P2X7R and P2X4R expression of mice submandibular gland in high-fat diet/streptozotocin-induced type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a chronic inflammatory disease that can compromise the functioning of various organs, including the salivary glands (SG). The purinergic system is one of the most important inflammatory pathways in T2DM condition, and P2X7R and P2X4R are the primary purinergic receptors in SG that regulate inflammatory homeostasis. This study aimed to evaluate P2X7R and P2X4R expression, and morphological changes in the submandibular gland (SMG) in T2DM. Twenty-four 5-week-old mice were randomly assigned to control (CON) and diabetes mellitus (DM) groups (n = 12 each). Body weight, diet, and blood glucose levels were monitored weekly. The histomorphology of the SMG and the expression of the P2X7R, and P2X7R was evaluated by immunohistochemistry (IHC) staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 11 and 13 weeks of age. Our findings indicate a significant increase in food consumption, body weight, and blood glucose levels in the DM group. Although a significant increase in P2X7R and P2X4R expression was observed in the DM groups, the receptor location remained unchanged. We also observed a significant increase in the acinar area in the DM13w group, and a significant decrease in the ductal area in the DM11w and DM13w groups. Targeting purinergic receptors may offer novel therapeutic methods for diabetic complications.

Functional role of P2X7 purinergic receptor in cancer and cancer-related pain.

Numerous studies have revealed that the ATP-gated ion channel purinergic 2X7 receptor (P2X7R) plays an important role in tumor progression and the pathogenesis of cancer pain. P2X7R requires activation by extracellular ATP to perform its regulatory role functions. During tumor development or cancer-induced pain, ATP is released from tumor cells or other cells in the tumor microenvironment (such as tumor-associated immune cells), which activates P2X7R, opens ion channels on the cell membrane, affects intracellular molecular metabolism, and regulates the activity of tumor cells. Furthermore, peripheral organs and receptors can be damaged during tumor progression, and P2X7R expression in nerve cells (such as microglia) is significantly upregulated, enhancing sensory afferent information, sensitizing the central nervous system, and inducing or exacerbating pain. These findings reveal that the ATP-P2X7R signaling axis plays a key regulatory role in the pathogenesis of tumors and cancer pain and also has a therapeutic role. Accordingly, in this study, we explored the role of P2X7R in tumors and cancer pain, discussed the pharmacological properties of inhibiting P2X7R activity (such as the use of antagonists) or blocking its expression in the treatment of tumor and cancer pain, and provided an important evidence for the treatment of both in the future.

Involvement of P2X7R-mediated microglia polarization and neuroinflammation in the response to electroacupuncture on post-stroke memory impairment.

PURPOSE: Post-stroke cognitive impairment (PSCI) is a common complication of ischemic stroke episodes. Memory impairment is an important component of the poststroke cognitive syndrome. Microglial activation plays a critical role in stroke-induced neuroinflammation. Previous studies have reported that electroacupuncture (EA) provides neuroprotective effects by reducing the expression levels of the Purinergic receptor P2X ligand-gated ion channel 7 (P2X7) and inhibiting neuroinflammation in rat model of ischemic stroke. Further understanding of the role and connections between P2X7R and microglial activation in EA-induced anti-inflammatory can reveal novel targets for post-stroke memory impairment treatment. METHODS: A Middle cerebral artery occlusion and reperfusion (MCAO/R) model was established. We used 2'(3')-O-(4-benzoyl) benzoyl ATP (BzATP) as a P2X7R agonist. Following MCAO/R injury, the rats underwent EA therapy at the Baihui (DU20) and Shenting (DU24) acupoints for seven consecutive days. The Barnes maze test was used to evaluate memory function. Following intervention, a T2 weighted images (T2WI) scan was performed to identify changes in cerebral infarction volume in MCAO/R rats. The levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6) and Interleukin-4 (IL-4), Interleukin-10 (IL-10) in the peri-infarct hippocampal were examined by ELISA. Immunofluorescence was employed to evaluate Iba-1+ / P2X7R+, Iba-1+/ iNOS+ and Iba-1+/ Arg-1+ cell populations in the peri-infarct hippocampal DG area. The protein expression of P2X7R, Nuclear factor E2-related factor 2 (Nrf2), Recombinant nlr family, pyrin domain containing protein 3 (NLRP3), Inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1) in the peri-infarct hippocampal were investigated using western blot assays. Besides, we also measured the levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). RESULTS: We found EA treatment reduced inflammation and oxidative stress, which is consistent with a decrease in P2X7R expression and improved learning and memory functions. In contrast, we found BzATP enhanced inflammation and oxidative stress. Moreover, our results showed EA treatment up-regulated Nrf2, down-regulated NLRP3, and promoted microglia M2 polarization. Finally, EA-mediated positive effects were reversed by intracerebroventricular injection of BzATP, which is consistent with an increase in P2X7R expression. CONCLUSION: EA ameliorates memory impairment in a rat model of ischemic stroke by reducing inflammation and ROS through the inhibition of P2X7R expression. In turn, this mechanism regulates Nrf2 and NLRP3 expression, suggesting EA is beneficial for ischemic stroke treatment using P2X7R as target.

The human P2X7 receptor alters microglial morphology and cytokine secretion following immunomodulation.

Introduction: In recent years, purinergic signaling via the P2X7 receptor (P2X7R) on microglia has repeatedly been implicated in depression genesis. However, it remains unclear which role the human P2X7R (hP2X7R) plays in regulating both microglia morphology and cytokine secretion upon different environmental and immune stimuli, respectively. Methods: For this purpose, we used primary microglial cultures derived from a humanized microglia-specific conditional P2X7R knockout mouse line to emulate different gene-environment interactions between microglial hP2X7R and molecular proxies of psychosocial and pathogen-derived immune stimuli. Microglial cultures were subjected to treatments with the agonists 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP) and lipopolysaccharides (LPS) combined with specific P2X7R antagonists (JNJ-47965567, A-804598). Results: Morphotyping revealed overall high baseline activation due to the in vitro conditions. Both BzATP and LPS + BzATP treatment increased round/ameboid microglia and decreased polarized and ramified morphotypes. This effect was stronger in hP2X7R-proficient (CTRL) compared to knockout (KO) microglia. Aptly, we found antagonism with JNJ-4796556 and A-804598 to reduce round/ameboid microglia and increase complex morphologies only in CTRL but not KO microglia. Single cell shape descriptor analysis confirmed the morphotyping results. Compared to KO microglia, hP2X7R-targeted stimulation in CTRLs led to a more pronounced increase in microglial roundness and circularity along with an overall higher decrease in aspect ratio and shape complexity. JNJ-4796556 and A-804598, on the other hand, led to opposite dynamics. In KO microglia, similar trends were observed, yet the magnitude of responses was much smaller. Parallel assessment of 10 cytokines demonstrated the proinflammatory properties of hP2X7R. Following LPS + BzATP stimulation, IL-1ß, IL-6, and TNFα levels were found to be higher and IL-4 levels lower in CTRL than in KO cultures. Vice versa, hP2X7R antagonists reduced proinflammatory cytokine levels and increased IL-4 secretion. Discussion: Taken together, our results help disentangle the complex function of microglial hP2X7R downstream of various immune stimuli. In addition, this is the first study in a humanized, microglia-specific in vitro model identifying a so far unknown potential link between microglial hP2X7R function and IL-27 levels.

Spotlight on P2X7 Receptor PET Imaging: A Bright Target or a Failing Star?

The homotrimeric P2X7 receptor (P2X7R) is expressed by virtually all cells of the innate and adaptive immune system and plays a crucial role in various pathophysiological processes such as autoimmune and neurodegenerative diseases, inflammation, neuropathic pain and cancer. Consequently, the P2X7R is considered a promising target for therapy and diagnosis. As the development of tracers comes hand-in-hand with the development of potent and selective receptor ligands, there is a rising number of PET tracers available in preclinical and clinical studies. This review analyzes the development of P2X7R positron emission tomography (PET) tracers and their potential in various PET imaging applications.

Effect of Biling Qutong Prescription on NLRP3， P2X7R， FN， and Hepatic Steatosis in Type 2 Diabetes Mellitus Complicated with Gouty Arthritis / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Biling Qutong prescription （BLQT） on serum levels of NOD-like receptor thermal protein domain associated protein 3 （NLRP3）， purinergic ligand-gated ion channel 7 receptor （P2X7R）， fibronectin （FN）， and hepatic steatosis in patients with type 2 diabetes mellitus （T2DM） complicated with gouty arthritis （GA）. MethodSixty-four patients diagnosed with T2DM comorbid with GA and treated at the First Affiliated Hospital of Anhui University of Chinese Medicine from January 2019 to December 2022 were enrolled and randomly divided into a BLQT group （Chinese medicine group， 32 cases） and the ibuprofen group （western medicine group， 32 cases）. Thirty healthy individuals who underwent routine health examinations during the same period were assigned to the control group. The BLQT group and the western medicine group received basic treatment along with BLQT and ibuprofen， respectively. After 8 weeks of continuous treatment， the traditional Chinese medicine syndrome score （TCMSS） of the patients was evaluated before and after treatment. The differences in fasting plasma glucose （FPG）， 2-hour postprandial plasma glucose （2 h PG）， glycated hemoglobin （HbA1c）， fasting insulin （FINS）， homeostasis model assessment of insulin resistance （HOMA-IR）， serum uric acid （SUA）， serum creatinine （SCr）， total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， controlled attenuation parameter （CAP）， liver stiffness measurement （LSM）， NLRP3， P2X7R， and FN levels before and after treatment were compared. Adverse drug reactions that occurred during treatment were recorded. ResultThe TCMSS for joint redness， swelling， pain， joint burning， yellow urine， and red tongue with yellow and greasy coating， as well as total score were significantly reduced in both the BLQT group and the western medicine group as compared with those before treatment （P<0.05， P<0.01）. The BLQT group also showed a significant reduction in symptom scores such as dry mouth， polyuria， polydipsia， and slippery and rapid pulse （P<0.01）. Compared with the western medicine group after treatment， the BLQT group exhibited a more significant reduction in all symptom scores and total score （P<0.05， P<0.01）. The BLQT group and the western medicine group showed a decrease in FPG， 2 h PG， HbA1c， SCr， SUA， TG， TC， and LDL-C levels （P<0.05， P<0.01） after treatment， and the BLQT group showed decreased HOMA-IR， ALT， AST， and HDL-C levels （P<0.05， P<0.01） compared with those before treatment. When compared with the western medicine group after treatment， the BLQT group showed a more significant reduction in all laboratory parameters except for HDL-C （P<0.05， P<0.01）. Before treatment， NLRP3， P2X7R， and FN levels in both the BLQT group and the western medicine group were higher than those in the control group （P<0.01）. After treatment， NLRP3 and P2X7R levels in both groups significantly decreased （P<0.01）， and FN levels in the BLQT group also decreased significantly （P<0.01）. When compared with the western medicine group after treatment， the BLQT group showed a more significant reduction in NLRP3， P2X7R， and FN levels （P<0.01）. Before treatment， CAP and LSM levels in both the BLQT group and the western medicine group were higher than those in the control group （P<0.01）. After treatment， CAP and LSM levels in both groups decreased （P<0.05， P<0.01）. Compared with the western medicine group after treatment， the BLQT group showed a more significant reduction in CAP and LSM （P<0.01）. The incidence of adverse reactions was 3.13% （1/32） in the BLQT group and 15.63% （5/32） in the western medicine group， with no significant difference. ConclusionBLQT has good efficacy in patients with T2DM complicated with GA， which can significantly alleviate joint redness， swelling， heat， pain， limited mobility， dry mouth， and polydipsia， reduce blood glucose， uric acid， and lipid levels， suppress the high expression of NLRP3， P2X7R， and FN， and improve hepatic steatosis.

Gene-Level Analysis of Anthracycline-Induced Cardiomyopathy in Cancer Survivors: A Report From COG-ALTE03N1, BMTSS, and CCSS.

Background: Anthracyclines are highly effective in treating cancer, albeit with increased cardiomyopathy risk. Although risk is attributed to associations with single nucleotide polymorphisms (SNPs), multiple SNPs on a gene and their interactions remain unexamined. Objectives: This study examined gene-level associations with cardiomyopathy among cancer survivors using whole-exome sequencing data. Methods: For discovery, 278 childhood cancer survivors (129 cases; 149 matched control subjects) from the COG (Children's Oncology Group) study ALTE03N1 were included. Logic regression (machine learning) was used to identify gene-level SNP combinations for 7,212 genes and ordinal logistic regression to estimate gene-level associations with cardiomyopathy. Models were adjusted for primary cancer, age at cancer diagnosis, sex, race/ethnicity, cumulative anthracycline dose, chest radiation, cardiovascular risk factors, and 3 principal components. Statistical significance threshold of 6.93 × 10-6 accounted for multiple testing. Three independent cancer survivor populations (COG study, BMTSS [Blood or Marrow Transplant Survivor Study] and CCSS [Childhood Cancer Survivor Study]) were used to replicate gene-level associations and examine SNP-level associations from discovery genes using ordinal logistic, conditional logistic, and Cox regression models, respectively. Results: Median age at cancer diagnosis for discovery cases and control subjects was 6 years and 8 years, respectively. Gene-level association for P2RX7 (OR: 0.10; 95% CI: 0.04-0.27; P = 2.19 × 10-6) was successfully replicated (HR: 0.65; 95% CI: 0.47-0.90; P = 0.009) in the CCSS cohort. Additional signals were identified on TNIK, LRRK2, MEFV, NOBOX, and FBN3. Individual SNPs across all discovery genes, except FBN3, were replicated. Conclusions: In our study, SNP sets having 1 or no copies of P2RX7 variant alleles were associated with reduced risk of cardiomyopathy, presenting a potential therapeutic target to mitigate cardiac outcomes in cancer survivors.

PET Imaging of P2X7 Receptor (P2X7R) for Neuroinflammation with Improved Radiosynthesis of Tracer [18F]4A in Mice and Non-human Primates.

The P2X7 receptor (P2X7R) is a key neuroinflammation target in a variety of neurodegenerative diseases. Improved radiosynthesis was developed according to the previously reported P2X7R antagonist GSK1482160. Biodistribution, radiometabolite, and dynamic positron emission tomography/computed tomography-magnetic resonance imaging (PET/CT-MRI) of the lipopolysaccharide (LPS) rat model and the transgenic mouse model of Alzheimer's disease (AD) revealed a stable, low uptake of [18F]4A in the brain of healthy rats but a higher standardized uptake value ratio (SUVR) in LPS-treated rats (1.316 ± 0.062, n = 3) than in sham (1.093 ± 0.029, n = 3). There were higher area under curves (AUCs) in the neocortex (25.12 ± 1.11 vs 18.94 ± 1.47), hippocampus (22.50 ± 3.41 vs 15.90 ± 1.59), and basal ganglia (22.26 ± 0.81 vs 15.32 ± 1.76) of AD mice (n = 3) than the controls (n = 3) (p < 0.05). Furthermore, 50 min dynamic PET in healthy nonhuman primates (NHPs) indicated [18F]4A could penetrate the blood-brain barrier (BBB). In conclusion, [18F]4A from this study is a potent P2X7R PET tracer that warrants further neuroinflammation quantification in human studies.

Hetero-aryl bromide precursor fluorine-18 radiosynthesis and preclinical evaluation of a novel positron emission tomography (PET) tracer [18F]GSK1482160.

The purinergic P2X7 receptor (P2X7R), an ATP gated ion channel, is an important therapeutic target for various inflammatory immune and neurodegenerative diseases. A novel P2X7R targeting radiotracer GSK1482160 was radiosynthesized by hetero-aryl bromides precursor 10 with [18F]Et4NF, 20-30 % radiochemical yield, > 68 GBq/µmol specific activity, >98 % radiochemical purity. Evaluation in healthy male Sprague-Dawley rats revealed that [18F]GSK1482160 ([18F]11) was stably retained 87.81 %, 72.45 %, and 56.32 % in brain, blood and liver respectively 60-min post-injection. Ex-vivo biodistribution of [18F]11 proved that it was able to target the P2X7R in vivo and there was no defluorination in the major organs. PET/MRI imaging and autoradiography revealed that [18F]11 was able to penetrate the blood-brain barrier (BBB) and to be a promising P2X7R PET radioligand for clinical translation.

Identification of Follicular T Cells in the Gut.

Humoral adaptive immune responses trigger the establishment of plasma B cells secreting antibodies of various isotypes that bind antigen specifically and with high affinity. Moreover, memory B cells will be generated. To accomplish this, B cells need assistance from a special subset of CD4 T cells, the so called follicular T cells that differentiate from naïve T cells in the course of the immune response. Therefore, the study of follicular T cells is of primordial interest when investigating the molecular and cellular determinants of adaptive immune responses. This is done by direct analysis of the cells isolated from mice following an immunological challenge but in many instances such analyses must involve follow-up studies in cell culture requiring living cells. Especially, in vitro experimentation necessitates isolation and sorting of follicular T cells. However, follicular T cells are generally difficult to handle because they are prone to apoptosis and cell death. This is particularly evident when dealing with follicular T cells residing in the gut since we observed that isolation and processing from murine gut notoriously results in very high loss rates when compared for example to cells obtained from immunized peripheral lymph nodes. To bypass these limitations, we developed a protocol that allows for efficient isolation of intact follicular T cells. The protocol introduced here illustrates isolation and handling of follicular T cells using murine Peyer's Patches as an example because they constantly harbor significant amounts of these cells.

Phillygenin Mitigates LPS/ATP-induced L02 Cell Inflammation by Regulating P2X7R/NF-κB/NLRP3 Inflammasome Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effects of phillygenin （PHI） on the inflammation in L02 cells induced by lipopolysaccharide （LPS） and adenosine triphosphate （ATP） and the expression of purinergic 2X7 receptor （P2X7R）， NOD-like receptor family pyrin domain containing 3 （NLRP3）， and nuclear factor kappa B （NF-κB） expression. MethodIn this study， the inflammation model was induced in L02 cells by 100 μg·L-1 LPS treatment for 24 h and 5 mmoL·L-1 ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI （100， 50， 25 mg·L-1） for 6 h in the LPS treatment period， followed by LPS treatment for another 18 h. After ATP treatment for 5 h， the mRNA and protein expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， P2X7R， NLRP3， Caspase-1 precursor （pro-Caspase-1）， cleaved Caspase-1， NF-κB， and NF-κB inhibitor protein α （IκBα） in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Molecular docking was used to predict whether P2X7R could bind to PHI， and DCFH-DA was employed to detect the accumulation of reactive oxygen species （ROS） in cells. P2X7R was silenced by small interfering ribonucleic acid （siRNA）， and then the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was detected by Real-time PCR. ResultReal-time PCR and Western blot showed that compared with the normal group， the model group showed increased expression of IL-1β and IL-18 （P<0.05）， and compared with the model group， the PHI groups showed down-regulated IL-1β， IL-18 mRNA and protein expression （P<0.05）. Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group （P<0.05）， and compared with the model group， the PHI groups showed down-regulated mRNA and protein expression of P2X7R （P<0.05）. DCFH-DA results showed that compared with the normal group， the model group showed increased content of ROS （P<0.05）， and compared with the model group， the PHI groups decreased the accumulation of ROS （P<0.05）. As demonstrated by Real-time PCR and Western blot， compared with the normal group， the model group showed increased expression of NLRP3 inflammasome and NF-κB （P<0.05）， and compared with the model group， the PHI groups significantly decreased the mRNA and protein expression of NLRP3 and cleaved Caspase-1， and up-regulated the mRNA and protein expression of NF-κB and IκBα （P<0.05）. Real-time PCR analysis showed that compared with the results in the model group， after silencing P2X7R by siRNA， the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was decreased （P<0.05）. PHI exerted the same effect， and the mRNA expression was further reduced after the combination of them. ConclusionPHI is presumed to suppress the expression of the NLRP3/NF-κB signaling pathway by down-regulating upstream P2X7R to alleviate the LPS/ATP-induced inflammation in L02 cells， suggesting that P2X7R may be the target of PHI against inflammation.

A influência de macrófagos M1, M2 e TAM, e do receptor P2X7 na invasividade e resistência de neuroblastoma / The influence of different macrophages' phenotypes, and of the P2X7 receptor, on the invasiveness and chemoresistance of neuroblastoma

O neuroblastoma é um tumor sólido muito comum em crianças. O estágio mais avançado da doença é altamente agressivo e invasivo, além de pouco responsivo à terapia, que é limitada por mecanismos de resistência e reincidência relacionados à metástase. Muitos estudos tem sido feitos para identificar mecanismos de invasão e quimioresistência de células tumorais, afim de aumentar a sobrevida dos pacientes com câncer. Nesse trabalho, nós estudamos o efeito dos macrófagos, as células imunes mais abundantes no microambiente tumoral, os TAMs (do inglês tumor-associated macrophage) e do receptor P2X7, um purinoreceptor acionado por ATP, nesses processos. Os TAMs respondem e atuam de acordo com a miríade de fatores que encontram, podendo gerar populações heterogêneas e com funções distintas, tanto antitumorais, como pró-tumorais. Altos níveis de ATP extracelular são encontrados no microambiente tumoral, podendo então ativar o receptor P2X7. Este receptor tem sido relacionado tanto a funções inflamatórias como funções na resolução da inflamação de macrófagos. Além disso, o receptor P2X7 está envolvido em uma variedade de eventos celulares, incluindo a secreção de mediadores pró-inflamatórios, a proliferação celular e a apoptose de células tumorais. Primeiramente, foi avaliado o papel do receptor P2X7 na polarização de macrófagos da derivados medula óssea de camundongos wild-type e nocaute para o P2X7 na presença e ausência de fatores secretados por células de neuroblastoma, e então foi estudada a influência desses diferentes macrófagos polarizados em eventos celulares de grande relevância clínica para o neuroblastoma: a invasividade e quimiorresistência. Os resultados demonstraram que, apesar do reconhecido envolvimento do receptor P2X7 na inflamação, a ausência deste receptor não atenua a expressão de marcadores característicos do fenótipo inflamatório, M1. O aumento da expressão do receptor P2Y2, também envolvido na inflamação, nessas células, sugere um mecanismo genético de compensação para não atenuação da inflamação em macrófagos que não expressam o receptor P2X7. Contudo, a ausência do receptor P2X7 levou a alterações no fenótipo alternativo, M2, de modo que a expressão de Tnf, marcador de M2, não foi reprimido. TAMs noucates para P2X7 tiveram a expressão de arg1, marcador de M2, suprimida, reforçando a importância do receptor P2X7 no estabelecimento de fenótipos ativados alternativamente. Nossos dados também sugerem que ausência do receptor P2X7 em TAMs permite a aquisição de um fenótipo capaz de tornar as células de neuroblastoma que expressam P2X7 mais invasivas e mais quimioresistentes à vincristina. Por outro lado, TAMs, independentemente da presença ou ausência do receptor P2X7, induziram a proliferação e quimioresistência das células de neuroblastoma silenciadas para o receptor P2X7, o que nos leva a concluir que o receptor P2X7 em TAMs é desfavorável à progressão de tumores expressando P2X7

Neuroblastoma is a highly common childhood solid tumor. The most advanced stage of the disease is highly aggressive and invasive, besides from being poorly responsive to therapies, which are limited by resistance and recurrence mechanisms related to metastasis. Several studies attempt to identify invasion and resistance mechanisms of the tumor cells in order to increase overall survival of the patients. On the present work, we investigated the effect of macrophages, the most abundant immune cells on the tumor microenvironment, called TAMs (tumor-associated macrophages), and of the P2X7 receptor, an ATP-gated purinoceptor, on these processes. TAMs and cancer cells crosstalk, and behave accordingly to a miriad of factors present at the TME, generating heterogeneous populations with distinct functionalities, either pro- or antitumor. High extracellular levels of ATP are found in the TME, being able to activate the P2X7 receptor. This receptor mediates both pro- and anti-inflammatory functions in macrophages. In addition, it is involved in several cellular events, including the secretion of pro-inflammatory mediators, cell proliferation and tumor cell apoptosis. At first, we evaluated the role of the P2X7 receptor on the polarization of bone marrow-derived macrophages (BMDM), either wild-type or knockout for the P2X7 receptor, in presence or absence or factors secreted by neuroblastoma cells. Next, we investigated the influence of the polarized macrophages in highly relevant cellular events for neuroblastoma, such as invasiveness and chemoresistance. Our results showed that, despite the known involvement of P2X7 receptor on inflammation, its absence did not decrease the expression if inflammatory markers of M1 macrophage populations. An increase in the expression of the P2Y2 receptor, also involved in inflammation, on these cells suggest a genetic compensation mechanism for preventing attenuation of inflammation when P2X7 is lacking. However, P2X7 receptor absence did compromise the M2 phenotype, driving the expression of Tnf. TAMs knockout for the P2X7 receptor were not able to express arg1, also an M2 marker, reinforcing a role of the P2X7 receptor on establishing alternative macrophage phenotypes. Our data also suggest that TAMs lacking the P2X7 receptor acquire a phenotype capable of turning P2X7R-expressing neuroblastoma cells more invasive and chemoresistant to vincristine. On the other hand, TAMs, independently on the presence of the P2X7 receptor, induced proliferation and resistance of neuroblastoma cells silenced for P2X7 receptor expression, leading us to the conclusion that the P2X7 receptor in TAMs is unfavorable for the progression of P2X7R-expressing tumors

Activation of the purinergic receptor P2X7 improves hepatosteatosis by promoting lipophagy.

Nonalcoholic fatty liver disease (NAFLD) is a global health problem that develops through unclear molecular mechanisms. The P2X7 purinergic receptor (P2RX7) is an ATP-gated ion channel that belongs to the P2XR family. Thus far, studies on P2RX7 in NAFLD have been largely contradictory. Integrating experiments and modeling, we elucidate the dynamic processes of lipid droplet fusion and degradation following regulation of P2RX7. We show that activation of P2RX7 can activate the AMPK/ULK1 pathway to promote autophagosome generation and lysosomal degradation of autophagosomes. Inhibiting P2RX7 has the opposite effect. Notably, we find that lipid droplets become larger by the fusion of dysfunctional lysosomes but cannot be degraded by them following P2RX7 inhibition. Our study provides evidence that P2RX7 activation improves NAFLD by promoting lipophagy.

P2X7R antagonists in chronic stress-based depression models: a review.

Depression affects around 320 million people worldwide. Growing evidence proposes the immune system to be the core interface between psychosocial stress and the neurobiological and behavioural features of depression. Many studies have identified purinergic signalling via the P2X7 receptor (P2X7R) to be of great importance in depression genesis yet only a few have evaluated P2X7R antagonists in chronic stress-based depression models. This review summarizes their findings and analyses their methodology. The four available studies used three to nine weeks of unpredictable, chronic mild stress or unpredictable, chronic stress in male mice or rats. Stress paradigm composition varied moderately, with stimuli being primarily psychophysical rather than psychosocial. Behavioural testing was performed during or after the last week of stress application and resulted in depressive-like behaviours, immune changes (NLRP3 assembly, interleukin-1ß level increase, microglia activation) and neuroplasticity impairment. During the second half of each stress paradigm, a P2X7R antagonist (Brilliant Blue G, A-438079, A-804598) was applied. Studies differed with regard to antagonist dosage and application timing. Nonetheless, all treatments attenuated the stress-induced neurobiological changes and depressive-like behaviours. The evidence at hand underpins the importance of P2X7R signalling in chronic stress and depression. However, improvements in study planning and reporting are necessary to minimize experimental bias and increase data purview. To achieve this, we propose adherence to the Research Domain Criteria and the STRANGE framework.

Depression Induced by Chronic Unpredictable Mild Stress Increases Susceptibility to Parkinson's Disease in Mice via Neuroinflammation Mediated by P2X7 Receptor.

The relationship between depression and Parkinson's disease (PD) is complicated and still not fully understood. We investigated whether depression increased the susceptibility to PD and whether this resulted from neuroinflammation mediated by purinergic ligand-gated ion channel 7 receptor (P2X7R) of microglia in mice. Depression was induced by a 14-day chronic unpredictable mild stress (CUMS), and PD was induced by 1-day acute injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Before MPTP administration, some mice were given brilliant blue G (BBG), a P2X7R inhibitor. Changes in depression and motor function were assessed by sucrose preference, tail suspension, open field, and rotating rod tests. Differences in P2X7R, caspase-1, NLRP3 inflammasome, interleukin (IL)-1ß, tyrosine hydroxylase (TH), and microglial activation among experimental groups were detected by immunofluorescence, immunohistochemistry, western blotting, and ELISA. CUMS-induced depression-like behavior, and MPTP induced PD in mice. CUMS mice had no motor dysfunction, but the dyskinesia and loss of TH-positive neurons in the substantia nigra after MPTP treatment were more serious than with MPTP treatment alone. With behavioral changes, neuroinflammatory markers, such as caspase-1, NLRP3 and IL-1ß increased, and microglia were activated as well as expression of P2X7R increased. Additionally, BBG partly reversed the above abnormalities. Summarily, we suggest that CUMS aggravates dyskinesia and death of dopaminergic neurons in an MPTP-PD model via promoting activation of microglia and neuroinflammation, which may be mediated by P2X7R. Inhibition of P2X7R could be a new control strategy for PD associated with depression.

Recent developments on PET radiotracers for TSPO and their applications in neuroimaging.

The 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is predominately localized to the outer mitochondrial membrane in steroidogenic cells. Brain TSPO expression is relatively low under physiological conditions, but is upregulated in response to glial cell activation. As the primary index of neuroinflammation, TSPO is implicated in the pathogenesis and progression of numerous neuropsychiatric disorders and neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), major depressive disorder (MDD) and obsessive compulsive disorder (OCD). In this context, numerous TSPO-targeted positron emission tomography (PET) tracers have been developed. Among them, several radioligands have advanced to clinical research studies. In this review, we will overview the recent development of TSPO PET tracers, focusing on the radioligand design, radioisotope labeling, pharmacokinetics, and PET imaging evaluation. Additionally, we will consider current limitations, as well as translational potential for future application of TSPO radiopharmaceuticals. This review aims to not only present the challenges in current TSPO PET imaging, but to also provide a new perspective on TSPO targeted PET tracer discovery efforts. Addressing these challenges will facilitate the translation of TSPO in clinical studies of neuroinflammation associated with central nervous system diseases.

Mini-TrpRS is essential for IFNγ-induced monocyte-derived giant cell formation.

Truncated tryptophanyl-tRNA synthetase (mini-TrpRS), like any other aminoacyl-tRNA synthetases, canonically functions as a protein synthesis enzyme. Here we provide evidence for an additional signaling role of mini-TrpRS in the formation of monocyte-derived multinuclear giant cells (MGCs). Interferon-gamma (IFNγ) readily induced monocyte aggregation leading to MGC formation with paralleled marked upregulation of mini-TrpRS. Small interfering (si)RNA, targeting mini-TrpRS in the presence of IFNγ prevented monocyte aggregation. Moreover, blockade of mini-TrpRS, either by siRNA, or the cognate amino acid and decoy substrate D-Tryptophan to prevent mini-TrpRS signaling, resulted in a marked reduction in expression of the purinergic receptor P2X 7 (P2RX7) in monocytes activated by IFNγ. Our findings identify mini-TrpRS as a critical signaling molecule in a mechanism by which IFNγ initiates monocyte-derived giant cell formation.

Effect of Yinxing Mihuan Oral Solutionon P2RX7/NLRP3 Signaling Pathway in Depression Model Rats Induced by Isolation Combined with Chronic Unpredictable Mild Stress / 中国实验方剂学杂志

Objective:To explore the antidepressant mechanism of Yinxing Mihuan oral solution （YMO） by investigating its effect on depression model rats. Method:The depression rats were induced by isolation combined with chronic unpredictable mild stress （CUMS） and then randomly divided into model group， fluoxetine group （10 mg·kg^-1） and high-dose （618 mg·kg^-1） and low-dose （309 mg·kg^-1） YMO groups. A blank control group was also set up and ten rats were included in each group. Modeling lasted for 21 consecutive days， and rats were administered the 8th day after stimulation at a dose of 10 mL·kg^-1 for 14 days， except those in the blank control and model groups which were given distilled water. Afterward， the sucrose preference test， open field test， tail suspension test were carried out. The pathological changes of hippocampus in depression rats were observed after hematoxylin-eosin （HE） staining. The content of interleukin-1<italic>β </italic>（IL-1<italic>β</italic>）， interleukin-6 （IL-6） and tumor necrosis factor-<italic>α </italic>（TNF-<italic>α</italic>） in the hippocampus of rats in each group and the expression of NOD-like receptor 3 （NLRP3） and other proteins in its related activation signaling pathways were detected with multi-factor detection （Luminex） and Western blot. Result:After 14 days of continuous administration， compared with the blank control group， the model group witnessed significantly reduced sugar water consumption rate and the times of rearing and significantly prolonged cumulative time of immobility during tail suspension （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the fluoxetine group and the high-dose YMO group saw increases in the times of rearing， times of crossing and sugar water consumption rate and a significant decrease in the cumulative time of immobility during tail suspension （<italic>P</italic><0.05， <italic>P</italic><0.01）. The results of HE staining showed that the neurons in the hippocampus of rats in the high-dose YMO group were arranged in order and slightly loosened， without obvious microglia infiltration observed. The levels of IL-1<italic>β</italic>， IL-6 and TNF-<italic>α</italic> in the hippocampus of the model group increased significantly as compared with the blank control group （<italic>P</italic><0.05， <italic>P</italic><0.01）， and their content in the high-dose YMO group was significantly lowered in the comparison with the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）. Molecular biology experiments demonstrated that compared with the results of blank group， the expression of purinergic receptor P2X7 （P2RX7）， NLRP3， apoptosis-associated speck-like protein （ASC）， Caspase-1 and IL-1<italic>β</italic> remarkably increased in the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）. Additionally， the expression of P2RX7， NLRP3， ASC， Caspase-1 and IL-1<italic>β </italic>was significantly inhibited in the fluoxetine group and the high-dose YMO group compared with the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:YMO can improve the depression-like behaviors of rats induced by isolation combined with CUMS， and its mechanism of action is related to the regulation of the P2RX7/NLRP3 signaling pathway.

Transplantation of microencapsulated neural stem cells inhibits neuropathic pain mediated by P2X7 receptor overexpression.

BACKGROUND: Neuropathic pain (NPP) is a common clinical symptom, its pathological mechanism is complex, and there is currently no good treatment method. Therefore, exploring the treatment method of NPP is a critical issue that needs to be urgently solved. METHODS: Neural stem cells (NSC) and microencapsulated neural stem cells (MC-NSC) were transplanted into the site of sciatic nerve injury, and behavioral methods were used to detect changes in pain. Expression levels of P2X7R were detected in the dorsal root ganglion (DRG) by molecular biological methods. RESULTS: After sciatic nerve injury, mechanical withdrawal thresholds (MWT) and thermal withdrawal latency (TWL) of rats were significantly reduced, the expression levels of P2X7R in the DRG were significantly increased. After transplantation of NSC and MC-NSC, it was found that expression levels of P2X7R were significantly reduced and pain was significantly suppressed. Importantly, compared with NSC transplantation, MC-NSC could better reduce the expression levels of P2X7R and inhibit pain. CONCLUSION: MC-NSC can better decrease the expression levels of P2X7R and relieve NPP. Our results provide a novel method and data support for the treatment of NPP.

Transplantation of olfactory ensheathing cells combined with chitosan down-regulates the expression of P2X7 receptor in the spinal cord and inhibits neuropathic pain.

BACKGROUND: Neuropathic pain (NPP) is the common symptom of most clinical diseases, and its treatment has always been a difficult problem at present. Therefore, the purpose of this study is to explore a new method for the treatment of NPP by transplanting olfactory ensheathing cells combined with chitosan (OECs-CS). METHODS: Animal model of chronic compression sciatic nerve injury (CCI) was made, olfactory ensheathing cells (OECs) were cultured, chitosan (CS) biomaterials were prepared, and biocompatibility of OECs and CS were detected by MTT method, OECs and OECs-CS were transplanted into the site of the injured sciatic nerve respectively, behavioral method was used to measured the mechanical withdrawal thresholds (MWT) and thermal withdrawal latency (TWL) of rats. On days 7 and 14 after surgery, the expression level of P2X7 receptor (P2X7R) in the L4-5 spinal cord was measured by using in situ hybridization, western-blotting and qRT-PCR. To explore the therapeutic effect of OECs-CS transplantation on pain suppression. RESULTS: After chronic compression sciatic nerve injury, the MWT and TWL of rats were significantly reduced, and the expression levels of P2X7R protein and mRNA in the L4-5 spinal cord was significantly increased. After the transplantation of OECs and OECs-CS, the expression levels of P2X7R was significantly reduced, and the MWT and TWL of rats were significantly increased. Importantly, compared with the transplantation of OECs, OECs-CS transplantation could better reduce the expression levels of P2X7R, and relieve hyperalgesia in rats. Moreover, compared with the CCI + OECs-CS group on days 7 after surgery, the expression levels of P2X7R in the CCI + OECs-CS group was reduced on days 14 after surgery, and the pain in rats was relieved. CONCLUSION: OECs and OECs-CS transplantation can inhibit P2X7R overexpression mediated NPP, while OECs-CS transplantation has better therapeutic effect than OECs transplantation alone. Our results provide a novel method and theoretical basis for the treatment of NPP.