ry score of astaxanthin in the treatment of fungal keratitis in mice

图1. 虾青素治疗小鼠真菌性角膜炎的临床表现及炎症评分

3.2. ASX在基因水平抑制烟曲霉诱导的真菌性角膜炎小鼠角膜中炎性细胞因子的产生

在真菌性角膜炎小鼠模型角膜中,于处理后第3天检测ASX治疗组与对照组的IL-1β、IL-6和TNF-α的mRNA的表达(图2A-C)。PCR结果显示,在第3天,ASX治疗组的IL-1β、IL-6和TNF-α的表达水平在基因水平显著低于DMSO组。提示ASX可以抑制烟曲霉诱导的真菌性角膜炎小鼠模型中炎性细胞因子的产生。

3.3. ASX在蛋白水平抑制烟曲霉诱导的真菌性角膜炎小鼠角膜中炎性细胞因子的产生

在真菌性角膜炎小鼠模型角膜中,于处理后第3天检测ASX治疗组与对照组的IL-1β、IL-6和TNF-α的mRNA的表达(图3A-C)。ELISA结果显示,在第3天,ASX治疗组的IL-1β、IL-6和TNF-α的表达水平在蛋白水平显著低于DMSO对照组。提示ASX可以抑制烟曲霉诱导的真菌性角膜炎小鼠模型中炎性细胞因子的产生。

注:A-C.虾青素治疗组在感染后第3天,较DMSO处理组相比,IL-1β、IL-6和TNF-α的相对表达显著下调。(bP < 0.01)。

Figure 2. The mRNA relative expression of inflammatory factors (IL-1β, IL-6 and TNF-α) in the mouse model of fungal keratitis induced by Aspergillus fumigatus

图2. 烟曲霉菌诱导的真菌性角膜炎小鼠模型中炎症因子(IL-1β、IL-6和TNF-α)的mRNA的相对表达

注:A-C. ELISA结果显示,虾青素处理组IL-1β、TNF-α和HMGB-1蛋白水平在感染后第3天明显低于DMSO处理组。(bP < 0.01) (N = 正常组;DMSO = DMSO组;ASX = 虾青素组)。

Figure 3. The expression of inflammatory factors (IL-1β, IL-6 and TNF-α) in the mouse model of fungal keratitis induced by Aspergillus fumigatus in protein level

图3. 烟曲霉菌诱导的真菌性角膜炎小鼠模型中炎症因子(IL-1β、IL-6和TNF-α)的蛋白水平的表达

3.4. ASX在基因水平抑制烟曲霉菌感染的HCECs中的炎症因子的上调

烟曲霉菌处理HCECs细胞后8 h,我们设置了6个不同处理条件的组,来排除药物溶剂DMSO对细胞的影响,结果示,正常组及加菌组和加了DMSO处理的正常组及加菌组相比,结果无统计学意义(图4A-C),说明了药物溶质DMSO对实验结果无统计学意义的影响。烟曲霉菌处理的HCECs中IL-1β、IL-6和TNF-α都较正常组有显著升高,说明烟曲霉菌诱导了HCECs中炎症的发生,而ASX处理组的相关因子与单纯加菌组相比较有着显著的下调(图4A-C),说明了ASX在人角膜上皮细胞中抑制烟曲霉诱导的真菌性角膜炎小鼠模型中炎性细胞因子的产生,抑制炎症表达。

3.5. ASX在蛋白水平抑制HCECs中因烟曲霉菌而上调的炎性细胞因子

细胞系的ELISA实验结果在蛋白水平检测了炎症因子的表达水平,表明了ASX能够下调因烟曲霉菌诱导而升高的相关炎症因子(图5A-C),进一步验证了ASX在人角膜上皮细胞中抑制烟曲霉诱导的真菌性角膜炎小鼠模型中炎性细胞因子蛋白水平的表达,抑制炎症。

注:A.加真菌后8 h后各组HCECs中QT-PCR检测炎症因子IL-1β,IL-6和TNF-α的表达。(bP < 0.01) (Normal = 正常组;N + D = 正常HCEC细胞 + DMSO;ASX = 虾青素处理组;AF = 烟曲霉菌组;AF + D = 烟曲霉菌 + DMSO;AF + ASX = 烟曲霉菌 + 虾青素)。

Figure 4. Detection of the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) by QT-PCR in HCECs

图4. HCECs中QT-PCR检测炎症因子 (IL-1β、IL-6和TNF-α)的表达

注:A-C.加真菌后24 h后各组HCECs中ELISA检测炎症因子IL-1β、IL-6和TNF-α的表达。(bP < 0.01) (Normal = 正常组;N + D = 正常HCEC细胞 + DMSO;ASX = 虾青素处理组;AF = 烟曲霉菌组;AF + D = 烟曲霉菌 + DMSO;AF + ASX = 烟曲霉菌 + 虾青素)。

Figure 5. Detection of the expression of inflammatory factors (IL-1β, IL-6 and TNF-α) by ELISA in HCECs

图5. HCECs中ELISA检测炎症因子(IL-1β、IL-6和TNF-α)的表达

4. 讨论

真菌性角膜炎(真菌性角膜炎)是眼科学临床方面面临的巨大挑战 [25]。而烟曲霉菌是世界范围内真菌性角膜炎的主要病原之一,感染后往往会导致视力损害甚至失明 [26]。

通常来说,炎症是机体的一种抗病反应,对机体往往是有利的,例如炎性充血,能使组织得到更多的氧、营养物质等,使得表面组织代谢和抗击力增加;渗出液能稀释毒素,其中所含的抗体能清除病原体并中和毒素;渗出的纤维蛋白元凝结而成假膜,设立成一道遮蔽,能阻止病原菌向深部蔓延;渗出物中的中性白细胞和巨噬细胞能吞噬病原体及坏死崩解的细胞碎屑;炎症部位的浆细胞和淋巴细胞能产生抗体中和毒素 [5]。

但是,我们要从两个方面来分析炎症。炎症反应中的某些利于因素,在一定条件下,可以向着各自相反的方向转化而变成对机体有害的因素 [27]。真菌性角膜炎即使及时给予适当的治疗,临床疗效也往往不佳,其中一个最重要的原因是过度的免疫反应,它会招募过量的各种炎性细胞因子和免疫细胞,导致角膜组织蛋白质沉淀,降低角膜透明度。所以在临床治疗过程中,在合适的时机联合用药是治疗真菌性角膜炎的最佳方案 [28]。因此,控制真菌性角膜炎中晚期的炎症反应是非常关键的,因为在真菌性角膜炎中晚期,过度炎症反应引起的组织损伤大于其保护作用,甚至在联合用药的情况下,早期控制炎症对角膜的保护都是积极的 [29]。

本篇研究探讨ASX在真菌性角膜炎中的抗炎作用。ASX是一种具有强抗氧化性的类胡萝卜素,研究表明它在肝炎、胰腺炎、皮炎、神经病变、乳腺炎和葡萄膜炎等多种急慢性疾病中都能起到重要的生物学作用。我们的实验结果发现,ASX改善了烟曲霉菌诱导的小鼠真菌性角膜炎的临床表现,降低了临床炎症评分,提高了角膜透明度,提示ASX在真菌性角膜炎病程中对角膜组织起保护作用。

炎性细胞因子如IL-1β、IL-6和TNF-α是反映炎症反应严重程度的重要指标,是真菌性角膜炎中最重要的炎症因子,在炎症过程中起着至关重要的作用 [6]。它们激活免疫系统,趋化及活化巨噬细胞中性粒细胞及其他炎症因子,在真菌性角膜炎中,这些作用往往会破化角膜基质细胞,影响内皮细胞的正常功能,从而导致了角膜透明度的损失 [30] [31]。而减轻炎症的过程,就是保护角膜透明度的过程。大量研究表明ASX能够抑制各种急慢性炎症中炎症因子的表达。Suzuki等的研究表明ASX可通过阻断NF-kB依赖性信号通路抑制葡萄膜炎大鼠IL-1β、IL-6和TNF-α的表达 [17]。Zhou L.等在小鼠败血症模型中发现ASX通过下调炎症因子如IL-1β和TNF-α来防止组织损伤 [32]。这些结论说明ASX能够在体内外抑制真菌性角膜炎中IL-1β和TNF-α的高表达,提示ASX在真菌性角膜炎的病理过程中对角膜组织起到保护作用。

而我们的实验结果表明,不仅在细胞实验中,而且在动物模型中,ASX处理组的炎症因子(IL-1β、IL-6和TNF-α)都较对照组在基因和蛋白水平都有明显的下调。这些结果都提示ASX在真菌性角膜炎病理过程中同样能发挥抗炎作用,可作为一种有潜力的治疗真菌性角膜炎的药物,甚至可考虑与其他药物联合治疗,发挥有效保护角膜组织,防止角膜组织细胞的过度损坏,保护角膜透明度的作用,在临床治疗中可考虑进一步实验和应用。

5. 结论

总而言之,ASX在各种疾病中发挥了积极的生物学作用。越来越多的研究数据也表明了ASX在各种急慢性炎症疾病中能有效地发挥抗炎作用,保护机体正常的组织结构,本实验的研究结果也表明了ASX有成为真菌性角膜炎新型抗炎药物的潜力,而关于ASX对真菌性角膜炎的治疗的具体作用机制则需要进一步的研究。

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