Antineuroinflammatory Effect of Amburana cearensis and Its Molecules Coumarin and Amburoside A by Inhibiting the MAPK Signaling Pathway in LPS-Activated BV-2 Microglial Cells.

Microglia plays an important role in the neuroinflammatory response, identified as one of the major factors in the development and progression of neurodegenerative diseases. Amburana cearensis and its bioactive compounds, including coumarin (CM), vanillic acid (VA), and amburoside A (AMB), exert antioxidant, anti-inflammatory, and neuroprotective activities, on 6-OHDA-induced neurotoxicity in rat mesencephalic cells determined by our group. The present study investigated the anti-inflammatory effect of the dry extract from A. cearensis (DEAC), CM, AMB, and VA on lipopolysaccharide- (LPS-) stimulated microglial cells and elucidated the possible molecular mechanism of action. The DEAC was characterized by HPLC-PDA (chemical markers: CM, AMB, and VA). The BV-2 microglial cell line was pretreated with increasing concentrations of DEAC, CM, AMB, or VA in the presence or absence of LPS to evaluate the toxicity and anti-inflammatory activity. The cytotoxicity of DEAC, CM, AMB, or VA on BV-2 cells was evaluated by the MTT test, the free radical scavenging activity of test drugs was investigated, and the nitric oxide (NO) production was determined using the Griess reagent, while cytokine levels were measured by ELISA. The expressions of toll-like receptor 4 (TLR-4), nuclear factor kappa B (NF-κB), MAPK members (JNK and ERK1/2), and iNOS were determined through Western blot analysis. DEAC, CM, AMB, or VA (5-100 µg/mL) did not induce any detectable cytotoxicity in BV-2 cells. All test drugs (100 µg/mL) showed free radical scavenging activity (hydroxyl and superoxide radicals); however, only DEAC, CM, and AMB (5-100 µg/mL) significantly reduced NO production. DEAC (100 µg/mL), as well as CM (50 and 100 µg/mL) and AMB (25 µg/mL), reduced at least 50% of NO produced and markedly decrease the production of TNF-α and IL-6 but they did not significantly affect IL-10 levels. Only DEAC (100 µg/mL) and AMB (25 µg/mL) reduced the expression of iNOS, and they did not affect arginase activity. DEAC (100 µg/mL) suppressed the activation of the MAPKs JNK and ERK1/2 in LPS-activated BV-2 cells but it did not suppress the expression of TLR-4 nor the phosphorylation of NF-κB. In conclusion, DEAC, CM, and AMB exerted anti-inflammatory activity in LPS-activated microglial cells as observed by the reduction in the production of inflammatory mediators and the expression of iNOS. We identified the MAPK signaling pathway as a probable mechanism of action to the anti-inflammatory effects observed.

Uncaria tomentosa reduces osteoclastic bone loss in vivo.

BACKGROUND: The genus Uncaria (Rubiaceae) has several biological properties significant to human health. However, the mechanisms underlying the protective effect of this plant on bone diseases are uncertain. PURPOSE: The present study investigated the role of Uncaria tomentosa extract (UTE) on alveolar bone loss in rats and on osteoclastogenesis in vitro. MATERIALS: UTE was characterized by an Acquity UPLC (Waters) system, coupled to an Electrospray Ionization (ESI) interface and Quadrupole/Flight Time (QTOF, Waters) Mass Spectrometry system (MS). The effect of UTE treatment for 11 days on the ligature-induced bone loss was assessed focusing on several aspects: macroscopic and histological analysis of bone loss, neutrophil and osteoclast infiltration, and anabolic effect. The effect of UTE on bone marrow cell differentiation to osteoclasts was assessed in vitro. RESULTS: The analysis of UTE by UPLC-ESI-QTOF-MS/MS identified 24 compounds, among pentacyclic or tetracyclic oxindole alkaloids and phenols. The administration of UTE for 11 days on ligature-induced rat attenuated the periodontal attachment loss and alveolar bone resorption. It also diminished neutrophil migration to the gingiva tissue, demonstrated by a lower level of MPO. UTE treatment also decreased the level of RANKL/OPG ratio, the main osteoclast differentiation-related genes, followed by reduced TRAP-positive cell number lining the alveolar bone. Additionally, the level of bone-specific alkaline phosphatase, an anabolic bone marker, was elevated in the plasma of UTE treated rats. Next, we determined a possible direct effect of UTE on osteoclast differentiation in vitro. The incubation of primary osteoclast with UTE decreased RANKL-induced osteoclast differentiation without affecting cell viability. This effect was supported by downregulation of the nuclear factor activated T-cells, cytoplasmic 1 expression, a master regulator of osteoclast differentiation, and other osteoclast-specific activity markers, such as cathepsin K and TRAP. CONCLUSION: UTE exhibited an effective anti-resorptive and anabolic effects, which highlight it as a potential natural product for the treatment of certain osteolytic diseases, such as periodontitis.