The role of Glabridin in antifungal and anti-inflammation effects in Aspergillus fumigatus keratitis.

PURPOSE: To investigate the effect of Glabridin (GLD) in Aspergillus fumigatus keratitis and its associated mechanisms. METHODS: Aspergillus fumigatus (A. fumigatus) conidia was inoculated in 96-well plate, and minimal inhibitory concentration (MIC) and biofilm formation ability were evaluated after GLD treatment. Spore adhesion ability was evaluated in conidia infected human corneal epithelial cells (HCECs). Keratitis mouse model was created by corneal intrastromal injection with A. fumigatus conidia, and GLD treatment started at the day after infection. The number of fungal colonies was calculated by plate count, and degree of corneal inflammation was assessed by clinical score. Flow cytometry, myeloperoxidase (MPO), and immunofluorescence staining (IFS) experiments were used to assess neutrophil infiltrations. PCR, ELISA and Western blot were conducted to determine levels of TLR4, Dectin-1 as well as downstream inflammatory factors. RESULTS: GLD treatment suppressed the proliferation, biofilm formation abilities and adhesive capability of A. fumigatus. In mice upon A. fumigatus infection, treatment of GLD showed significantly decreased severity of corneal inflammation, reduced number of A. fumigatus in cornea, and suppressed neutrophil infiltration in cornea. GLD treatment obviously inhibited mRNA and protein levels of Dectin-1, TLR4 and proinflammatory mediators such as IL-1ß, HMGB1, and TNF-α in mice corneas compared to the control group. CONCLUSION: GLD has antifungal and anti-inflammatory effects in fungal keratitis through suppressing A. fumigatus proliferation and alleviating neutrophil infiltration, and repressing the expression of TLR4, Dectin-1 and proinflammatory mediators.

Baicalein Protects Against Aspergillus fumigatus Keratitis by Reducing Fungal Load and Inhibiting TSLP-Induced Inflammatory Response.

Purpose: To investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms. Methods: The noncytotoxic antifungal concentration of baicalein was determined using CCK8, cell scratch assay, minimum inhibitory concentration, biofilm formation, scanning electron microscopy, propidium iodide uptake test and adherence assay in vitro and Draize test in vivo. In fungal keratitis (FK) mouse models, clinical score and plate count were used to evaluate FK severity, and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity. Real-time PCR, ELISA, and Western blot were performed to explore the anti-inflammatory activity of baicalein and the underlying mechanisms in vivo and in vitro. Results: Baicalein at 0.25 mM (noncytotoxic) significantly inhibited A. fumigatus growth, biofilm formation, and adhesion in vitro. In A. fumigatus keratitis mice, baicalein mitigated FK severity, reduced fungal load, and inhibited neutrophil infiltration and activity. Baicalein not only suppressed mRNA and protein levels of proinflammatory factors IL-1ß, IL-6, and TNF-α, but also inhibited the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in vivo and in vitro. In HCECs, mRNA and protein levels of IL-1ß, IL-6, and TNF-α were significantly lower in the TSLP siRNA-treated group, while higher in the rTSLP-treated group than in the corresponding control. Baicalein treatment significantly inhibited rTSLP induced the expression of IL-1ß, IL-6, and TNF-α. Conclusions: Baicalein plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal growth, biofilm formation, and adhesion, and suppressing inflammatory response via downregulation of the TSLP/TSLPR pathway.

Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load, Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines.

Purpose: Isorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis. Methods: In vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR. Results: In HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1ß, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro. Conclusions: Isorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.

Clinical characteristics of 66 patients with novel coronavirus (2019-nCoV) -infected pneumonia (NCIP) in Enshi, Hubei / 中华急诊医学杂志

Objective:To describe the epidemiological and clinical characteristics of patients with novel coronavirus (2019-nCoV) -infected pneumonia in Enshi, Hubei, and to improve the awareness of the disease, which is key for surveillance and control measures of 2019-nCoV pneumonia in the region.Methods:The first 66 laboratory-confirmed 2019-nCoV patients in Enshi between January 23, 2020 and February 1, 2020 were included. Their epidemiological data, demographic data, clinical data and therapeutic effect were retrospectively analyzed.Results:Among the first 66 patients with confirmed 2019-nCoV, patients had an average age of 46±9 years, 35 (53.0%) patients were male and 31 (47.0%) female; 38 (57.6%) patients were Han, 18 (27.3%) were Tujia, and 10 (15.1%) were Miao. Thirty-seven (56%) patients had chronic diseases such as coronary heart disease, chronic bronchus, inflammation, diabetes, hypertension, hypothyroidism, and rheumatoid arthritis. All the patients had a history of exposure or indirect exposure in Wuhan epidemic area, mainly history of Wuhan sojourner. The mean incubation period varied from 2.5-16 days, with the 95th percentile of the distribution at 7 days. The main clinical manifestation were fever [66 (100%) patients], dry cough [56 (84.8%)], chest tightness [21 (31.8%)], shortness of breath [8 (12.1%)], massive fatigue [23 (34.8%)], muscle ache [6 (9.1%)], headache [4 (6.1%)], sore throat [13 (19.7%) ], rhinorrhoea [11 (16.7%) ], and diarrhea [ 5 (7.6%)]. Six (9.1%) critically ill patients and 4 (6.1%) critically ill patients had dyspnea. White blood cell counts were normal or decreased in 51 (77.3%) patients, and increased in 15 (22.7%). Lymphocyte counts were reduced or normal in 58 (87.9%) patients, and increased in 8 (12.1%) patients. Most patients had elevated CRP and erythrocyte sedimentation, and some patients had normal ranges of liver function, renal function, and electrolytes. Ten severely ill patients had elevated liver enzymes and cardiac enzymes (especially K and CKMB). There were significant differences in inflammatory markers among patients of different nationalities. Arterial blood gas analysis in 56 (84.8%) mild patients showed no significant abnormalities. Chest CT scans of 66 patients showed single or multiple small patchy shadows and interstitial changes, which were evident in the extrapulmonary band. All patients were hospitalized and isolated for treatment. Of the 66 patients received oxygen therapy, most of them received antiviral therapy (abidol/Kaletra, 0.2 g po tid/2 pills po bid) and interferon (aerosol inhalation), few patient received antibacterial therapy (Tanreqing, 20 mL ivgtt qd) and glucocorticoid therapy (methylprednisolone, 40 mg ivgtt bid), and supplemented by traditional Chinese medicine treatment. Six (9.1%) severely ill patients and 4 (6.1%) critically ill patients treated with non-invasive or invasive ventilator.Conclusions:The patients in this study are all imported cases. Most patients have mild clinical symptoms. The lungs show single or multiple small patchy shadows and interstitial changes, which are obvious with extrapulmonary bands. Seek medical treatment as soon as possible, and the above-mentioned symptomatic treatment scheme is effective. The overall morbidity and mortality in this region are lower than those in other parts of the province, and the mortality and critical illness rate of ethnic minority patients are lower than those of Han patients. There are significant differences in inflammatory markers such as leukocytes and lymphocytes among patients of different nationalities.