Protective roles of apremilast via Sirtuin 1 in atherosclerosis.

Atherosclerosis is an inflammatory disease with a high level of cholesterol in the blood. Apremilast is a new anti-inflammatory drug that possesses a potential anti-atherosclerosis effect. RT-qPCR and western blot were undertaken to assay the levels of Sirtuin 1 (SIRT1), oxidized low density lipoprotein receptor 1 (LOX-1), and CD36 molecule (CD36). Reactive oxygen species (ROS) levels were evaluated by 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and Oil Red O staining was performed to show lipid accumulation. The result showed that apremilast treatment reduced the expression levels of pro-inflammatory factors and p-p65, as well as lipid accumulation. Meanwhile, triglyceride (TG), total cholesterol (TC) and free cholesterol (FC) levels declined in oxidized low density lipoprotein (ox-LDL)-treated macrophages. Mechanistically, apremilast targets SIRT1 and increases SIRT1 expression. The efficacy of apremilast on inflammatory response and lipid formation required the involvement of SIRT1. Additionally, apremilast treatment reduced scavenger receptors, LOX-1, and CD36 levels. These findings suggest the protective effects of apremilast via SIRT1 in atherogenesis and highlight the need for translational research from bench to bedside.

Perillaldehyde Protects Against Aspergillus fumigatus Keratitis by Reducing Fungal Load and Inhibiting Inflammatory Cytokines and LOX-1.

PURPOSE: The purpose of this research was to explore the antifungal and anti-inflammatory effects of perillaldehyde (PAE) in Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanism. METHODS: The biofilm formation, adherence assay, and propidium iodide uptake test were used to determine the possible mechanism of PAE in terms of antifungal effects in vitro. The severity of corneal infection was evaluated by clinical scores. The immunofluorescence staining (IFS) was adopted to detect the number of macrophages in infected corneas. Draize test was performed to assess the ocular toxicity of PAE. Real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot reflected the expression of inflammatory cytokines and Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) in mice corneas and RAW264.7 cells. RESULTS: PAE was able to inhibit the formation of biofilm, reduce conidial adhesion, and damage the integrity of membranes to exert antifungal activity. In C57BL/6 mice models, PAE alleviated the severity of infected corneas, reduced the recruitment of macrophages and had low ocular toxicity. In addition, the mRNA and protein levels of TNF-α, CCL-2, and LOX-1 could be significantly decreased by the application of PAE after A. fumigatus infection in vivo and in vitro. CONCLUSION: Our study indicated that PAE protected against A. fumigatus keratitis by reducing fungal load, accumulation of macrophages, and inhibiting the expression of inflammatory cytokines.

Plaque-Targeted Rapamycin Spherical Nucleic Acids for Synergistic Atherosclerosis Treatment.

Atherosclerosis with unstable plaques is the dominant pathological basis of lethal cardio-cerebrovascular diseases, which can cause acute death due to the rupture of plaques. Plaque-targeted drug delivery to achieve promoted treatment remains the main challenge because of the systemic occurrence of atheroma. Herein, a rapamycin (RAP) spherical nucleic acid (SNA) structure, capable of specifically accumulating in plaques for synergistic atherosclerosis treatment is constructed. By designing consecutive phosphorothioate (PS) at 3' terminus of the deoxyribonucleic acid (DNA) strand, multiple hydrophobic RAPs are covalently grafted onto the PS segment to form an amphiphilic drug-grafted DNA (RAP-DNA), which successively self-assembles into micellar SNA (RAP-SNA). Moreover, the phosphodiester-DNA segment constitutes the outer shell of RAP-SNA, enabling further hybridization with functional siRNA (targeting lectin-like oxidized low-density lipoprotein receptor-1, LOX-1) to obtain the drug codelivered SNA (LOX-1/RAP-SNA). With two active ingredients inside, LOX-1/RAP-SNA can not only induce robust autophagy and decrease the evil apoptosis of the pathological macrophages, but also simultaneously prohibit the LOX-1-mediated formation of damageable foam cells, realizing the effect of synergistic therapy. As a result, the LOX-1/RAP-SNA significantly reduces the progression of atheroma and stabilizes the plaques, providing a new strategy for synergistically targeted atherosclerosis treatment.

Thymol Protects against Aspergillus Fumigatus Keratitis by Inhibiting the LOX-1/IL-1ß Signaling Pathway.

OBJECTIVE: To explore the anti-inflammatory effects and mechanisms of action of thymol in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The minimum inhibitory concentration of thymol against A. fumigatus was detected. To characterize the anti-inflammatory effects of thymol, mouse corneas and human corneal epithelial cells were pretreated with thymol or dimethyl sulfoxide (DMSO) before infection with A. fumigatus spores. Slit-lamp microscopy, immunohistochemistry, myeloperoxidase detection, quantitative real-time polymerase chain reaction, and Western blotting were used to assess infection. Neutrophil and macrophage recruitment, in addition to the secretion of LOX-1 and IL-1ß, were quantified to evaluate the relative contribution of thymol to the inflammatory response. RESULTS: We confirmed that the growth of A. fumigatus was directly inhibited by thymol. In contrast with the DMSO group, there was a lower degree of inflammation in the mouse corneas of the thymol-pretreated group. This was characterized by significantly lower clinical scores, less inflammatory cell infiltration, and lower expression of LOX-1 and IL-1ß. Similarly, in vitro experiments indicated that the production of LOX-1 and IL-1ß was significantly inhibited after thymol treatment, in contrast with the DMSO-pretreated group. CONCLUSION: Our findings demonstrate that thymol exerted a direct fungistatic activity on A. fumigatus. Furthermore, thymol played a protective role in fungal keratitis by inhibiting LOX-1/IL-1ß signaling pathway and reducing the recruitment of neutrophils and macrophages.

α- MSH Plays Anti-inflammatory and Anti-fungal Role in Aspergillus Fumigatus Keratitis.

PURPOSE: To investigate the anti-inflammatory and antifungal role of α-melanocyte stimulating hormone (α-MSH) in Aspergillus Fumigatus (A. fumigatus) keratitis. METHOD: Corneas of C57BL/6 mice were infected with A. Fumigatus. α-MSH (5 ul, 1×10-4 mmol/ml) was given by subconjunctival injection from day 1 to day 3 post infection (p.i.). After 3 days p.i., clinical score was recorded and HE staining was tested. Fungal load in mice corneas was observed by plate counting. Proinflammatory mediators and pattern recognition receptors (PRRs) were detected. The number of neutrophils and macrophages was tested by immunofluorescence staining. The role of α-MSH in RAW264.7 cells after A. fumigatus stimulation were evaluated by PCR and Western blot, and MPKA protein levels including total-JNK (T-JNK), phosphorylated-JNK (P-JNK), total-ERK (T-ERK), and phosphorylated-ERK (P-ERK) were tested via Western blot with or without α-MSH treatment. RESULTS: Compared with PBS control group, α-MSH treatment alleviated disease response and decreased clinical score at 3 days p.i. HE staining showed less infiltration in corneal tissue after α-MSH treatment. Plate counting experiment showed that number of viable fungus in corneas of α-MSH treated group was less than control group. mRNA levels of IL-1ß, TNF-α, IL-6, MIP-2, LOX-1, Dectin-1, and iNOS were decreased. Protein levels of IL-1ß, TNF-α, IL-6, and Dectin-1 were decreased. α-MSH treatment also decreased the infiltrating neutrophils and macrophages. The levels of proinflammatory cytokines, Dectin-1 and LOX-1 stimulated by A. fumigatus, were also suppressed by pretreatment of α-MSH in RAW264.7 cells. The ratio of P-JNK/T-JNK and P-ERK/T-ERK was downregulated in α-MSH group compared with PBS control group. CONCLUSION: α-MSH alleviates the severity and decreases fungal load of A. fumigatus keratitis in mice. Migration of neutrophils and macrophages are restrained. α-MSH downregulates the expression of dectin-1 and the ratio of P-JNK/T-JNK and P-ERK/T-ERK in A. fumigatus infection.

Improved scFv Anti-LOX-1 Binding Activity by Fusion with LOX-1-Binding Peptides.

The oxidized low-density lipoprotein receptor-1 (LOX-1) targeted single-chain variable fragment (scFvs) is a promising molecule for the targeted delivery of imaging and therapeutic molecules of atherosclerotic diseases; however, its applications are limited by the inherent low antigen affinity. In this study, the three-dimensional (3D) model of the anti-LOX-1 scFv was constructed and its docking with the LOX-1 protein was developed. To improve the LOX-1-binding activity, the anti-LOX-1 scFv was designed to fuse with one of three LOX-1-binding heptapeptides, LTPATAI, FQTPPQL, and LSIPPKA, at its N-terminus and C-terminus and in the linker region, which have different LOX-1-binding interfaces with the anti-LOX-1 scFv analyzed by an array of computational approaches. These scFv/peptide fusions were constructed, successfully expressed in Brevibacillus choshinensis hosts, and purified by a two-step column purification process. The antigen binding activity, structural characteristics, thermal stability, and stability in serum of these fusion proteins were examined. Results showed that the scFv with N-terminal fusing peptides proteins demonstrated increased LOX-1-binding activity without decrease in stability. These findings will help increase the application efficacy of LOX-1 targeting scFv in LOX-1-based therapy.