麦冬多糖对烟曲霉菌性角膜炎小鼠炎症反应的影响

Effects of Ophiopogon japonicus polysaccharides on inflammatory response in mice with Aspergillus fumigatus keratitis

目的探讨麦冬多糖(OJP)对烟曲霉菌性角膜炎(AFK)小鼠的治疗作用及机制。方法通过对小鼠角膜注射烟曲霉菌(AF)悬浮液建立AFK模型,将54只建模成功的AFK小鼠随机分为5组:模型组(n=11)、低剂量组(n=11)、中剂量组(n=11)、高剂量组(n=11)和激动剂组(n=10);取未建模的小鼠作为对照组(n=10)。低剂量组、中剂量组和高剂量组小鼠分别用50μL浓度为5,10,20 g/L的OJP溶液滴眼。激动剂组小鼠用50μL浓度为20 g/L的OJP溶液滴眼,同时用0.25 mg/kg的Toll样受体4(TLR4)激动剂CRX-527溶液灌胃。对照组和模型组小鼠分别用50μL生理盐水滴眼。各组小鼠均给药1周。治疗结束24 h后,在裂隙灯显微镜下进行角膜炎症评分。采用苏木素伊红(HE)染色观察角膜形态;采用ELISA法检测角膜组织上清液中促炎因子白介素(IL)-1β、肿瘤坏死因子-α(TNF-α)、IL-6水平;采用qRT-PCR分析角膜IL-1β、TNF-α、IL-6、TLR4、髓样分化因子88(MyD88)、核因子-κB(NF-κB)p65的mRNA水平;采用Western blot分析角膜TLR4、MyD88、NF-κB p65蛋白相对表达。结果与对照组比较,模型组大鼠的角膜炎症评分升高(P<0.05),角膜出现明显病变,角膜IL-1β、TNF-α、IL-6蛋白表达和mRNA水平均升高(P<0.05),角膜TLR4、MyD88和NF-κB p65的mRNA水平和蛋白表达均升高(P<0.05)。与模型组比较,低剂量组、中剂量组和高剂量组大鼠的角膜炎症评分降低(P<0.05),角膜病变减轻,角膜IL-1β、TNF-α、IL-6蛋白表达和mRNA水平均降低(P<0.05),角膜TLR4、MyD88和NF-κB p65的mRNA水平和蛋白表达均降低(P<0.05)。与低剂量组比较,中剂量组和高剂量组大鼠的角膜炎症评分降低(P<0.05),角膜病变减轻,角膜IL-1β、TNF-α、IL-6蛋白表达和mRNA水平均降低(P<0.05),角膜TLR4、MyD88和NF-κB p65的mRNA水平和蛋白表达均降低(P<0.05)。与高剂量组比较,激动剂组大鼠的角膜炎症评分升高(P<0.05),角膜病变加重,角膜IL-1β、TNF-α、IL-6蛋白表达和mRNA水平均升高(P<0.05),角膜TLR4、MyD88和NF-κB p65的mRNA水平和蛋白表达均升高(P<0.05)。结论OJP可能通过抑制TLR4/MyD88/NF-κB信号通路减轻AFK小鼠角膜炎症损伤。

Objective To investigate the therapeutic effect and its mechanism of Ophiopogon japonicus polysaccharides(OJP)on Aspergillus fumigatus(AF)-induced keratitis(AFK)in mice.Methods The animal model of AFK was established by injecting AF suspension into the cornea of mice.Fifty-four AFK mice were randomly divided into 5 groups:model group(n=11),low dose group(n=11),medium dose group(n=11),high dose group(n=11)and agonist group(n=10).The unmodeled mice were selected as control group(n=10).Mice in low dose group,medium dose group and high dose group were given 50μL eyedrops of 5,10,20 g/L OJP solution.Mice in agonist group were given 50μL eyedrops of 20 g/L OJP solution and gavaged with 0.25 mg/kg Toll-like receptor 4(TLR4)activator CRX-527 solution.Mice in control group and model group were given 50μL normal saline as eyedrops.All mice in each group were treated for one week.At 24 h after treatment,the corneal inflammation was scored under slit lamp microscope.Corneal morphology was observed by hematoxylin eosin(HE)staining.The levels of pro-inflammatory factors interleukin(IL)-1β,tumor necrosis factor-α(TNF-α)and IL-6 in the supernatant of corneal tissue were detected by ELISA method.The mRNA levels of IL-1β,TNF-α,IL-6,TLR4,myeloid differentiation factor 88(MyD88)and nuclear factor kappa B(NF-κB)p65 in cornea were analyzed by qRT-PCR.The relative levels of TLR4,MyD88 and NF-κB p65(nucleus)proteins in cornea were analyzed by Western blot.Results Compared with control group,the corneal inflammation score was increased in model group(P<0.05),the corneal lesions were obvious,the protein and mRNA levels of IL-1β,TNF-α,IL-6 in corneal were increased(P<0.05),and the mRNA and protein levels of TLR4,MyD88,NF-κB p65 in corneal were increased(P<0.05).Compared with model group,the corneal inflammation score was decreased in low dose group,medium dose group and high dose group(P<0.05),the corneal lesions were improved,the protein and mRNA levels of IL-1β,TNF-α,IL-6 in corneal were decreased(P<0.05),and the mRNA and protein levels of TLR4,MyD88,NF-κB p65 in corneal were decreased(P<0.05).Compared with low dose group,the corneal inflammation score was decreased in medium dose group and high dose group(P<0.05),the corneal lesions were alleviated,the protein and mRNA levels of IL-1β,TNF-α,IL-6 in corneal were decreased(P<0.05),and the mRNA and protein levels of TLR4,MyD88,NF-κB p65 in corneal were decreased(P<0.05).Compared with high dose group,the corneal inflammation score was increased in agonist group(P<0.05),the corneal lesions were worsened,the protein and mRNA levels of IL-1β,TNF-α,IL-6 in corneal were increased(P<0.05),and the mRNA and protein levels of TLR4,MyD88,NF-κB p65 in corneal were increased(P<0.05).Conclusion OJP can partly attenuate the corneal inflammatory injury in AFK mice by inhibiting TLR4/MyD88/NF-κB signaling pathway.