Thioperamide Alleviates Lipopolysaccharide-Induced Neuroinammation and Promotes Neurogenesis by Histamine Dependent Activation of H2R/PKA/CREB Pathway

Adult neurogenesis in hippocampus dentate gyrus (DG) is associated with numerous neurodegenerative diseases such as aging and Alzheimer's disease (AD). Overactivation of microglia induced neuroinammation is well acknowledged to contribute to the impaired neurogenesis in pathologies of these diseases and then leading to cognitive dysfunction. Histamine H3 receptor (H3R) is a presynaptic autoreceptor regulating histamine release via negative feedback way. Recently, studies show that H3R are highly expressed not only in neurons but also in microglia to modulate inammatory response. However, whether inhibition of H3R is responsible for the neurogenesis and cognition in chronic neuroinammation induced injury and the mechanism remains unclear. AMP response element-binding protein (CREB) pathway but inhibited nuclear factor kappa-B (NF-κB) signaling. Activation of CREB by thioperamide promoted interaction of CREB-CREB Binding Protein (CBP) to increase anti-inammatory cytokines (Interleukin-4 and Interleukin-10) and brain-derived neurotrophic factor (BDNF) release but inhibited NF-κB-CBP interaction to decrease pro-inammatory cytokines (Interleukin-1β, Interleukin-6 and Tumor necrosis factor α) release. H89, an inhibitor of PKA/CREB signaling, abolished effects of thioperamide on neuroinammation and neurogenesis. extracts was determined by the Bradford assay (Thermo, Hercules, CA). The precipitates were denatured with SDS sample loading bu ﬀ er and separated on 10% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes using a Bio-Rad mini-protein-III wet transfer unit overnight at 4°C. Transfer membranes were then incubated with blocking solution (5% nonfat dried milk dissolved in tris bu ﬀ ered saline tween (TBST) bu ﬀ er (in mM): 10 Tris-HCl, 150 NaCl, and 0.1% Tween-20) for 2 h at room temperature, and incubated with primary antibody overnight at 4°C. The primary antibodies used in this experiment were phospho-PKA (Cell Signaling Technology, 1:1,000), PKA (Cell Signaling Technology, 1:1,000), phospho-CREB (Cell Signaling Technology, 1:1,000), CREB (Cell Signaling Technology, 1:1,000), phospho-P65 NF-kB (abcam, 1:1000), P65 NF-kB (abcam, 1:1000), CBP (abcam, 1:1000) and GAPDH (Boster, 1:3,000). Membranes were washed three times in TBST bu ﬀ er and incubated with the appropriate secondary antibodies (LI-COR, Odyssey, 1:5,000) for 2 h. Images were acquired with the Odyssey infrared imaging system and analyzed as specied in the Odyssey software manual. The results were expressed as the target protein/GAPDH ratio and then normalized to the values measured in the control groups (presented as 100%).

Taken together, these results suggested under LPS induced neuroin ammation, the H3R antagonist thioperamide inhibited microglia activity and in ammatory response, and ameliorated impairment of neurogenesis and cognitive dysfunction via enhancing histamine release. Histamine activated H2R and reinforced CREB-CBP interaction but weakened NF-κB-CBP interaction to exert anti-in ammatory effects. This study uncovered a novel histamine dependent mechanism behind the therapeutic effect of thioperamide on neuroin ammation.

Background
Adult neurogenesis, a process of generating functional neurons from adult neural precursors, occurs throughout life in restricted brain regions in mammals [1,2]. Within the hippocampal dentate gyrus (DG), newly born neurons in the granule cell layer (GL) send axonal projections to the CA3 sub eld of the hippocampus and spineless dendrites to the molecular layer [3]. The new neurons then integrate into the existing hippocampal tri-synaptic circuitry by establishing synapses in the molecular layer with other neurons, [4] and then promote the ability of learning and memory [5]. Dysregulation of hippocampal neurogenesis has been shown to be an important mechanism underlying the cognitive impairment associated with normal aging, as well as the cognitive de cits in Alzheimer's disease (AD) and other neurodegenerative diseases [6]. Chronic neuroin ammation is a common pathological feature in normal aging as well as in these neurodegenerative conditions and has been shown to negatively affect hippocampal neurogenesis and cognitive processes across the lifespan [7]. Conversely, neurotrophic factors, environmental enrichment, learning, and exercise could positively regulate adult hippocampal neurogenesis and associated cognitive function [6,8,9]. Therefore, clarify the fundamental mechanisms regulating adult neurogenesis in physiological and pathological conditions will thus provide the basis for cell replacement therapy for treatment of disorders of central nervous system (CNS).
Microglia account for approximately 10% of cells in the CNS, and help shape neural circuits by modulating the strength of synaptic transmissions and sculpting neuronal synapses [10]. Overactivation of glial cells is commonly found in numerous neurodegenerative diseases such as normal aging and AD in CNS [11,12]. Microglia activation is often categorized as either classical (M1) or alternative (M2). M1 microglia produce proin ammatory cytokines and chemokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) whereas M2 microglia produce anti-in ammatory cytokines such as interleukin-4 (IL-4), interleukin-10 (IL-10) and neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1(IGF-1) [13]. Recent studies show that microglia are involved in neurogenesis [14]. IL4-driven M2 microglia polarization in the hippocampus trigger BDNF-dependent neurogenesis [15]. However, mediators of M1 microglia, including IL-1β, IL-6 and TNF-α have been shown to have a negative effect on hippocampal neurogenesis by reducing the proliferation and survival of newborn cells [6,16,17]. Thus, it is of great importance to study focusing on therapeutic agents capable of inducing M2 or preventing M1 polarization in chronic neurodegenerative diseases.
Histamine is a heterocyclic amine formed by decarboxylation of the amino acid l-histidine. It is involved in the local regulation of physiological processes but also can occur exogenously in the food supply [18]. In the CNS, histamine is an endogenous neurotransmitter regulating many physiological processes including temperature regulation, emesis, food intake, and avoidance behavior [19,20]. Accumulating evidence suggests that the activating histaminergic system in CNS may regulate brain injury [21,22]. However, the direct application of histamine is clinically limited due to its poor penetration of the bloodbrain barrier and its pro-in ammatory effect in the peripheral system [22]. Histamine H3 receptor (H3R) is a presynaptic autoreceptor that regulates histamine release from histaminergic neurons via negative feedback way [23,24], as well as a heteroreceptor that regulates the release of other neurotransmitters [25][26][27][28][29][30]. A number of experiments have also provided evidences that inhibition of H3R could alleviate cognitive de cit in chronic in ammation related neurodegenerative diseases such as normal aging and AD [31][32][33][34][35][36][37]. H3R antagonist rescues autistic spectrum disorder-like behaviors through attenuating the proin ammatory cytokines [38]. Thioperamide, a histamine H3R antagonist, also suppresses in ammatory cell recruitment through histamine dependent mechanism [39]. Recent studies indicate that histamine and H3R are also involved in modulation of neurogenesis. Histamine enhances neurogenesis, and promotes neuronal differentiation and dendritic arbor complexity [40,41]. Inhibition of H3R promotes neurogenesis in preterm white matter injury, traumatic brain injury and aging mice [36, [42][43][44]. Moreover, H3R is also involved in migration of neural stem cells (NSCs) [45]. Therefore, H3R antagonist may serve to develop new therapeutic approaches to overcome chronic in ammation related neurodegenerative disorders. However, the mechanism underlying the anti-in ammatory effect of H3R antagonist on microglial phenotypes and its effect on in ammation-related impairment of neurogenesis has not been reported.
In this study, we hypothesized that thioperamide, a H3R antagonist has a microglia-dependent antiin ammatory effect, which is involved with neurogenesis. We investigated the effects of thioperamide on microglial phenotypes and hippocampal neurogenesis in LPS-induced in ammation in mice. We found that thioperamide inhibited in ammation and promoted neurogenesis through histamine-dependent H2R/cAMP/PKA/CREB pathway. Furthermore, this study showed that thioperamide promoted secretion of anti-in ammatory cytokines including IL-4, IL-10 and BDNF from M2 microglia via enhancing CREB-CREB binding protein (CBP) interaction but inhibited secretion of pro-in ammatory cytokines IL-1β, IL-6 and TNFα from M1 microglia via suppressing CREB-nuclear factor kappa-B (NF-κB) interaction. We would uncover a novel mechanism that H3R antagonist-mediated neurogenesis in chronic in ammation related neurodegenerative diseases.

Ethical statement
All animal studies were carried out according to protocols approved by the Institutional Animal Care and Use Committee of Binzhou Medical University Hospital, and conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize any pain or discomfort, and the minimum number of animals was used.

Animals
Adult male C57BL/6 mice (Pengyue Laboratory, Jinan, China) weighing 22-25g were used in this study. The mice were housed in a temperature-and humidity-controlled animal facility, which was maintained on a 12 h light/dark cycle, food and water were given ad libitum.

Stereotaxic surgery
Stereotaxic surgery was performed as previously described [46] . Mice were anesthetized with the intraperitoneal injection of 1% chloral hydrate and then immobilized on a stereotactic frame. The gauge guide cannula was implanted into the lateral ventricle (0.2 mm posterior, 1.1 mm lateral and 2.7 mm ventral to the bregma). After surgery, mice were housed individually and allowed to recover for 7 d.

Drug treatments
For in vivo study, a stainless-steel injector connected to a 5-ml syringe was inserted into the guide cannula and extended 1 mm beyond the tip. The administration of the chemicals was administrated on the basis of previous studies [47] . LPS (i.p., 1 mg/kg) or vehicle was administrated daily at 7 days after stereotaxic surgery for 5 days. Thioperamide (i.p., 5 mg/kg) or vehicle was administrated daily at 7 days after stereotaxic surgery until the beginning of the behavior tests on day 14. H89 or vehicle (i.c.v., 2 µg of 2 µl) was administrated 0.5 h before thioperamide injection. 5-bromo-2'-deoxyuridine (BrdU) (i.p., 50mg/kg) was administrated for 4 times every 4 h at 7 days after stereotaxic surgery, and then administrated every other day before the behavior experiments [48] . At 14 days after stereotaxic surgery, the novel object recognition (NOR) test was carried on for 4 days. At 18 days after stereotaxic surgery, Y maze (YM) test was carried on for 1 day. At 19 days after stereotaxic surgery, morris water maze (MWM) test was carried on for 6 days. During the period of behavior test, thioperamide or vehicle was administrated 0.5 h, whereas H89 or vehicle was administrated 1 h before the test at day 14, day 18 and day 19.
BrdU/DCX/NeuN staining BrdU was used to label newly born cells, DCX was used to label immature neurons and NeuN was used to label mature neurons. The frozen brain sections (40 μm) were incubated for 30 min in 2 N HCl at 37°C, and neutralized with 0.1 M borate buffer (Sinopharm Chemical Reagent, PH=8.5) for 10 min. After incubating in 1% H2O2 (30% H1009, Sigma) for 10 min, the sections were blocked with PBS containing 1% BSA and 0.3% (w/v) Triton X-100 (T8787, Sigma) for 1 h at room temperature. They were then reacted with rat monoclonal anti-BrdU (1:50, Abcam), rabbit monoclonal anti-DCX (1:50, Cell Signaling Technology) and mouse monoclonal antibody against NeuN (1:300, Millipore) at 4°C overnight. After washing with PBS, they were incubated with secondary antibody for 2 h at room temperature. The stained cells were observed under a laser scanning confocal microscope (Leica TCS SPE, Germany). Image analysis was performed using Image J software.

Stereological cell counting of BrdU+ cells
We used the stereological techniques to count the number of BrdU+ cell as described previously [49] .
BrdU+ and BrdU+/DCX+ cells from every eighth section covering the entire rostrocaudal axis of the DG were counted using a high-power (40×) microscope. Cells were counted in a blind manner. At least eight sections from both sides of the DG were counted per animal. The number for each group of animals is indicated in gure legends.

ELISA
Mice were anaesthetized by i.p. injection of 1% chloral hydrate, sacri ced and the brain was quickly removed. The separated tissues were lysed in ice-cold RIPA lysis buffer (R0020, Solarbio), then centrifuged at 14,000×g at 4°C for 20 min, and the supernatants were measured for soluble IL-1β, IL-6, TNF-α, IL-4 and BDNF ELISAs (R&D) according to the manufacturer's instructions. The values were expressed as amount per total protein.

Western blot
Western blot was performed as previously described [50] . Brie y, mice were anaesthetized by i.p. injection of chloral hydrate (400 mg/kg), sacri ced and the brain was quickly removed. The separated tissues were lysed in ice-cold RIPA lysis buffer (R0020, Solarbio), then centrifuged at 14,000×g at 4°C for 20 min, and the protein concentration in the extracts was determined by the Bradford assay (Thermo, Hercules, CA). The precipitates were denatured with SDS sample loading buffer and separated on 10% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes using a Bio-Rad mini-protein-III wet transfer unit overnight at 4°C. Transfer membranes were then incubated with blocking solution (5% nonfat dried milk dissolved in tris buffered saline tween (TBST) buffer (in mM): 10 Tris-HCl, 150 NaCl, and 0.1% Tween-20) for 2 h at room temperature, and incubated with primary antibody overnight at 4°C. The primary antibodies used in this experiment were phospho-PKA (Cell Signaling Technology, 1:1,000), PKA (Cell Signaling Technology, 1:1,000), phospho-CREB (Cell Signaling Technology, 1:1,000), CREB (Cell Signaling Technology, 1:1,000), phospho-P65 NF-kB (abcam, 1:1000), P65 NF-kB (abcam, 1:1000), CBP (abcam, 1:1000) and GAPDH (Boster, 1:3,000). Membranes were washed three times in TBST buffer and incubated with the appropriate secondary antibodies (LI-COR, Odyssey, 1:5,000) for 2 h. Images were acquired with the Odyssey infrared imaging system and analyzed as speci ed in the Odyssey software manual. The results were expressed as the target protein/GAPDH ratio and then normalized to the values measured in the control groups (presented as 100%).

Novel object recognition (NOR)
The NOR test was performed 14 days after stereotaxic surgery as previously described [46,51] . Brie y, mice received 2 d of habituation in a 45 × 45 cm square arena, and on the third day, they were allowed to explore two identical objects made from large Lego bricks for 10 min (training trial). They were returned to their home cage, and 24 h later, a different shape and color object replaced one of the objects and the mice were returned to the arena for 10 min (testing trial). The time spent on each object was then calculated as a percentage of total object exploration.
The Y maze test was performed 18 days after stereotaxic surgery as previously described [46,51] . Brie y, the apparatus for YM was made of gray plastic, with each arm 40 cm long, 12 cm high, 3 cm wide at the bottom and 10 cm wide at the top. The three arms were connected at an angle of 120°. Mice were individually placed at the end of an arm and allowed to explore the maze freely for 8 min. The total arm entries and spontaneous alternation percentage (SA%) were measured. SA% was de ned as a ratio of the arm choices that differed from the previous two choices ('successful choices') to total choices during the run ('total entry minus two' because the rst two entries could not be evaluated). For example, if a mouse made 10 entries, such as 1-2-3-2-3-1-2-3-2-1, there are 5 successful choices in 8 total choices (10 entries minus 2). Therefore, SA% in this case is 62.5%.

Morris water maze (MWM)
The MWM maze test was performed 19 days after stereotaxic surgery as previously described [46,52] . Brie y, the water maze of 1.50 m in diameter and 0.50 m in height was lled with water (20 ± 1°C) to maintain the water surface 1.50 cm higher than the platform (10 cm in diameter). Water was dyed white and the tank was divided into four quadrants by four points: North (N), South (S), East (E), and West (W). The platform was placed at the center of either quadrant and video tracking software was used to automatically track the animals. Learning and memory acquisition lasts for ve days. Animals were put into the water from four points in random order every day until they found the platform and stayed for 10 s within 1 min. If the mice cannot nd the platform within 1 min, they were guided to the platform. Following acquisition test, on the sixth day, learning and memory maintenance test was carried on. The platform was removed, and the mice were placed in water from the opposite quadrant of the platform, and then the times crossing the platform was recorded within 1 min.

Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVAs followed by Tukey's post hoc comparisons or two-way ANOVAs followed by Bonferroni post hoc comparisons, using prism software. P value <0.05 was considered statistically signi cant.

Thioperamide decreases LPS-induced microglial activation and pro-in ammatory cytokines production
The study of the microglia response in the in ammatory process has been copiously supported by the use of LPS, a gram-negative cell wall component [53]. LPS binds to the CD14/TLR4/MD2 receptor complex, located on the cell membrane, triggering classical microglial responses such as proliferation, migration, phagocytosis and release of in ammatory mediators [54,55]. Therefore, LPS was used to evaluate the effect of thioperamide, a H3R antagonist on neuroin ammation in mice. We examined the activated microglia in DG of the hippocampus by Iba1 immunostaining. Results showed that the area of Iba1 + -cells in the hippocampal DG of LPS treated mice increased dramatically compared with the vehicle treated mice (from 0.9187 ± 0.08736 to 5.200 ± 0.4575, P <0.001, Figure 1A, B), which was reversed by administration of thioperamide (to 2.258 ± 0.2632, P <0.01, Figure 1A, B).
The activation of microglia may promote the pathological process of chronic neuroin ammation-related diseases through releasing of pro-in ammatory cytokines which could lead to neuronal damage and cognitive impairments [56,57]. Moreover, the in ammatory cytokines in neurodegenerative diseases are thought to lead to an impairment of neurogenesis [6]. Therefore, we investigated transcriptional and expressional level of pro-in ammatory factors in LPS treated mice. To con rm the effect of thioperamide on the gene transcription of in ammatory cytokines, we tested the effects of thioperamide on mRNA expression of IL-1β, IL-6 and TNF-α by RT-PCR. We found that LPS up-regulated the mRNA level of proin ammatory cytokines IL-1β (increased to 363.8 ± 39.05% of control, p < 0.01, Figure 1C), IL-6 (increased to 431.7 ± 84.46% of control, p < 0.01, Figure 1D) and TNF-α (increased to 571.1 ± 83.20% of control, p < 0.001, Figure 1E) in hippocampus. Interestingly, thioperamide decreased the mRNA level of IL-1β (decreased to 122.0 ± 37.85% of control, p < 0.05, Figure 1C), IL-6 (decreased to 150.0 ± 15.90% of control, p < 0.01, Figure 1D) and TNF-α (decreased to 187.8 ± 63.97% of control, p < 0.001, Figure 1E). We further con rmed the effect of thioperamide on protein levels of in ammatory cytokines by ELISA. Similarly, we found that the upregulated protein level of IL-1β (from 311.0 ± 44.29% to 109.5 ± 23.29% of control, p < 0.01, Figure 1F), IL-6 (from 280.8 ± 42.87% to 110.3 ± 26.32% of control, p < 0.05, Figure 1G) and TNF-α (from 262.8 ± 18.67% to 116.3 ± 22.52% of control, p < 0.001, Figure 1H) induced by LPS in hippocampus was signi cantly reversed by thioperamide. Taken together, all these results suggested that thioperamide could effectively suppress both activation of microglia and secretion of pro-in ammatory cytokines in LPS treated mice.
Thioperamide ameliorates LPS-induced impairment of neurogenesis Normal aging might prime microglia towards the classic M1 phenotype, and increase basal release of the pro-in ammatory cytokines IL-1β, interleukin-6 and TNFα, which have been shown to reduce hippocampal neurogenesis [58]. Therefore, we next assessed the effect of thioperamide on adult neurogenesis in the DG region of the hippocampus. We examined the newborn cells labeled by BrdU in DG 1 day after the last time of LPS administration. The results indicated that the BrdU + cells in the DG region of hippocampus decreased in the vehicle group compared to the control group (decreased to 51.62 ± 1.475% of control, P <0.001, Figure 2A, D), and thioperamide rescued the decreased BrdU+ cells to 85.84 ± 4.788% signi cantly (P <0.001, Figure 2A, D).
To further investigate the effect of thioperamide on neurogenesis in LPS-treated mice, we analyzed the presence of neurogenesis in DG of hippocampus. Firstly, Doublecortin (DCX) + neuroblasts and immature neurons were detected. The results indicated that the number of DCX + cells decreased signi cantly after LPS treatment (decreased to 63.36 ± 3.342% of control, P <0.001, Figure 2B, E), which was reversed dramatically by administration of thioperamide (increased to 92.47 ± 5.029% of control, P <0.01, Figure   2B, E). The number of BrdU + /DCX + cells was also analyzed to assess the effect of thioperamide on immature newborn neurons. Results showed that the number of BrdU + /DCX + cells decreased signi cantly after LPS treatment (decreased to 37.25 ± 1.307% of control, P <0.001, Figure 2B, F), and thioperamide remarkedly rescued the impairment of immature newborn neurons (increased to 85.62 ± 4.999% of control, P <0.001, Figure 2B, F). Moreover, the percentage of BrdU + /DCX + newborn neuronal cells over BrdU + newborn cells was further analyzed, and the results showed that the decreased percentage of BrdU + /DCX + cells over BrdU + cells in the LPS treated mice was signi cantly reversed by administration of thioperamide (from 32.66 ± 1.37% to 45.09 ± 1.543%, P <0.05, Figure 2B, G).
We further examined the effect of thioperamide on mature newborn neurons in DG of hippocampus by BrdU + /NeuN + staining. Results showed that, number of BrdU + /NeuN + cells decreased signi cantly in the LPS vehicle group compared to the control group (decreased to 42.36 ± 2.778% of control, P <0.001, Figure 2C, H), and thioperamide rescued the impairment of mature newborn neurons signi cantly (increased to 93.06 ± 5.534% of control, P <0.001, Figure 2C, H). In addition, the percentage of BrdU + /NeuN + newborn mature neurons over BrdU + newborn cells were also analyzed, and the results showed that the decreased percentage of BrdU + /NeuN + cells over BrdU + cells in the LPS treated mice was signi cantly reversed by administration of thioperamide (from 31.44 ± 0.8138% to 43.39 ± 1.269%, P <0.01, Figure 2B, G).
Taken together, results above showed that thioperamide promoted neurogenesis in LPS induced neuroin ammation.

Thioperamide alleviates LPS-induced cognitive dysfunction
Neuroin ammation plays an important role in the onset and progression of neurodegenerative diseases such as aging and AD. LPS level is higher in the brains of AD patients and is associated with neuroin ammation and cognitive dysfunction [59]. in this research, we studied the effect of thioperamide on LPS-induced cognitive decline. The NOR test indicated that time spending on novel objection decreased signi cantly in the LPS group compared with the control group (from 70.15 ± 2.384% to 51.57 ± 4.737%, P <0.01, Figure 3A). Administration of thioperamide signi cantly increased the time spending on novel object (to 66.07 ± 2.282%, P <0.05, Figure 3A) in LPS treated mice. In the YM test, we observed a decreased spontaneous alternation % (SA%) in the LPS group compared with the control group (from 83.20 ± 3.751% to 57.08 ± 7.202%, P <0.01, Figure 3B). Administration of thioperamide increased the SA% to 81.40 ± 4.007% signi cantly in LPS treated mice (P <0.05, Figure 3A). In morris water maze (MWM) test, the escape latency increased signi cantly in the LPS group on day 3 to day 5 (P <0.05, Figure 3C). Administration of thioperamide signi cantly decreased the escape latency (P <0.01, Figure 3C) in LPS treated mice. Moreover, times crossing the platform decreased in LPS treated mice (from 7.000 ± 0.5669 to 3.500 ± 0.4226, P <0.01, Figure 3D) on day 6, and administration of thioperamide increased it signi cantly (to 6.250 ± 0.5901, P <0.05, Figure 3D) in LPS treated mice. Results above suggested that thioperamide improved the cognitive impairments in in LPS treated mice.
The effects of thioperamide on neuroin ammation, neurogenesis and cognition involve histamine dependent H2R activation Histamine has been shown to counteract LPS-induced glial activation and release of pro-in ammatory cytokines release as well as neurogenesis impairment [40,53]. Moreover, numerous evidences indicated that central histamine have an important role in cognitive function as it has been shown to enhance memory [60]. As a presynaptic receptor on histaminergic neurons, H3R suppresses histamine synthesis and releases in a negative feedback way. Therefore, inhibition of H3R by thioperamide leads to enhanced synaptic histamine release [47]. In order to con rm whether or not the effects of thioperamide are histamine dependent, pyrilamine or cimetidine, antagonist of H1R or H2R, was applied.
We further examined the role of histamine in the enhanced neurogenesis offered by thioperamide in LPS treated mice. As expect, we found that the BrdU + cells in the DG region of hippocampus were reversed by administration of cimetidine (from 85.84 ± 4.788 to 50.15 ± 3.789 of control, P <0.001, Figure 2A Finally, we tested whether histamine was involved in the alleviated cognitive impairment offered by thioperamide in LPS treated mice. The NOR test showed that time spending on novel objection was reversed by administration of cimetidine (from 66.07 ± 2.282% to 52.62 ± 3.254% of control, P <0.05, Figure 3A) but not pyrilamine (from 66.07 ± 2.282% to 65.33 ± 2.483% of control, P >0.05, Figure  3A). The YM test also showed that SA% was reversed by administration of cimetidine (from 81.40 ± 4.007% to 58.59 ± 5.367% of control, P <0.05, Figure 3B) but not pyrilamine (from 81.40 ± 4.007% to 81.27 ± 3.499% of control, P >0.05, Figure 3B). In the MWM test, the escape latency was reversed by administration of cimetidine (P <0.05, Figure 3C) but not pyrilamine (P >0.05, Figure 3C) on day 4 to day 5.
In addition, increased times crossing the platform offered by thioperamide was reversed by cimetidine (from 6.250 ± 4.007% to 3.375 ± 0.8004% of control, P <0.05, Figure 3D) but not pyrilamine (P >0.05, Figure  3D). In all, results above showed that thioperamide alleviated LPS induced cognitive dysfunction through histamine dependent H2R activation.
Thioperamide reverses LPS-induced inactivation of PKA/CREB pathway via histamine dependent H2R activation Results above showed that histamine dependent H2R activation is involved in the protection against LPS induced in ammatory response. Recent reports indicate that H2R and its downstream activation of cAMP/PKA is also necessary to the inhibited immune response of histamine [61, 62]. Moreover, cAMP/PKA/CREB signaling is considered to play an important role in the suppression of microglia activation and its related neuroin ammation by inhibiting NF-κB activation [63, 64]. Thus, in order to elucidate the mechanisms of the anti-in ammatory offered by thioperamide in LPS treated mice, we investigated the H2R downstream protein level of PKA and CREB. In consistent with the previous reports, decreased p-PKA and p-CREB level were observed in hippocampus in LPS treated mice (p-PKA decreased to 55.89 ± 7.068% of control group, P < 0.01, Figure 4A, B; p-CREB decreased to 55.87 ± 3.954% of control group, P < 0.001, Figure 4A, C). As expect, thioperamide up-regulated the p-CREB expression (p-PKA increased to 97.94 ± 18.63% of control group, P < 0.05, Figure 4A, B; p-CREB increased to 90.00 ± 6.892% of control group, P < 0.01, Figure 4A, C), which was reversed by cimetidine (p-PKA decreased to 59.38 ± 8.18% of control group, P < 0.05, Figure 4A, B; p-CREB decreased to 59.32 ± 3.999% of control group, P < 0.01, Figure 4A, C) but not pyrilamine, suggesting thioperamide activated the H2R downstream PKA/CREB signaling.
The phosphorylated CREB exerts a dual function in in ammatory response. First, by forming a complex with CBP to activate transcription of anti-in ammatory cytokines such as IL-4, IL-10 as well as BDNF.
Thioperamide promotes polarization of M2 microglia from M1 microglia via activating PKA/CREB pathway in LPS-treated mice In order to further investigate the involvement of PKA/CREB signaling in the effects of thioperamide on the activation of microglia, H89, the inhibitor of PKA/CREB was administrated to inhibit p-CREB. The results showed that the area of Iba1 + -cells in hippocampus DG markedly increased in the thioperamide + H89 group compared with the thioperamide group in LPS treated mice (from 2.542 ± 0.3964 to 5.114 ± 0.4003, P <0.001, Figure 5A, B).
Reports have shown that CREB is involved in polarizing microglia from M1 to M2 phenotype. M1 microglia produce pro-in ammatory cytokines, such as IL-1β, IL-6 and TNF-α, whereas M2 microglia produce anti-in ammatory cytokines such as IL-4, IL-10 and BDNF [13]. Thus, we further investigated the involvement of PKA/CREB signaling in the effect of thioperamide on microglia phenotypes. Firstly, we examined the role of CREB activation in the secretion of M1 microglia related pro-in ammatory cytokines, including IL-1β, IL-6 and TNF-α. Results showed that the decreased mRNA level of all the three proin ammatory cytokines offered by thioperamide in hippocampus were reversed signi cantly after H89 treatment (IL-1β: from 40.28 ± 11.50 to 86.62 ± 1.721 of vehicle, P <0.01; IL-6: from 37.12 ± 4.813 to 88.47 ± 4.688 of vehicle, P <0.01; TNFα: from 43.27 ± 13.97 to 91.37 ± 7.308 of vehicle, P <0.05; Figure  5C) in LPS treated mice. Otherwise, a reversed protein level of three pro-in ammatory cytokines were also observed in the Thio+H89 group compared with the thio group in hippocampus (IL-1β: from 40 Figure 5D) in LPS treated mice.
Taken together, these results indicated that thioperamide promoted switch of microglia from M1 to M2 phenotype through activating H2R downstream PKA/CREB signaling.

Thioperamide increases the dendritic complexity via activating PKA/CREB pathway in LPS-treated mice
The previous studies have shown that the pro-in ammatory cytokines can induce abnormal neuronal morphology and promote the loss of synapses in AD [40,[72][73][74]. Therefore, we explored the effects of thioperamide on morphologies of neurons and further analyzed the involvement of PKA/CREB signaling.

Discussion
In the present study, we have shown that inhibition of H3R by thioperamide inhibited microglial activation, suppressed in ammatory response and further enhanced neurogenesis in LPS induced neuroin ammation in mice. Importantly, the alleviated effects of thioperamide on reactivity of microglia, neuroin ammation, neurogenesis and cognitive function were all compromised by cimetidine but not pyrilamine, suggesting a mechanism involvement of histamine dependent H2R activation. Moreover, H89 reversed the decreased phosphorylation of NF-κB, and the increased interaction of CREB-CBP offered by thioperamide, suggesting an underlying mechanism involving H2R-cAMP/PKA/CREB signaling.
Up to now, the population of aged individuals is increasing worldwide with signi cant health and socioeconomic implications. Studies on aging have discovered myriad changes in the brain, including reduced neurogenesis, increased synaptic aberrations, higher metabolic stress associating with cognitive decline [11]. Accumulating evidence suggests that the pathological changes occurs in part because the environment is affected during disease in a cascade of processes collectively termed neuroin ammation [75]. Microglia are the resident immune cells in CNS and play a pivotal role in maintaining brain homeostasis. In the aging brain, microglia lose their homeostatic molecular signature and show profound functional impairments, such as increased production of pro-in ammatory cytokines and buildup of dysfunctional lysosomal deposits indicative of impaired phagocytosis [76]. In aging pathogenesis, microglia activation plays a dual role: on one side, activation of pro-in ammatory M1 microglia contributes to neurotoxicity and synapse loss by triggering several proin ammatory cascades. In contrary, acute activation of anti-in ammatory M2 microglia induces increased phagocytosis or clearance [77].
Up to now, no therapy is available to block or slow down aging related diseases such as AD, and the involved mechanisms are still not fully understood [78]. In the CNS, the histaminergic system is involved in regulating many basic physiological functions including cognition [79]. H3R is a presynaptic autoreceptor negatively regulating histamine release from histaminergic neurons [23,24], which suggesting inhibition of H3R induces increased histamine release. Therefore, we studied the effect of H3R antagonist in neuroin ammation induced pathology in mice. We found that under LPS induced neuroin ammatory condition, inhibition of H3R by thioperamide signi cantly inhibited activation of microglia. Meanwhile, thioperamide also inhibited the pro-in ammatory cytokines transcription and expression. The mechanism might be related to its upregulated release of histamine and subsequent activation of H2R, because inhibiting histaminergic neurons by H2R antagonist cimetidine but not H1R antagonist pyrilamine reversed the microglia activation and anti-in ammatory effects of thioperamide. In accordance with these results, our previous study showed that thioperamide offered anti-in ammatory effects in AD [37]. Moreover, other study also shows that H3R antagonist JNJ10181457 reduces the upregulation of microglial pro-in ammatory cytokines and improved depression-like behavior, which is consistent with our results [80]. Conversely, there is also report shows activation but not inhibition of microglial H3R suppresses acute LPS-induced proin ammatory activities in primary cultured microglia [81]. Concerning that acute moderate activation of microglia promotes anti-in ammation M2 microglia phenotypes, whereas chronic microglial activation exerts pro-in ammation M1 phenotypes [82].
The differences might be attributed to the regulation of two different polarization of microglia by different factors.
Based on these studies, we found for the rst time that the mechanism by which thioperamide offered anti-in ammatory effects was related to up-regulated histamine release and subsequent H2R activation.
This is in accordance with other reports showing that histamine was able to counteract LPS-induced glial activation and release of pro-in ammatory molecules [40]. Depletion of histaminergic neurons in the hypothalamus induces potentiated microglial response to challenge with LPS in histidine decarboxylase (HDC) knockout mice [83]. In contrast, other reports show that histamine promotes microglia activation and induces phagocytosis [84,85]. These contradictory studies may be due to the different regulation of two microglia polarization under different circumstance through activating different receptors. Although histamine is acknowledged to be involved in promoting in ammatory effects in the peripheral system, accumulating evidences show that in the CNS, histamine has a dual role in the modulation of microglial in ammatory responses. 85 Histamine per se triggers microglia activation and in ammation, whereas exerts anti-in ammatory effects under stress induced pathological conditions [40,53,86]. Moreover, activation of H1R induces activation of microglia and pro-in ammatory effects [84, 85] but H2R induces inhibition of microglia and anti-in ammatory effects [39,62,87].
Neuroin ammation is a signi cant pathological feature affecting cognition in aging, and recent evidence demonstrates that it also negatively affects hippocampal neurogenesis [6]. We observed that under LPS induced physiological conditions, thioperamide promoted hippocampal neurogenesis signi cantly through a histamine dependent mechanism. In concomitant with our results, either H3R antagonist or histamine has been shown to upregulate neurogenesis [40,41,43], showing that activating histaminergic neurons may represent a new tool for brain repair strategies in CNS. Interestingly, we found that the effects of thioperamide on neurogenesis involves activating histamine H2R, because they were reversed by cimetidine but not pyrilamine. In support with our results, other studies also show that histamine play a vital role in cerebral neurogenesis by activation of H2R to promote proliferation of neural precursors [88]. However, some reports also show that H1R is involved in the promoted neurogenesis offered by histamine in traumatic brain injury [43]. Moreover, de ciency of H1R leads to a reduced neurogenesis and cognition [89]. The different mechanism offered by histamine on neurogenesis might be related to different pathologies of diseases. It is possible that under chronic neuroin ammation, in ammatory blockade by histamine rescues impaired neurogenesis via activating H2R. Because a majority of studies indicate that anti-in ammatory drug restores neurogenesis [17,90,91]. Moreover, we also observed a relationship of histamine dependent H2R activating and neuroin ammation induced cognition. Although reports show that both H1R and H2R mediates effects of histamine on cognition. In chronic neuroin ammation related diseases such as aging, AD, schizophrenia, and autism spectrum disorder, histamine promotes cognition mainly through modulating microglial function [92]. Meanwhile, histamine exerts its effects on microglia and neuroin ammation mostly through activating H2R [64,89]. Therefore, the alleviated cognitive impairment by H2R activating might be related to its suppression on microglia activation and neuroin ammation.
To further understand the molecular mechanism underlying the effect mediated by H2R, we examined the H2R downstream signaling PKA/CREB. Reduced p-CREB level has been observed in neurodegenerative diseases [93,94]. In addition, the decreased p-CREB expression is also thought to be important for regulation of microglia activation and neuroin ammation in CNS [95][96][97]. Stimulating CREB pathway enhances M2 microglia polarization[98] and inhibits microglia-mediated neuroin ammation [63,99]. Therefore, CREB might play an important role in mediating the effect of H2R activation on neuroin ammation by histamine. Moreover, It is well acknowledged that activation of H2R induces activating cAMP/PKA/CREB pathway [100]. Therefore, we investigated the effect of thioperamide on PKA/CREB phosphorylation in LPS induced neuroin ammation. In agreement with previous studies, we found that LPS induced neuroin ammation was accomplished by decreased levels of p-PKA and p-CREB, which were both reversed by thioperamide treatment, showing thioperamide activated the PKA/CREB pathway in LPS induced neuroin ammation. Specially, both cimetidine, an H2R antagonist and H89, an inhibitor of PKA/CREB compromised the effects offered by thioperamide, including decreased microglia activity, decreased in ammatory cytokines levels and neurogenesis, further suggesting that the antiin ammatory effects and promoted neurogenesis offered by thioperamide involves activation of H2R-PKA/CREB pathway.
The expression of pro-in ammatory cytokines requires NF-κB activation and its nuclear translocation to interact with DNA [101]. Studies indicate that activation of CREB induces reduced expression of p-NF-κB and decreased production of pro-in ammatory cytokines in brain injury [102]. We found that LPS induced activation of NF-κB, and thioperamide mitigated this effect, showing thioperamide inhibited LPS induced activation of NF-κB. A critical step in the transcriptional regulation mediated by NF-κB or CREB is the interaction of each of these transcription factors with the co-activator CBP [103]. Interaction of NF-κB-CBP mediates pro-in ammatory cytokines release whereas CREB-CBP mediates anti-in ammatory cytokines release [64]. Interestingly, we found that LPS induced an increased interaction of NF-κB-CBP but decreased CREB-CBP. Thioperamide promoted CREB-CBP but inhibited NF-κB-CBP interaction, which were both reversed by cimetidine, suggesting that thioperamide promoted CREB-CBP combination via histamine dependent activating H2R. Studies indicate that activation of CREB induces reduced expression of p-NF-κB and decreased production of pro-in ammatory cytokines in brain injury [102].
Consistent with these results, we found that thioperamide inhibited transcriptional and protein levels of pro-in ammatory cytokines, including IL-1, IL-6 and TNFα from M1 microglia but promoted antiin ammatory cytokines and neurotrophic factor including IL-4, IL-10 and BDNF from M2 microglia, which were both reversed by H89, suggesting that thioperamide regulated in ammatory cytokines transcription through activating CREB. Phosphorylation of CREB induced by thioperamide might promoted CREB-CBP interaction but inhibited NF-κB-CBP interaction to transcriptional regulate the in ammatory cytokines levels.

Conclusions
In conclusion, the present study indicates that H3R antagonist thioperamide improved cognitive impairment in LPS treated mice via histamine dependent H2R downstream up-regulating CREB-CBP interaction mediated inhibited M1 microglia related pro-in ammatory cytokines and M2 microglia related anti-in ammatory cytokines transcription, which contributed to neurogenesis (Figure 9).These results uncovered a novel mechanism behind the therapeutic effect of thioperamide in neuroin ammation and further provided an experimental basis for starting a clinical trial for H3R antagonists as a treatment for chronic neuroin ammation related diseases such aging and AD.