Acute brain slices represent a workhorse model for studying the central nervous system (CNS) from nanoscale events to complex circuits. While slice preparation inherently involves tissue damage, it is unclear how microglia, the main immune cells and damage sensors of the CNS react to this injury and shape neuronal activity ex vivo. To this end, we investigated microglial phenotypes and contribution to network organization and functioning in acute brain slices. We reveal time-dependent microglial phenotype changes influenced by complex extracellular ATP dynamics through P2Y12R and CX3CR1 signalling, which is sustained for hours in ex vivo mouse brain slices. Downregulation of P2Y12R and changes of microglia-neuron interactions occur in line with alterations in the number of excitatory and inhibitory synapses over time. Importantly, functional microglia modulate synapse sprouting, while microglial dysfunction results in markedly impaired ripple activity both ex vivo and in vivo. Collectively, our data suggest that microglia are modulators of complex neuronal networks with important roles to maintain neuronal network integrity and activity. We suggest that slice preparation can be used to model time-dependent changes of microglia-neuron interactions to reveal how microglia shape neuronal circuits in physiological and pathological conditions.

This study aimed to determine whether trimethylamine N-oxide (TMAO) was involved in sympathetic activation in aging and the underlying mechanisms. Our hypothesis is TMAO reduces P2Y12 receptor (P2Y12R) and induces microglia-mediated inflammation in the paraventricular nucleus (PVN), then leading to sympathetic activation in aging. This study involved 18 young adults and 16 old adults. Aging rats were established by injecting D-galactose (D-gal, 200 mg/kg/d) subcutaneously for 12 weeks. TMAO (120 mg/kg/d) or 1% 3, 3-dimethyl-l-butanol (DMB) was administrated via drinking water for 12 weeks to investigate their effects on neuroinflammation and sympathetic activation in aging rats. Plasma TMAO, NE and IL-1β levels were higher in old adults than in young adults. In addition, standard deviation of all normal to normal intervals (SDNN) and standard deviation of the average of normal to normal intervals (SDANN) were lower in old adults and negatively correlated with TMAO, indicating sympathetic activation in old adults, which is associated with an increase in TMAO levels. Treatment of rats with D-gal showed increased senescence-associated protein levels and microglia-mediated inflammation, as well as decreased P2Y12R protein levels in PVN. Plasma TMAO, NE and IL-1β levels were increased, accompanied by enhanced renal sympathetic nerve activity (RSNA). While TMAO treatment exacerbated the above phenomenon, DMB mitigated it. These findings suggest that TMAO contributes to sympathetic hyperactivity in aging by downregulating P2Y12R in microglia and increasing inflammation in the PVN. These results may provide promising new target for the prevention and treatment of aging and aging-related diseases.

BACKGROUND: Atherosclerosis (AS) is a chronic disease characterized by lipid accumulation in the aortic wall and the formation of foam cells overloaded with large lipids inclusions. Currently, Western medicine is primarily used to improve lipid metabolism disorders and reduce inflammatory reactions to delay AS progression, but these medicines come with serious side effects and drug resistance. Gualou-Xiebai (GLXB) is a renowned herb pair that has been proven effective against AS. However, the potential molecular mechanism through which GLXB exerts the anti-atherosclerotic effects of increasing lipophagy in vascular smooth muscle cells (VSMCs) remains unknown.
OBJECTIVE: This study aims to explore the role of lipophagy and the therapeutic mechanism of GLXB in AS.
METHODS: UPLC-Q-TOF-MS for the determination of the main components of GLXB-containing serum. An AS mouse model was established by feeding a high-fat diet (HFD) to ApoE-/- mice for 12 weeks. Ultrasonography monitoring was used to confirm the successful establishment of the AS model. Plaque areas and lipid deposition were evaluated using HE staining and aorta imagingafter GLXB treatment. Immunofluorescence staining and Western blotting were utilized to observe the P2RY12 and lipophagy levels in AS mice. VSMCs were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce foam cell formation. The degree of lipophagy and the related molecular mechanisms were assessed after treating the VSMCs with GLXB-containing serum or si-P2RY12 transfection. The active components of GLXB-containing serum that act on P2RY12 were screened and verified by molecular docking and dual-luciferase reporter assays.
RESULTS: Seventeen components of GLXB were identified in rat serum by UPLC-Q-TOF-MS. GLXB significantly reduced lipid deposition in HFD-fed ApoE-/- mice and ox-LDL-induced VSMCs. GLXB strikingly increased lipophagy levels by downregulating P2RY12, p62, and plin2, upregulating LC3Ⅱ protein expression, and increasing the number of autophagosomes. Notably, the lipophagy inhibitor CQ and the P2RY12 receptor agonist ADPβ abolished the GLXB-induced increase in lipophagy. Last, we confirmed that albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin from GLXB significantly inhibited P2RY12.
CONCLUSIONS: GLXB activates lipophagy and inhibits lipid accumulation-associated VSMC-derived foam cell formation through suppressing P2RY12 activation, resulting in anti-atherosclerotic effects. The GLXB components albiflorin, apigenin, luteolin, kaempferol, 7,8-dihydroxyflavone, and hesperetin are the potential active effectors against P2RY12.

Increasing evidence suggests that immunometabolism has started to unveil the role of metabolism in shaping immune function and autoimmune diseases. In this study, our data show that purinergic receptor P2Y12 (P2RY12) is highly expressed in concanavalin A (ConA)-induced immune hepatitis mouse model and serves as a potential metabolic regulator in promoting metabolic reprogramming from oxidative phosphorylation to glycolysis in T cells. P2RY12 deficiency or inhibition of P2RY12 with P2RY12 inhibitors (clopidogrel and ticagrelor) are proved to reduce the expression of inflammatory mediators, cause CD4+ and CD8+ effector T cells hypofunction and protect the ConA-induced immune hepatitis. A combined proteomics and metabolomics analysis revealed that P2RY12 deficiency causes redox imbalance and leads to reduced aerobic glycolysis by downregulating the expression of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway, indicating that HK2 might be a promising candidate for the treatment of diseases associated with T cell activation. Further analysis showed that P2RY12 prevents HK2 degradation by activating the PI3K/Akt pathway and inhibiting lysosomal degradation. Our findings highlight the importance of the function of P2RY12 for HK2 stability and metabolism in the regulation of T cell activation and suggest that P2RY12 might be a pivotal regulator of T cell metabolism in ConA-induced immune hepatitis.

Ulinastatin, a broad spectrum of serine protease inhibitor, has been found to alleviate neuropathic pain (NPP). However, its mechanism is not completely clear. Here, a sciatic nerve ligation rat model and BV2 microglial cells were used to investigate the effect of Ulinastatin on the activation of microglia and P2Y12 receptors in vivo and in vitro. Levels of P2Y12 receptor and NF-κB (P65) expression in the dorsal horn of the lumbar enlargement region of the spinal cord and BV2 cells were assessed by immunohistochemistry and double-label immunofluorescence assays. Levels of IL-1β and TNF-α in cell culture medium and cerebrospinal fluid (CSF) were examined by ELISA. The results showed that Ulinastatin reduced the release of inflammatory IL-1β and TNF-α by inhibiting the activation of spinal microglia. Ulinastatin down-regulated P2Y12 receptor and NF-κB (P65) expression in the spinal microglia of the chronic constrictive injury model. The results indicated that Ulinastatin may attenuate the activation of spinal microglia after peripheral nerve injury by inhibiting the activation of P2Y12 receptor signal pathway in microglia. NF-kB may play a key role in the mechanism of Ulinastatin.

Remarkable interindividual variability in clopidogrel response is observed, genetic polymorphisms in P2RY12 and its signal pathway is supposed to affect clopidogrel response in CHD patients.
539 CHD patients treated with clopidogrel were recruited. The platelet reaction index (PRI) indicated by VASP-P level were detected in 12-24 h after clopidogrel loading dose or within 5-7 days after initiation of maintain dose clopidogrel. A total of 13 SNPs in relevant genes were genotyped in sample A (239 CHD patients). The SNPs which have significant differences in PRI will be validated in another sample (sample B, 300 CHD patients).
CYP2C19*2 increased the risk of clopidogrel resistance significantly. When CYP2C19*2 and CYP2C19*3 were considered, CYP2C19 loss of function (LOF) alleles were associated with more obviously increased the risk of clopidogrel resistance; P2RY12 rs6809699C > A polymorphism was also associated with increased risk of clopidogrel resistance (AA vs CC: P = 0.0398). This difference still existed after stratification by CYP2C19 genotypes. It was also validated in sample B. The association was also still significant even in the case of stratification by CYP2C19 genotypes in all patients (sample A + B).
Our data suggest that P2RY12 rs6809699 is associated with clopidogrel resistance in CHD patients. Meanwhile, the rs6809699 AA genotype can increase on-treatment platelet activity independent of CYP2C19 LOF polymorphisms.

BACKGROUND: Acute lung injury (ALI) is characterized by abnormal inflammatory response without effective therapies. P2Y12 receptor (P2Y12R) plays a vital role in inflammatory response. This study intends to explore whether P2Y12R antagonists can inhibit LPS-induced inflammatory injury of human pulmonary microvascular endothelial cells (HPMVECs) and endothelial cell dysfunction.
METHODS: Using a cell model of ALI, the role of P2Y12R was investigated in LPS-induced HPMVECs. The expression of P2Y12R was detected by RT-qPCR and Western blot analysis assay and TNF-α, IL-1β, and IL-6 levels were analyzed by RT-qPCR. NO levels were also analyzed through NO kit. The levels of NF-κB p65, P-IκB-α, and IκB-α, as well as p-AKT and eNOS levels were detected by Western blot analysis assay. Wound healing assay was performed to evaluate HPMVECs migration. FITC-dextran was used to evaluate endothelial cell permeability, and the analysis of adherens junction protein VE-cadherin and endothelial cell tight junction proteins ZO-1, Claudin 5 and Occludin expression was performed by RT-qPCR and Western blot analysis assay.
RESULTS: In vitro, LPS increased the expression levels of P2Y12R and pro-inflammatory mediators (TNF-α, IL-1β, and IL-6), followed by a decrease in HPMVECs migration. In addition, LPS led to an increase in endothelial cell permeability. P2Y12R antagonists Ticagrelor or clopidogrel treatment significantly reversed these effects of LPS.
CONCLUSIONS: The inhibitor of P2Y12R was able to decrease inflammatory response, promote migration and improve endothelial cell function and permeability, suggesting a key role of P2Y12R in ALI.

A series of novel thienopyridine derivatives were designed and synthesized as P2Y12 receptor inhibitors. Several solid compounds were assessed for inhibitory effect where they exhibited stronger potency than clopidogrel. Compound 6b and 6g were evaluated for metabolism to verify that they could overcome clopidogrel resistance and for toxicity where they showed lower toxicity than prasugrel. Compound 6b exhibited lower risk of bleeding than prasugrel and showed good stability under stress testing. Overall, as a promising antiplatelet agent, representative compound 6b showed the following advantages: (1) no drug resistance for CYP2C19 poor metabolizers; (2) higher potency than clopidogrel; (3) lower toxicity than prasugrel; (4) lower risk of bleeding than prasugrel; (5) good stability as a non-salt solid.

Depression is a class of important mental illness, which has become a severe health problem perplexing the world due to its high morbidity rate, high disability rate, and great disease burden. This study aimed to evaluate the role and possible mechanisms of P2RY12 in the depression-like behaviors model.
Serum samples of patients with depression-like behaviors were used to analyze the expression of P2RY12. Models of mice were given LPS via intraperitoneal injection for 7 days. Behavioral tests were executed in this experiment.
The expression of P2RY12 in models of depression-like behaviors or mice with depression- like behaviors were induced. The inhibition of P2RY12 presents depression-like behaviors and reduces inflammation in the model of depression-like behaviors. P2RY12 induced NLRP3 expression and suppressed NLRP3 ubiquitination in a model of depression-like behavior. The inhibition of NLRP3 reduced the effects of P2RY12 in mice model of depression-like behaviors. The regulation of NLRP3 controlled the effects of the P2RY12 in vitro model of depression-like behaviors.
We conclude that P2RY12 increased neuroinflammation to accelerate depression-like behaviors by NLPR3 inflammasome, providing novel information for the treatment of depressionlike behaviors.

Purinergic 2Y12 (P2Y12) receptor antagonists are used as platelet aggregation inhibitors. Long non-coding RNAs (lncRNAs) play an important role in neuropathological events. Satellite glial cells (SGCs) in the superior cervical ganglia (SCGs) encircle the somata of neurons. This study explored if the upregulated P2Y12 receptor in SCGs was relevant to lncRNA uc.48+ during myocardial ischemia (MI). The results showed that upregulation of P2Y12 receptor was accompanied by increased expression of uc.48+ in the SCGs of MI rats which displayed abnormal changes in cervical sympathetic nerve activity, blood pressure, heart rate, electrocardiograms and cardiac tissue structure. The P2Y12 antagonist clopidogrel improved abnormal alterations in cardiac function and tissue structure in MI rats. Short hairpin RNA (shRNA) against uc.48+ significantly inhibited P2Y12 receptor upregulation and its co-expression with glial fibrillary acidic protein (GFAP) in SCGs, and ameliorated the cardiac dysfunction in MI rats. By contrast, overexpression of uc.48+ increased the expression of P2Y12 in SCGs and enhanced cervical sympathetic nerve activity in control rats. Direct interaction between uc.48+ and the P2Y12 receptor was predicted using the bioinformatic tool CatRAPID and confirmed by RNA immunoprecipitation. Moreover, overexpression of the P2Y12 receptor reversed the protective effect of uc.48+ shRNA on cardiac dysfunction in MI rats. Uc.48 shRNA treatment also inhibited the enhanced rise of intracellular free Ca2+ level ([Ca2+]i) evoked by the P2Y12 agonist 2-methylthio-adenosine-5\'-diphosphate (2-MeSADP) in SGCs of SCGs after oxygen-glucose deprivation (OGD) treatment. These data demonstrated that uc.48+ shRNA could counteract the P2Y12 upregulation and improve P2Y12-implicated cardiac dysfunction due to MI.