronger effects than other compounds such as NR and 78c to suppress Il6 and Nos2 in cultured microglia and Il6 in cultured astrocytes (Fig. 7). Moreover, apigenin decreased cytokine expression even in LPS-injected CD38 KO mice ( Fig. S2), suggesting that it has targets beyond CD38, which could include MAPK, Akt, JNK [55] or the GSK3b/Nrf2 signaling pathway [56].

Future prospects
Neuroin ammation is a contributing factor in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease [55,57]. Importantly, the vulnerability of hippocampal neurons that leads to cognitive impairment is strongly associated with neuroin ammation [58,59]. In fact, apigenin and NR have been reported to have bene cial effects on neurodegenerative disease models [31,60]. Apigenin has been reported to cross the blood-brain barrier (BBB) and increase NAD + concentration in the brain [61]. NR can also increase NAD + levels in the brains of Alzheimer's disease model mice when orally administered [30]. In ongoing work, we aim to clarify the neuroprotective mechanisms in disease models in order to better identify biomarkers that will enable successful clinical translation.

Conclusions
We identi ed novel effects of NR and apigenin on LPS-induced neuroin ammation. Testing these compounds in neurodegenerative diseases, aging, and neurodevelopmental disease models may be important for the development of new therapies for these diseases. Availability of data and materials All data generated or analyzed during this study are included in this article.
Ethics approval and consent to participate All animals used in this study were treated humanely and were performed in strict accordance with the National Institutes of Health guidelines. (green) and Iba1 (red) of 0 and 24 h after LPS injection in the HPC (CA1 and CA3) of WT and CD38 KO mice. Nuclei were counterstained with DAPI. Scale bars: 100 µm. (d) The graphs represent the intensity of GFAP (left) and Iba1 (right) 0 and 24 h after LPS injection in the CA1 and CA3 of WT and CD38 KO mice. n = 5. Data represent means ± SEM. P values are determined by two-way ANOVA followed by Scheffe's F test. +p < 0.05 and ++p < 0.01 vs 0 h of LPS in the WT mice. #p < 0.05 and ##p < 0.01 vs 0 h of LPS in the CD38 KO mice. *p < 0.05 and **p < 0.01 between WT and CD38 KO mice.  NR, apigenin and 78c reduced in ammatory response in vitro. RT-qPCR analysis for the expression levels of in ammatory genes in glial cultures. (a-j) Microglia (a-d) or astrocyte (e-j) cultures were treated with saline, 78c, apigenin, NAD+ or NR for 4 h, then stimulated with LPS (100 ng/ml) for 6 h. n = 6. Data represent means ± SEM. P values are determined by two-way ANOVA followed Scheffe's F test. *p < 0.05 and **p < 0.01 vs LPS-treated control cells.

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