Anti-inflammatory activity of Jatropha curcas L. in brain glial cells primary cultures.

ETHNOPHARMACOLOGICAL RELEVANCE: Jatropha curcas L. (Euphorbiaceae), a medicinal plant known in Brazil as "Pinhão Manso", is highly adaptable, being cultivated in different tropical and subtropical regions of the world. Antimicrobial, antioxidant and antiinflammatory activities have been attributed to different parts of the plant. In the central nervous sytem (CNS), neuroinflammation is mediated by glial cells, mainly by astrocytes and microglia, a process that plays an important role in neurodegenerative diseases and other CNS disorders. In this study, we investigated the anti-inflammatory activity of the methanolic extract obtained from the leaves of J. curcas L. (MEJc) in primary cultures of glial cells submited to inflammatory stimulus. MATERIALS AND METHODS: Primary cultures of glial cells obtained from the cerebral cortex of neonate Wistar rats were treated with MEJc (0.1-50,000 µg mL-1) and its fractions (FnJc) (0.1 µg mL-1) with or without lipopolysaccharide of Escherichia coli (LPS) (1 µg mL-1). Cell viability was determined with MTT test. Modifications in glial cell morphology were investigated by means of phase contrast microscopy and May-Grünwald staining. The reactivity of astrocytes and microglia were investigated with immunocytochemistry for GFAP, Iba1 and transcription factor NF-kB, as well as with Greiss reaction to determine the nitric oxide (NO) production. RESULTS: MEJc at 0.1-1000 µg mL-1 was non-toxic to glial cells and the DE50 was 10.794 µg mL-1. The treatment with LPS induced the activation of astrocytes and microglia marked by morphological modifications and changes in the expression of GFAP and Iba1, as well as the increase in NF-kB expression and NO production. Treatment with MEJc inhibited the morphological modifications, changes in GFAP and Iba1 expression, and the increase in NF-kB and NO production induced by LPS. CONCLUSION: This study demonstrates that the MEJc and its fractions modulate inflammatory response of astrocytes and microglia to LPS and may be considered as a potential therapeutic strategy for neuroinflammation-related diseases.

GANT61 Reduces Hedgehog Molecule (GLI1) Expression and Promotes Apoptosis in Metastatic Oral Squamous Cell Carcinoma Cells.

Due to its importance in the pathogenesis of oral squamous cell carcinoma (OSCC), the Hedgehog (HH) pathway is considered a potential therapeutic target. We investigated the effects of GANT61, a GLI inhibitor, on HH gene expression, as well as on metastatic OSCC cell proliferation and death. Following culture in DMEM medium, cytotoxicity of GANT61 against different tumor and non-tumor cell types was assessed by alamarBlue assays. Cytotoxicity analysis revealed that the metastatic HSC3 cell line was the most sensitive (IC50: 36 µM) to the tested compound. The compound's effects on the expression of HH pathways components were analyzed by qPCR and Western blot; cell viability was analyzed by trypan blue assay and flow cytometry were used to investigate cell cycle phase, morphology, and death patterns in HSC3 cells. A significant reduction in mRNA levels of the GLI1 transcription factor was found after 12 h of treatment withGANT61. Protein expression levels of other HH pathway components (PTCH1, SHH, and Gli1) and HSC3 cell viability also decreased after 24 h of treatment. Cell cycle analysis and death pattern evaluations revealed significantly increased nuclear fragmentation in sub-G1 phase, as well as cell death due to apoptosis. In conclusion, the significantly reduced GLI1 gene expression seen in response to the GLI inhibitor indicates diminished downstream activation in HH pathway components. GANT61 significantly reduced cell viability in the metastatic cell line of OSCC and promoted a significant increase in nuclear fragmentation and cell death by apoptosis.

The flavonoid rutin and its aglycone quercetin modulate the microglia inflammatory profile improving antiglioma activity.

Microglia cells are the immune effector in the Central Nervous System (CNS). However, studies have showed that they contribute more to glioma progression than to its elimination. Rutin and its aglycone quercetin are flavonoids present in many fruits as well as plants and have been demonstrated to bear anti-inflammatory, antioxidant and antitumor properties also to human glioblastoma cell lines. Previous studies also demonstrated that rutin, isolated from the Brazilian plant Dimorphandra mollis Bent., presents immunomodulatory effect on astrocytes and microglia. In this study, we investigate the antitumor and immunomodulatory properties of rutin and its aglycone quercetin on the viability of glioma cells alone and under direct and indirect interaction with microglia. Flavonoid treatment of rat C6 glioma cells induced inhibition of proliferation and migration, and also induced microglia chemotaxis that was associated to the up regulation of tumor necrosis factor (TNF) and the down regulation of Interleukin 10 (IL-10) at protein and mRNA expression levels, regulation of mRNA expression for chemokines CCL2, CCL5 and CX3CL1, and Heparin Binding Growth Factor (HDGF), Insulin-like growth factor (IGF) and Glial cell-derived neurotrophic factor (GDNF) growth factors. Treatment of human U251 and TG1 glioblastoma cells with both flavonoids also modulated negatively the expression of mRNA for IL-6 and IL-10 and positively the expression of mRNA for TNF characterizing changes to the immune regulatory profile. Treatment of microglia and C6 cells either in co-cultures or during indirect interaction, via conditioned media from glioma cells treated with flavonoids or via conditioned media from microglia treated with flavonoids reduced proliferation and migration of glioma cells. It also directed microglia towards an inflammatory profile with increased expression of mRNA for IL-1ß, IL-6, IL-18 and decreased expression of mRNA for nitric oxide synthase 2 (NOS2) and prostaglandin-endoperoxide synthase 2 (PTGS2), arginase and transforming growth factor beta (TGF-ß), as well as Insulin-like growth factor (IGF). Treatment of U251 cells with flavonoids also reduced tumorigenesis when the cells were xenotransplanted in rat brains, and directed microglia and also astrocytes in the microenvironment of tumor cell implantation as well as in the brain parenchyma to a not favorable molecular inflammatory profile to the glioma growth, as observed in cultures. Together these results demonstrate that the flavonoid rutin and its aglycone quercetin present antiglioma effects related to the property of modulating the microglial inflammatory profile and may be considered for molecular and preclinical studies as adjuvant molecules for treatment of gliomas.

The flavonoid rutin modulates microglial/macrophage activation to a CD150/CD206 M2 phenotype.

Rutin is a glycosylated flavonoid present in many fruits and plants that has been demonstrated to have anti-inflammatory and antioxidant properties. However, little is known about the mechanisms underlying microglial activation and its effects on the regulation of cytokines and chemokines associated with inflammatory responses in the central nervous system. In this study we examined the effect of rutin on resting or lipopolysaccharide (LPS)-stimulated microglia and characterized their modulation to an activated M1 phenotype or an alternatively activated M2 phenotype. Microglial cells were treated with rutin (1-100 µM); alternatively, microglial cells were stimulated with LPS and the cells were then treated with rutin (50 µM). The results revealed that rutin treatment was not toxic to microglial cells and induced a dose-dependent increase in microglial proliferation associated with changes in morphology after 24 h of treatment. Rutin also induced microglial activation characterized by an increase in OX-42 positive cells and a large proportion of cells with a CD150/CD206-positive M2 phenotype. Rutin also induced a decrease in the mRNA levels of TNF, IL1ß, IL6 and iNOS, reduced the production of IL6, TNF, and nitric oxide, and increased production of the M2 regulatory cytokine IL10 and arginase. Rutin also significantly inhibited the LPS-induced expression of PTGS2, IL18 and TGFß mRNA. These findings show that rutin has the ability to promote microglial proliferation and induces microglial polarization to the M2 profile when cells are stimulated with LPS. These results point this flavonoid as a possible alternative in the treatment or prevention of neurodegenerative disorders.