Efficacy of malachite green mediated photodynamic therapy on treatment of Cutaneous Leishmaniasis: In vitro study.

BACKGROUND: Leishmaniasis is a common zoonotic disease that is transmitted by phlebotomus and causes several clinical conditions, from self healing lesion to deadly internal organ involvement. Photodynamic therapy (PDT) is a treatment method that leads to the generation of cytotoxic species and consequently to cell death and tissue destruction by visible light in the presence of a photosensitizer and oxygen. The aim of this study was to investigate effect of malachite green (MG)-mediated PDT in Leishmania tropica (L. tropica) promastigotes. MATERIAL AND METHODS: Parasites were incubated with 0.19, 0.39, 1.56, 3.25 and 6.25 µM of MG for one hour and subjected to 46.4 J/cm2 light irradiation. Trypan blue assay was used to evaluate the viability of the cells and mitochondirial activity alteration was determined by MTT. Morphological changes were analyzed by Giemsa staining and Scanning electron microscopy (SEM) analyses. Flow cytometry was used to quantify the fluorescence emitted by cell volume, JC-1, Cell Cycle and Annexin V/PI staining reagents. RESULTS: Malachite green mediated photodynamic therapy at 1.56 and 3.125 µM decreased the viability of the L. tropica promastigotes and induced changes in the mitochondrial membrane potential. L.tropica promastigotes was bloked in G0/G1 phase. The morphology of the parasite was affected at the 1.56 and 3.125 µM MG+PDT, resulting in rounded cells with loss of flagellum and irregular shape. CONCLUSIONS: This study demonstrated that antileishmanial effects through mitochondrial dysfunction, cell cycle arrest, and apoptosis-like cell death to parasites. This work showed PDT with MG effectedparasites. Therefore, MG-mediated PDT may provide a promising approach for L. tropica promastigotes.

Study of the correlation between the noncanonical pathway of pyroptosis and idiopathic inflammatory myopathy.

BACKGROUND: The pathogenesis of idiopathic inflammatory myopathy (IIM) is complex and unclear. The purpose of this study was to investigate whether the noncanonical pathway of pyroptosis is involved in the pathogenesis of IIM, and the intervention effect of drugs glyburide and bright blue G (BBG). METHODS: After the drug intervention, we detected the expression of the caspase-4, caspase-5, caspase-11, GSDMD, pannexin-1, NLRP3 and P2X7R proteins in skeletal muscle tissues from the six groups using Western blotting. We detected the expression of the caspase-11, GSDMD, pannexin-1, NLRP3 and P2X7R mRNAs in skeletal muscle tissues from the six groups using RT-qPCR and detected the serum IL-18 and IL-1ß levels in the six groups using ELISAs. RESULT: Lower expression levels of the P2X7R and NLRP3 proteins were observed in the EAM + BBG group than in the EAM1 group (P < 0.05). The expression of NLRP3 in the EAM + glyburide group was lower than in the EAM2 group (P < 0.05). Lower expression levels of the P2X7R and NLRP3 mRNAs were detected in the EAM + BBG group than in the EAM1 group (P < 0.05). NLRP3 was expressed at lower levels in the EAM + glyburide group than in the EAM2 group (P < 0.05). Lower serum IL-1ß levels were detected in the EAM + BBG group than in the EAM1 group (P < 0.05), and serum IL-1ß and IL-18 levels in the EAM + glyburide group were lower than those in the EAM2 group (P < 0.05). CONCLUSION: Our results suggest that the noncanonical pathway of pyroptosis may be involved in the pathogenesis of IIM, and glyburide and BBG exert certain intervention effects on its pathogenesis.

Comparative proteomic analysis provides insight into the key proteins as potential targets underlying the effect of malachite green against Ichthyophthirius multifiliis.

Target identification is important for drug discovery. Unfortunately, no drug targets have been found in Ichthyophthirius multifiliis until now and further limited development of the novel drug for Ichthyophthiriasis. In this study, an iTRAQ-based quantitative proteomic analysis was used to find the target of malachite green (MG), exhibiting greater efficacy than the existing drugs, against I. multifiliis trophonts in situ. We also verified the proteomic results by RT-qPCR, TEM and cell apoptosis assay. Our results showed that major variations in protein abundance were found among many of the ribosome proteins, indicating ribosome might be a candidate target. Furthermore, GO and KEGG pathway analyses of differentially expressed proteins (DEPs) revealed that ribosome and PI3K-Akt signalling pathway were remarkably enriched. Taken together, the above DEPs were also verified by RT-qPCR and morphological observations. This study provides insights into the key proteins enriched in PI3K-Akt signal pathway and ribosome pathway as potential targets of MG killing I. multifiliis, which could be served as targets for other less toxic drugs and be tested as potential treatments for I. multifiliis.

Combination therapy with dipeptidyl peptidase-4 and P2X7 purinoceptor inhibitors gives rise to antiepileptic effects in rats.

OBJECTIVE(S): Although the available therapeutic agents alleviate the symptoms in patients with temporal lobe epilepsy (TLE), these antiepileptic drugs do not provide adequate control of seizures in 30-40 % of patients. This study was conducted to evaluate anti-epileptic effects of simultaneous inhibition of dipeptidyl peptidase-4 and P2 × 7 purinoceptors in Kainate treated rats. MATERIALS AND METHODS: Brilliant Blue G)BBG(, linagliptin)lin(and lin + BBG were administrated 30 min prior to induction of the intrahippocampal kainate model of epilepsy in male Wistar rats. In the case of valproic acid group, the animals intraperitoneally received valproic acid for 7 consecutive days prior to induction of the model. We carried out histological evaluations, monitoring of behavior, recording of intracranial electroencepholography (IEEG), and determination of astrogliosis and DNA fragmentation using ELISA methods. RESULTS: Our results showed that BBG and lin combination therapy had better effects on decrease in astrogliosis, DNA fragmentation and cognitive disturbances than ones whereas its effects on neuronal survival and seizure severity was similar to only BBG or lin. Likewise, the effects of lin + BBG on decrease in DNA fragmentation and cognitive disturbances were better than valproic acid group. CONCLUSION: Our findings suggest that simultaneous inhibition of dipeptidyl peptidase-4 and P2 × 7 purinoceptors might more efficiently provide protection against progression of the kainate-induced TLE in rats.

Brilliant blue G, a P2X7 receptor antagonist, attenuates early phase of renal inflammation, interstitial fibrosis and is associated with renal cell proliferation in ureteral obstruction in rats.

BACKGROUND: Previous study showed that purinergic P2X7 receptors (P2X7R) reach the highest expression in the first week after unilateral ureteral obstruction (UUO) in mice, and are involved in the process of inflammation, apoptosis and fibrosis of renal tissue. We, herein, document the role of purinergic P2X7 receptors activation on the third day of UUO, as assessed by means of BBG as its selective inhibitor. METHODS: We investigated the effects of brilliant blue G (BBG), a P2X7R antagonist, in the third day of kidney tissue response to UUO in rats. For this purpose, male Wistar rats submitted to UUO or sham operated, received BBG or vehicle (V), comprising four groups: UUO-BBG, UUO-V, sham-BBG and sham-V. The kidneys were harvested on day 3 UUO and prepared for histology, immunohistochemistry (P2X7R, PCNA, CD-68, α-sma, TGF-ß1, Heat-shock protein-47, TUNEL assay), quantitative real-time PCR (IL-1ß, procollagens type I, III, and IV) for mRNA quantification. RESULTS: The group UUO-V presented an enhancement in tubular cell P2X7-R expression, increase influx of macrophages and myofibroblasts, HSP-47 and TGF- ß1 expression. Also, upregulation of procollagen types I, III, and IV, and IL-1ß mRNAs were seen. On the other hand, group UUO-BBG showed lower expression of procollagens and IL-1ß mRNAs, as well as less immunoreactivity of HSP-47, TGF-ß, macrophages, myofibroblasts, and tubular apoptosis. This group also presented increased epithelial cell proliferation. CONCLUSION: BBG, a known highly selective inhibitor of P2X7R, attenuated renal inflammation, collagen synthesis, renal cell apoptosis, and enhanced renal cell proliferation in the early phase of rat model of UUO.

Preoperative computerized tomography-guided blue dye localization for metastatic lymphadenopathy and peritoneal tumor implants during laparoscopic surgery: Two case reports.

RATIONALE: Computerized tomography (CT)-guided blue dye localization has been widely discussed for preoperative localization of pulmonary nodules. However, few studies have investigated this technique for intra-abdominal lesions. Although preoperative localization is not commonly required in laparotomy, it may assume importance with advancements in the field of laparoscopic surgery. PATIENT CONCERNS: Herein, we report the cases of 2 patients diagnosed with colon cancer who underwent hemicolectomy with extended lymphadenectomy and subsequent chemotherapy. DIAGNOSES: Follow-up CT scans showed newly developed metastatic lymphadenopathy and peritoneal tumor implants. INTERVENTIONS: Considering the difficulty in identification of and access to the target lesions during laparoscopic surgery, preoperative CT-guided blue dye localization was performed in both cases. OUTCOMES: All the target lesions were identified by the dye marker and removed successfully. The pathologic results revealed adenocarcinoma. LESSONS: We established the following strategy for preoperative CT-guided dye localization of intra-abdominal lesions:Intra-abdominal lesions that are hard to identify due to their size or morphology, and difficult to approach due to their location or surrounding structures, maybe the candidates for this procedure, especially in cases of laparoscopic surgery.Operators should adjust their localization planning based on the surgery method, cutting path, and location of port sites. The target dye marker should be clearly visible in the presumed intra-operative field of view.A second dye marker should be made to ensure surgical success when the target dye marker is obscured by the surrounding structures in the presumed intra-operative field of view.

MCCK1 enhances the anticancer effect of temozolomide in attenuating the invasion, migration and epithelial-mesenchymal transition of glioblastoma cells in vitro and in vivo.

Chemotherapy with temozolomide (TMZ) is the traditional treatment for glioblastoma (GBM). Nevertheless, majority of GBM patients have recurrence from resistance to the chemotherapy. Herein, we examined combinational effects of MCCK1 (a specific and effective IKKε inhibitor) with TMZ in GBM U251MG and U-87MG cell lines as well as U251MG xenograft models to overcome the therapeutic limitation of chemotherapy for GBM. Although MCCK1 alone showed inhibitory effects on in vitro proliferation, migration, invasion, and EMT of U251MG and U-87MG cells, combination of MCCK1 and TMZ showed enhanced inhibitory effects. In the U251MG GBM xenograft models, MCCK1 showed synergistic therapeutic effects in combination with TMZ to reduce tumor volumes significantly. These data indicated that MCCK1 could be a candidate sensitizer to potentiate therapeutic effects of conventional cytotoxic treatment for GBM.

Long-term treatment with the P2X7 receptor antagonist Brilliant Blue G reduces liver inflammation in a humanized mouse model of graft-versus-host disease.

Allogeneic haematopoietic stem cell transplantation (HSCT) is a frequent curative therapy for numerous haematological malignancies. However, HSCT is limited by the occurrence of graft-versus-host disease (GVHD), with current therapies restricted to general immunosuppression. Activation of the P2X7 receptor by extracellular adenosine triphosphate (ATP) causes inflammation and tissue damage in GVHD. Short-term pharmacological blockade of P2X7 has been shown to reduce clinical disease and/or reduce inflammatory markers in allogeneic and humanized mouse models of GVHD. The current study demonstrates that long-term P2X7 blockade by intra-peritoneal injection of Brilliant Blue G (BBG) thrice weekly for up to 10 weeks did not impact human (h) peripheral blood mononuclear cell (PBMC) engraftment, predominantly T cells, in blood at 3 weeks post-hPBMC injection or in spleens at end-point in humanized mice. Histological analysis demonstrated long-term BBG treatment reduced leukocyte infiltration in the livers of humanized mice. Immunohistochemical analysis demonstrated that BBG treatment reduced liver apoptosis. Long-term BBG treatment did not alter clinical disease, mRNA expression of pro-inflammatory markers in tissues or serum human interferon (IFN)-γ concentrations. Therefore, this study demonstrates that P2X7 activation plays a role in GVHD pathogenesis in the livers of humanized mice, supporting a role for this receptor in GVHD development in HSCT recipients.

Comparison of ion channel inhibitor combinations for limiting secondary degeneration following partial optic nerve transection.

Following neurotrauma, secondary degeneration of neurons and glia adjacent to the injury leads to further functional loss. A combination of ion channel inhibitors (lomerizine + oxATP + YM872) has been shown to be effective at limiting structural and functional loss due to secondary degeneration. Here we assess efficacy of the combination where oxATP is replaced with Brilliant Blue G (BBG), a more clinically applicable P2X7 receptor inhibitor. Partial optic nerve transection was used to model secondary degeneration in adult female rats. Animals were treated with combinations of lomerizine + YM872 + oxATP or lomerizine + YM872 + BBG, delivered via osmotic mini-pump directly to the injury site. Outcomes assessed were Iba1 + and ED1 + microglia and macrophages, oligodendroglial cell numbers, node/paranode structure and visual function using the optokinetic nystagmus test. The lomerizine + BBG + YM872 combination was at least as effective at the tested concentrations as the lomerizine + oxATP + YM872 combination at preserving node/paranode structure and visual function when delivered locally. However, neither ion channel inhibitor combination significantly improved microglial/macrophage nor oligodendroglial numbers compared to vehicle-treated controls. In conclusion, a locally delivered combination of ion channel inhibitors incorporating lomerizine + BBG + YM872 is at least as effective at limiting secondary degeneration following partial injury to the optic nerve as the combination incorporating oxATP.

Purinergic P2X7 receptor blockade mitigates alcohol-induced steatohepatitis and intestinal injury by regulating MEK1/2-ERK1/2 signaling and egr-1 activity.

The P2X7 receptor is an ATP-binding cation channel involved in a broad range of inflammatory diseases. However, little is known about the potential role of P2X7R in alcohol-induced steatohepatitis and intestinal injury. In our study, C57BL/6 mice were intraperitoneally injected with P2X7R antagonists Brilliant Blue G and A438079 from the 4th day to the 10th day during the induction of chronic plus binge alcohol feeding model. Our results showed that alcohol feeding induced significant steatohepatitis and liver injury, which were mitigated by P2X7R blockade as evidenced by decreased serum levels of ALT, AST, T-CHO and TG, reduced lipid accumulation, and less inflammation. The increased intestinal inflammatory cytokines production and the prominent intestinal barrier disruption caused by alcohol were also modulated by P2X7R antagonism. Interestingly, alcohol feeding increased the relative abundance of phylum Bacteroidetes while decreased the number of phylum Verrucomicrobia and genus Akkermansia in the cecal content, which were reversed by P2X7R antagonist. Importantly, the improvement of intestinal barrier function and the restoration of partial taxonomic alterations in the gut microbiota might contribute to protect the liver from gut microbiota dysbiosis-induced second hit. Furthermore, P2X7R blockade inhibited MEK1/2-ERK1/2 phosphorylation and egr-1 expression in both liver and intestine from alcohol-fed mice. Collectively, P2X7R blockade mitigates alcohol-induced steatohepatitis and intestinal injury by inhibiting MEK1/2-ERK1/2 signaling and egr-1 expression. These studies strongly suggest that P2X7R blockade may be a promising therapeutic approach for treating alcoholic liver disease.

Two decades of experience with laparoscopic varicocele repair in children: Standardizing the technique.

BACKGROUND: Controversy still exists about the indications and the gold standard approach for varicocele treatment in pediatric population. OBJECTIVE: The authors report their 23 years of experience in laparoscopic varicocele repair in the pediatric population. STUDY DESIGN: We retrospectively evaluated the data of 345 consecutive patients who underwent laparoscopic left varicocelectomy from January 1993 to December 2015. Average patient age was 12.5 years (range 8-17). Seven out of 345 patients (2%) had a recurrent varicocele, and five out of 345 patients (1.4%) had a varicocele on a single testis. In 335/345 patients (97.1%) we performed a Palomo procedure, and in 10/345 patients (2.9%) an artery-sparing Palomo procedure. After 2010, in 105/345 patients (30.4%) we performed a lymphatic sparing procedure using isosulfan blue injection preoperatively. RESULTS: All procedures were completed in laparoscopy (Figure), without conversions or intraoperative complications. The average operative time was 17 min (range 14-45) for the Palomo procedure and 26 min (range 18-50) for artery-sparing Palomo. In 45/345 patients (13%) we performed additional procedures. We recorded 4/345 (1.3%) recurrences/persistences in patients undergoing Palomo, while we recorded 1/10 (10%) recurrence/persistence after artery-sparing Palomo. On 230 Palomo procedures performed in the pre-isosulfan blue era, we recorded 25 cases of hydrocele (10.8%), 13 of these were treated with transcrotal puncture and 12 required surgical operation. The last 105 patients undergoing isosulfan blue injection had no postoperative hydrocele. We also reported 10 other complications (I grade Clavien-Dindo) such as umbilical granuloma or instrumental problems. DISCUSSION: Analyzing the international literature of the last 25 years, most papers focused on the minimally invasive treatment of pediatric varicocele. There are several reasons to perform laparoscopic repair of pediatric varicocele. First of all, it is technically easy to perform, the average operative time is very short, and it has excellent outcome in regard to varicocele persistence/recurrence. In addition it has a very low complication rate, and in particular adopting the intradartoic/intratesticular isosulfan blue injection before surgery we recorded no postoperative hydrocele. CONCLUSION: On the basis of our 23 years of experience with varicocele repair, we clearly believe that laparoscopic Palomo lymphatic sparing varicocelectomy should be considered the standard of care for the treatment of pediatric patients with varicocele. Laparoscopic varicocelectomy is technically easy and quick to perform, painless, and scarless, with a recurrence rate of about 1%. The use of a preoperative injection of isosulfan blue completely eliminates postoperative hydrocele formation.

[Effect of BBG on Acute Graft-Versus-Host Disease in Mice after Allogeneic Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To investigate the effect of P2X7R antagonist brilliant blue G (BBG) on aGVDH of mice after allo-HSCT. METHODS: aGVHD mouse model after HSCT was established and treated with the P2X7R antagonist BBG of different dosages (50 mg/kg and 75 mg/kg). After treatment, the survival, body weight, pathological and liver function of aGVDH mice were abserved, and the expression levels of P2X7, NLRP3, caspase-1, IL-1ß, IL-18 mRNA and protein were evaluated by real-time PCR and Western blot. RESULTS: The allo-HSCT aGVHD mouse model was successfully established, the intraperitoneal injection of BBG alleviated the aGVHD clinical manifestations including roachback, ruffled fur, skin peeling and weight loss of recipient mice, decreased P2X7R and IL-1ß expression and reduced the mRNA levels of P2X7R, NLRP3, Caspase-1, IL-1ß and IL-18. Furthermore, GVHD group receiving 75 mg/kg BBG showed most significant difference of these indexes. CONCLUSION: BBG alleviates liver inflammatory damage induced by aGVHD after allo-HSCT, and decreases the expression of proinflammatory cytokines. Moreover, the protective effect of that of BBG 75 mg/kg group is better than that of BBG 50 mg/kg group.

The P2X7 receptor antagonist Brilliant Blue G reduces serum human interferon-γ in a humanized mouse model of graft-versus-host disease.

Graft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.

Repression of acetaminophen-induced hepatotoxicity by a combination of celastrol and brilliant blue G.

The sterile inflammatory response is an eminent contributor to acetaminophen (APAP)-hepatotoxicity in humans. Recent advances unraveled an axial role of the NLRP3-inflammasome in APAP-post injury inflammation. Nevertheless, the role of signaling events preceded the NLRP3-inflammasome activation, like the transcription factor NF-κB and the purinergic receptor P2X7, is still unclear and needs further elucidation. Here, we investigated the pharmacological inhibition of these upstream signaling molecules by celastrol and brilliant blue G (BBG) (separately or simultaneously) in APAP-hepatotoxicity in mice. The results indicated that both celastrol and BBG pretreatments, especially when combined together, curbed APAP-induced hepatocellular injury (ALT, AST and LDH) and death (necrosis and apoptosis). The underlying mechanisms of protection of such combination against APAP-challenge were attributed to their efficient cooperation in: i) preventing the consumption of hepatic antioxidants (reduced glutathione and superoxide dismutase); ii) limiting the overproduction of lipid peroxidation aldehydes (malondialdehyde and 4-hydroxynonenal) and total nitrate/nitrite products; iii) attenuating the inflammatory cells accumulation in the liver, as evidenced by reducing the number of F4/80 positive cells/field in immunostaining and myeloperoxidase activity; iv) reversing the dysregulation in production of pro-inflammatory (TNF-α, IL-17A and IL-23) and anti-inflammatory (IL-10) cytokines; and v) enhancing the reparative capacity of injured hepatocytes, as demonstrated by increasing the percentage of PCNA positive hepatocytes per field of immunostaining. In conclusion, this murine study elicits a potential clinical applicability and therapeutic utility of celastrol and BBG combination in human cases of APAP-overdose hepatotoxicity.

Brilliant Blue G, But Not Fenofibrate, Treatment Reverts Hemiparkinsonian Behavior and Restores Dopamine Levels in an Animal Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by the loss of dopaminergic neurons in the substantia nigra and their projections to the striatum. Several processes have been described as potential inducers of the dopaminergic neuron death, such as inflammation, oxidative stress, and mitochondrial dysfunction. However, the death of dopaminergic neurons seems to be multifactorial, and its cause remains unclear. ATP-activating purinergic receptors influence various physiological functions in the CNS, including neurotransmission. Purinergic signaling is also involved in pathological scenarios, where ATP is extensively released and promotes sustained purinergic P2X7 receptor (P2X7R) activation and consequent induction of cell death. This effect occurs, among other factors, by oxidative stress and during the inflammatory response. On the other hand, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is involved in energy metabolism and mitochondrial biogenesis. Expression and activity upregulation of this protein has been related with reduction of oxidative stress and neuroprotection. Therefore, P2X7R and PGC-1α are potential targets in the treatment of PD. Here hemiparkinsonism was induced by unilateral stereotactic injection of 6-OHDA in a rat model. After 7 days, the establishment of PD was confirmed and followed by treatment with the P2X7R antagonist Brilliant Blue G (BBG) or PGC-1α agonist fenofibrate. BBG, but not fenofibrate, reverted hemiparkinsonian behavior accompanied by an increase in tyrosine hydroxylase immunoreactivity in the substantia nigra. Our results suggest that the P2X7R may be a therapeutic target in Parkinson's disease.

Radiosensitizing Effect of P2X7 Receptor Antagonist on Melanoma in Vitro and in Vivo.

Melanoma is highly malignant, and generally exhibits radioresistance, responding poorly to radiation therapy. We previously reported that activation of P2X7, P2Y6, and P2Y12 receptors is involved in the DNA damage response after γ-irradiation of human lung adenocarcinoma A549 cells. However, it is not clear whether these receptors are also involved in the case of melanoma cells, although P2X7 receptor is highly expressed in various cancers, including melanoma. Here, we show that P2X7 receptor antagonist enhances radiation-induced cytotoxicity in B16 melanoma cells in vitro and in vivo. We confirmed that these cells express P2X7 receptor mRNA and exhibit P2X7 receptor-mediated activities, such as ATP-induced pore formation and cytotoxicity. We further examined the radiosensitizing effect of P2X7 receptor antagonist Brilliant Blue G (BBG) in vitro by colony formation assay of B16 cells. γ-Irradiation dose-dependently reduced cell survival, and pretreatment with BBG enhanced the radiation-induced cytotoxicity. BBG pretreatment also decreased the number of DNA repair foci in nuclei, supporting involvement of P2X7 receptor in the DNA damage response. Finally, we investigated the radiosensitizing effect of BBG on B16 melanoma cells inoculated into the hind footpad of C57BL/6 mice. Neither 1 Gy γ-irradiation alone nor BBG alone suppressed the increase of tumor volume, but the combination of irradiation and BBG significantly suppressed tumor growth. Our results suggest that P2X7 receptor antagonist BBG has a radiosensitizing effect in melanoma in vitro and in vivo. BBG, which is used as a food coloring agent, appears to be a promising candidate as a radiosensitizer.

Inhibiting purinergic P2X7 receptors with the antagonist brilliant blue G is neuroprotective in an intranigral lipopolysaccharide animal model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder, which is characterized by the selective and progressive death of dopaminergic (DA) neurons in the substantia nigra. Increasing evidence suggests that inflammation is important in the degeneration of DA neurons. The purinergic receptor subtype P2X7 receptor (P2X7R) is key in the activation and proliferation of microglia. The present study aimed to examine whether inhibiting purinergic P2X7 receptors is neuroprotective in a rat model of PD, specifically via inhibiting p38 mitogen­activated protein kinase (MAPK). In an intranigral lipopolysaccharide (LPS) rat model of PD, immunohistochemical analysis revealed enhanced expression of P2X7R was observed in microglia. The administration of the P2X7R antagonist, brilliant blue G (BBG), reduced activation of the microglia and the loss of nigral DA neurons. In addition, immunohistochemistry and western blot analysis revealed the phosphorylation level of p38 MAPK increased in the microglia of the LPS­injected rats, which was inhibited by BBG treatment. The p38 MAPK inhibitor, SB203580, reduced microglial activation and the loss of DA neurons. Thus, these findings suggested that inhibition of P2X7R by BBG attenuated microglial activation and the loss of substantia nigra DA neurons via p38 MAPK in the rat LPS model of PD.

Early P2X7R-related astrogliosis in autoimmune encephalomyelitis.

Astrocytes are the main cells responsible for maintenance of brain homeostasis. Undisturbed action and signaling with other cells are crucial for proper functioning of the central nervous system (CNS). Dysfunctional astrocytes may determine the degree of neuronal injury and are associated with several brain pathologies, among which are multiple sclerosis (MS) and the animal model of this disease which is known as experimental autoimmune encephalomyelitis (EAE). One of the many functions of astrocytes is their response to CNS damage when they undergo reactive gliosis. Our data reveal that activation of astrocytes occurs in forebrains of immunized rats at a very early stage of EAE, well before the symptomatic phase of the disease. We have noted enhanced expression of GFAP and S100ß starting from day 4 post-immunization. Temporal coincidence between the expression of astrocyte activation markers and the expression of connexin 43 and purinergic P2X7 receptor (P2X7R) was also observed. Administration of Brilliant blue G, an antagonist of P2X7R, significantly decreases astrogliosis as confirmed by immunohistochemical analysis and observation of decreased levels of GFAP and S100ß. The condition of the treated animals was improved and the neurological symptoms of the disease were alleviated. With the knowledge that cerebral astroglia represent the main source of ATP and glutamate which are potentially neurotoxic substances released through P2X7R and connexin hemichannels, we suggest that astroglia may be involved in pathogenesis of MS/EAE at a very early stage through the purinergic/glutamatergic mechanisms.