Fungal keratitis (FK) is a persistent and vision-threatening disease. The potential of vitamin C (VC) in tissue protection is well-recognized; however, its specific role in FK and its interaction with antifungal drugs are not well defined. In this study, we investigated the role of VC in FK in vitro and in vivo. It revealed that VC substantially decreases clinical scores and corneal perforation rates in a murine model of FK. VC promoted a concentration-dependent increase in neutrophil infiltration via mast cells in vitro and confirmed this effect in in vivo experiments. Moreover, VC enhanced fungicidal activity by boosting infiltrated neutrophils, without influencing reactive oxygen species (ROS) production or neutrophil extracellular traps (NETs) formation. VC also mitigated the cytokine storm within bone marrow-derived macrophages and increased neutrophil apoptosis, thereby facilitating the efficient clearance of senescent neutrophils. The combination of VC with amphotericin B (AmB) demonstrated additive antifungal effects, reducing fungal load and corneal perforation. These results indicate that VC is pivotal in defending against fungal corneal infections by promoting neutrophil chemotaxis through mast cells (MCs) and modulating the inflammatory response, without suppressing neutrophils' antifungal capability. The combination of VC with AmB may present a new therapeutic avenue for FK.

Objective:To investigate the mechanism of tissue damage caused by neutrophil matrix metalloproteinase-8 (MMP-8) in Fusarium keratitis. Methods:A total of 108 male C57BL/6J SPF grade mice, 6-8 weeks old, were selected to establish a model of Fusarium keratitis (FK) in the right eyes.Corneal inflammation in mice was observed and scored under a slit lamp microscope.Based on the corneal inflammation scores, the modeling eyes were divided into 0, 12, 24, 48, and 72-hour groups post-modeling.At the corresponding time points, mice were euthanized, and corneal tissues were collected.The expressions of MMP-8, adenylate-activated protein kinase (AMPKα) and its serine 172-site phosphorylated form (p-AMPKα) proteins in corneal tissues were detected by Western blot.The neutrophil count in mice corneal tissues at each time point was determined using hematoxylin and eosin staining.The co-localization of neutrophils and MMP-8 protein in the cornea was observed by immunofluorescence staining.In the in vitro corneal collagen degradation experiment, corneal tissues were divided into MMP-8 group, buffer group, and normal saline group, which were treated with 100 μl of activated recombinant MMP-8, detection buffer, and normal saline, respectively.Hydroxyproline content in corneal tissues was determined using a hydroxyproline assay kit, and the mass fractions of hydroxyproline were compared among the groups.Peripheral blood neutrophils were isolated from human blood samples, and Fusarium spores were collected for experiments.Human neutrophils were divided into four groups, negative control group (cultured neutrophils), co-culture group (neutrophils co-cultured with spores), AICAR-treated group (neutrophils co-cultured with spores and treated with p-AMPK protein kinase activator AICAR), and compound C-treated group (neutrophils co-cultured with spores and treated with the inhibitor compound C).The MMP-8 protein expression levels in each group of human neutrophils were assessed via immunofluorescence staining.The use and care of animals complied with the ARVO statement and Regulations for the Administration of Affairs Concerning Experimental Animals.The animal experiment protocol was approved by the Animal Ethics Committee of Henan Eye Hospital (No.HNEECA-2017-04-02).One healthy adult volunteer was selected and 10 ml of peripheral venous blood was collected.The clinical study protocol was approved by the Clinical Ethics Committee of Henan Eye Hospital (No.HNEECKY-2019[16]). Results:At 24 hours post-modeling, corneal opacification was observed in the modeling eyes, and corneal perforation occurred in 72-hour post-modeling group.The corneal inflammation scores in 24, 48, and 72-hour post-modeling groups were all higher than those in 12-hour post-modeling group, and the differences were statistically significant (all at P<0.001).The relative expression levels of MMP-8 protein in the cornea were higher in 12, 24, and 48-hour post-modeling groups compared to 0-hour group, with statistically significant differences (all at P<0.001).There was a moderate positive correlation between the relative expression level of MMP-8 protein in the cornea and the inflammation scores of the modeling eye ( rs=0.50, P<0.05).In the cornea, the p-AMPKα (Thr 172)/AMPKα ratio was higher in 24, 48, and 72-hour post-modeling groups than in 0-hour group, and the differences were statistically significant (all at P<0.05).The p-AMPKα(Thr 172)/AMPKα ratio in the cornea was moderately positively correlated with the relative expression level of MMP-8 protein ( r=0.54, P<0.01).The number of neutrophils in the cornea was significantly higher in 24, 48, and 72-hour post-modeling groups than in 0-hour group, with statistically significant differences (all at P<0.001).The number of neutrophils in the cornea was strongly positively correlated with the inflammation score ( rs=0.77, P<0.001), and was moderately positively correlated with the relative expression level of MMP-8 protein ( r=0.56, P<0.05).MMP-8 protein expression in the cornea of the modeling eyes showed a high degree of co-localization with neutrophils.The hydroxyproline content in the cornea was (0.52±0.02)μg/mg, (0.51±0.03)μg/mg, and (0.27±0.02)μg/mg in buffer group, normal saline group and MMP-8 group, respectively, with a significant overall difference among them ( F=156.63, P<0.01).The corneal hydroxyproline content was lower in MMP-8 group compared to buffer and normal saline groups, and the differences were statistically significant (all at P<0.05).In the experiment involving the infection of cultured Fusarium spores with human neutrophils, the fluorescence intensity of MMP-8 expression was significantly higher in AICAR-treated group than in negative control group and compound C-treated group, with statistically significant differences (all at P<0.05). Conclusions:The MMP-8 secreted by neutrophils in mice with fungal keratitis can degrade corneal stromal collagen fibers, leading to corneal opacification or perforation.The variations in MMP-8 protein expression levels in human neutrophils may be associated with AMPK activation.

Purpose To determine whether Activated 5'AMP-activated protein kinase (AMPK) activation enhances Fusarium solani (F. solani) fungicidal capacity of neutrophils.Methods The expression of AMPK and phosphorylated-AMPK (p-AMPK) proteins was tested using Western Blot. Plate counting studied the effects of the fungicidal capacity of neutrophils enhanced by AMPK activation. Phagocytized spores by neutrophils were assessed by immunostaining and inhibited hyphal growth images were captured by JULI Stage real-time cell history recorder. Flow cytometry assay tested Reactive Oxygen Species (ROS) production and the percentage of apoptosis neutrophils. ROS Assay Kit also tested ROS production at different time points. The F. solani keratitis murine model was established, and slit-lamp microscopy captured corneal photographs.Results Our experiments were divided into the following groups. Neutrophils (N), neutrophils + spores (N + S), neutrophils + spores+ 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (N + S + A), neutrophils + spores + Compound C (N + S + C). AMPK activator AICAR significantly increased the expression of p-AMPK in neutrophils. The plate counting experiment showed that the number of colonies in the N + S + A was significantly less than in the N + S group. Immunostaining results showed phagocytized spores were significantly increased in the N + S + A group compared with the N + S group. Captured photographs by a real-time cell history recorder camera showed F. solani hyphal growth in the N + S + A group was significantly inhibited than in the N + S group. ROS release in the N + S + A group was significantly higher in the N + S + A group than in other groups. The percentage of apoptosis neutrophils in the N + S + A group was decreased than in the N + S group. Captured photographs by slit-lamp showed that AICAR eye drop treatment alleviated the severity and decreased clinical score at 12 and 24 hours post-infection (h.p.i)Conclusion AMPK activation enhances the efficacy of neutrophils in killing F. solani in vitro and in vivo.

Objectie To investigate the susceptibility of drug-resistant staphylococci isolated from different parts of the anterior segment to levofloxacin, tobramycin, cefazolin sodium, fusidic acid and clindamycin. Methods: Experimental Study. A total of 67 patients with anterior segment infection (33 cases of conjunctivitis, 6 cases of bacterial keratitis, 7 cases of blepharitis, 9 cases of neonatal dacryocystitis, 9 cases of neonatal dacryocystitis, 1 case of adult dacryocystitis and 11 cases of other infectious eye diseases) were collected from the conjunctival sac, cornea, eyelid margin and lacrimal sac. Minimum inhibitory concentration (MIC) determination of methicillin-resistant Staphylococcus (MRS) strains and β-lactamase-producing (β-Lac) strains by a micro-liquid-based method, according to the M100 standard of the American Institute for Clinical and Laboratory Standardization Susceptibility and resistance determinations were made. Data were statistically analyzed using Chi-square or Fisher's exact test. Results: Thirty-five MRS, 30 β-Lac and 2 β-Lac MRS isolates were identified from 67 multidrug-resistant Staphylococcus . There were 3, 9, 4, and 19 MRS isolates isolated from the lacrimal sac, cornea, eyelid margin and conjunctival sac, accounting for 3/4, 9/12, 4/8, 19/43 (44.2%) of the isolated sites respectively. There were 1, 3, 3, and 23 β-Lac isolates, accounting for 1/4, 3/12, 3/8 and 23/43 (53.5%) of the isolated sites, respectively. The highest proportion of β-Lac isolates isolated from patients with a diagnosis of conjunctivitis was 17 (25.3%) from the conjunctival sac. Among the MRS strains isolated from the cornea and lacrimal sac, 5 (7.5%) and 3 (4.5%) were from patients diagnosed with bacterial keratitis and neonatal tear, respectively. The number of MRS strains and β-Lac isolates isolated from patients with a diagnosis of blepharitis were both 3 (4.5%) from the lid margin.Among the strains isolated from the eyelid margin and the conjunctival sac, drug-resistant Staphylococcus epidermidis was the main strain, the drug-resistant Staphylococcus aureus was the major isolates in lacrimal sac and cornea. Among the 35 MRS isoaltes, 25, 24, 12, 12, and 11 were sensitive to cefazolin sodium, fusidic acid, levofloxacin, clindamycin and tobramycin, and the sensitivity rates were 71.4%, 68.6%, 34.3%, 34.3% and 31.4%, the difference was statistically significant (χ2=22.756, P<0.001), The sensitivity rates of levofloxacin, tobramycin, cefazolin sodium, fusidic acid and clindamycin against MRS isolates from the anterior segment were both statistically significant differences (χ2=18.493, 11.594, 8.906, 9.841, 16.059; all P<0.05). The susceptibility rates of MRS isolates against five antibiotics was statistically significant differences (χ2=33.080, P<0.001). Among the 30 β-Lac isolates, 27, 22, 19, 16, and 8 were sensitive to cefazolin sodium, fusidic acid, levofloxacin, tobramycin and clindamycin, and the sensitivity rates were 90.0 % , 73.3%, 63.3%, 53.3% and 26.7%, the difference was statistically significant (χ2=28.280, P<0.001). The sensitivity rates of five antibiotics against β-Lac isolates from the anterior segment were both statistically significant differences (χ2=50.971, 24.543, 48.147, 44.899, 18.676; all P<0.001). The susceptibility rates of β-Lac isolates against five antibiotics was statistically significant differences (χ2=23.383, P<0.001). The sensitivity of cefazolin sodium and fusidic acid against β-Lac isolates were higher than MRS isolates. Conclusions: Cefazolin sodium and fusidic acid may be the best choice for the treatment of drug-resistant Staphylococcus isolated from anterior conjunctival sac, cornea, eyelid margin and lacrimal sac, especially for β-Lac-producing drug-resistant Staphylococcus infection.

Objective:To study the antimicrobial activity of diacerein on common pathogens of the ocular surface in vitro.Methods:Pathogens were collected from patients with ocular surface infections in Henan Eye Hospital, including Gram-positive cocci and bacilli, Gram-negative bacilli, filamentous fungi, and Candida.The antimicrobial activity of diacerein was determined by the K-B agar diffusion method, and its minimum inhibitory concentration (MIC) was determined by the micro-liquid method.Levofloxacin and voriconazole were used as the control of antibacterial and antifungal drug, respectirely.Results:Diacerein showed antibacterial activity against 42 strains of Gram-positive cocci and 10 strains of Gram-positive bacilli, its inhibition zone diameters for Staphylococcus epidermidis, S.aureus, S.intermedius and Gram-positive Corynebacterium were not significantly different from those of levofloxacin (all at P>0.05). Its MIC range of diacetate against Staphylococcus epidermidis, S. aureus, S. intermedius and other Staphylococci was 1-32 μg/ml, and its respective MIC 90 was 16, 8, 16, and 32 μg/ml.Diacerein had no bacteriostatic effect on 23 strains of Gram-negative bacilli, 10 strains of filamentous fungi and 3 strains of candida. Conclusions:Diacerein has antibacterial effects against Gram-positive Staphylococcus and Corynebacterium isolated from the ocular surface, but shows no antimicrobial activity against Gram-negative bacilli and fungi.Diacerein offers a new drug option and method for the treatment of bacterial keratitis.

Objective To study the effects of platelets on macrophages phagocytosis and inhibition of fungi. Methods Macrophages were cultured, Fusarium Pythium spores were extracted and platelets were isolated from blood of mouse.Simple spore group, spore+ macrophage group and mixed platelet group were set, and were inoculated with fungal spore, equal proportion spore+ macrophage and platelet+ spore+ macrophage, respectively.The prepared plate was placed on a spinning disk laser scanning confocal microscope at 1 hour, 2, 3 and 4 hours after culture, five visual fields were randomly selected at the corresponding time points for photography.The phagocytic rate, phagocytic index and fungal spore germination rate were calculated.Fungal hyphae length of each group at 4, 6 and 8 hours after culture were calculated.The single macrophage group, spore+ macrophage group and mixed platelet group were set and the cytotoxicity was measured by real-time cell analyzer.The breeding and use of mice was in according with the ARVO statement.This study was approved by the Experimental Animal Ethics Committee of Henan Eye Institute (HNEECA-2017-04). Results The phagocytic rate and phagocytic index of macrophages in mixed platelet group at 1 hour, 2, 3 and 4 hours after culture were significantly higher than those in spore+ macrophage group at corresponding time point (all at P 0.05). There were significant differences in cell index among different groups at 30, 36, 42 and 48 hours after culture (F=10.81, 8.08, 5.61, 5.72; all at P<0.05). The cell indexes in spore+ macrophage group at different time points were significantly lower than those in simple macrophage group (all at P<0.01). Conclusions Platelets can promote the phagocytosis and inhibition of macrophages on fungi, and platelets may have antagonistic effect on fungal cytotoxicity. Key words: Keratitis/pathology; Macrophage; Fungi; Platelets; Phagocytosis; Germination rate

Bacterial keratitis is an aggressive infectious corneal disease. With the continuing rise in antibiotic resistance and a decline in the discovery of new antibiotics, new antimicrobial drugs are now required. In the present study, we determined the antibacterial activity of diacerein, an anti-inflammatory drug, against 76 Gram-positive cocci isolated from bacterial keratitis patients in vitro and anti-Staphylococcus aureus activity in a mouse bacterial keratitis model in vivo The MICs of diacerein were tested using the broth microdilution method in vitro A BALB/c Staphylococcus aureus keratitis animal model was selected and the corneal clinical observation, viable bacteria, and hematoxylin-eosin and Gram staining of infected corneas were measured to evaluate the antibacterial efficacy of diacerein eye drops in vivo An in vivo eye irritation study was carried out by a modified Draize test in rabbits. Our in vitro results showed that diacerein possesses satisfactory antibacterial activity against the majority of Gram-positive cocci (60/76), including all 57 tested Staphylococcus spp. and 3 Enterococcus spp. The in vivo experiment showed that diacerein eye drops reduced bacterial load and improved ocular clinical scores after topical administration of diacerein drops on infected corneas. The ocular irritation test revealed that diacerein eye drop had excellent ocular tolerance. These results indicated that diacerein possesses in vivo anti-Staphylococcus aureus activity. We suggest that diacerein is a possible topically administered drug for Staphylococcus aureus-infected patients, especially those with ocular surface inflammatory disorders.

目的 探讨双醋瑞因减轻小鼠角膜碱烧伤炎症反应的作用.方法 将174只小鼠角膜碱烧伤造模成功的小鼠随机分为三组:双醋瑞因组、地塞米松组和生理盐水组,分别给予双醋瑞因、地塞米松、生理盐水滴眼制剂滴眼,比较造模后2d、3d、4d、5d各组小鼠的角膜混浊度、病灶面积、上皮缺损面积及炎症细胞的浸润情况.结果 双醋瑞因组造模后4d、5d角膜临床评分明显较生理盐水组低(均为P＜0.01),地塞米松组造模后3d、4d、5d角膜临床评分较生理盐水组低(均为P＜0.05).对于角膜上皮缺损而言,双醋瑞因组碱烧伤后各时间点角膜上皮缺损面积均小于生理盐水组(均为P ＜0.05),地塞米松组与生理盐水组角膜上皮缺损无明显差异.对角膜中性粒细胞进行统计发现,双醋瑞因组碱烧伤后各时间点中性粒细胞计量明显少于生理盐水组(均为P＜0.01);地塞米松组造模后3d、5d中性粒细胞浸润少于生理盐水组(均为P＜0.05).对巨噬细胞进行统计发现,双醋瑞因组造模后3d、5d巨噬细胞均较生理盐水组浸润少(均为P＜0.05);造模后5d地塞米松组浸润少于生理盐水组(P＜0.05).通过MPO活性测定发现,双醋瑞因组和地塞米松组造模后2d、3d、5dMPO活性均较生理盐水组明显降低(均为P＜0.01).结论 双醋瑞因能够减轻小鼠角膜碱烧伤的炎症反应,改善角膜碱烧伤的预后.

目的 探究高糖饮食对小鼠真菌性角膜炎的影响.方法 选取健康无眼疾的雄性C57BL/6J小鼠78只,随机分为高糖饮食组和模型对照组,每组36只,模型对照组给予正常饮用水,高糖饮食组给予含体积分数10％果糖溶液,每2d测量两组小鼠体质量及血糖,10 d后建立真菌性角膜炎模型.造模后24h、36 h、48 h、72 h、96 h、120 h、168 h裂隙灯显微镜下对角膜进行临床评分并拍照.处死小鼠后,取角膜组织进行HE染色和PAS染色;测定角膜内中性粒细胞和巨噬细胞浸润体积.利用酶联免疫吸附实验对小鼠角膜内的白细胞介素-1β含量进行测定.结果 造模后0～14 d,两组小鼠体质量与血糖差异均无统计学意义(均为P＞0.05).造模后24h、36 h、48 h、120 h、168 h,高糖饮食组小鼠角膜临床评分均明显高于模型对照组,差异均有统计学意义(均为P＜0.05).高糖饮食组小鼠角膜穿孔率79.5％,高于模型对照组的40.9％,差异有统计学意义(P =0.000).造模后各时间点,高糖饮食组中性粒细胞浸润体积均高于模型对照组,差异均有统计学意义(均为P =0.000).造模后72 h、96h、120 h、168 h,高糖饮食组巨噬细胞浸润体积均高于模型对照组(均为P=0.000).角膜组织病理学检查结果示,高糖饮食组炎症反应更重,角膜组织破坏更早且更为严重.造模后24h、48h高糖饮食组白细胞介素-1β含量均明显高于模型对照组(均为P＜0.05).结论 高糖饮食加重了真菌性角膜炎感染的严重程度,增强了中性粒细胞、巨噬细胞的趋化,促进了IL-1β的分泌.