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.

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

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.

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.

Spinal P2X7R contributes to streptozotocin-induced mechanical allodynia in mice / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Painful diabetic neuropathy (PDN) is a diabetes mellitus complication. Unfortunately, the mechanisms underlying PDN are still poorly understood. Adenosine triphosphate (ATP)-gated P2X7 receptor (P2X7R) plays a pivotal role in non-diabetic neuropathic pain, but little is known about its effects on streptozotocin (STZ)-induced peripheral neuropathy. Here, we explored whether spinal cord P2X7R was correlated with the generation of mechanical allodynia (MA) in STZ-induced type 1 diabetic neuropathy in mice. MA was assessed by measuring paw withdrawal thresholds and western blotting. Immunohistochemistry was applied to analyze the protein expression levels and localization of P2X7R. STZ-induced mice expressed increased P2X7R in the dorsal horn of the lumbar spinal cord during MA. Mice injected intrathecally with a selective antagonist of P2X7R and P2X7R knockout (KO) mice both presented attenuated progression of MA. Double-immunofluorescent labeling demonstrated that P2X7R-positive cells were mostly co-expressed with Iba1 (a microglia marker). Our results suggest that P2X7R plays an important role in the development of MA and could be used as a cellular target for treating PDN.

Papel dos receptores purinérgicos em modelo animal de doença de Parkinson / Role of purinergic receptors in an animal model of Parkinsons Disease

A Doença de Parkinson é uma doença altamente incapacitante e de grande prevalência. Pouco se sabe sobre sua etiologia e os tratamentos atuais consistem na diminuição dos sintomas, uma vez que ainda não foi encontrada uma maneira de reverter o déficit de neurônios dopaminérgicos observados nos pacientes acometidos. Sabe-se que os receptores purinérgicos são encontrados por todo o sistema nervoso central, não só no indivíduo adulto como também em diferentes estágios do desenvolvimento embrionário e estão envolvidos com proliferação e diferenciação celular. Este trabalho estudou a participação dos receptores purinérgicos em modelo animal de doença de Parkinson por lesão dos neurônios dopaminérgicos da via nigroestriatal com 6-OH dopamina (6-OHDA). Realizamos a análise do perfil de expressão gênica dos diferentes receptores após a lesão e subsequente modulação. Observamos expressão gênica alterada dos receptores P2X7 e P2Y6 até 5 semanas após a lesão. O uso do antagonista do receptor P2X7 Brilliant Blue G (BBG) induziu a regeneração da via nigroestriatal e o uso do antagonista do receptor P2Y6 MRS2578 preveniu a morte dos neurônios. Como esses efeitos foram acompanhados pela inativação de células microgliais, supõe-se que o controle do microambiente neuroinflamatório causado pela injeção de 6-OHDA seja a principal causa do efeito antiparkinsoniano observado pelo tratamento com BBG e MRS2578. Além disso, o transplante celular com células precursoras neuraisnão foi capaz de reverter o comportamento hemiparkinsoniano dos animais lesionados. Apesar do uso concomitante com BBG reduzir o comportamento, parece que esse efeito deve-se ao BBG per se, uma vez que o tratamento somente com o antagonista de P2X7 foi mais eficaz. De maneira geral, a modulação da atividade dos receptores purinérgicos se mostrou uma ferramenta promissora na pesquisa de cura e compreensão das bases moleculares da Doença de Parkinson

Parkinson's disease is a highly disabling and prevalent disease. Little is known about its etiology and the current treatments consist in the reduction of the symptoms, since there is no known method to reverse the dopaminergic neurons deficit observed in patients. Purinergic receptors are found throughout the central nervous system, not only in the adult individual but also at different stages of embryonic development, and are involved in proliferation and differentiation. This work investigated the role of purinergic receptors in the animal model of Parkinson's disease induced by 6-OH dopamine (6-OHDA) injection and consequent death of dopaminergic neurons of the nigrostriatal pathway. Patterns of purinergic receptors gene expression after the lesion and subsequent modulation were analyzed. We observed altered gene expression of P2X7 and P2Y6 receptors within 5 weeks of injury. The use of the P2X7 receptor antagonist Brilliant Blue G (BBG) induced the regeneration of the nigrostriatal pathway and treatment with P2Y6 receptor antagonist MRS2578 prevented the death of the neurons. Since these effects were accompanied by the inactivation of microglial cells, it is assumed that the control of neuroinflammatory milieu caused by the 6-OHDA injection is the main cause of the antiparkinsonian effect observed by the treatment with BBG and MRS2578. In addition, transplantation with neural precursor cells was not able to reverse the hemiparkinsonian behavior of injured animals. Although concomitant use with BBG improved cell engraftment, it appears that this effect is due to BBG per se, since treatment with only this P2X7receptor antagonist was more effective. In general, modulation of purinergic receptor activity showed to be a promising tool in the research of cure and understanding of the molecular bases of Parkinson's Disease