α-黑素细胞刺激素联合羧甲基纤维素钠对干眼大鼠眼表的保护作用

摘要

背景 研究表明,α-黑素细胞刺激素(α-MSH)具有抗炎作用,可缓解氢溴酸东莨菪碱诱导的大鼠干眼模型的眼表异常,而人工泪液羧甲基纤维素钠(CMC)能够润滑干眼眼表,但α-MSH能否在大鼠干眼模型中促进人工泪液CMC的治疗效果尚有待进一步研究. 目的 探讨α-MSH联合CMC对氢溴酸东莨菪碱诱导的大鼠干眼模型中眼表的保护作用. 方法 选取清洁级雌性Wistar大鼠60只,采用随机数字表法随机分为正常对照组、模型对照组、NaCl点眼组、CMC点眼组、α-MSH点眼组和α-MSH+CMC点眼组,每组10只.除正常对照组外,其他各组大鼠分别于每日9:00、12:00、15:00、18:00接受0.5 ml(6 mg/ml)氢溴酸东莨菪碱皮下注射,共28 d,以诱导干眼模型,NaCl点眼组、CMC点眼组、α-MSH点眼组和α-MSH+CMC点眼组于注射后第1天分别用质量分数0.9％ NaCl溶液、质量分数0.5％ CMC滴眼液、1×10-3 mg/ml α-MSH溶液、1×10-3 mg/ml α-MSH+0.5％ CMC混合液点右眼,每日2次(8:00和17:00).各组大鼠分别于用药后0、7、14、21和28 d行Shirmer试验Ⅰ检测(SⅠt)、泪膜破裂时间(BUT)检测、角膜上皮荧光素染色评分,并于用药后28 d完成检查后处死,取包含上下眼睑的整个大鼠眼球,取角膜组织行苏木精-伊红染色,光学显微镜下观察大鼠角膜的形态学变化,大鼠结膜组织行过碘酸希夫染色,光学显微镜下观察各组大鼠结膜杯状细胞的数量.结果 随着造模后时间的延长,大鼠SⅠt值和BUT值逐渐下降,角膜荧光素染色评分逐渐增加,造模后各时间点模型对照组与正常对照组比较,差异均有统计学意义(均P＜0.01).大鼠用药后7、14和21和28 d,NaCl点眼组SⅠt值、BUT值和角膜荧光素染色评分与模型对照组比较,差异均无统计学意义(均P＞0.05).用药后7、14和21 d,α-MSH+CMC点眼组大鼠SⅠt值分别为(4.800±0.789)、(4.100±0.516)和(4.300±0.856) mm,明显高于相应时间点NaCl点眼组的(2.875±0.719)、(2.375±0.619)和(2.532±0.957) mm,差异均有统计学意义(均P＜0.01).用药后7d,α-MSH点眼组和α-MSH+CMC点眼组的BUT值分别为(4.938±1.843)s和(5.000±1.491)s,明显高于NaCl点眼组的(3.250±1.000)s,差异均有统计学意义(均P＜0.01).用药后各时间点CMC点眼组、α-MSH点眼组和α-MSH+CMC点眼组大鼠角膜荧光素染色评分均明显低于NaCl点眼组,差异均有统计学意义(均P＜0.05),以α-MSH+CMC点眼组角膜荧光素染色评分最低.苏木精-伊红染色显示,模型对照组和NaCl点眼组大鼠出现角膜上皮层增厚、角膜水肿、角膜上皮粗糙和角膜基质层纤维排列紊乱,α-MSH+CMC点眼组大鼠角膜水肿和上皮细胞的增生轻微,角膜形态接近正常对照组.大鼠结膜组织过碘酸希夫染色显示,CMC点眼组、α-MSH点眼组和α-MSH+CMC点眼组大鼠结膜杯状细胞数量明显多于模型对照组和NaCl点眼组(均P＜0.01). 结论 单独使用o-MSH和CMC可一定程度上改善干眼大鼠的角膜组织损伤、角膜形态学异常以及结膜杯状细胞数量的减少,但相对于单独用药,α-MSH和CMC联合应用在造模后早期对改善干眼模型大鼠泪液分泌量和泪膜稳定性作用更明显.%Background Reasearches showed that α-melanocyte-stimulating hormone (α-MSH) inhibits inflammation and ameliorates the ocular surface abnormalities in a scopolamine-induced dry eye rat model,and the managing effect of sodium carboxymethylcellulose (CMC) on dry eyes also has been determined.However,whether α-MSH can enhance the therapeutic effects of CMC remains to be investigated.Objective This study was to investigate the protective effects of α-MSH combined with CMC on ocular surface in a scopolamine-induced dry eye rat model.Methods Sixty clean female Wistar rats were randomly divided into normal control group,model control group,NaCl group,CMC group,α-MSH group and α-MSH+CMC group,and 10 rats for each group.The dry eye models were established by subcutaneous injection of scopolamine hydrobromide at 9:00,12:00,15:00 and 18:00 per day for 28 days.0.9％ NaCl solution,1×10 3 mg/ml α-MSH solution,0.5％ CMC eye drop,and 1 ×10-3 mg/ml α-MSH+0.5％ CMC solution were topically administered twice a day (8:00,17:00) since the initial day of modeling according to grouping.Shirmer Ⅰ test (S Ⅰ t),breakup time of tear film (BUT) and corneal fluorescence staining were performed before and 7,14,21,28 days after the application of drugs.At 28 days following the administration of drugs,the eyeballs of the rats were collected.Hemotoxylin and eosin staining was employed to examine the morphology of corneas,and periodic acid schiff (PAS) staining was used to count the conjunctival goblet cells.This study protocol was approved by Experimental Animal Ethic Committee of Tianjin Medical University (SYXK 2009-0001),and the use and care of the rats complied with ARVO Statement.Results The S Ⅰ t and BUT values were significantly reduced,and the corneal fluorescence staining scores were significantly increased over time following modeling in the model control group (all at P＜0.01).No significant differences were found in the S Ⅰ t,BUT and corneal fluorescence staining scores between model control group and NaCl group at various time points (all at P＞0.05).At 7,14 and 21 days after intervention,the S Ⅰ t values were (4.800±0.789),(4.100±0.516) and (4.300±0.856) mm in the α-MSH+CMC group,which were considerably higher than (2.875 ±0.719),(2.375 ±0.619) and (2.532±0.957)mm in the NaCl group (all at P＜0.01).At 7 days after intervention,the BUT values were (4.938± 1.843) seconds and (5.000±1.491) seconds in the α-MSH group and α-MSH+CMC group,which were significantly higher than (3.250±1.000) seconds in the NaCl group (both at P＜0.01).The corneal fluorescence staining scores in the CMC group,α-MSH group and α-MSH+CMC group were significantly lower than that in the NaCl group,with the lowest score in the α-MSH +CMC group (all at P＜0.05).The thickening of corneal epithelial layer,corneal edema and arrangement disorder of corneal stroma were found in the model control group and NaCl group;while slight corneal edema and epithelial cell proliferation were exhibited in the α-MSH+CMC group by hemotoxylin and eosin staining.PAS staining showed that the number of goblet cells was much more in the CMC group,α-MSH group and α-MSH+ CMC group than that in the model control group and NaCl group (all at P ＜ 0.01).Conclusions The sole application of α-MSH or CMC alleviates ocular surface damage and morphological abnormality to certain extent,and the combination of α-MSH and CMC generates more effective protection in comparison with sole administration of α-MSH or CMC.The early application of the drugs plays an improvement role in tear secretion and tear film stability in dry eyes.