Abstract Currently, nicotine withdrawal symptoms pose a significant challenge in tobacco cessation efforts, particularly withdrawal affective symptoms, such as anxiety and depression like behavior. However, the underlying mechanisms of these symptoms remain poorly understood. Emerging evidence implicates the hippocampus, a key region in the limbic system, involved in emotional regulation. In this study, we employed transcriptome sequencing, untargeted metabolomics, and integrative multi-omics analysis to elucidate the molecular mechanisms underlying nicotine withdrawal-induced affective symptoms in the hippocampus of male C57BL/6J mice. Our findings corroborate previous research linking nicotine withdrawal symptoms to dysregulation of neuroendocrine pathways and inflammatory processes within the brain. Importantly, we identify impaired glutathione metabolism as a significant contributing factor to the development of these symptoms. Furthermore, our investigation reveals that theobromine, a principal psychoactive compound found in cocoa, exerts a potent therapeutic effect in alleviating nicotine withdrawal affective symptoms through diverse mechanisms. In addition to its modulation of neuroendocrine pathways and inflammation, theobromine's ability to restore glutathione metabolism in the hippocampus emerges as a pivotal aspect of its pharmacological action. Keywords: Nicotine withdrawal symptoms, Theobromine, Glutathione metabolism, Integrative multi-omics analysis, Hippocampus Introduction Tobacco smoke is linked to numerous diseases and is recognized as the second leading risk factor for mortality globally ([33]Zhang et al., 2023). Approximately 70 % of smokers desire to quit, however, individuals attempting to quit smoking make an average of about 6 quit attempts before achieving successful cessation. Cessation failures are often attributed to intense withdrawal symptoms, particularly psychiatric manifestations such as anxiety and depression ([34]Hughes, 2007). The hippocampus, a critical component of the limbic system, plays a pivotal role in memory, learning, language ability, emotional regulation, and spatial cognition ([35]Covington and Duff, 2016, [36]Lisman et al., 2017). Recent research has revealed that the hippocampus is also implicated in drug addiction and withdrawal symptoms ([37]Akimoto et al., 2018, [38]Deji et al., 2022, [39]Garcia-Fuster et al., 2012, [40]Zeid et al., 2018). For instance, the hippocampus is involved in the neuroadaptations that occur during prolonged withdrawal ([41]Garcia-Fuster et al., 2012). Additionally, the ventral hippocampus circuit regulates anxiety-like behaviors induced by morphine withdrawal ([42]Deji et al., 2022). Nicotine exposure activates nicotinic acetylcholine receptors (nAChRs) in the hippocampus, impacting both excitatory and inhibitory circuits ([43]Zeid et al., 2018). Metabolomics studies have indicated that nicotine withdrawal disrupts amino acid metabolism in the hippocampus, including levels of N-acetylaspartate, glutamate, and γ-aminobutyric acid (GABA) ([44]Akimoto et al., 2018). However, a clear and convincing mechanism underlying the hippocampus's involvement in nicotine withdrawal symptoms has yet to be elucidated. Cocoa, a primary ingredient in cocoa-rich products like chocolate, has long been associated with various health benefits, including its mood-enhancing effects ([45]Fusar-Poli et al., 2022). Clinical investigations with 13,626 US adults have shown significant improvements in depression symptoms with cocoa consumption, particularly dark chocolate ([46]Jackson et al., 2019). Theobromine and caffeine are the primary psychopharmacological compounds in cocoa. While the effects of caffeine on anxiety are inconclusive, some studies indicate its potential to exacerbate anxiety, supported by animal models confirming caffeine's anxiety-inducing properties ([47]Nehlig, 2016, [48]Klevebrant and Frick, 2022). However, cocoa products contain a higher concentration of theobromine compared to caffeine. Theobromine also has a longer half-life in the blood compared to caffeine in both humans and rodents ([49]Martinez-Pinilla et al., 2015). Moreover, numerous studies have highlighted theobromine's crucial role in mood regulation ([50]Martinez-Pinilla et al., 2015, [51]Zhang et al., 2024a, [52]Judelson et al., 2013). However, there is currently no research on whether theobromine has an improved effect on affective symptoms of nicotine withdrawal. In the present study, we employed transcriptome sequencing (RNA-seq) and untargeted metabolomics analysis to investigate the mechanisms by which the hippocampus is involved in affective symptoms of nicotine withdrawal. We subsequently examined theobromine's effect on these symptoms and explored its underlying mechanisms. Our findings suggest that affective symptoms of nicotine withdrawal are not only linked to abnormalities in neuroendocrine and immune systems but also to disruptions in glutathione metabolism. Theobromine may ameliorate the symptoms through multiple pathways, including the restoration of glutathione metabolism. Materials and methods (S)-(-)-Nicotine (N412450, 99 % purity) was obtained from Toronto Research Chemicals (Toronto, Canada). It was then dissolved and diluted to the desired concentration through a physiological saline solution. Theobromine ([53]T45040, 99 % purity) was obtained from Shanghai Acmec Biochemical Technology Co., Ltd. The reagents used for metabolomics mass spectrometry analysis were all from Merck (Darmstadt, Germany). All other reagents used were of analytical grade and commercially available. Animals and drug treatment Male C57BL/6J mice (aged 7–8 weeks) were obtained from the Charles River Laboratories (Beijing, China). The experimental animals were housed in cages under a 12 h light/12 h dark cycle, 60 ± 5 % humidity, and a temperature of 25 ± 1 °C with access to water and food freely. All experimental procedures were conducted in accordance with the Ethics Committee of Zhengzhou University (ZZUIRB-2024-167). Model of nicotine withdrawal A nicotine withdrawal model was established based on previous literature reports ([54]Chellian et al., 2021, [55]Jackson et al., 2008). Twenty adult mice, divided into a control group (Con) and a nicotine withdrawal group (Nic) with 10 mice each, were allowed to acclimate for 7 days. Subsequently, the Nic group received subcutaneous injections of nicotine at a dose of 2 mg/kg body weight four times daily for 14 days, while the Con group received equivalent volumes of physiological saline. The injection time was started at 6:00AM, the next was 11:00 and 16:00PM, the last was 21:00PM. The interval of time is 5 h and 9 h. Following a 24-h cessation of nicotine injections, behavioral tests were conducted on the mice. Upon completion of the tests, the mice were euthanized via overdose anesthesia, and their brains were perfused by cold PBS and dissected to obtain hippocampal tissue. The tissue samples were divided into two portions, with one portion undergoing transcriptome sequencing (RNA-seq) and the other undergoing untargeted metabolomics analysis. Theobromine treatment for nicotine withdrawal Thirty adult mice were divided into three groups: a control group (Con), a nicotine withdrawal group (Nic), and a theobromine treatment group (TN), each consisting of 10 mice. The model establishment followed the same procedure as above. However, the TN group received a daily oral administration of 100 mg/kg body weight theobromine (a mixture with 0.5 % sodium carboxymethyl cellulose) in addition to nicotine injections. The dosage of the chemical compound was determined based on previous literature ([56]Cova et al., 2019), and the Con and Nic groups were given the same amount of 0.5 % sodium carboxymethyl cellulose without theobromine. Animal behavioral methodologies All animal behavior studies utilize equipment fitted with cameras and ANY-maze 5.0 software. Open Field Test (OFT): To evaluate the locomotor activity and anxiety-like behavior of mice, a plain, 44 cm × 44 cm × 30 cm open field arena was employed. After a 30-second adaptation period, a segment was taken every 5 min to record the total distance and time traveled by the animals in the center and corner arenas (14.7 cm×14.7 cm). The experiment lasted for a total of 15 min. Elevated Plus Maze (EPM): Anxiety-like behavior in mice was measured using the EPM test. The apparatus consists of two closed arms (30 cm × 6 cm × 15 cm) and two open arms (30 cm × 6 cm). Mice were allowed to explore the maze for 5 min, and the time spent in the closed and open arms, as well as the center, was calculated. Tail Suspension Test (TST): Mice were suspended by their tails using adhesive tape, positioned 1 cm from the tip of the tail and 15 cm above the ground. Small plastic tubes were affixed to their tails to prevent climbing. The test lasted for 6 min, and immobility time was measured and analyzed during the final 4 min. Forced Swim Test (FST): Individual mice were placed into a transparent glass cylinder (15 cm in diameter, 30 cm in height) filled with water (23 ± 1 °C) to a depth of 15 cm for 6 min. Immobility time was calculated during the last 4 min of the test. RNA-seq and bioinformatic analysis RNA extraction, library preparation, RNA sequencing, and bioinformatic analysis were performed by GENEWIZ (Suzhou, China) following standard protocols. The process is done to the methods of Jingyu [57]Chen et al. (2021). The quality of RNA was assessed using the Agilent 2100 Bioanalyzer System prior to library preparation. Libraries for sequencing were constructed following the protocol outlined in the NEBNext® UltraTM RNA Library Prep Kit for Illumina manual. These libraries were sequenced using 150-bp paired-end reads on an Illumina Hiseq platform (Novogene Co., Ltd, Beijing, China). Cleaned reads from each sample were aligned to the reference genomes (mouse: GRCm38) using HISAT2 software with default parameters. Gene read counts were then calculated using the Genomic Features and Genomic Alignments packages in R. Differential expression analysis and significance tests for genes were performed using the DESeq2 package (p < 0.05; absolute fold change > 2; FPKM ≥ 1 in at least one sample). Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) was conducted using the R package cluster Profiler. Visualization of the enrichment results was accomplished using the R packages GOplot and enrichplot. Untargeted metabolomics analysis Sample preparation and metabolic profiling followed established protocols as described in previous literature ([58]Pang et al., 2021). In briefly, Tissue was homogenized in 1 mL of 80 % methanol, incubated on ice for 10 min, and centrifuged at 14,000g for 15 min at 4°C. The supernatant was freeze-dried, re-dissolved in 100 µL of 12 % acetonitrile, centrifuged again, and stored at −80 °C. the separation was performed on a UHPLC system (Agilent 1290) with an ACQUITY UPLC BEH C18 column. Mobile phases were 0.1 % formic acid in H₂O (A) and acetonitrile (B) with a gradient from 2 % to 100 % B. Full MS was scanned from 100 to 1000 m/z (60,000 resolution), and MS/MS analysis used collision energies of 20, 40, 60, and 80 eV. Samples were randomized with three technical replicates. Raw MS data was converted to mzXML and processed with Progenesis QI. Metabolites were identified using the in-house MS2 and HMDB databases. Identification confidence was based on Progenesis QI scores (threshold 35.0). Statistical analysis was performed using MetaboAnalyst and visualized in Origin 2022. Data were considered significantly different if p < 0.05 and the absolute fold change was greater than 1.2. The detailed process is displayed in the supplemental file. Real-time quantitative PCR Hippocampus samples from different groups were used for gene expression analysis by an RT-PCR. Amplification and detection using TIANGEN Talent qPCR PreMix（FP209, Beijing, China）were performed with an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). All the primers used in this study have been listed in [59]Supplemental Table 1. All the expression levels of the target genes were normalized to the expression of GAPDH. The relative expression of the target genes was calculated using the ΔΔCT method. The difference in CT values between the target gene and the GAPDH gene for each sample was calculated (ΔCT = CT_target − CT_reference). The ΔΔCT was then calculated by subtracting the ΔCT value of the control group from the ΔCT value of the experimental group (ΔΔCT = ΔCT_experimental − ΔCT_control). The relative gene expression was determined by the formula 2^(-ΔΔCT). Statistical analysis Data were expressed as mean ± standard error of the mean (SEM). Statistical significance was assessed using either a two-tailed unpaired Student's t-test for comparisons between two groups, or one-way ANOVA for comparisons among multiple groups. For datasets analyzed with ANOVA, the Bonferroni post-hoc test was applied. A p-value less than 0.05 was considered statistically significant. All statistical analyses were conducted using Prism version 8.0 (GraphPad, La Jolla, CA, USA). Results Nicotine withdrawal causes significant affective symptoms and changes in gene expression In this research, nicotine withdrawal induced-affective symptoms were checked after 24 h nicotine discontinuation. The Nic-mice spent less time in the center and more time in the corners during the OFT (t [(18)]= 7.868, P < 0.0001; t [(18)] = 3.648, P = 0.0018, center zone, corner zone, respectively, [60]Fig. 1A); and the Nic-mice spent less time in open arms and more time in closed arms of the EPM compared with control mice (t [(18)] = 3.407, P = 0.0031; t [(18)] = 4.219, P = 0.0005, open arms, closed arms, respectively, [61]Fig. 1B). Additionally, whether in OFT or EPM experiments, the Nic-mice showed the less traveled distance compared with control mice (t [(18)] = 4.591, P = 0.0002; t [(18)] = 3.393, P = 0.0032, OFT, EPM, respectively, [62]Fig. 1A and B). These findings showed the mice with nicotine withdrawal exhibited significant anxiety-like behavior compared to the control mice. On the other hand, in the TST and FST, the immobile time reflects the duration during which the mouse ceases struggling, and this time is inversely correlated with the level of depression. The data demonstrated a significant increment in the immobile times in the Nic-mice compared to the control, suggesting that the mice with nicotine withdrawal exhibited more depression-like behavior than their control counterparts (t [(18)] = 2.494, P = 0.0226; t [(18)] = 3.133, P = 0.0057, FST, TST, respectively, [63]Fig. 1C and D). In addition, we also analyzed the somatic symptoms of the model mice, including rearing, scratching, forelimb tremors, body shakes, headshakes, abdominal constrictions, jumps, genital licks, and grooming; and found that their somatic symptom scores were much higher than those of the control group [64](Supplemental Fig. 1A, supplemental table 3). In summary, our animal model shows significant nicotine withdrawal symptoms, which can be used for further mechanism exploration. Fig. 1. [65]Fig. 1 [66]Open in a new tab Nicotine withdrawal causes significant affective symptoms and changes in gene expression within the hippocampus. A. An open field test (OFT) was conducted to study the anxiety-like behavior after nicotine withdrawal, which showed the animal's movement distance, time spent in corners and center, respectively. B. An elevated plus maze (EPM) was conducted to study the anxiety-like behavior after nicotine withdrawal, which showed the animal's movement distance, time spent in close arms and open arms, respectively. C and D. Forced swimming test (FST) and tail suspension test (TST) experiments were used to detect depressive-like behavior after nicotine withdrawal, and the percentage of immobility time indicated the strength of depressive symptoms. E. Comparison of RNA-seq gene expression in hippocampal tissue between control (Con) and nicotine withdrawal (Nic) groups through Partial least squares Discriminant Analysis (PLS-DA). F. Volcano plot showing differential gene expression between the Con and Nic groups, and the differential expression multiples were more than 2. The genes with significant changes are colored. The dotted lines indicate the cut-off for significant changes (p < 0.05). G, H, I and J. KEGG functional enrichment analysis for the differentially expressed genes and the detailed signaling pathway in the four KEGG categories are displayed. All the data are expressed as mean ± SEM (n = 10 per group in behaviors test, n = 5 in RNA-seq), *p < 0.05, Control vs nicotine withdrawal. Partial least squares Discriminant Analysis (PLS-DA) of the RNA-seq data from hippocampal tissue showed two distinct clusters, signifying substantial transcriptional changes between the Nic-mice and control mice ([67]Fig. 1E). Differential expression analysis unveiled 203 genes with significant differences between the two groups (p < 0.05; absolute fold change > 2; FPKM ≥ 1 in at least one sample). Among these, 62 genes were up-regulated, while 141 genes were down-regulated in Nic-mice ([68]Fig. 1F). To determine the key pathways affected by nicotine withdrawal, we performed KEGG functional enrichment analysis on the differentially expressed genes. These genes were primarily associated with five KEGG categories: human disease, organismal systems, environmental information processing, metabolism and cellular processes ([69]Supplemental Fig. 2). Within the organismal systems, the top three enriched systems are the endocrine system, immune system, and nervous system. This aligns with existing literature, which highlights the critical roles of the endocrine and nervous systems in nicotine withdrawal ([70]Pickworth and Fant, 1998, [71]Paolini and De Biasi, 2011); moreover, there is already an established association between major depression disorder and brain inflammation ([72]Setiawan et al., 2015). In the environmental information processing, neuroendocrine and immune signaling pathways remain the main enriched pathways. In the cellular processes, no significant pathways related to nicotine withdrawal symptoms were identified. In the metabolism, there was a notable enrichment in oxidative phosphorylation and glutathione metabolism pathways. Previous studies have documented both increases and decreases in brain glutathione levels in individuals with major depressive disorder ([73]Rae and Williams, 2017). Furthermore, nicotine addiction is known to influence energy balance in various human organs ([74]Seoane-Collazo et al., 2021). In summary, animals undergoing nicotine withdrawal display affective symptoms, including anxiety and depression-like behavior. RNA-seq reveals that nicotine withdrawal induces changes in the neuroendocrine and immune systems, as well as in energy metabolism within the hippocampus. Moreover, the glutathione metabolic pathway has been identified for the first time as playing a role in nicotine withdrawal. Untargeted metabolomics suggests the glutathione metabolic pathway may play a crucial role in nicotine withdrawal RNA sequencing offers insights into potential molecular mechanisms at the gene expression level, yet biological processes are primarily carried out by proteins and small molecule metabolites. To further explore the mechanisms underlying how nicotine withdrawal induces the physiological and psychological changes, we employed untargeted metabolomics to analyze the change of small molecule metabolites in the hippocampus. The data is presented in [75]Fig. 2. The raw data from LC–MS/MS were processed and annotated manually, resulting in a total of 419 distinct annotated metabolites. Through PLS-DA analysis, the samples could be clearly grouped into two clusters, indicating substantial changes in metabolites in nicotine withdrawal mice ([76]Fig. 2A). Metabolites were categorized into chemical super-classes using the MetaboAnalyst enrichment module ([77]Fig. 2B). The largest number of defined metabolites belonged to alkaloids and derivatives, followed by benzenoids and homogeneous non-metal compounds, then lipids and lipid-like molecules. Comparing all defined metabolites between the control and nicotine withdrawal groups, we found 101 significantly changed metabolites, with 48 having difference multiples of more than 1.2. Among these, 32 were down-regulated, and 16 were up-regulated ([78]Fig. 2C). the top two increasing metabolites are Pyridoxamine 5′-phosphate and Indole-3-carboxylic acid (t [(28)] = 2.909, P = 0.007; t [(28)] = 3.004, P = 0.0056, respectively, [79]Supplemental Fig. 3A); the top two decreasing metabolites are D-Mannose and Beta-D-Glucosamine (t [(28)] = 6.754, P < 0.0001; t [(28)] = 6.200, P < 0.0001, respectively, [80]Supplemental Fig. 3B). KEGG functional enrichment analysis of the differentially expressed metabolites identified the TCA cycle, fructose and mannose metabolism, and glutathione metabolism among the top 25 enriched signaling pathways ([81]Supplemental Fig. 4). The results also hint that energy and glutathione metabolism participate in the occurrence of nicotine withdrawal symptoms. Fig. 2. [82]Fig. 2 [83]Open in a new tab Untargeted metabolomics analysis demonstrates that nicotine withdrawal leads to alterations in the glutathione metabolic pathway. A. PLS-DA was used to assess the differences in small molecule metabolites between the Con and Nic group in hippocampal tissue. B. Volcano plot showing differential gene expression between the Con and Nic group, and the differential multiples were more than 1.2. The metabolites with significant changes are colored. The dotted lines indicate the cut-off for significant changes (p < 0.05). C. The classification for significantly changed chemical compositions between the Con and Nic group in hippocampal tissue. D. Enrichment analysis of metabolic signaling pathways after joint analysis of RNA-seq and untargeted metabolomics differential molecules. E. After joint analysis, the alterations in identified small molecule metabolites belonging to the glutathione metabolic pathway. F. After joint analysis, the alterations in identified genes belonging to the glutathione metabolic pathway. All the data are expressed as mean ± SEM (n = 15 per group in the changed metabolites, n = 5 in the changed genes), *p < 0.05, Control vs nicotine withdrawal. Next, we conducted a joint-pathway analysis using RNA-seq and untargeted metabolomics data, focusing on significant changes observed between the control and nicotine withdrawal groups, with fold differences exceeding 2 or 1.2. A total of 48 metabolic signaling pathways have been enriched ([84]Fig. 2D). Surprisingly, the most significantly enriched signaling pathway is glutathione metabolism, followed by linoleic acid metabolism and alanine, aspartate, and glutamate metabolism. Further analysis of the glutathione metabolism pathway revealed that all identified genes and metabolites were downregulated in the nicotine withdrawal group compared to the control group (t [(8)] = 5.046, P = 0.00099; t [(8)] = 3.986, P = 0.00403, t [(8)] = 3.153, P = 0.0135, t [(28)] = 6.243, P < 0.0001, t [(8)] = 12.28, P < 0.0001, Ggt1, Gpx1, Gstt3, Pyroglutamic acid, γ-glutamylcysteine, respectively, [85]Fig. 2E, F and [86]Supplemental Fig. 5). The pyroglutamic acid is formed through the catalysis of Ggt1, involving the involvement of glutathione in biological transformations, whereas the γ-glutamylcysteine serves as the substrate for Gss, catalyzing the production of glutathione. Ggt1, Gpx1, and Gstt3 are the enzymes involved in the biological transformation process of glutathione, and their decrease indicates a weakened involvement of glutathione in the biological reaction process. In summary, untargeted metabolomics and joint-pathway analysis with RNA-seq indicated that nicotine withdrawal significantly affects energy metabolism and glutathione metabolism in hippocampal tissue. Theobromine significantly prevents nicotine withdrawal affective symptoms Theobromine, a major psychoactive substance found in commonly consumed chocolate, has the potential to regulate emotions. Our next step is to investigate whether theobromine can mitigate the affective symptoms associated with nicotine withdrawal and to explore its underlying mechanisms. As depicted in [87]Fig. 3A, the nicotine-withdrawn mice (Nic-mice) spent less time in the center and more time in the corners compared to the control mice. However, when fed with theobromine (TN group), the mice spent more time in the center and less time in the corners than the Nic-mice (F [(2,8)] = 14.71, P = 0.0021; F [(2,8)] = 16.96, P = 0.0013, center zone, corner zone, respectively). This pattern was also evident in the elevated plus maze experiments, where Nic-mice spent more time in the closed arm, and less time in the open arm, but TN-mice spent more time in the open arm and less time in the closed arm compared to the Nic-mice (F [(2,8)] = 10.19, P = 0.0063; F [(2,8)] = 7.137, P = 0.0166, closed arms, open arms, respectively, [88]Fig. 3B). Additionally, the total movement distance also indicated that theobromine can counteract the reduced motility observed in nicotine-withdrawn mice (F [(2,8)] = 15.07, P = 0.0019; F [(2,8)] = 12.67, P = 0.0037, OFT, EPM, respectively, [89]Fig. 3A and B). The TST and FST further showed theobromine feeding eliminated the depressive behavior caused by nicotine withdrawal (F [(2,8)] = 14.28, P = 0.0293; F [(2,8)] = 14.32, P = 0.0023, TST, FST, respectively, [90]Fig. 3C and D). Overall, the data from these behavioral methodologies indicated that theobromine feeding ameliorated anxiety/depression-like behaviors induced by nicotine withdrawal. To exclude the possibility that the observed phenomenon is attributable to the influence of theobromine on nicotine addiction behavior, we further examined whether theobromine affects the establishment of nicotine addiction models. As depicted in [91]Supplemental Fig. 1B, continuous nicotine administration for one week significantly induces nicotine preference in mice. The 50 mg/kg and 100 mg/kg doses of theobromine do not modify the nicotine-induced effect, while the 150 mg/kg dose significantly attenuates nicotine preference. This reduction may be related to the inherent bitterness of theobromine. Moreover, consistent with literature reports ([92]Picciotto et al., 2002), the use of nicotine can prevent anxiety and depression like behavior in mice, while theobromine did not further affect the effect of nicotine at this time ([93]Supplemental Fig. 1B). Additionally, to confirm that the observed effects stem from the neuropharmacological properties of theobromine itself, rather than any confounding factors, we investigated the effects of theobromine alone on the behavior of healthy mice. As shown in [94]Supplemental Fig. 1C, theobromine administration alone significantly ameliorates anxiety- and depression-like behaviors in baseline healthy mice. Therefore, the alleviation of affective symptoms associated with nicotine withdrawal by theobromine is likely due to its intrinsic neuropharmacological effects. Fig. 3. [95]Fig. 3 [96]Open in a new tab Theobromine treatment significantly improves effective symptoms caused by nicotine withdrawal. A. An OFT was conducted to study the anxiety-like behavior after nicotine withdrawal and theobromine treatment, which showed the animal's movement distance, time spent in corners and center, respectively. B. An EPM was conducted to study the anxiety-like behavior after nicotine withdrawal and theobromine treatment, which showed the animal's movement distance, time spent in close arms and open arms, respectively. C and D. The FST and TST experiments were used to detect depressive-like behavior after nicotine withdrawal and theobromine treatment, and the percentage of immobility time indicated the strength of depressive symptoms. E. Comparison of RNA-seq gene expression in hippocampal tissue between the Con, Nic and TN group through PLS-DA. F. One-way ANOVA analysis for the Con, Nic and TN group, considering significant changes at p < 0.05. All the data are expressed as mean ± SEM (n = 5 per group), *p < 0.05, Con vs Nic, #p < 0.05, Nic vs TN. After completing the animal behavioral testing, hippocampal tissue was sequenced again. PLS-DA analysis showed that the data was effectively grouped into two clusters between the Nic and TN groups ([97]Fig. 1E). We can then conduct differential gene analysis on it. The genes significantly altered by theobromine feeding are still mainly enriched in the neuroendocrine, immune system, and glutathione metabolism As shown in the [98]Fig. 4A. Theobromine treatment induced a significant change in gene expression compared with the Nic group. Differential expression analysis unveiled 285 genes with significant differences between the two groups (p < 0.05; absolute fold change > 2; FPKM ≥ 1 in at least one sample). Among these, 141 genes were up-regulated, while 144 genes were down-regulated in theobromine-treated mice. To identify the predominant pathways influenced by theobromine treatment, we also conducted a KEGG functional enrichment analysis for the differentially expressed genes. The results were similar to previous findings, with the differential genes mainly belonging to five KEGG categories: human disease, organismal systems, environmental information processing, metabolism, and cellular processes. Within the organismal systems category, the nervous system, endocrine system, and immune system were prominent, with the endocrine and immune systems being among the top three. In the environmental information processing category, the top two enriched signaling pathways are cytokine-cytokine receptor interaction and neuroactive ligand-receptor interaction, consistent with previous analyses (control vs nicotine withdrawal). In the metabolism category, the top two enriched signaling pathway are drug metabolism-cytochrome p450 and glutathione metabolism, similar to previous analyses, except for the absence of the oxidative phosphorylation pathway. In the cellular processes category, the findings were also consistent with previous analyses, with no surprising results. Fig. 4. [99]Fig. 4 [100]Open in a new tab Theobromine treatment significantly altered gene expression in the hippocampus of nicotine withdrawal mice. A. Volcano plot showing differential gene expression between the Nic and TN group, and the differential expression multiples were more than 2. The genes with significant changes are colored. The dotted lines indicate the cut-off for significant changes (p < 0.05). B. KEGG functional enrichment analysis for the differentially expressed genes C, D, E and F. The detailed signaling pathway in the four KEGG categories. In summary, we observed significant changes in gene expression when comparing the Nic group to the Con group, as well as the TN group to the Nic group. Notably, the enriched signaling pathways associated with these gene alterations exhibited similarities across both comparisons. Animal behavioral tests demonstrated that theobromine treatment significantly prevents the affective symptoms of nicotine withdrawal. Based on the aforementioned bioinformatics analysis, we hypothesize that theobromine may mitigate nicotine withdrawal symptoms by modulating these enriched signaling pathways. Theobromine improves affective symptoms of nicotine withdrawal by regulating the glutamate and prolactin signal pathway To further validate our previous inference, we examined the genes that showed significant changes in the neuroactive-ligand-receptor interaction signaling pathway: Pth2r, Prl, Lhcgr, Grik4, Grid1, Galr3, and Chrna1 ([101]Fig. 5). Fig. 5. [102]Fig. 5 [103]Open in a new tab All the significantly changed genes in the neuroactive-ligand receptor interaction signaling pathway among the Con, Nic and TN group. A. Relative changes in parathyroid hormone 2 receptor (Pth2r) gene mRNA among the three experimental groups. B. Relative changes in prolactin (Prl) gene mRNA among the three experimental group. C. Relative changes in Luteinizing hormone/choriogonadotropin receptor (Lhcgr) gene mRNA among the three experimental groups. D. Relative changes in glutamate receptor, ionotropic, kainate 4 (Grik4) gene mRNA among the three experimental groups. E. Relative changes in glutamate ionotropic receptor delta type subunit 1 (Grid1) gene mRNA among the three experimental groups. F. Relative changes in Galanin receptor 3 (Galr3) gene mRNA among the three experimental groups. G. Relative changes in Neuronal acetylcholine receptor subunit alpha-1 (Chrna1) gene mRNA among the three experimental groups. All the data are expressed as mean ± SEM (n = 5 per group), *p < 0.05, Con vs Nic, #p < 0.05, Nic vs TN. In [104]Fig. 5A (F [(2,8)] = 16.11, P = 0.0016), Pth2r encodes the parathyroid hormone (PTH) 2 receptor, which plays a vital role in calcium regulation. While serum PTH concentrations haven't been directly associated with depression in clinical studies ([105]Zhao et al., 2010), primary hyperparathyroidism (PHPT), characterized by elevated PTH levels, has been linked to psychiatric symptoms ([106]Kunert et al., 2020), possibly mediated through Pth2r activation ([107]Diskin and Diskin, 2020). In [108]Fig. 5B (F [(2,8)] = 4.75, P = 0.0437), Prl, encoding prolactin, is recognized for its diverse roles in stress modulation, pregnancy, and lactation ([109]Torner, 2016). Hyperprolactinemia has been shown to significantly impact psychological well-being, contributing to symptoms such as anxiety and depression ([110]Fava et al., 1982, [111]Oliveira et al., 2000). In [112]Fig. 5C (F [(2,8)] = 5.415, P = 0.0326), Lhcgr, the luteinizing hormone (LH) receptor, traditionally linked with reproductive functions, has recently been implicated in mental health. Elevated LH levels are now being associated with mood disorders, including anxiety and depression ([113]Sims et al., 2023). In [114]Fig. 5D and E (F [(2,8)] = 12.4, P = 0.0035, F [(2,8)] = 70.46, P < 0.0001, Grik4, Grid1, respectively), Grik4 and Grid1 encode glutamate receptors, with glutamate dysregulation being linked to depression and anxiety ([115]Murrough et al., 2017, [116]Bergink et al., 2004). Studies have demonstrated that disrupting GluK4 can lead to behaviors resembling those of anxiolytics and antidepressants in animal models ([117]Catches et al., 2012, [118]Paddock et al., 2007). In [119]Fig. 5F (F [(2,8)] = 32.35, P < 0.0001), Galr3 encodes the galanin receptor, part of a neuropeptide system implicated in depression and anxiety pathophysiology. Inhibiting GalR1 and GalR3, while activating GalR2, has shown promise in alleviating depressive symptoms ([120]Demsie et al., 2020). In [121]Fig. 5G (F [(2,8)] = 8.916, P = 0.0092), Chrna1, encoding the α1 subunit of the nicotinic acetylcholine receptor, has been implicated in the complex relationship between anxiety and depression ([122]Terry et al., 2023). Targeting these receptors in the brain has shown efficacy in treating major depression and concurrent alcohol or nicotine addiction ([123]Rahman, 2015). In summary, considering the functions of these genes and the observed changes in behavior and gene expression patterns, we infer that theobromine treatment may prevent nicotine withdrawal symptoms by modulating prolactin, glutamate, and nicotinic acetylcholine signaling within the neuroendocrine system. Theobromine may improve nicotine withdrawal affective symptoms by attenuating brain inflammation The next, the several genes related to pro-inflammatory and anti-inflammatory were also checked ([124]Fig. 6). The significantly changed pro-inflammatory genes include Cxcl10, Cxcl1, Il12a, Cd40, and Cd74 (F [(2,8)] = 16.16, P = 0.0016, F [(2,8)] = 10.78, P = 0.0054, F [(2,8)] = 9.537, P = 0.0076, F [(2,8)] = 20.56, P = 0.0007, F [(2,8)] = 9.081, P = 0.0087, Cxcl10, Cxcl1, Il12a, Cd40 and Cd74, respectively). The expression levels of Cxcl10, Cxcl1, Il12a and Cd40 remarkably increased in the Nic group, but not Cd74. This is consistent with literature in which nicotine withdrawal and major depression disorder are associated with brain inflammation ([125]Beurel et al., 2020, [126]Cruz et al., 2023). Theobromine treatment significantly inhibited the expression of these elevated inflammatory factors. The significantly changed anti-inflammatory genes are Pparg, Mrc1 and Arg1 (F [(2,8)] = 5.055, P = 0.0381, F [(2,8)] = 8.115, P = 0.0119, F [(2,8)]= 15.22, P = 0.0019, Pparg, Mrc1, Arg1, respectively). The expression levels of Arg1 and Mrc1 remarkably decreased in the Nic group, but not Pparg. After treatment with theobromine, their expressions were partially restored. Based on these data, we infer that theobromine may prevent symptoms of depression and anxiety by inhibiting brain inflammation caused by nicotine withdrawal. Fig. 6. [127]Fig. 6 [128]Open in a new tab All the significantly changed pro-inflammatory and anti-inflammatory factor genes among the Con, Nic and TN group. A. Relative changes in C-X-C motif chemokine 10 (Cxcl10) gene mRNA among the three experimental groups. B. Relative changes in C-X-C motif chemokine 1 (Cxcl1) gene mRNA among the three experimental groups. C. Relative changes in interleukin 12α subunit (Il12α) gene mRNA among the three experimental groups. D. Relative changes in CD40 gene mRNA among the three experimental groups. E. Relative changes in CD74 gene mRNA among the three experimental groups. F. Relative changes in Arginase 1 (Arg1) gene mRNA among the three experimental groups. G. Relative changes in Peroxisome proliferator-activated receptor γ(Pparg) gene mRNA among the three experimental groups. H. Relative changes in Mannose receptor 1 (Mrc1) gene mRNA among the three experimental groups. All the data are expressed as mean ± SEM (n = 5 per group), *p < 0.05, Con vs Nic, #p < 0.05, Nic vs TN. Theobromine improves nicotine withdrawal affective symptoms by enhancing glutathione metabolism signal pathway To further determine the impact of theobromine on the glutathione metabolism, several genes with significant differences in the glutathione metabolism signaling pathway were checked at last: Gss, Gsr, Gclc, Gstt2, Ggt5, Idh1, G6pdx, Gpx1, Gstm7, ([129]Fig. 7). Fig. 7. [130]Fig. 7 [131]Open in a new tab All the significantly changed genes in glutathione metabolism signaling pathway among the Con, Nic and TN group. A. Relative changes in glutathione synthetase (Gss) gene mRNA among the three experimental groups. B. Relative changes in glutathione-disulfide reductase (Gsr) gene mRNA among the three experimental groups. C. Relative changes in glutamate-cysteine ligase catalytic subunit (Gclc) gene mRNA among the three experimental groups. D. Relative changes in gamma-glutamyltransferase 5 (Ggt5) gene mRNA among the three experimental groups. E. Relative changes in glutathione s-transferase theta 2 (Gstt2) gene mRNA among the three experimental groups. F. Relative changes in glutathione s-transferase Mu 7 (Gstm7) gene mRNA among the three experimental groups. G. Relative changes in isocitrate dehydrogenase 1 (Idh1) gene mRNA among the three experimental groups. H. Relative changes in glucose-6-phosphate 1-dehydrogenase (G6pdx) gene mRNA among the three experimental groups. I. Relative changes in glutathione peroxidase 1 (Gpx1) gene mRNA among the three experimental groups. All the data are expressed as mean ± SEM (n = 5 per group), *p < 0.05, Con vs Nic, #p < 0.05, Nic vs TN. Glutathione synthetase (Gss) and glutathione reductase (Gsr) are enzymes responsible for producing GSH from its oxidized form (GSSG) and L-γ-glutamylcysteine, respectively. Glutamate-cysteine ligase catalytic subunit (Gclc) is involved in synthesizing L-γ-glutamylcysteine from L-cysteine and L-glutamate. Our findings indicate that the expression levels of these enzymes were significantly reduced during nicotine withdrawal, but theobromine treatment restored their expression (F [(2,8)] = 10.43, P = 0.0059, F [(2,8)] = 11.24, P = 0.0047, F [(2,8)] = 11.64, P = 0.0043, Gss, Gsr, Gclc, respectively). Glutathione S-transferase theta-2 (Gstt2) and glutathione S-transferase, mu 7 (Gstm7) are the enzymes that aid in attaching GSH to xenobiotic substrates, while gamma-glutamyltransferase 5 (Ggt5) transfers gamma-glutamyl groups from glutathione to an acceptor, forming glutamate. These processes are crucial for biotransformation and detoxification. Our data demonstrate that Ggt5 and Gstm7 expression were markedly decreased during nicotine withdrawal but were restored by theobromine treatment (F [(2,8)] = 27.07, P = 0.0003, F [(2,8)] = 9.635, P = 0.0074, Ggt5, Gstm7, respectively). Conversely, Gstt2 expression was elevated during nicotine withdrawal, but theobromine treatment decreased its expression level (F [(2,8)] = 11.78, P = 0.0041). Isocitrate dehydrogenase (Idh1) and glucose-6-phosphate 1-dehydrogenase (G6pdx) are enzymes crucial for converting NADP+ to NADPH within the animal body. NADPH acts as a key component for providing reducing power in various metabolic reactions. Additionally, NADPH plays a critical role as a coenzyme in the reduction of GSSG to GSH, which is essential for cellular antioxidant defense mechanisms. Our data demonstrate that Idh1 and G6pdx expression were markedly decreased during nicotine withdrawal but were restored by theobromine treatment (F [(2,8)] = 14.19, P = 0.0023, F [(2,8)] = 13.48, P = 0.0027, Idh1, G6pdx, respectively). Glutathione peroxidase 1 (Gpx1) is an enzyme possessing peroxidase activity, primarily tasked with safeguarding the organism against oxidative harm by interacting with GSH. Its fundamental biochemical function involves the conversion of lipid hydroperoxides into their respective alcohols and the reduction of free hydrogen peroxide into water. Our data demonstrate the Gpx1 expression was markedly decreased during nicotine withdrawal but was restored by theobromine treatment (F [(2,8)] = 6.601, P = 0.0203). In summary, our analysis reveals that while there were some inconsistencies, there was a notable decrease in glutathione metabolism observed in mice undergoing nicotine withdrawal. However, administering theobromine significantly reversed this reduction, indicating that theobromine may have the potential to prevent the oxidative stress induced by nicotine withdrawal. Discussion Currently, nicotine withdrawal symptoms have become a significant obstacle in tobacco use control, especially withdrawal affective symptoms. Therefore, exploring its mechanism of occurrence and developing new treatment methods is crucial. In this study, we employed non-targeted metabolomics and RNA sequencing to explore the underlying mechanisms. During data analysis, partial least squares discriminant analysis (PLS-DA) was performed as an exploratory tool, which revealed clear distinctions between the groups, indicating that nicotine withdrawal and theobromine treatment induced alterations in the profiles of small molecule metabolites associated with changes in the expression of multiple genes. PLS-DA results and KEGG analysis showed that the differential genes were mostly enriched in the neuroendocrine, immune and glutathione metabolic signaling pathways such as Pth2r, Prl, Ggt1 and Gstt3; the differential metabolites also suggested that abnormal glutathione metabolism is an important phenomenon accompanying nicotine withdrawal, such as Pyroglutamic acid and Y-glutamylcysteine; and theobromine supplementation restored glutathione metabolism and reduced inflammation. Conventionally, it's believed that these symptoms primarily stem from alterations in the neurotransmitter system, involving nAChR, the dopaminergic system, glutamate, and serotonin ([132]Jackson et al., 2015). The hippocampus has been identified as a brain region involved in nicotine withdrawal mechanisms ([133]Paolini and De Biasi, 2011). Galantamine, an acetylcholinesterase inhibitor and positive allosteric modulator of nAChRs, has been shown to reverse the nicotine withdrawal deficits in contextual fear conditioning in mice, possibly through enhanced levels of acetylcholine via acetylcholinesterase inhibition and/or actions at hippocampal nAChRs ([134]Wilkinson and Gould, 2011). However, our RNA-seq data showed among the 19 nAChR genes identified, only Chrna1 showed a significant increase in expression level, while the expression levels of other genes remained unchanged ([135]Fig. 5, [136]Supplemental Table 2). Moreover, we did not find any significant changes in the receptors of dopamine and serotonin. However, two ionotropic glutamate receptors showed significant changes in nicotine withdrawal. Glutamate is the major excitatory neurotransmitter in the mammalian brain, and its actions are mediated by fast-acting ionotropic receptors and slow-acting G-protein-coupled metabotropic receptors. Research has shown that inhibition of metabotropic glutamate receptors may be novel therapeutics for nicotine dependence and depression ([137]Markou, 2007, [138]Cross et al., 2018). Although studies have shown that the drugs targeting metabotropic glutamate receptors have fewer side effects ([139]D'Souza and Markou, 2013), our results suggest that ionotropic glutamate receptors may play a greater role in the development of nicotine withdrawal symptoms, and theobromine can weaken its expression and improve withdrawal symptoms. Numerous studies have indicated the involvement of the endocrine system in nicotine withdrawal symptoms ([140]Leach et al., 2015). In our research, we specifically observed significant alterations in three hormone systems—parathyroid hormone, prolactin, and luteinizing hormone—within the hippocampus. Although there is limited data suggesting fluctuations in the levels of these three hormones during nicotine use, there is insufficient evidence to substantiate their connection with nicotine withdrawal symptoms. However, it is noteworthy that there exists a significant correlation between prolactin levels and the onset of anxiety and depression ([141]Zamorano et al., 2014, [142]Elgellaie et al., 2021). So, according to our findings, prolactin may be associated with affective symptoms of nicotine withdrawal, while theobromine improves these symptoms by reducing prolactin expression. Moreover, a well-established connection exists between brain inflammation, depression, and nicotine withdrawal ([143]Beurel et al., 2020, [144]Cruz et al., 2023, [145]Amiry et al., 2023), and literature has shown that theobromine has significant anti-inflammatory functions ([146]Zhang et al., 2024b). Our RNA-Seq data indicated that multiple inflammatory factors were increased in the nicotine withdrawal group, while theobromine consumption reduced the expression of these genes. These results suggest that theobromine may mitigate brain inflammation induced by nicotine withdrawal. However, sequencing results alone cannot accurately reflect changes in inflammation, and further experiments are needed to confirm these findings. Two studies have presented contradictory conclusions regarding the effects of nicotine on oxidative stress ([147]Ozdemir-Kumral et al., 2017, [148]Oyeyipo et al., 2014). However, a significant finding in our research is the pivotal involvement of the glutathione metabolism pathway in the hippocampus during nicotine withdrawal symptoms. We observed a decrease in blood glutathione levels among smokers, which returned to normal after one year of smoking cessation ([149]Mons et al., 2016). While multiple factors may contribute to the reduction of blood glutathione during tobacco use, this finding indirectly suggests a potential correlation between glutathione levels and nicotine withdrawal. Furthermore, our study not only revealed that theobromine can aid in the restoration of glutathione metabolism in the hippocampus also shows a positive correlation with the alleviation of withdrawal symptoms. Glutathione, the most crucial antioxidant in the body, serves a vital function in detoxification. Several studies have indicated that depression is linked to reduced levels of glutathione in the brain ([150]Rae and Williams, 2017, [151]Freed et al., 2017), suggesting that glutathione enhancers may hold therapeutic potential in stress-related psychopathologies ([152]Zalachoras et al., 2020). Our RNA-seq and untargeted metabolomics data revealed that during nicotine withdrawal, the expression of numerous genes related to the production and utilization of glutathione in the hippocampus was notably decreased, suggesting a compromised antioxidant and detoxification role of glutathione. The metabolism of chemicals such as nicotine in the body depends on the body's ability to biotransform, and the biotransformation reactions involved in glutathione are a very important part. Theobromine enhances the body's detoxification function, namely biotransformation capacity, which may be an important mechanism for alleviating withdrawal symptoms. However, administration of theobromine reversed the expression of these genes, indicating that theobromine enhanced the antioxidant and detoxification capabilities of glutathione. To date, there have been no reports directly linking theobromine to glutathione metabolism. However, research on theobromine's alleviation of oxidative stress has been documented ([153]Gu et al., 2020). Nevertheless, further investigation is required to elucidate the underlying mechanisms. In summary, this study extensively explored the mechanism underlying nicotine withdrawal affective symptoms in the hippocampus using multiple omics techniques. Our findings not only validate previous research linking nicotine withdrawal affective symptoms to neuroendocrine function and brain inflammation but also highlight weakened glutathione metabolism as a significant factor in their occurrence. Moreover, we discovered that theobromine, a major psychoactive compound in cocoa, effectively prevents nicotine withdrawal affective symptoms through multifaceted mechanisms. Beyond its regulation of neuroendocrine function and brain inflammation, theobromine's ability to restore glutathione metabolism in the hippocampus emerges as a crucial aspect of its action. Considering the edible nature and low toxicity of theobromine, it holds promise as a potential food additive or over-the-counter medication for treating nicotine withdrawal symptoms in the future. Nonetheless, our research results have several limitations, primarily as they were derived from male mice and are therefore only applicable to male subjects. It is well established that nicotine withdrawal affective symptoms exhibit sex differences, along with variations in associated neural activity ([154]Correa et al., 2019). Consequently, comparative studies involving both male and female animals will be essential in future research. CRediT authorship contribution statement Yu Tian: Visualization, Resources, Methodology, Investigation, Formal analysis. Qi Zhang: Writing – review & editing, Project administration, Investigation, Funding acquisition, Data curation, Conceptualization. Jufang Hao: Writing – review & editing, Writing – original draft, Supervision, Resources, Investigation, Funding acquisition, Data curation, Conceptualization. Xingyu Liu: Supervision, Resources, Investigation, Formal analysis, Data curation. Wenjuan Zhang: Visualization, Software, Methodology, Investigation, Funding acquisition. Baojiang He: Resources, Methodology, Investigation, Data curation. Funding This work was supported by the grants of Science and Technology Projects of Beijing Life Science Academy (BLSA [2024] No. 2, 2024300CC0030), Special Project on Stable Support for Innovation Platform of the State Tobacco Monopoly Administration of China (National Tobacco Company [2021] No. 171) and China Tobacco Industry Development Center Science and Technology Plan Project (ZYSYM-2022-3). Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements