Anti-Inflammatory Activity of a Demineralized Bone Matrix: An In Vitro Pilot Study

Demineralized bone matrix (DBM) is commonly used for the reconstruction of bone defects. Early graft consolidation involves a transient inflammatory process. It is, however, unclear whether DBM can modulate this process. To test this possibility, we prepared acid lysates of demineralized ground cortical (DGC) and moldable demineralized fibers (MDF). Murine RAW 264.7 and primary bone marrow macrophages were exposed to acid lysates of DGC and MFD prior to provoking an inflammatory response with lipopolysaccharide (LPS). Similarly, murine ST2 mesenchymal cells were exposed to DGC and MFD with and without interleukin 1β (IL1) and TNFα. We show here that acid lysates of DGC and MFD reduced the expression of IL1 and IL6 in RAW 264.7 macrophages, as determined by RT-PCR and, for IL6, by immunoassay. This response was confirmed with primary macrophages. Likewise, desalted acid lysates exert anti-inflammatory properties on RAW 264.7 cells and in ST2 cells, the forced expression of IL6, inducible nitric oxide synthase (iNOS) and chemokine ligand 5 (CCL5) was reduced. These in vitro findings suggest that DGC and MFD lower the inflammation-induced expression of inflammatory mediators in murine cell-based bioassays.

Novel benzofurane-pyrazole derivatives with anti-inflammatory, cyclooxygenase inhibitory and cytotoxicity evaluation

Novel benzofurane-pyrazolone hybrids have been synthesized for evaluating their anti-inflammatory and cytotoxic properties. 4-(2-chloroacetyl)-1,5-dimethyl-2-phenyl-1,2-dihydro-3H-pyrazol-3-one were reacted with α-hydroxy aldehyde or α-hydroxy ketone derivatives to obtain nine novel pyrazolone derivatives. Structures were successfully elucidated by 1H NMR, 13C NMR, IR and HRMS. Enzyme inhibitory activity was measured on cyclooxygenases (COXs) as considered to address anti-inflammatory activity. Compound 2 showed the highest activity on both COX-1 and COX-2 subtypes with 12.0 μM and 8.0 μM IC50, respectively. This activity was found close to indomethacin COX-2 inhibition measured as 7.4 μM IC50. Rest of the compounds (1, 3–9) showed 10.4–28.1 μM IC50 on COX-2 and 17.0–35.6 μM IC50 on COX-1 (Compound 1 has no activity on COX-1). Tested compounds (1–9) showed activity on NO production. Only compound was the 4, which showed a low inhibition on IL-6 levels. Cell viability was up to 60% at 100 μM for all compounds (1–9) on RAW 264.7 and NIH3T3 cell lines, thus compounds were reported to be noncytotoxic.

Flavopiridol Protects Against Fungal Keratitis by Alleviating Inflammation Through the Promotion of Autophagy

Background: Fungal keratitis is a serious infectious keratopathy related to fungal virulence and excessive inflammatory responses. Autophagy exhibits a potent ability to resolve inflammation during fungal infection. This study aimed to investigate the protective function of flavopiridol in fungal keratitis and explore its effects on autophagy.Methods: A mouse model of fungal keratitis was established and then treated with 5 μM flavopiridol. RAW 264.7 cells were treated with 200 nM flavopiridol before fungal stimulation. The severity of corneal diseases was evaluated by slit-lamp microscopy. The expression levels of cytokines were detected by RT-PCR and ELISA. The protein levels of LC3, Beclin-1 and Atg7 were determined by western blot and immunofluorescence. A Cell Counting Kit-8 assay was used to test cell viability. Autolysosomes were detected by transmission electron microscopy (TEM). An inhibitor of autophagy, 3-methyladenine (3-MA), was used to pretreat RAW 264.7 cells. Phagocytosis of RAW 264.7 cells was evaluated by counting colony forming units. A. fumigatus was incubated with flavopiridol, and the hyphae were stained with calcofluor white. Absorbance assay, crystal violet staining and adherence assay were used to detect the antifungal activity of flavopiridol.Results: Flavopiridol treatment notably reduced corneal opacity and the clinical scores of infected corneas. Compared with DMSO treatment, flavopiridol treatment greatly downregulated IL-1β, IL-6 and TNF-a expression in infected corneas. In RAW 264.7 cells, flavopiridol treatment inhibited IL-1β, IL-6 and TNF-a expression but promoted IL-10 expression. TEM images showed that more autolysosomes were presented in infected corneas and RAW 264.7 cells after flavopiridol treatment than after DMSO treatment. Flavopiridol treatment notably upregulated the protein expression of LC3, Beclin-1 and Atg7 in infected corneas as well as in RAW 264.7 cells. 3-MA pretreatment counteracted the cytokine regulation induced by flavopiridol. Moreover, flavopiridol promoted the phagocytosis of RAW 264.7 cells. Flavopiridol also exhibited antifungal activity by restricting fungal growth and limiting fungal biofilm formation and conidial adhesion. Conclusions: Flavopiridol significantly alleviated the inflammation of fungal keratitis by activating autophagy. In addition, flavopiridol promoted the phagocytosis of RAW 264.7 cells and exhibited antifungal function, indicating the potential therapeutic role of flavopiridol in fungal keratitis.

Sulfated Chinese yam polysaccharide enhances immunomodulatory activity of RAW 264.7 cells via TLR4-MAPK/NF-κB signaling pathway

In this study, Chinese yam polysaccharide (CYP) was isolated from yam by hydroextraction and alcoholic precipitation. Subsequently, the chlorosulfate-pyridine (CSA-Pyr) method was used to obtain the sulfated Chinese yam polysaccharide...