Triphosphate Release Research Articles

Baicalin inhibited PANX-1/P2Y6 signaling pathway activation in porcine aortic vascular endothelial cells infected by Glaesserella parasuis

Glaesserella parasuis can induce endothelial barrier damage in piglets, although the mechanism by which this pathogen triggers inflammatory damage remains unclear. Baicalin possesses anti-inflammatory and anti-oxidant activities. However, whether baicalin can relieve endothelial barrier damage caused by Glaesserella parasuis infection has not yet been studied. Hence, we evaluated the ability of baicalin to counteract the changes induced by Glaesserella parasuis in porcine aortic vascular endothelial cells. The results showed that Glaesserella parasuis could upregulate the expression of pannexin 1 channel protein and promote the release of adenosine triphosphate, adenosine diphosphate, adenosine 3′-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate in porcine aortic vascular endothelial cells. The expression level of purinergic receptor P2Y6 was upregulated in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. In addition, Glaesserella parasuis could activate phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells. Baicalin could inhibit pannexin 1 channel protein expression, reduce adenosine triphosphate, adenosine diphosphate, adenosine 3′-monophosphate, uridine triphosphate, uridine diphosphate, and uridine monophosphate release, and attenuate the expression level of P2Y6 in porcine aortic vascular endothelial cells induced by Glaesserella parasuis. Baicalin could also reduce the activation of phospholipase C-protein kinase C and myosin light chain kinase-myosin light chain signaling pathways in porcine aortic vascular endothelial cells triggered by Glaesserella parasuis. Our study report that Glaesserella parasuis could promote pannexin 1 channel protein expression, induce nucleosides substance release, and P2Y6 expression in porcine aortic vascular endothelial cells and baicalin could inhibit the expression levels of pannexin 1, nucleosides substance, and P2Y6 in the porcine aortic vascular endothelial cells induced by Glaesserella parasuis, which might be served as some targets for treatment of inflammation disease caused by Glaesserella parasuis.

Extracellular adenosine triphosphate regulates inflammatory responses of periodontal ligament cells.

Various stimuli, that is, mechanical stresses or inflammation, induce the release of adenosine triphosphate (ATP) by human periodontal ligament cells (HPDLCs). Extracellular adenosine triphosphate (eATP) affects HPDLCs' functions such as immunosuppressive action and inflammatory responses. Lipopolysaccharide (LPS) is the key factor involved in periodontal inflammation. However, the possible correlation and detailed mechanism of inflammation-mediated eATP by LPS and inflammatory cascade formation in HPDLCs is unclarified. This study aims to examine the role of eATP on the HPDLCs' responses concerning inflammatory actions after LPS treatment. HPDLCs were stimulated with Porphyromonas gingivalis LPS and polyinosinic:polycytidylic acid (poly I:C). The amount of ATP release was measured at different time points using a bioluminescence assay. HPDLCs were treated with eATP. The expression of pro-inflammatory and anti-inflammatory genes was determined. Specific P2X purinoreceptor 7 (P2X7) inhibitors (brilliant blue G [BBG] and KN62), a specific P2Y purinoreceptor 1 (P2Y1) inhibitors (MRS2179), calcium chelator (EGTA), protein kinase C (PKC) inhibitors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF𝜅B) activation inhibitors, and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) inhibitors (H89 dihydrochloride) and activators (forskolin) were used to dissect the mechanism of eATP-induced HPDLCs' inflammatory responses. LPS and poly I:C induced ATP release. A low concentration of eATP (50µM) increased pro-inflammatory genes (COX2, IL1B, IL6, IL8, IL12, and TNFA), while a high concentration (500µM) enhanced anti-inflammatory genes (IL4 and IL10). BBG, KN62, and NF𝜅B activation inhibitors impeded eATP-induced pro-inflammatory genes. MRS2179 and H89 markedly suppressed eATP-induced anti-inflammatory genes. Forskolin induced IL4 and IL10. HPDLCs respond to LPS by releasing ATP. eATP has dose-dependent dual functions on HPDLCs' inflammatory responses via different pathways. As regulation of inflammation is important in regeneration, eATP may help to limit inflammation and trigger periodontal regeneration.

Aging is associated with impaired triggering of TRPV3-mediated cutaneous vasodilation: a crucial process for local heat exposure.

Sensing temperature is vitally important to adapt our body to environmental changes. Local warm detection is required to initiate regulation of cutaneous blood flow, which is part of the peripheral thermoregulatory mechanisms, and thus avoid damage to surrounding tissues. The mechanisms mediating cutaneous vasodilation during local heat stress are impaired with aging. However, the impact of aging on the ability of the skin to detect subtle thermal changes is unknown. Among heat-activated cation channels, transient receptor potential vanilloid 3 (TRPV3) is a thermo-sensor predominantly expressed on keratinocytes and involved in local vascular thermoregulatory mechanisms of the skin in young mice. In the present study, using a murine in vivo model of local heat exposure of the skin, we showed that heat-induced vasodilation was reduced in old mice associated with reduced expression of TRPV3 channels. We also found a decrease in expression and activity of TRPV3 channel, as well as reduced TRPV3-dependent adenosine tri-phosphate release in human primary keratinocytes from old donors. This study shows that aging alters the epidermal TRPV3 channels, which might delay the detection of changes in skin temperature, thereby limiting the mechanisms triggered for local vascular thermoregulation in the old skin.

Adeno-Associated Viral Vector-Delivered Pannexin-1 Mimetic Peptide Alleviates Airway Inflammation in an Allergen-Sensitized Mouse Model.

Asthma is a chronic inflammatory disease around the world. Extracellular adenosine triphosphate works as a dangerous signal in responding to cellular stress, irritation, or inflammation. It has also been reported its association with the pathogenicity in asthma, with increased level in lungs of asthmatics. Pannexin-1 is one of the routes that contributes to the release of adenosine triphosphate form intracellular to extracellular. The aim of this study was to apply pannexin-1 peptide antagonist 10Panx1 into adeno-associated viral (AAV) vectors on ovalbumin (OVA)-induced asthmatic mouse model. The results demonstrated that this treatment was able to reduce the adenosine triphosphate level in bronchoalveolar lavage fluid and downregulate the major relevant to the symptom of asthma attack, airway hyperresponsiveness to methacholine. The histological data also gave a positive support with decreased tissue remodeling and mucus deposition. Other asthmatic related features, including eosinophilic inflammation and OVA-specific T helper type 2 responses, were also decreased by the treatment. Beyond the index of inflammation, the proportion of effector and regulatory T cells was examined to survey the potential mechanism behind. The data provided a slightly downregulated pattern in lung GATA3+ CD4 T cells. However, an upregulated population of CD25+FoxP3+ CD4 T cells was seen in spleens. These data suggested that exogeneous expression of 10Panx1 peptide was potential to alleviated asthmatic airway inflammation, and this therapeutic effect might be from 10Panx1-mediated disruption of T cell activation or differentiation. Collectively, AAV vector-mediated 10Panx1 expression could be a naval therapy option to develop.

Nanobody-based pannexin1 channel inhibitors reduce inflammation in acute liver injury

BackgroundThe opening of pannexin1 channels is considered as a key event in inflammation. Pannexin1 channel-mediated release of adenosine triphosphate triggers inflammasome signaling and activation of immune cells. By doing so, pannexin1 channels play an important role in several inflammatory diseases. Although pannexin1 channel inhibition could represent a novel clinical strategy for treatment of inflammatory disorders, therapeutic pannexin1 channel targeting is impeded by the lack of specific, potent and/or in vivo-applicable inhibitors. The goal of this study is to generate nanobody-based inhibitors of pannexin1 channels.ResultsPannexin1-targeting nanobodies were developed as potential new pannexin1 channel inhibitors. We identified 3 cross-reactive nanobodies that showed affinity for both murine and human pannexin1 proteins. Flow cytometry experiments revealed binding capacities in the nanomolar range. Moreover, the pannexin1-targeting nanobodies were found to block pannexin1 channel-mediated release of adenosine triphosphate. The pannexin1-targeting nanobodies were also demonstrated to display anti-inflammatory effects in vitro through reduction of interleukin 1 beta amounts. This anti-inflammatory outcome was reproduced in vivo using a human-relevant mouse model of acute liver disease relying on acetaminophen overdosing. More specifically, the pannexin1-targeting nanobodies lowered serum levels of inflammatory cytokines and diminished liver damage. These effects were linked with alteration of the expression of several NLRP3 inflammasome components.ConclusionsThis study introduced for the first time specific, potent and in vivo-applicable nanobody-based inhibitors of pannexin1 channels. As demonstrated for the case of liver disease, the pannexin1-targeting nanobodies hold great promise as anti-inflammatory agents, yet this should be further tested for extrahepatic inflammatory disorders. Moreover, the pannexin1-targeting nanobodies represent novel tools for fundamental research regarding the role of pannexin1 channels in pathological and physiological processes.Graphical

Adenosine Triphosphate Release From Influenza-Infected Lungs Enhances Neutrophil Activation and Promotes Disease Progression.

Adenosine triphosphate (ATP) enhances neutrophil responses, but little is known about the role of ATP in influenza infections. We used a mouse influenza model to study if ATP release is associated with neutrophil activation and disease progression. Influenza infection increased pulmonary ATP levels 5-fold and plasma ATP levels 3-fold vs healthy mice. Adding ATP at those concentrations to blood from healthy mice primed neutrophils and enhanced CD11b and CD63 expression, CD62L shedding, and reactive oxygen species production in response to formyl peptide receptor stimulation. Influenza infection also primed neutrophils in vivo, resulting in formyl peptide receptor-induced CD11b expression and CD62L shedding up to 3 times higher than that of uninfected mice. In infected mice, large numbers of neutrophils entered the lungs. These cells were significantly more activated than the peripheral neutrophils of infected mice and pulmonary neutrophils of healthy mice. Plasma ATP levels of infected mice and influenza disease progression corresponded with the numbers and activation level of their pulmonary neutrophils. Findings suggest that ATP release from the lungs of infected mice promotes influenza disease progression by priming peripheral neutrophils, which become strongly activated and cause pulmonary tissue damage after their recruitment to the lungs.

Structure-Based Design and Synthesis of Stapled 10Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases.

Following a rational design, a series of macrocyclic ("stapled") peptidomimetics of 10Panx1, the most established peptide inhibitor of Pannexin1 (Panx1) channels, were developed and synthesized. Two macrocyclic analogues SBL-PX1-42 and SBL-PX1-44 outperformed the linear native peptide. During in vitro adenosine triphosphate (ATP) release and Yo-Pro-1 uptake assays in a Panx1-expressing tumor cell line, both compounds were revealed to be promising bidirectional inhibitors of Panx1 channel function, able to induce a two-fold inhibition, as compared to the native 10Panx1 sequence. The introduction of triazole-based cross-links within the peptide backbones increased helical content and enhanced in vitro proteolytic stability in human plasma (>30-fold longer half-lives, compared to 10Panx1). In adhesion assays, a "double-stapled" peptide, SBL-PX1-206 inhibited ATP release from endothelial cells, thereby efficiently reducing THP-1 monocyte adhesion to a TNF-α-activated endothelial monolayer and making it a promising candidate for future in vivo investigations in animal models of cardiovascular inflammatory disease.

Tumor suppressor p53 mediates interleukin-6 expression to enable cancer cell evasion of genotoxic stress

The tumor suppressor p53 primarily functions as a mediator of DNA damage-induced cell death, thereby contributing to the efficacy of genotoxic anticancer therapeutics. Here, we show, on the contrary, that cancer cells can employ genotoxic stress-induced p53 to acquire treatment resistance through the production of the pleiotropic cytokine interleukin (IL)-6. Mechanistically, DNA damage, either repairable or irreparable, activates p53 and stimulates Caspase-2-mediated cleavage of its negative regulator mouse double minute 2 (MDM2) creating a positive feedback loop that leads to elevated p53 protein accumulation. p53 transcriptionally controls the major adenosine triphosphate (ATP) release channel pannexin 1 (Panx1), which directs IL-6 induction via a mechanism dependent on the extracellular ATP-activated purinergic P2 receptors as well as their downstream intracellular calcium (iCa2+)/PI3K/Akt/NF-ĸB signaling pathway. Thus, p53 silencing impairs Panx1 and IL-6 expression and renders cancer cells sensitive to genotoxic stress. Moreover, we confirm that IL-6 hampers the effectiveness of genotoxic anticancer agents by mitigating DNA damage, driving the expression of anti-apoptotic Bcl-2 family genes, and maintaining the migratory and invasive properties of cancer cells. Analysis of patient survival and relevant factors in lung cancer and pan-cancer cohorts supports the prognostic and clinical values of Panx1 and IL-6. Notably, IL-6 secreted by cancer cells during genotoxic treatments promotes the polarization of monocytic THP-1-derived macrophages into an alternative (M2-like) phenotype that exhibits impaired anti-survival activities but enhanced pro-metastatic effects on cancer cells as compared to nonpolarized macrophages. Our study reveals the precise mechanism for genotoxic-induced IL-6 and suggests that targeting p53-mediated IL-6 may improve the responsiveness of cancer cells to genotoxic anticancer therapy.

Pannexin 1 targets mitophagy to mediate renal ischemia/reperfusion injury

Renal ischemia/reperfusion (I/R) injury contributes to the development of acute kidney injury (AKI). Kidney is the second organ rich in mitochondrial content next to the heart. Mitochondrial damage substantially contributes for AKI development. Mitophagy eliminates damaged mitochondria from the cells to maintain a healthy mitochondrial population, which plays an important role in AKI. Pannexin 1 (PANX1) channel transmembrane proteins are known to drive inflammation and release of adenosine triphosphate (ATP) during I/R injury. However, the specific role of PANX1 on mitophagy regulation in renal I/R injury remains elusive. In this study, we find that serum level of PANX1 is elevated in patients who developed AKI after cardiac surgery, and the level of PANX1 is positively correlated with serum creatinine and urea nitrogen levels. Using the mouse model of renal I/R injury in vivo and cell-based hypoxia/reoxygenation (H/R) model in vitro, we prove that genetic deletion of PANX1 mitigate the kidney tubular cell death, oxidative stress and mitochondrial damage after I/R injury through enhanced mitophagy. Mechanistically, PANX1 disrupts mitophagy by influencing ATP-P2Y-mTOR signal pathway. These observations provide evidence that PANX1 could be a potential biomarker for AKI and a therapeutic target to alleviate AKI caused by I/R injury.

PIEZO1 promotes ATP release from periodontal ligament cells following compression force.

Orthodontic mechanical force on the periodontal ligament induces extracellular adenosine triphosphate (ATP) release. However, mechanosensitive molecules have not been confirmed functionally in periodontal ligament cells. In the present study, we examined the roles of mechanosensitive PIEZO channels in the mechanically stimulated release of ATP in human periodontal ligament fibroblasts (HPdLFs). To examine PIEZO expression in HPdLFs, we performed reverse transcription-quantitative polymerase chain reaction, fluorescent immunostaining, and Ca2+ imaging. ATP concentrations were measured in culture medium after applications of the PIEZO1 agonist Yoda1 and compression force in a newly developed in vitro weight-loaded cell model (IVWLC) using balance weights and a 48-well plate. The mechanosensitive channel inhibitor GsMTx4 and the ATP-releasing route inhibitors clodronic acid, meclofenamic acid, and probenecid were used. To suppress PIEZO1 expression, short interference RNA (siRNA) treatment of the PIEZO1 gene was performed. PIEZO1 mRNA was expressed more abundantly than PIEZO2 mRNA in HPdLFs. HPdLF cell bodies were immunoreactive to anti-PIEZO1 antibody. Yoda1 increased intracellular Ca2+ and extracellular ATP concentrations in a dose-dependent manner. ATP release was inhibited by GsMTx4 and inhibitors of ATP release routes. In the IVWLC, HPdLFs released ATP in response to compression force but not in response to hypoxic stimulation that was simultaneously applied to cells. Mechanically stimulated ATP release was inhibited by GsMTx4, inhibitors of ATP-releasing routes and siRNA treatment of PIEZO1. PIEZO1 on the cell membranes of HPdLFs is activated by compression force and then induces ATP release via intracellular Ca2+-dependent exocytosis and ATP-permeable channels.

Translocation of telomerase reverse transcriptase coincided with ATP release in postnatal cochlear supporting cells.

The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate (ATP) by supporting cells in the Kölliker's organ. However, the mechanisms responsible for initiating spontaneous ATP release have not been determined. Our previous study revealed that telomerase reverse transcriptase (TERT) is expressed in the basilar membrane during the first postnatal week. Its role in cochlear development remains unclear. In this study, we investigated the expression and role of TERT in postnatal cochlea supporting cells. Our results revealed that in postnatal cochlear Kölliker's organ supporting cells, TERT shifts from the nucleus into the cytoplasm over time. We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo. Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis, suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions. We observed increased ATP synthesis, release and activation of purine signaling systems in supporting cells during the first 10 postnatal days. The phenomenon that TERT translocation coincided with changes in ATP synthesis, release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system. Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period.

Ratiometric Fluorescent Detection of Ultrasound-Regulated ATP Release: An Ultrasound-Resistant Cu,N-Doped Carbon Nanosphere.

Focused ultrasound, as a protocol of cancer therapy, might induce extracellular adenosine triphosphate (ATP) release, which could enhance cancer immunotherapy and be monitored as a therapeutic marker. To achieve an ATP-detecting probe resistant to ultrasound irradiation, we constructed a Cu/N-doped carbon nanosphere (CNS), which has two fluorescence (FL) emissions at 438 and 578 nm to detect ultrasound-regulated ATP release. The addition of ATP to Cu/N-doped CNS was conducted to recover the FL intensity at 438 nm, where ATP enhanced the FL intensity probably via intramolecular charge transfer (ICT) primarily and hydrogen-bond-induced emission (HBIE) secondarily. The ratiometric probe was sensitive to detect micro ATP (0.2-0.6 μM) with the limit of detection (LOD) of 0.068 μM. The detection of ultrasound-regulated ATP release by Cu,N-CNS/RhB showed that ATP release was enhanced by the long-pulsed ultrasound irradiation at 1.1 MHz (+37%, p < 0.01) and reduced by the short-pulsed ultrasound irradiation at 5 MHz (-78%, p < 0.001). Moreover, no significant difference in ATP release was detected between the control group and the dual-frequency ultrasound irradiation group (+4%). It is consistent with the results of ATP detection by the ATP-kit. Besides, all-ATP detection was developed to prove that the CNS had ultrasound-resistant properties, which means it could bear the irradiation of focused ultrasound in different patterns and detect all-ATP in real time. In the study, the ultrasound-resistant probe has the advantages of simple preparation, high specificity, low limit of detection, good biocompatibility, and cell imaging ability. It has great potential to act as a multifunctional ultrasound theranostic agent for simultaneous ultrasound therapy, ATP detection, and monitoring.

TRPV4 Regulates the Development of Psoriasis by Controlling Adenosine Triphosphate Expression in Keratinocytes and the Neuroimmune System

TRPV4 is a calcium ion channel that is widely expressed in various cells. It is also involved in physiological and pathological processes. However, the role of TRPV4 in psoriasis remains unknown. We aimed to investigate the role of TRPV4 in psoriasis using human psoriasis skin samples and an imiquimod-induced psoriasis-like mouse model. Keratinocytes in human psoriasis skin had high TRPV4 expression. Trpv4-knockout mice had less severe dermatitis than wild-type mice in the imiquimod-induced mouse model. Knockout mice had significantly reduced epidermal thickness and a low number of infiltrated CD3+ T cells and CD68+ macrophages on the basis of histopathological studies and decreased mRNA expression of Il17a, Il17f, and Il23, as detected through qPCR. Furthermore, knockout mice had a significantly low expression of neuropeptides and the neuron marker PGP9.5. Adenosine triphosphate release was significantly suppressed by TRPV4 knockdown in both human and mouse keratinocytes invitro. Finally, treatment with TRPV4 antagonist was significantly effective in preventing the progression of psoriasis-like dermatitis. In conclusion, TRPV4 mediates the expression of keratinocyte-derived adenosine triphosphate and increases the secretion of neuropeptides, resulting in the activation and amplification of IL-23/Th17 responses. Hence, TRPV4 can serve as a novel therapeutic target in psoriasis.

Determination of structural features that underpin the pannexin1 channel inhibitory activity of the peptide 10Panx1

Pannexin1 channels facilitate paracrine communication and are involved in a broad spectrum of diseases. Attempts to find appropriate pannexin1 channel inhibitors that showcase target-selective properties and in vivo applicability remain nonetheless scarce. However, a promising lead candidate, the ten amino acid long peptide mimetic 10Panx1 (H-Trp1-Arg2-Gln3-Ala4-Ala5-Phe6-Val7-Asp8-Ser9-Tyr10-OH), has shown potential as a pannexin1 channel inhibitor in both in vitro and in vivo studies. Nonetheless, structural optimization is critical for clinical use. One of the main hurdles to overcome along the optimization process consists of subduing the low biological stability (10Panx1 t1/2 = 2.27 ± 0.11 min). To tackle this issue, identification of important structural features within the decapeptide structure is warranted. For this reason, a structure–activity relationship study was performed to proteolytically stabilize the sequence. Through an Alanine scan, this study demonstrated that the side chains of Gln3 and Asp8 are crucial for 10Panx1′s channel inhibitory capacity. Guided by plasma stability experiments, scissile amide bonds were identified and stabilized, while extracellular adenosine triphosphate release experiments, indicative of pannexin1 channel functionality, allowed to enhance the in vitro inhibitory capacity of 10Panx1.

Functional DNA Superstructures Exhibit Positive Homotropic Allostery in Ligand Binding

AbstractInspired by intrinsically disordered proteins in nature, DNA aptamers can be engineered to display strongly homotropic allosteric (or cooperative) ligand binding, representing a unique feature that could be of great utility in applications such as biosensing, imaging and drug delivery. The use of an intrinsic disorder mechanism, however, comes with an inherent drawback of significantly reduced overall binding affinity. We hypothesize that it could be addressed via the design of multivalent supramolecular aptamers. We built functional DNA superstructures (denoted as 3D DNA), made of long‐chain DNA containing tandem repeating DNA aptamers (or concatemeric aptamers). The 3D DNA systems exhibit highly cooperative binding to both small molecules and proteins, without loss of binding affinities of their parent aptamers. We further produced a highly responsive sensor for fluorescence imaging of glutamate stimulation‐evoked adenosine triphosphate (ATP) release in neurons, as well as force stimulus‐triggered ATP release in astrocytes.

Functional DNA Superstructures Exhibit Positive Homotropic Allostery in Ligand Binding.

Inspired by intrinsically disordered proteins in nature, DNA aptamers can be engineered to display strongly homotropic allosteric (or cooperative) ligand binding, represents a unique feature that could be of great utility in applications such as biosensing, imaging and drug delivery. The use of intrinsic disorder mechanism, however, comes with an inherent drawback: the overall binding affinity is significantly reduced. We hypothesize that this issue could be addressed via the design of multivalent supramolecular aptamers. To confirm this idea, we engineered a DNA aptameric assembly, which we denote "functional DNA superstructures" (3D DNA). This functional 3D DNA is made of long chain DNA containing tandem repeating DNA aptamers (or concatemeric aptamers). We demonstrate that functional 3D DNA systems exhibit highly cooperative binding to both small molecules and proteins, without loss of binding affinities of their parent aptamers. We further use this to produce a highly responsive sensor for fluorescence imaging of glutamate stimulation-evoked adenosine triphosphate (ATP) release in neurons, as well as force stimulus-triggered ATP release in astrocytes.

The anti-platelet activity of panaxadiol fraction and panaxatriol fraction of Korean Red Ginseng in vitro and ex vivo

BackgroundThe anti-platelet activity of the saponin fraction of Korean Red Ginseng has been widely studied. The saponin fraction consists of the panaxadiol fraction (PDF) and panaxatriol fraction (PTF); however, their anti-platelet activity is yet to be compared. Our study aimed to investigate the potency of anti-platelet activity of PDF and PTF and to elucidate how well they retain their anti-platelet activity via different administration routes. MethodsFor ex vivo studies, Sprague-Dawley rats were orally administered 250 mg/kg PDF and PTF for 7 consecutive days before blood collection via cardiac puncture. Platelet aggregation was conducted after isolation of the washed platelets. For in vitro studies, washed platelets were obtained from Sprague-Dawley rats. Collagen and adenosine diphosphate (ADP) were used to induce platelet aggregation. Collagen was used as an agonist for assaying adenosine triphosphate release, thromboxane B2, serotonin, cyclic adenosine monophosphate, and cyclic guanosine monophosphate (cGMP) release. ResultsWhen treated ex vivo, PDF not only inhibited ADP and collagen-induced platelet aggregation, but also upregulated cGMP levels and reduced platelet adhesion to fibronectin. Furthermore, it also inhibited Akt phosphorylation induced by collagen treatment. Panaxadiol fraction did not exert any anti-platelet activity in vitro, whereas PTF exhibited potent anti-platelet activity, inhibiting ADP, collagen, and thrombin-induced platelet aggregation, but significantly elevated levels of cGMP. ConclusionOur study showed that in vitro and ex vivo PDF and PTF treatments exhibited different potency levels, indicating possible metabolic conversions of ginsenosides, which altered the content of ginsenosides capable of preventing platelet aggregation.

Role of GPR56 in Platelet Activation and Arterial Thrombosis.

The adhesion G protein-coupled receptor GPR56 mediates cell-cell and cell-extracellular matrix interactions. To examine the function of GPR56 in platelet activation and arterial thrombosis, we generated GPR56-knockout mice and evaluated GPR56 expression in human and mouse platelets. The results revealed that the levels of the GPR56 N-terminal fragment were significantly higher on the first day after myocardial infarction than on the seventh day in the plasma of patients with ST-segment-elevation myocardial infarction. Next, we investigated the effects of GPR56 on platelet function in vitro and in vivo. We observed that collagen-induced aggregation and adenosine triphosphate release were reduced in Gpr56 -/- platelets. Furthermore, P-selectin expression on the Gpr56 -/- platelet surface was also reduced, and the spreading area on immobilized collagen was decreased in Gpr56 -/- platelets. Furthermore, collagen-induced platelet activation in human platelets was inhibited by an anti-GPR56 antibody. Gpr56 -/- mice showed an extended time to the first occlusion in models with cremaster arteriole laser injury and FeCl3-induced carotid artery injury. GPR56 activated the G protein 13 signaling pathway following collagen stimulation, which promoted platelet adhesion and thrombus formation at the site of vascular injury. Thus, our study confirmed that GPR56 regulated the formation of arterial thrombosis. Inhibition of the initial response of GPR56 to collagen could significantly inhibit platelet activation and thrombus formation. Our results provide new insights for research into antiplatelet drugs.

Microglial VDAC1 signaling modulates sleep deprivation-induced transition to chronic postsurgical pain.

This study verified that sleep deprivation before and after skin/muscle incision and retraction (SMIR) surgery increased the risk of chronic pain and investigated the underlying roles of microglial VDAC1 signaling. Adult mice received six hours of total sleep deprivation from one day prior to SMIR until the third days after surgery. Mechanical and heat-evoked pain was assessed before and within 21 days after surgery. Microglial activation and changes of VDAC1 expression and oligomerization were measured. Minocycline was injected to observe the effects of inhibiting microglial activation on pain maintenance. The VDAC1 inhibitor DIDS and oligomerization inhibitor VBIT-4 were used to determine the roles of VDAC1 signaling on microglial ATP release, inflammation (IL-1β and CCL2), and chronicity of pain. Sleep deprivation significantly increased the pain duration after SMIR surgery, activated microglia and enhanced VDAC1 signaling in the spinal cord. Minocycline inhibited microglial activation and alleviated sleep deprivation-induced pain maintenance. Lipopolysaccharide (LPS)-induced microglial activation was accompanied by increased VDAC1 expression and oligomerization, and more VDAC1 was observed on the cell membrane surface compared with control. DIDS and VBIT-4 rescued LPS-induced microglial ATP release and IL-1β and CCL2 expression. DIDS and VBIT-4 reversed sleep loss-induced microglial activation and pain chronicity in mice, similar to the effects of minocycline. No synergistic effects were found for minocycline plus VBIT-4 or DIDS. Perioperative sleep deprivation activated spinal microglia and increases the risk of chronic postsurgical pain in mice. VDAC1 signaling regulates microglial activation-related ATP release, inflammation, and chronicity of pain.

Connexin 43: An Interface Connecting Neuroinflammation to Depression

Major depressive disorder (MDD) is a leading chronic mental illness worldwide, characterized by anhedonia, pessimism and even suicidal thoughts. Connexin 43 (Cx43), mainly distributed in astrocytes of the brain, is by far the most widely and ubiquitously expressed connexin in almost all vital organs. Cx43 forms gap junction channels in the brain, which mediate energy exchange and effectively maintain physiological homeostasis. Increasing evidence suggests the crucial role of Cx43 in the pathogenesis of MDD. Neuroinflammation is one of the most common pathological features of the central nervous system dysfunctions. Inflammatory factors are abnormally elevated in patients with depression and are closely related to nearly all links of depression. After activating the inflammatory pathway in the brain, the release and uptake of glutamate and adenosine triphosphate, through Cx43 in the synaptic cleft, would be affected. In this review, we have summarized the association between Cx43 and neuroinflammation, the cornerstones linking inflammation and depression, and Cx43 abnormalities in depression. We also discuss the significant association of Cx43 in inflammation and depression, which will help to explore new antidepressant drug targets.