目的探讨复方野菊花眼贴对蓝光诱导小鼠睑板腺功能异常的防治作用及其机制。
方法将64只15周龄雄性C57BL/6J小鼠按照随机数字表法分成2个部分，每部分32只，分别进行预防实验和治疗实验。预防实验和治疗实验各32只小鼠分别按照随机数字随机分为正常组、蓝光组、溶剂组和眼贴组，每组8只。预防实验中，除正常组外，各组小鼠采用波长460 nm、光照度2 000 lx的蓝光，每天暴露6 h，连续暴露15 d建立小鼠睑板腺功能异常模型；溶剂组和眼贴组每天在蓝光暴露前和暴露后，分别用相对应眼贴敷贴预防25 min，连续15 d，蓝光组仅接受蓝光暴露15 d处理，并于第15天行睑板腺开口照相观察小鼠睑板腺功能。治疗实验中，除正常组外，各组小鼠均采用上述方法建立小鼠睑板腺功能异常模型，溶剂组和眼贴组在结束15 d的蓝光暴露后，每天早上和下午分别用相对应眼贴敷贴治疗25 min，连续15 d，蓝光组置于标准环境中观察15 d，并在第15天行睑板腺开口照相观察小鼠睑板腺功能变化。预防实验及治疗实验的各组小鼠在处理完成后进行离体睑板腺拍照、油红O染色、苏木精－伊红染色观察小鼠睑板腺组织学变化；采用实时荧光定量PCR法检测各组睑板腺组织中炎性因子白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)mRNA相对表达量；采用Western blot法检测睑板腺组织中核因子(NF)-κB和磷酸化NF-κB(p-NF-κB)蛋白的表达，评估复方野菊花眼贴对蓝光诱导小鼠睑板腺炎症的改善程度。
结果与正常组相比，蓝光组暴露后15 d小鼠睑板腺开口阻塞数量逐渐增加，睑板腺下睑相对剩余面积逐渐减小，差异均有统计学意义(均 P<0.05)。预防实验中，眼贴组睑板腺开口阻塞数量为1.833±0.753，明显少于溶剂组的3.667±1.033，眼贴组睑板腺下睑相对剩余面积为0.718±0.091，明显大于溶剂组的0.624±0.130，差异均有统计学意义(均 P<0.05)。苏木精－伊红染色结果显示，蓝光组和溶剂组睑板腺有炎性细胞浸润；眼贴组无炎性细胞浸润，腺泡形态接近正常组；油红O染色结果显示各组睑板腺均无明显脂质沉积。眼贴组IL-1β、IL-6、TNF-α、IFN-γ mRNA相对表达量明显低于溶剂组，眼贴组NF-κB、p-NF-κB蛋白相对表达量明显低于溶剂组，差异均有统计学意义(均 P<0.05)。治疗实验中，眼贴组和溶剂组的睑板腺开口阻塞数量分别为4.333±1.211和4.833±1.722，睑板腺下睑相对剩余面积分别为0.572±0.151和0.588±0.154，2个组比较差异均无统计学意义(均 P>0.05)。苏木精－伊红染色结果显示，蓝光组和溶剂组睑板腺有炎性细胞浸润，眼贴组无炎性细胞浸润，腺泡形态接近正常组；油红O染色结果显示各组睑板腺均无明显脂质沉积。眼贴组IL-1β、IL-6、IFN-γ mRNA相对表达量明显低于溶剂组，眼贴组NF-κB、p-NF-κB蛋白相对表达量明显低于溶剂组，差异均有统计学意义(均 P<0.05)。
结论复方野菊花眼贴可通过抑制NF-κB信号通路减轻睑板腺组织炎症反应，对蓝光诱导的睑板腺功能异常具有防治作用。
ObjectiveTo investigate the preventive and therapeutic effects of compound wild chrysanthemum eye pad on blue light-induced alteration of meibomian gland function in mice and its mechanism.
MethodsSixty-four 15-week-old male C57BL/6J mice were divided into two groups of 32 mice each according to random numbers for the prevention test and the treatment test.The respective 32 mice in the prevention and treatment experiments were randomly divided into normal group, blue light group, solvent group and eye pad group according to random numbers, with eight mice in each group, respectively.In the prevention experiments, mice in each group were exposed to blue light at a wavelength of 460 nm and a light intensity of 2 000 lx for 6 hours per day for 15 consecutive days to establish a mouse model of meibomian gland function changes except for the normal group.The solvent group and the eye pad group were treated with the corresponding eye pad before and after the blue light exposure for 25 minutes daily for the 15 consecutive days.The blue light group was treated with blue light exposure only for 15 days, and the mice were photographed at the edge of the meibomian gland on day 15 to observe the function of the meibomian gland except for the normal group.In the treatment test, all groups of mice except the normal group were induced the altered function of the mouse meibomian gland by the above method.The solvent and eye pad groups were treated with corresponding eye pads for 25 minutes in the morning and afternoon of each day for 15 consecutive days after blue light exposure.The blue light group was kept in a standard environment for 15 days and the changes in meibomian gland function of mice were detected by meibomian gland photographs on day 15.Photography of the eyelid margin in vitro, oil red O staining, and hematoxylin-eosin staining were performed to observe the histologic changes in the meibomian glands of mice after the preventive and experimental treatment.The relative expression of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) mRNA in mouse meibomian gland tissues was detected by real-time fluorescence quantitative PCR.The expression of nuclear factor-κB (NF-κB) and phosphorylation of NF-κB (p-NF-κB) proteins in mice meibomian gland tissues was detected by Western blot to assess the degree of amelioration of blue light-induced inflammation in mouse meibomian glands by the compound wild chrysanthemum eye pad.This study was conducted in accordance with the Statement of the Association for Research in Vision and Ophthalmology on the Use of Animals in Ophthalmology and Vision Research, and was approved by the Animal Ethics Committee of Xiamen University (No.XMULAC20220258).
ResultsCompared with the normal group, a gradually increased number of blocked meibomian gland openings, and a gradually decreased remaining area of lower meibomian gland, were observed in the mice after 15 days of blue light group, and all the differences were statistically different (all at P<0.05). In the prevention test, the number of obstructed opening in the eye pad group was 1.833±0.753, which was significantly less than 3.667±1.033 in the solvent group ( P<0.05). The relative remaining area of the lower lid meibomian gland in the eye pad group was 0.718±0.091, which was significantly greater than 0.624±0.130 in the solvent group ( P<0.05). Hematoxylin-eosin staining showed inflammatory cell infiltration in mouse meibomian gland in the blue light and solvent groups.There was no inflammatory cell infiltration in eye pad group, and the morphology of the acini was similar to that of the normal group.Oil red O staining showed that there was no significant lipid deposition in the groups.The relative expressions of IL-1β, IL-6, TNF-α, and IFN-γ mRNA were significantly lower, and the relative expressions of NF-κB and p-NF-κB proteins were significantly lower in the eye pad group than in the solvent group, showing statistically significant differences (all at P<0.05). In the treatment test, the number of obstructed openings in the eye pad group and solvent group was 4.333±1.211 and 4.833±1.722, respectively, and the relative remaining area of the lower meibomian gland was 0.572±0.151 and 0.588±0.154, respectively, showing no statistically significant differences (both at P>0.05). Hematoxylin-eosin staining showed inflammatory cell infiltration in mouse meibomian glands in the blue light and solvent groups, with a similar morphology of acini as in the normal group.There was no inflammatory cell infiltration in eye pad group.Oil red O staining showed that there was no significant lipid deposition in the groups.The relative expressions of IL-1β, IL-6, and IFN-γ mRNA were significantly lower and the relative expressions of NF-κB and p-NF-κB proteins were significantly lower in the eye pad group than in the solvent group (all at P<0.05).
ConclusionsCompound wild chrysanthemum eye pad may have preventive and therapeutic effects on blue light-induced changes in meibomian gland function by reducing the inflammatory response of meibomian gland tissue through the inhibition of the NF-κB signaling pathway.
李勇,黄彩虹,李清坚,等. 复方野菊花眼贴对蓝光诱导小鼠睑板腺功能异常的防治作用及其机制[J]. 中华实验眼科杂志,2024,42(02)：117-128.
DOI:10.3760/cma.j.cn115989-20230217-00055版权归中华医学会所有。
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李勇：参与研究设计、研究实施、收集数据、分析/解释数据、起草文章、论文修改；黄彩虹、李清坚、王玉倩：实施研究、收集数据、分析/解释数据；吕雨霏、张兆强、胡皎月：参与研究选题、分析/解释数据、起草文章；刘祖国：参与研究设计、论文内容修改和审核、最终定稿
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