In recent years, growing evidence indicates that the nervous system plays an indispensable role in tumor development and metastasis. Elucidating crosstalk between the nervous system and tumor progression has thrived as a hot topic and a new direction for understanding cancer pathogenesis. Notably, many novel discoveries have suggested that neurotransmitter receptors (NRs) are not only widely expressed in cancer cells, but also play key roles in regulating cancer initiation and progression by diverse approaches. In this review, we summarized the latest advance in cancer neuroscience, especially emphasizing the important roles of different NRs in cancer development and prevention. The exemplary studies presented herein illustrate the emerging view that NRs are profoundly influential, manifested in tumor growth, apoptosis, angiogenesis, metastasis, resistance to drugs, and participate in the formation of neural-cancer interactions. In addition, NRs also regulate cellular metabolic processes and tumor microenvironment (TME) remodeling. More importantly, numerous basic and clinical studies have suggested that NRs may be potential targets for cancer treatments, and corresponding agonists or antagonists have been identified effectively in controlling tumor growth and metastasis. In conclusion, NRs are emerging as novel targets for anti-cancer drug exploration and clinical cancer treatments, while trying to uncover deeper mechanisms and connections between NRs and cancer is of high clinical significance and translational value.
Humans ; Neoplasms/metabolism* ; Receptors, Neurotransmitter/physiology* ; Animals ; Tumor Microenvironment/physiology*

The study aimed to investigate the effect of the CD200R1 gene deletion on microglia activation and nigrostriatal dopamine neuron loss in the Parkinson's disease (PD) process. The CRISPR-Cas9 technology was applied to construct the CD200R1^-/- mice. The primary microglia cells of wild-type and CD200R1^-/- mice were cultured and treated with bacterial lipopolysaccharide (LPS). Microglia phagocytosis level was assessed by a fluorescent microsphere phagocytosis assay. PD mouse model was prepared by nigral stereotaxic injection of recombinant adeno-associated virus vector carrying human α-synuclein (α-syn). The changes in the motor behavior of the mice with both genotypes were evaluated by cylinder test, open field test, and rotarod test. Immunohistochemical staining was used to assess the loss of dopamine neurons in substantia nigra. Immunofluorescence staining was used to detect the expression level of CD68 (a key molecule involved in phagocytosis) in microglia. The results showed that CD200R1 deletion markedly enhanced LPS-induced phagocytosis in vitro by the microglial cells. In the mouse model of PD, CD200R1 deletion exacerbated motor behavior impairment and dopamine neuron loss in substantia nigra. Fluorescence intensity analysis results revealed a significant increase in CD68 expression in microglia located in the substantia nigra of CD200R1^-/- mice. The above results suggest that CD200R1 deletion may further activates microglia by promoting microglial phagocytosis, leading to increased loss of the nigrostriatal dopamine neurons in the PD model mice. Therefore, targeting CD200R1 could potentially serve as a novel therapeutic target for the treatment of early-stage PD.
Animals ; Microglia/physiology* ; Mice ; Phagocytosis ; Parkinson Disease/genetics* ; Disease Models, Animal ; Receptors, Cell Surface/physiology* ; Dopaminergic Neurons/pathology* ; Antigens, CD/metabolism* ; Gene Deletion ; Substantia Nigra ; Mice, Inbred C57BL ; Mice, Knockout ; Cells, Cultured ; Male ; alpha-Synuclein ; CD68 Molecule ; Orexin Receptors

Objective To explore the effects of Shuanglu Tongnao Formula on neuroinflammation in ischemic stroke (IS) rats via the P2X purinoceptor 7 receptor (P2X7R)/NLR family pyrin domain-containing 3 (NLRP3) pathway. Methods The rats were divided into five groups: the IS group, control group, Shuanglu Tongnao Formula group, P2X7R inhibitor brilliant blue G (BBG) group, and Shuanglu Tongnao Formula combined with P2X7R activator adenosine triphosphate (ATP) group, with 18 rats in each group. Except for the control group, rats in all other groups were used to construct an IS model using the suture method. After successful modeling, the drug was given once a day for 2 weeks. Neurological function scores and cerebral infarction volume ratios were measured in rats. Pathological examination of the ischemic penumbra brain tissue was performed. Immunofluorescence staining was used to quantify the proportions of microglia co-expressing both inducible nitric oxide synthase (iNOS) and ionized calcium-binding adapter molecule 1 (Iba1), as well as arginase 1 (Arg1) and Iba1, in the ischemic penumbra brain tissue. ELISA was used to detect tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), interleukin 6 (IL-6) and IL-10 in the ischemic penumbra brain tissue. Western blotting was used to measure P2X7R, NLRP3, and IL-1β proteins in the ischemic penumbra brain tissue. Results Compared with the control group, the IS group showed disordered neuronal arrangement, nuclear condensation, and obvious infiltration of inflammatory cells in the ischemic penumbra; significantly elevated neurological function scores, cerebral infarction volume ratios, proportions of microglia co-expressing iNOS and Iba1, and levels of TNF-α, IL-6, and P2X7R, NLRP3, IL-1β proteins; along with reduced proportions of microglia co-expressing Arg1 and Iba1 and levels of TGF-β and IL-10. Compared with the IS group, the Zhuang medicine Shuanglu Tongnao Formula and BBG groups demonstrated alleviated brain tissue damage; reduced neurological function scores, cerebral infarction volume ratios, proportions of microglia co-expressing iNOS and Iba1, and levels of TNF-α, IL-6, and P2X7R, NLRP3, IL-1β proteins; along with increased proportions of microglia co-expressing Arg1 and Iba1 and levels of TGF-β and IL-10. ATP reversed the effects of Zhuang medicine Shuanglu Tongnao Formula on microglial polarization and neuroinflammation in IS rats. Conclusion Zhuang medicine Shuanglu Tongnao Formula may promote the transformation of microglia from M1 type to M2 type by inhibiting the P2X7R/NLRP3 pathway, thereby improving neuroinflammation in IS rats.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Receptors, Purinergic P2X7/metabolism* ; Male ; Drugs, Chinese Herbal/pharmacology* ; Rats ; Ischemic Stroke/pathology* ; Rats, Sprague-Dawley ; Disease Models, Animal ; Signal Transduction/drug effects* ; Neuroinflammatory Diseases/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; Interleukin-10/metabolism* ; Brain Ischemia/drug therapy* ; Microglia/metabolism*

OBJECTIVE: To explore the therapeutic effects and underlying mechanisms of Dendrobium officinale (Tiepi Shihu) extract (DOE) on insomnia. METHODS: Forty-two male Sprague-Dawley rats were randomly divided into 6 groups (n=7 per group): normal control, model control, melatonin (MT, 40 mg/kg), and 3-dose DOE (0.25, 0.50, and 1.00 g/kg) groups. Rats were raised in a strong-light (10,000 LUX) and -noise (>80 db) environment (12 h/d) for 16 weeks to induce insomnia, and from week 10 to week 16, MT and DOE were correspondingly administered to rats. The behavior tests including sodium pentobarbital-induced sleep experiment, sucrose preference test, and autonomous activity test were used to evaluate changes in sleep and emotions of rats. The metabolic-related indicators such as blood pressure, blood viscosity, blood glucose, and uric acid in rats were measured. The pathological changes in the cornu ammonis 1 (CA1) region of rat brain were evaluated using hematoxylin and eosin staining and Nissl staining. Additionally, the sleep-related factors gamma-aminobutyric acid (GABA), glutamate (GA), 5-hydroxytryptamine (5-HT), and interleukin-6 (IL-6) were measured using enzyme linked immunosorbent assay. Finally, we screened potential sleep-improving receptors of DOE using polymerase chain reaction (PCR) array and validated the results with quantitative PCR and immunohistochemistry. RESULTS: DOE significantly improved rats' sleep and mood, increased the sodium pentobarbital-induced sleep time and sucrose preference index, and reduced autonomic activity times (P<0.05 or P<0.01). DOE also had a good effect on metabolic abnormalities, significantly reducing triglyceride, blood glucose, blood pressure, and blood viscosity indicators (P<0.05 or P<0.01). DOE significantly increased the GABA content in hippocampus and reduced the GA/GABA ratio and IL-6 level (P<0.05 or P<0.01). In addition, DOE improved the pathological changes such as the disorder of cell arrangement in the hippocampus and the decrease of Nissel bodies. Seven differential genes were screened by PCR array, and the GABA_A receptors (Gabra5, Gabra6, Gabrq) were selected for verification. The results showed that DOE could up-regulate their expressions (P<0.05 or P<0.01). CONCLUSION DOE demonstrated remarkable potential for improving insomnia, which may be through regulating GABA_A receptors expressions and GA/GABA ratio.
Animals ; Dendrobium/chemistry* ; Rats, Sprague-Dawley ; Male ; Sleep Initiation and Maintenance Disorders/blood* ; Plant Extracts/therapeutic use* ; Receptors, GABA-A/metabolism* ; Noise/adverse effects* ; Light/adverse effects* ; gamma-Aminobutyric Acid/metabolism* ; Sleep/drug effects* ; Rats ; Receptors, GABA/metabolism*

OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na⁺, K⁺, and especially Ca²⁺. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies. METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group. RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity. CONCLUSIONS We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.
Animals ; Zebrafish/genetics* ; Receptors, Purinergic P2X2/deficiency* ; CRISPR-Cas Systems/genetics* ; Gene Knockout Techniques ; Phenotype ; Zebrafish Proteins/genetics* ; Disease Models, Animal

OBJECTIVES: To observe the effect of 4-(arylethynyl)-pyrrolo[2,3-d] pyrimidine (10b) on post-traumatic stress disorder (PTSD)-like behaviors and ERK1/2-SGK1 signaling pathway in mice. METHODS: C57BL/6 mouse models exposed to single prolonged stress (SPS) were treated with daily gavage of saline, 10b at low, moderate and high doses, or paroxetine for 14 days. The changes in PTSD-like behaviors of SPS mice with different treatments were observed using behavioral tests. Western blotting and immunofluorescence assay were used to detect the protein expression levels of mGluR5, p-ERK, and SGK1 in the hippocampus of the mice. Pathological changes in the liver and kidney tissues of the mice were examined using HE staining. Molecular docking and molecular dynamics analyses were employed to evaluate the binding stability between the compound 10b and mGluR5. RESULTS: Compared to the normal control mice, the SPS mice exhibited obvious PTSD-like behaviors with increased hippocampal expressions of mGluR5 and p-ERK proteins and decreased SGK1 protein expression. Compound 10b significantly ameliorated behavioral abnormalities in SPS mice, inhibited mGluR5 expression, and reversed the dysregulation of p-ERK and SGK1. No obvious liver or kidney toxicity was observed after 10b treatment. Molecular docking and dynamics studies demonstrated a stable interaction between 10b and mGluR5. CONCLUSIONS The compound 10b ameliorates PTSD-like behaviors induced by SPS in mice possibly by inhibiting mGluR5 expression to modulate the ERK1/2-SGK1 signaling pathway.
Animals ; Stress Disorders, Post-Traumatic/drug therapy* ; Receptor, Metabotropic Glutamate 5/metabolism* ; Mice, Inbred C57BL ; Mice ; Protein Serine-Threonine Kinases/metabolism* ; Pyrimidines/pharmacology* ; Immediate-Early Proteins/metabolism* ; Signal Transduction/drug effects* ; MAP Kinase Signaling System/drug effects* ; Male ; Molecular Docking Simulation ; Hippocampus/metabolism*

OBJECTIVES: To investigate the effect of orexin-A-mediated regulation of ionotropic glutamate receptors for promoting motor function recovery in rats with spinal cord injury (SCI). METHODS: Thirty-six newborn SD rats (aged 7-14 days) were randomized into 6 groups (n=6), including a normal control group, a sham-operated group, and 4 SCI groups with daily intrathecal injection of saline, DNQX, orexin-A, or orexin-A+DNQX for 3 consecutive days after PCI. Motor function of the rats were evaluated using blood-brain barrier (BBB) score and inclined plane test 1 day before and at 1, 3, and 7 days after SCI. For patch-clamp experiment, spinal cord slices from newborn rats in the control, sham-operated, SCI, and SCI+orexin groups were prepared, and ventral horn neurons were acutely isolated to determine the reversal potential and dynamic indicators of glutamate receptor-mediated currents under glutamate perfusion. RESULTS: At 3 and 7 days after SCI, the orexin-A-treated rats showed significantly higher BBB scores and grip tilt angles than those with other interventions. Compared with those treated with DNQX alone, the rats receiving the combined treatment with orexin and DNQX had significantly higher BBB scores and grip tilt angles on day 7 after PCI. In the patch-clamp experiment, the ventral horn neurons from SCI rat models exhibited obviously higher reversal potential and greater rise slope of glutamate current with shorter decay time than those from sham-operated and orexin-treated rats. CONCLUSIONS Orexin-A promotes motor function recovery in rats after SCI possibly by improving the function of the ionotropic glutamate receptors.
Animals ; Spinal Cord Injuries/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Receptors, Ionotropic Glutamate/metabolism* ; Recovery of Function/drug effects* ; Orexins/pharmacology* ; Male ; Female ; Animals, Newborn ; Neuropeptides/pharmacology* ; Intracellular Signaling Peptides and Proteins/pharmacology*

OBJECTIVES: To investigate the therapeutic effect of Ziwuliuzhu acupuncture in a rat model of insomnia and its regulatory effect on the glutamic acid (Glu)/γ-aminobutyric acid (GABA)-glutamine (Gln) metabolic loop. METHODS: Forty male SD rats were randomly assigned to control group, model group, Najia group and Nazi group (n=10). In the latter 3 groups, rat models of insomnia were established by intraperitoneal injections of p-chlorophenylalanine and verified using a sodium pentobarbital-induced sleep test. After modeling, the rats in Najia and Nazi groups received acupuncture for 7 days at specifically chosen sets of acupoints based on the Ziwuliuzhu rationale in traditional Chinese medicine. Pathological changes in the hypothalamic tissue of the rats were examined with HE staining, and the levels of Glu and GABA in the hypothalamus were determined with high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS. Immunohistochemistry was used to detect the expressions of GABAA receptors (GABAARs) in the hypothalamus, and the expression levels of glutamate decarboxylase (GAD65/67) and glutamine synthetase (GS) were determined with Western blotting. RESULTS: Compared with the model group, the rats in Najia and Nazi groups exhibited decreased Glu levels and GABAA receptor expression and increased GABA levels with a decreased Glu/GABA ratio in the hypothalamus. Ziwuliuzhu acupuncture significantly increased the protein expressions of GAD65 and GAD67 and lowered the expression of GS in the hypothalamus in the rat models of insomnia. CONCLUSIONS Ziwuliuzhu acupuncture produces sedative and hypnotic effects in rat models of insomnia possibly by regulating Glu and GABA-Gln metabolism to restore the excitatory/inhibitory balance between Glu and GABA.
Animals ; Rats, Sprague-Dawley ; Male ; Rats ; gamma-Aminobutyric Acid/metabolism* ; Sleep Initiation and Maintenance Disorders/therapy* ; Glutamine/metabolism* ; Glutamic Acid/metabolism* ; Acupuncture Therapy ; Hypothalamus/metabolism* ; Receptors, GABA-A/metabolism* ; Acupuncture Points

OBJECTIVES: To explore the toxic mechanism of Clostridium perfringens Beta1 toxin mediated by P2X7 receptor-induced calcium dyshomeostasis. METHODS: Ten-day-old BALB/c mice were randomly divided into control group, recombinant Beta1 toxin (rCPB1) group, PD151746 group, and PD151746+rCPB1 group, and all the treatment agents were administered by gavage. The changes in expressions of inflammatory factors in the jejunum of the mice were detected using antibody chip technology to explore the regulatory role of calcium dyshomeostasis in Beta1 toxin-induced inflammatory injury level. In the cell experiment, THP-1 cells were transfected with a si-RNA targeting P2X7 receptor and treated with rCPB1, and the changes in cell survival rate, levels of Ca²⁺, ROS and ATP, and expressions of pyroptosis and ferroptosis markers were determined. RESULTS: Oral administration of rCPB1 significantly increased the levels of inflammatory cytokines in the jejunal tissue of the neonatal mice, but their levels were significantly decreased after treatment with PD151746. In THP-1 cells, rCPB1 treatment significantly decreased cell survival and increased the levels of Ca²⁺, ROS, ATP and the expressions of pyroptosis and ferroptosis markers, and these changes were obviously attenuated by P2X7 receptor knockdown. CONCLUSIONS P2X7 receptor-mediated functional pore formation by Beta1 toxin can further lead to calcium dyshomeostasis, thereby triggering excessive accumulation of ROS to subsequently induce the co-occurrence of pyroptosis and ferroptosis.
Animals ; Pyroptosis/drug effects* ; Receptors, Purinergic P2X7/metabolism* ; Mice ; Mice, Inbred BALB C ; Ferroptosis/drug effects* ; Humans ; Calcium/metabolism* ; Macrophages/drug effects* ; Bacterial Toxins/toxicity*

OBJECTIVE: To investigate the hemostatic effect of modified Sijunzi Granules (MSG) in primary immune thrombocytopenia (ITP) zebrafish model and explore the potential mechanism. METHODS: AB strain wild type zebrafish were treated with simvastatin (6 µmol/L) for 24 h to establish the hemorrhage model (model control group). The zebrafish were treated with MSG at different doses (55.6, 167, and 500 µg/mL), respectively. The hemostatic effect was assessed by examining the intestinal bleeding and hemostatic rate. 5-Hydroxytryptamine (5-HT) content was determined using enzyme-linked immunosorbent assay (ELISA) assay. The expressions of 5-HT2aR, 5-HT2bR, and SERT genes were detected by quantitative real-time polymerase chain reaction(PCR). The protein expressions of protein kinase B (Akt), p-Akt, extracellular regulated protein kinases (Erk), and p-Erk were examined using Western blot analysis. RESULTS: The intestinal bleeding rate was 37%, 40%, and 80% in the 55.6, 167, and 500 µg/mL dose of MSG, respectively, in which 55.6 and 167 µg/mL MSG dose groups were associated with significantly decreased intestinal bleeding rate when compared with the model control group (70%, P<0.05). Significantly higher hemostatic rates were also observed in the 55.6 (54%) and 167 (52%) µg/mL MSG dose groups (P<0.05). MSG increased the 5-HT content and mRNA expression levels of 5-HT2aR, 5-HT2bR, and SERT (P<0.05). In addition, caspase3/7 activity was inhibited (P<0.05). Significant increase in p-Akt and p-Erk was also detected after treatment with MSG (P<0.05). CONCLUSIONS MSG could reduce the incidence and severity of intestinal bleeding in zebrafish by activating MAPK/Erk and PI3K/Akt signal pathways through regulating the levels of 5-HT and its receptors, which may provide evidence for the treatment of ITP.
Animals ; Zebrafish ; Serotonin/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Hemostatics/pharmacology* ; MAP Kinase Signaling System/drug effects* ; Receptors, Serotonin/genetics* ; Extracellular Signal-Regulated MAP Kinases/metabolism* ; Signal Transduction/drug effects* ; Hemostasis/drug effects*