METHODS: The mammalian expression plasmid pCMV-p16INK4a was used to induce p16INK4a overexpression in THP-1 macrophages. Next, wild-type and p16INK4a-overexpressed macrophages were incubated with oxidized LDL to induce foam cells formation. Lipids accumulation was evaluated using Oil-red-O staining and cholesterol efflux assay, as well as expression of scavenger receptors CD36 and LOX-1. Cellular senescence in macrophage foam cells were determined through analysis of senescence-associated β-galactosidase activity and other SASP factors expression. Meanwhile, autophagy induction was assessed through detection of autophagosome formation and LC3B/p62 markers expression.
RESULTS: The findings showed that p16INK4a enhanced foam cells formation with increased scavenger receptors CD36 and LOX-1 expression and reduced cholesterol efflux in THP-1 macrophages. Besides, β-galactosidase activity was enhanced, and SASP factors such as IL-1α, TNF-α, and MMP9 were up-regulated. In addition, p16INK4a is also shown to induce autophagy, as well as increasing autophagy markers LC3B and p62 expression.
CONCLUSIONS: This study provides insights on p16INK4a in mediating macrophages foam cells formation, cellular senescence, and foam cells formation.
OBJECTIVE: This study aimed to investigate the immuno-modulatory effects of agarwood leaf extract (ALE) derived from Aquilaria malaccensis using RAW264.7 murine macrophages.
METHODS: In this study, RAW264.7 macrophages were incubated with ALE alone for 26 hours or ALE for 2 hours, followed by bacterial lipopolysaccharide for 24 hours. The nitrite and cytokine production (tumour necrosis factor-alpha (TNFα), interleukin (IL)-1β, IL-6, and IL-10), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) expression in the macrophages were assayed.
RESULTS: The study showed that ALE alone was immunostimulatory on the macrophages by increasing the nitrite, TNFα, and IL-6 production and COX2 expression (p<0.05 vs. untreated unstimulated cells). Pre-treatment of ALE suppressed nitrite level and iNOS expression but enhanced TNFα and IL-6 production and COX2 expression (p<0.05 vs. untreated lipopolysaccharides (LPS)-stimulated cells). ALE also increased IL-10 production regardless of LPS stimulation (p<0.05 vs. untreated cells).
CONCLUSION: ALE was able to promote the immune response of macrophages by upregulating pro-inflammatory cytokine levels and COX2 expression. It also regulated the extent of the inflammation by reducing iNOS expression and increasing IL-10 levels. Thus, ALE may have a role in enhancing the innate immune system against infection; however, its validation from in vivo studies is still pending.
MATERIALS AND METHODS: Root discs (2 mm thickness) were cut apical to CEJ and sectioned into quadrants. HIFU setup with bowl-shaped piezo ceramic transducer submerged in a water tank was used for exposure on each specimen for 15 s, 30 s or 60 s. The specimens of the control group were left without any HIFU exposure. HIFU was generated with a continuous sinusoidal wave of 120Vpp amplitude, 250 KHZ resonance-frequency and highest ultrasonic pressure of ∼10 bar at the focus. Specimens for SEM were viewed, and micro-topography characterization performed, using AFM and Ra parameter and surface area (SA) calculated by specialized SPM surface analysis software. For nano-indentation testing, experiments were carried out using AFM. Macrophage cell isolation and culturing was performed on cementum to receive the HIFU treatment at different time periods. Raman spectroscopy were scanned to create spectra perpendicular to the cementum substrate to analyze generation of standard spectra for Raman intensity ratio of hydroxyapatite normalized to the peaks ν1 960 cm-1. Data was expressed as means ± standard deviations and analyzed by one-way ANOVA in term of Ra, SA, H and Er. Different points for fluorescence intensity ratio were analyzed by Raman using Wilcoxon rank sum test.
RESULTS: HIFU exposure at 60 s removed the smear layer and most of cementum appeared smoothened. AFM characterisation, showed a slight decrease in the irregularity of the surface as exposure time increased. Intact macrophages can be identified in control and all experimental HIFU groups. The level of fluorescence for the control and HIFU 15 and 30 s were low as compared to HIFU 60 s.
CONCLUSION: If HIFU can be successfully implemented, it may be a possible alternative to current methods used in periodontal therapy to achieve smooth root surfaces.
AIM OF THE STUDY: To evaluate the anti-inflammatory activity as well as the preliminary mechanism of S. ferruginea parasitizing on Tecoma stans.
MATERIALS AND METHODS: The anti-inflammatory capability of freeze-dried stem aqueous extract was assessed via inhibition of inflammatory cytokines interleukin- (IL-) 1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulated RAW 264.7 macrophages. The underlying anti-inflammatory mechanism was deciphered through reverse transcriptase and real time quantitative polymerase chain reactions (RT-PCR and qPCR) for inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and TNF-α mRNA expression.
RESULTS: The results exhibited that aqueous extract of freeze-dried S. ferruginea stem sample concentration-dependently inhibited IL-1β protein production along with the down regulation of iNOS and IL-1β mRNA expression. Moreover, it significantly suppressed the protein release of IL-6 and IL-10 in a concentration-dependent manner. However, it slightly reduced TNF-α at higher sample concentration (250 μg/mL) without affecting the mRNA expression levels of COX-2 and TNF-α.
CONCLUSIONS: This study suggests that S. ferruginea parasitizing on Tecoma stans exerted anti-inflammatory capability attributed to inhibition of iNOS and IL-1β mRNA expression, NO creation, IL-1β, IL-6, IL-10, and TNF-α protein production, indicating this plant might be a useful plant-derived candidate against inflammation.