Rosmarinic acid alleviates fungal keratitis caused by Aspergillus fumigatus by inducing macrophage autophagy.

Fungal keratitis (FK) is an infectious keratopathy can cause serious damage to vision. Its severity is related to the virulence of fungus and response of inflammatory. Rosmarinic acid (RA) extracted from Rosmarinus officinalis exhibits antioxidant, anti-inflammatory and anti-viral properties. The aim of this study was to investigate the effect of RA on macrophage autophagy and its therapeutic effect on FK. In this study, we demonstrated that RA reduced expression of proinflammatory cytokine, lessened the recruitment of inflammatory cells in FK. The relative contents of autophagy markers, such as LC3 and Beclin-1, were significantly up-regulated in RAW 264.7 cells and FK. In addition, RA restored mitochondrial membrane potential (MMP) of macrophage to normal level. RA not only reduced the production of intracellular reactive oxygen species (ROS) but also mitochondria ROS (mtROS) in macrophage. At the same time, RA induced macrophage to M2 phenotype and down-regulated the mRNA expression of IL-6, IL-1ß, TNF-α. All the above effects could be offset by the autophagy inhibitor 3-Methyladenine (3-MA). Besides, RA promote phagocytosis of RAW 264.7 cells and inhibits spore germination, biofilm formation and conidial adherence, suggesting a potential therapeutic role for RA in FK.

Formononetin protects against Aspergillus fumigatus Keratitis: Targeting inflammation and fungal load.

PURPOSE: To investigate the potential treatment of formononetin (FMN) on Aspergillus fumigatus (A. fumigatus) keratitis with anti-inflammatory and antifungal activity. METHODS: The effects of FMN on mice with A. fumigatus keratitis were evaluated through keratitis clinical scores, hematoxylin-eosin (HE) staining, and plate counts. The expression of pro-inflammatory factors was measured using RT-PCR, ELISA, or Western blot. The distribution of macrophages and neutrophils was explored by immunofluorescence staining. The antifungal properties of FMN were assessed through minimum inhibitory concentration (MIC), propidium iodide (PI) staining, fungal spore adhesion, and biofilm formation assay. RESULTS: In A. fumigatus keratitis mice, FMN decreased the keratitis clinical scores, macrophages and neutrophils migration, and the expression of TNF-α, IL-6, and IL-1ß. In A. fumigatus-stimulated human corneal epithelial cells (HCECs), FMN reduced the expression of IL-6, TNF-α, IL-1ß, and NLRP3. FMN also decreased the expression of thymic stromal lymphopoietin (TSLP) and thymic stromal lymphopoietin receptor (TSLPR). Moreover, FMN reduced the levels of reactive oxygen species (ROS) induced by A. fumigatus in HCECs. Furthermore, FMN inhibited A. fumigatus growth, prevented spore adhesion and disrupted fungal biofilm formation in vitro. In vivo, FMN treatment reduced the fungal load in mice cornea at 3 days post infection (p.i.). CONCLUSION: FMN demonstrated anti-inflammatory and antifungal properties, and exhibited a protective effect on mouse A. fumigatus keratitis.

The therapeutic role and mechanism of 4-Methoxycinnamic acid in fungal keratitis.

Fungal keratitis is an infectious vision-threatening disease that has a poor prognosis, and the clinical therapeutic drugs have multiple limitations, such as epithelial toxicity and low bioavailability. Therefore, new antifungal treatment strategies must be developed. 4-Methoxycinnamic acid (MCA) is a widely occurring natural phenolic acid that has been proven to have multiple effects, such as antibacterial, antifungal, anti-inflammatory, neuroprotective, and inhibiting cancer. In this research, we explored the effects and underlying mechanisms of MCA on A. fumigatus keratitis and the antifungal effects of the combination of MCA and natamycin (NATA) on A. fumigatus. We found that MCA exerts antifungal effects by inhibiting the synthesis of the fungal cell wall, changing the permeability of fungal cell membranes. Moreover, the MCA-NATA combination exhibited synergy for A. fumigatus. In addition, MCA exerted an anti-inflammatory effect by downregulating the inflammatory factors (IL-1ß, TNF-α, IL-6, and iNOS) in C57BL/6 mice and RAW264.7 cells. The anti-inflammatory mechanism of MCA was associated with the Mincle signal pathway. In summary, MCA acts as a potential therapeutic drug for fungal keratitis and a potential antifungal sensitizer for natamycin. MCA inhibits fungal cell wall synthesis, destroys the permeability of fungal cell membranes, and mediates the anti-inflammatory, immune response of the host.

New insights into the prognosis of intraocular malignancy: Interventions for association mechanisms between cancer and diabetes.

The intraocular malignancies, which mostly originate from the retina and uvea, exhibit a high incidence of blindness and even death. Uveal melanoma (UM) and retinoblastoma (RB) are the most common intraocular malignancies in adults and children, respectively. The high risks of distant metastases lead to an extremely poor prognosis. Nowadays, various epidemiological studies have demonstrated that diabetes is associated with the high incidence and mortality of cancers, such as liver cancer, pancreatic cancer, and bladder cancer. However, the mechanisms and interventions associated with diabetes and intraocular malignancies have not been reviewed. In this review, we have summarized the associated mechanisms between diabetes and intraocular malignancy. Diabetes mellitus is a chronic metabolic disease characterized by prolonged periods of hyperglycemia. Recent studies have reported that the abnormal glucose metabolism, insulin resistance, and the activation of the IGF/insulin-like growth factor-1 receptor (IGF-1R) signaling axis in diabetes contribute to the genesis, growth, proliferation, and metastases of intraocular malignancy. In addition, diabetic patients are more prone to suffer severe complications and poor prognosis after radiotherapy for intraocular malignancy. Based on the common pathogenesis shared by diabetes and intraocular malignancy, they may be related to interventions and treatments. Therefore, interventions targeting the abnormal glucose metabolism, insulin resistance, and IGF-1/IGF-1R signaling axis show therapeutic potentials to treat intraocular malignancy.

Kaempferol ameliorate the prognosis of Aspergillus fumigatus keratitis by reducing fungal load and inhibiting the Dectin-1 and p38 MAPK pathway.

Fungal keratitis is one of leading reasons for blindness in the world, which causes corneal blindness mainly due to excessive inflammatory responses. Kaempferol (KAE) is a natural flavonoid which has potent anti-inflammatory effects. However, whether KAE plays protective roles in fungal keratitis and the potentially protective mechanisms are unrevealed. Here we first investigated the anti-inflammatory and antifungal effects of KAE on Aspergillus fumigatus (A. fumigatus) keratitis in C57BL/6 mice. We found that treatment of KAE ameliorated the severity of keratitis, inhibited macrophages and neutrophils recruitment, depressed corneal fungal load, and declined the expression of TLR4 and Dectin-1 in A. fumigatus infected mice corneas. And in activated hyphae or Curdlan stimulated macrophages, pretreatment of KAE also significantly decreased the mRNA and protein expression of IL-1ß, TNF-α, MIP-2 and the phosphorylated-p38 (p-p38)/p38 MAPK ratio. In summary, KAE ameliorated the prognosis of fungal keratitis in C57BL/6 mice by reducing corneal fungal load, depressing the inflammatory cells recruitment, and downregulating the expression of inflammatory factors, and those effects depended on the inhibition of Dectin-1 and p38 MAPK pathway.

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.

Galectin-3 plays an important pro-inflammatory role in A. fumigatus keratitis by recruiting neutrophils and activating p38 in neutrophils.

PURPOSE: To determine the role of galectin-3 (Gal-3) in cornea infected by Aspergillus fumigatus (A. fumigatus). METHODS: Gal-3 was tested in normal and infected corneas of C57BL/6 mice. Mice corneas were pretreated with or without rmGal-3 or Gal-3 siRNA and infected with A. fumigatus. Recombinant mouse (rm) Gal-3 stimulated polymorphonuclear neutrophilic leukocytes (PMNs). PMNs were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of Gal-3 siRNA. Disease severity was documented by clinical score and photographs with a slit lamp. PCR, Western blot, and ELISA tested expression of Gal-3, interleukin (IL)-1ß, IL-6, macrophage inflammatory protein 2 (MIP-2) and p-p38. PMNs infiltration was assessed by flow cytometry and myeloperoxidase (MPO) assay. RESULTS: Gal-3 expression was significantly elevated by A. fumigatus in mice corneas. rmGal-3 treatment increased clinical scores, PMNs infiltration, and cytokines expression, which were decreased by Gal-3 siRNA treatment. In PMNs, Gal-3 expression was also significantly increased by A. fumigatus. The rmGal-3 treatment upregulated proinflammatory cytokines secretion and p-p38 expression, which was significantly inhibited by Gal-3 siRNA. CONCLUSION: These data proved that A. fumigatus increased Gal-3 expression and elevated disease clinical scores, PMNs infiltration and cytokines expression through Gal-3. In PMNs, A. fumigatus upregulated IL-1ß and IL-6 secretion through the Gal-3 / p38 pathway.

Microsurgical Treatment of 86 Anterior Choroidal Artery Aneurysms: Analysis of Factors Influencing the Prognosis.

PURPOSE: To explore factors affecting the prognosis of choroidal anterior artery aneurysm (AChAA) and provide a reference for improving the postoperative outcome. METHODS: The clinical data of 86 patients with AChAA who underwent treatment by a single surgeon were collected and analyzed retrospectively. Univariate analysis and multivariate logistic regression analysis were conducted to examine 12 factors that possibly affected outcome. RESULTS: The five factors that affected the patient outcomes were times of subarachnoid hemorrhage (SAH), characteristics of SAH on computed tomography (CT), Hunt-Hess grade, aneurysm size, and presence or absence of postoperative complications. Characteristics of SAH on CT (odds ratio [OR]: 3.727; p = 0.000; 95% confidence interval [CI], 1.850-7.508), aneurysm size (OR: 6.335; p = 0.000; 95% CI, 2.564-15.647), and presence or absence of postoperative complications (OR: 4.141; p = 0.000; 95% CI, 1.995-8.599) were independent risk factors influencing the prognosis. In addition, the incidence of postoperative ischemia (caused by anterior choroidal artery syndrome) is related to the aneurysm emitting part and presence or absence of intraoperative rupture. CONCLUSIONS: The analysis of characteristics of SAH on CT, aneurysm size, and presence or absence of postoperative complications can roughly determine the outcome of patients with AChAAs.

Dimethyl itaconate protects against fungal keratitis by activating the Nrf2/HO-1 signaling pathway.

Dimethyl itaconate (DI) is a membrane-permeable itaconate derivative with anti-inflammatory functions. However, the anti-inflammatory effect of DI has never been studied in fungal keratitis. In this study, we tested the protective effect of DI against fungal keratitis and assessed the role of NF-E2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signaling in this process. Eyes of C57BL/6 (B6) mice were treated with 2 mm DI after infection with Aspergillus fumigatus. Human corneal epithelial cells (HCECs) were pretreated with 0.25 mm DI and then incubated with A. fumigatus. Clinical scoring, slit-lamp photography, myeloperoxidase determination, flow cytometry and immunostaining were used to assess the disease response and treatment efficacy. PCR, Western blot and ELISA were used to assess the expression of interleukin-1ß (IL-1ß), chemokine (C-X-C motif) ligand 1, IL-6, IL-8, Nrf2 and HO-1. In addition, quantification of viable fungi, absorbance assays and fluorimetry were used to measure DI fungistatic activity. We observed that DI-treated eyes showed decreased clinical scores, fungal loads, polymorphonuclear neutrophil (PMN) infiltration and cytokine expression, compared with phosphate-buffered saline-treated infected eyes. DI treatment decreased the cytokine levels in infected corneas and in HCECs stimulated with A. fumigatus. Moreover, DI treatment increased Nrf2 and HO-1 expression in corneas and nuclear Nrf2 accumulation in HCECs. DI-induced cytokine downregulation was inhibited by pretreatment with an Nrf2 or HO-1 inhibitor. Finally, DI treatment reduced the A. fumigatus absorbance and fungal mass. These data indicate that DI protects against fungal keratitis by limiting inflammation via the Nrf2/HO-1 signaling pathway and that DI inhibits the growth of A. fumigatus.