白介素1受体相关激酶1(IRAK1)和NF-κB在苯扎氯铵诱导的干眼症小鼠角膜和结膜组织中的表达

Expression of IRAK1 and NF-κB in keratoconjunctival tissue of mice with dry eye syndrome induced by benzalkonium chloride

目的探讨白介素-1受体相关激酶1(interleukin-1 receptor-associated kinase 1,IRAK1)和NF-κB在苯扎氯铵诱导的干眼症小鼠角膜和结膜组织中的表达。方法选取6～8周龄SPF级雄性C57BL/6小鼠60只。将小鼠分为对照组(15只)和干眼症模型组(45只)。向干眼症模型组小鼠双眼各滴入5μL浓度为0.75 g·L^(-1)的苯扎氯铵溶液,每天2次,持续7 d,诱导小鼠干眼模型;对照组小鼠不做处理。酚红棉线法测定泪液分泌量,分析各组小鼠各时间泪膜破裂时间,并行角膜和结膜HE染色。qRT-PCR检测角膜和结膜组织IL-1β、IL-6、IRAK1和NF-κB mRNA表达水平。免疫荧光染色检测IRAK1和NF-κB的表达和定位。结果干眼症模型组小鼠实验造模后1周和2周时,酚红棉线湿长降低,与对照组相比,差异均有统计学意义(均为P<0.05)。此外,干眼症小鼠泪液分泌量在造模后1周和2周增加了1.2～1.3倍,与对照组相比差异均有统计学意义(均为P<0.05)。HE染色结果显示,与对照组小鼠相比,干眼症模型组小鼠结膜上皮细胞数增加,细胞排列欠整齐,同时伴有缺损。对照组角膜上皮细胞、基质胶原纤维排列整齐。干眼症模型组小鼠结膜组织IL-6 mRNA表达水平在造模后2周高于对照组,但差异无统计学意义(P>0.05);4周时表达量比对照组增加了1倍,差异有统计学意义(P<0.05);干眼症模型组小鼠角膜组织中IL-6 mRNA表达量在造模后2周高于对照组,差异有统计学意义(P<0.01)。干眼症模型组小鼠结膜组织中IL-1βmRNA在造模后1周和2周均高于对照组,差异均有统计学意义(均为P<0.05);4周时下降,与对照组相比差异无统计学意义(P>0.05);而角膜组织中IL-1βmRNA在造模后1～4周内均高于对照组,差异均有统计学意义(P<0.05)。干眼症模型组小鼠角膜组织中IRAK1 mRNA的表达水平在造模后1周和2周时均高于对照组,差异均有统计学意义(均为P<0.05),4周时降低;而结膜组织中IRAK1 mRNA表达水平在造模后1周和2周时与对照组相比无明显变化(均为P>0.05),4周时低于对照组,差异有统计学意义(P<0.05)。干眼症小鼠角膜组织中NF-κB mRNA表达水平在造模后1周和2周时均高于对照组,差异均有统计学意义(均为P<0.05),4周时降低;而结膜组织中NF-κB mRNA表达水平仅在造模后2周时高于对照组,差异有统计学意义(P<0.01)。结论 IRAK1的表达随着干眼症病程动态变化,IRAK1可能通过促进NF-κB的核转移参与对干眼症炎症进程的调控。

Objective To investigate the expression of interleukin-1 receptor-associated kinase 1 (IRAK1) and nuclear factor κB (NF-κB) in corneal and conjunctival tissues of mice with dry eye syndrome induced by benzalkonium chloride. Methods Sixty SPF male C57BL/6 mice aged 6-8 weeks were selected.The mice were divided into a control group (15 mice) and a dry eye model group (45 mice).The mice in dry eye model group were injected with 5 μL of benzalkonium chloride solution at a concentration of 0.75 g·L^-1 to each eye twice a day for 7 days to induce dry eye symptoms.The mice in control group received no treatment.The amount of tear secretion was determined by phenol red cotton thread method.The tear film break-up time of each group was analyzed and HE staining of cornea and conjunctiva was performed.qRT-PCR was used to detect interleukin 1β(IL-1β),interleukin 1β(IL-6),IRAK1 and NF-κB mRNA in the cornea and conjunctiva.Immunofluorescence staining was used to detect the expression and localization of IRAK1 and NF-κB. Results The wet length of phenol red cotton line decreased at 1 week and 2 weeks after the establishment of dry eye model,which was significantly lower compared with that of the control group ( both P <0.05).In addition,the amount of tear secretion in dry eye mice increased by 1.2 to 1.3 times at 1 week and 2 weeks after modeling,and the difference was statistically significant in comparison with that of the control group (both P <0.05).HE staining showed that compared with the control group,the number of conjunctival epithelial cells in the dry eye model group increased and the cells were irregularly aligned,accompanied by structural defects.While the corneal epithelial cells and matrix collagen fibers of the control mice were arranged neatly.The expression level of IL-6 mRNA in the conjunctiva of the dry eye model group was higher than that of the control group at 2 weeks after model establishment,but their difference was not statistically significant ( P >0.05).At 4 weeks,the expression level of IL-6 mRNA increased by one fold over that of the control group with significant difference indicated ( P <0.05).The expression of IL-6 mRNA in the corneal tissue of the dry eye model group was significantly higher than that of the control group 2 weeks after model establishment ( P <0.01).The expression of IL-1β mRNA in the conjunctival tissue of the dry eye model group was significantly higher than that of the control group at 1 week and 2 weeks after model establishment (both P < 0.05) and it decreased at 4 weeks without significant difference ( P >0.05).The IL-1β mRNA in corneal tissue was significantly higher than that in the control group 1-4 weeks after model establishment ( P <0.05).The expression level of IRAK1 mRNA in the corneal tissue of the dry eye model group was significantly higher than that of the control group at 1 week and 2 weeks after model establishment (both P <0.05),and decreased at 4 weeks.However,the expression level of IRAK1 mRNA in conjunctival tissue did not change significantly at 1 week and 2 weeks after model establishment (both P >0.05),and was significantly lower than that of the control group at 4 weeks ( P <0.05).The expression of NF-κB mRNA in corneal tissue of dry eye mice was significantly higher than that of the control group at 1 week and 2 weeks after model establishment (both P <0.05),and decreased at 4 weeks.The expression level of NF-κB mRNA in conjunctival tissue was significantly higher than that of the control group only at 2 weeks after model establishment ( P <0.01). Conclusion The expression of IRAK1 has dynamic changes with the course of dry eye disease.IRAK1 may participate in the regulation of inflammatory process of dry eye by promoting the nuclear transfer of NF-κB.