大鼠烟曲霉菌性角膜炎角膜局部的免疫学研究

目的探讨大鼠角膜真菌感染后角膜局部的免疫反应机制。方法模拟角膜外伤制作大鼠的烟曲霉菌性角膜炎模型，在感染后不同阶段进行病理切片和免疫组化观察角膜局部中性粒细胞、总T淋巴细胞及T淋巴细胞亚群、树突状细胞、B淋巴细胞的动态变化。结果在大鼠烟曲霉菌性角膜炎早期，见大量中性粒细胞及少量CD3^＋T、CD4^＋T、CD8^＋T淋巴细胞和LC浸润，未检测到B淋巴细胞。中期，中性粒细胞浸润达高峰，T淋巴细胞浸润增加，其中CD4^＋T淋巴细胞多于CD8^＋T淋巴细胞。树突状细胞亦增多，B淋巴细胞开始出现。晚期，中性粒细胞数目有所减少，T淋巴细胞浸润达高峰，CD4^＋T淋巴细胞亦多于CD8^＋T淋巴细胞，B淋巴细胞少量表达。结论免疫反应与真菌性角膜炎的病程变化有密切的关系。     

烟曲霉菌性角膜炎大鼠角膜IL-6和IL-10的表达

目的 探讨大鼠角膜真菌感染后角膜局部细胞因子的表达.方法 在24只大鼠左眼角膜上制作烟曲霉菌性角膜炎(AFK)模型为实验组,右眼为对照组.在感染后早、中、晚期取下角膜组织,采用逆转录聚合酶链式反应(RT-PCR),检测白细胞介素-6(IL-6)、白细胞介素-10(IL-10)在不同病程角膜组织中的表达.结果 在实验组角膜中,IL-6早期即大量表达,中期炎症反应高峰时达高峰,以后逐渐下降;IL-10在早期表达最高以后逐渐下降.结论 IL-6在AFK病程中是一个敏感的炎性因子,与局部的炎症反应程度一致.IL-10在AFK病程中呈逐渐下降趋势.     

大黄素对烟曲霉菌性角膜炎模型大鼠的抗炎作用机制研究

目的：研究大黄素对烟曲霉菌性角膜炎模型大鼠的抗炎作用机制。

方法：建立烟曲霉菌性角膜炎模型,分为模型组和大黄素组,各8只,正常组10只。大黄素组采取大黄治疗,模型组和正常组采取等体积生理盐水治疗。观察大鼠角膜炎症指数、病理学特征,检测肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、细胞间黏附分子-1(ICAM-1)、过氧化物酶体增殖物激活受体(PPAR)、MAPK、NF-κB蛋白。

结果：大黄素组大鼠角膜炎症指数、角膜炎症细胞计数、TNF-α、IL-6、ICAM-1水平、MAPK、NF-κB蛋白低于模型组,PPAR蛋白表达高于模型组(P<0.05)。

结论：大黄素通过调控PPAR、MAPK、NF-κB蛋白,改善TNF-α、IL-6、ICAM-1炎症因子水平,对烟曲霉菌性角膜炎大鼠起到抗炎作用。     

大鼠烟曲霉菌性角膜炎角膜局部细胞间黏附分子-1的表达及其与局部炎症反应的关系

目的探讨大鼠角膜真菌感染后角膜局部细胞间黏附分子-1(ICAM-1)的表达及其与局部炎症反应的关系。设计实验研究。研究对象烟曲霉菌感染后的大鼠角膜。方法各组大鼠左眼为实验眼,右眼为对照眼。在建模后早期(1~3天)、中期(4~7天)、晚期(10~14天)三个不同阶段取下角膜组织,采用逆转录聚合酶链式反应(RT-PCR)检测ICAM-1在不同病程角膜组织中的表达。应用免疫组化及HE染色的方法,观察不同病程角膜组织中中性粒细胞、总T淋巴细胞的动态变化。主要指标ICAM-1及中性粒细胞、总T淋巴细胞。结果 ICAM-1早期开始表达(0.58±0.21),中期达高峰(1.22±0.12),至晚期时仍有少量表达(0.94±0.07)。这一变化趋势与角膜局部中性粒细胞的变化一致。总T淋巴细胞在病程中逐渐增多,病程后期达高峰。ICAM-1的表达在实验对照组与实验组早、中、晚期组四组间差异均有统计学意义(P均=0.000)。结论 ICAM-1参与了角膜烟曲霉菌性感染后角膜局部的炎症反应。     

血管内皮生长因子及其受体在大鼠碱烧伤后角膜新生血管中的表达

目的:探讨血管内皮生长因子(VEGF)及其受体Flk-1在大鼠角膜碱烧伤后新生血管中的表达。方法:应用1mol/LNaOH制作大鼠碱烧伤新生血管模型,随机分为3组,分别于1,4,7及14d处死大鼠取出角膜,免疫组织化学方法和RT-PCR方法检测大鼠角膜中的VEGF及其受体Flk-1的表达。结果:正常大鼠角膜中VEGF及Flk-1受体弱表达或不表达。二者均在角膜上皮层,基质层、内皮层及新生血管内皮细胞表达;大鼠角膜碱烧伤后各时间点VEGFmRNA和Flk-1mRNA相对表达量具有相关性。结论:大鼠角膜碱烧伤后,VEGF及其受体Flk-1结合诱导内皮细胞分化及角膜新生血管形成。     

The Role of SREC-Ⅰ in Innate Immunity to Aspergillus fumigatus Keratitis

PurposeTo determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-Ⅰ) in vitro and in a mouse model of Aspergillus fumigatus keratitis.MethodsSREC-Ⅰ mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-Ⅰ expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-Ⅰ small interfering RNA, then the mRNA levels of LOX-1, IL-1β, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-Ⅰ mRNA and protein expression were detected by RT-PCR, Western blot and immunofluorescence. The severity of FK was evaluated by clinical score. CLCX1, LOX-1, IL-1β, and TNF-α mRNA expression levels were tested before and after anti–SREC-Ⅰ treatment.ResultsSREC-Ⅰ expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-Ⅰ mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-Ⅰ small interfering RNA treatment inhibited the expressions of LOX-1, IL-1β, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1β, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-Ⅰ–neutralizing antibody treatment.ConclusionsSREC-Ⅰ is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-Ⅰ blockade could be a potential therapeutic approach for FK.     

Regulation of lipoxygenase-1 and Dectin-1 on interleukin-10 in mouse Aspergillus fumigatus keratitis

AIM: To investigate the regulation of lipoxygenase (LOX)-1 and Dectin-1 on interleukin-10 (IL-10) production in mice with Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: The corneas of C57BL/6 mice were pretreated with LOX-1 inhibitor Poly(I) or Dectin-1 siRNA separately before the infection of A. fumigatus. Polymerase chain reaction (PCR) and Western blot were used to detect the expression of IL-10. RESULTS: The mRNA and protein expressions of IL-10 were significantly increased in mice with A. fumigatus keratitis. Compared with the group pretreated with sterile water before infection, Poly(I) pretreatment suppressed IL-10 expression significantly. Compared with the group pretreated with scrambled siRNA before infection, Dectin-1 siRNA pretreatment significantly reduced IL-10 expression in response to A. fumigatus infection. CONCLUSION: LOX-1 and Dectin-1 regulate IL-10 production in mouse A. fumigatus keratitis.     

Inhibition of LOX-1 alleviates the proinflammatory effects of high-mobility group box 1 in Aspergillus fumigatus keratitis

AIM: To investigate the inflammatory amplification effect of high-mobility group box 1 (HMGB1) in Aspergillus fumigatus (A. fumigatus) keratitis and the relationship between lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and HMGB1 in keratitis immune responses. METHODS: Phosphate buffer saline (PBS), and Boxb were injected into BALB/c mice subconjunctivally before the corneas were infected with A. fumigatus. RAW264.7 macrophages and neutrophils were pretreated with PBS and Boxb to determine the HMGB1 inflammatory amplification effects. Abdominal cavity extracted macrophages were pretreated with Boxb and Poly (I) (a LOX-1 inhibitor) before A. fumigatus hyphae stimulation to prove the the relationship between the two molecules. LOX-1, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2) and IL-10 were assessed by polymerase chain reaction and Western blot. RESULTS: Pretreatment with Boxb exacerbated corneal inflammation. In macrophages and neutrophils, A. fumigatus induced LOX-1, IL-1β, TNF-α and MIP-2 expression in Boxb group was higher than those in PBS group. Poly (I) treatments before infection alleviated the proinflammatory effects of Boxb in abdominal cavity extracted macrophages. Pretreatment with Boxb did not influence Dectin-1 mRNA levels in macrophages and neutrophils. CONCLUSION: In fungal keratitis, HMGB1 is a proinflammatory factor in the first line of immune response. HMGB1 mainly stimulates neutrophils and macrophages to produce inflammatory cytokines and chemokines during the immune response. LOX-1 participates in HMGB1 induced inflammatory exacerbation in A. fumigatus keratitis.     

Expression of macrophage migration inhibitory factor in Aspergillus fumigatus keratitis

AIM: To investigate the expression of macrophage migration inhibitory factor (MIF) and detect its role in the innate immune response of fungal keratitis (FK). METHODS: We collected the paraffin-embedded cornea tissues from 10 FK and 6 ocular trauma patients to explore the MIF expression by immunohistochemistry. Then we cultured telomease-immortalized human corneal epithelial cells (THCEs), stimulated by the hyphae suspension of Aspergillus fumigatus (A. fumigatus) to detect the change of MIF with or without the pretreatment of MIF inhibitor [4-Iodo-6-phenylpyrimidine (4-IPP)] by real-time polymerase chain reaction (PCR). The protein level of MIF was also tested by immunohistochemistry, and the level of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) mRNA were compared between normal, hyphae stimulated and 4-IPP pretreated groups by real-time PCR to study the influence of MIF on the expression of TNF-α and IL-6. Corneal severity of rats’ FK models was documented by clinical scores, and real-time PCR. Western blot and immunohistochemistry were used to test the expression of MIF, TNF-α and IL-6 in rats’ corneas. RESULTS: In the corneas of FK patients, there was much stronger expression of MIF than that in the normal group showed by immunohistochemistry. In cultured THCEs stimulated by A. fumigatus, the expression of MIF became stronger in both immunohistochemistry and PCR at 16, 24, 32 and 48h post infection (p.i.; P<0.01, P<0.01, P<0.01, P<0.05). After pretreated with 4-IPP, the expression of MIF reduced at 4, 8, 16h p.i. (P<0.05, P<0.05, P<0.05) and the downstream TNF-α and IL-6 decreased obviously (P<0.05, P<0.01). In rats with A. fumigatus keratitis, the relative mRNA and protein level of MIF increased than those in the normal group by PCR (at 1d: P<0.01, 3d: P<0.01, 5d: P<0.01), Western blot and immunohistochemistry. After blocked MIF with 4-IPP, the clinical outcomes of rat keratitis showed markedly reduced inflammatory response (P<0.01), with TNF-α and IL-6 decreased in accordance with those in THCEs by PCR (P<0.05, P<0.01). CONCLUSION: The expression of MIF increased significantly in FK patients, THCEs and rats stimulated by A. fumigatus. After blocked with 4-IPP, the expression of MIF reduced, and so did its downstream cytokines: TNF-α and IL-6. The inflammation reaction of the rats’ corneas lightened after pretreated with 4-IPP. MIF may play a role in the innate immune response of the corneal resistance against A. fumigatus.     

两性霉素B缓释系统治疗兔烟曲霉菌眼内炎的药效学实验研究

目的探讨两性霉素B缓释系统（AmB—DDS）玻璃体腔植入对烟曲霉菌性眼内炎的疗效、AmB-DDS的药物释放规律及最佳释药量。方法选取40只新西兰白兔作为实验动物。（1）Amb-DDS治疗烟曲霉菌性眼内炎疗效学观察：动物玻璃体腔内注入烟曲霉菌悬液,48h后随机分为5组,A组为空白对照组（6只眼）,B组为空白DDS组（6只眼）,C组为两性霉素B玻璃体腔内注射组（6只眼）,D组为AmB—DDS250μg植入联合玻璃体切除术组（8只眼）,E组为AmB—DDS500μg植入联合玻璃体切除术组（8只眼）。术后不同时间点检测前房闪辉、细胞及玻璃体混浊程度,取玻璃体腔内容物行涂片检查和真菌培养,2个月时取眼球标本行病理学检查;（2）AmB—DDS玻璃体腔内药物浓度检测：H组玻璃体切除术后植入500μg AmB—DDS1个（6只眼）,术后第1、3、7天及2、4、6、8周取玻璃体液,高效液相色谱分析法检测药物浓度。结果A、B组全部发生严重眼内炎,伴眶内感染,组间比较差异无统计学意义（P〉0．05）;C、D、E组炎性反应较A、B组轻,差异有统计学意义（P≤0．005）;E组玻璃体混浊程度较C组轻,7～14d前房反应较C组轻,差异均有统计学意义（P≤0．005）;D组5只眼、E组8只眼治愈,差异有统计学意义（x^2=10．494,P=0．003）。不同时间点取玻璃体腔内容物涂片,所有标本6周内均见菌丝,真菌培养仅A、B组为阳性。病理学检查示治愈眼结构正常,感染未控制眼均萎缩,球壁结构被破坏。H组术后第1天即有释药,药物浓度迅速升高超出有效抑菌浓度,观察期内释药较平稳。结论AmB—DDS玻璃体腔内植入治疗烟曲霉菌性眼内炎安全有效,释药恒定,速率得当;以含药量为500μg的AmB—DDS治疗效果最佳。（中华腰科杂志,2007,43：546-553）     

NADPH oxidase 2 plays a protective role in experimental Aspergillus fumigatus keratitis in mice through killing fungi and limiting the degree of inflammation

AIM: To explore whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) is expressed in fungal keratitis in mice and investigate its role in this disease. METHODS: NOX2 expression was detected in C57BL/6 mice. After testing the inhibitory effect of diphenyleneiodonium chloride (DPI) on NOX2, its impact on clinical performance, myeloperoxidase levels, the number of colonies forming units, the level of H3, the generation of reactive oxygen species (ROS) and the release of cytokines [NF-κB, interleukin-17A (IL-17A), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), Nrf2, IL-10, and TGF-β] were compared. A one-way ANOVA and an unpaired, two-tailed Student’s t-test was used to determine the statistical significance. RESULTS: NOX2 expression was significantly increased after Aspergillus fumigatus injection in corneas and that this increase could be reduced by treatment with DPI. DPI treatment produced more severe inflammation and resulted in higher clinical scores, more neutrophils infiltration, a weakened ability to clear fungi, the release of fewer ROS and the formation of neutrophil extracellular traps. Treatment with DPI increased the expression of the proinflammatory cytokines NF-κB, IL-17A, IL-6, and TNF-α and decreased the expression of the anti-inflammatory cytokines Nrf2, IL-10 and TGF-β compared to their expression levels without DPI treatment. CONCLUSION: NOX2 plays an important role against Aspergillus fumigatus in the mouse cornea through killing fungi and limiting the degree of inflammation.