枸杞糖肽对角膜碱烧伤小鼠角膜炎症反应及愈合的作用及其机制研究

Effects and mechanisms of lycium barbarum glycopeptide on corneal inflammation and healing in alkali-burned mice

摘要:

目的 探讨枸杞糖肽（LbGp）在角膜碱烧伤损伤愈合中的作用及其机制。方法 根据治疗方式不同将45只健康、SPF级、6～8周、雄性C57BL/6J小鼠随机分为正常对照组（N组）、损伤对照组（PBS组）、LbGp治疗组（LbGp组），每组15只。PBS组和LbGp组小鼠右眼复制碱烧伤模型，采用点眼联合结膜下注射的方式分别给予PBS溶液或LbGp溶液（10 mg/mL）治疗。治疗后第3天采用裂隙灯显微镜及HE染色观察各组小鼠角膜上皮修复情况和角膜组织结构；实时荧光定量聚合酶链反应、Western blotting及免疫组织化学染色技术检测各组小鼠角膜组织NOD样受体热蛋白结构域相关蛋白3（NLRP3）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、髓过氧化物酶（MPO）的表达及核转录因子κB（NF-κB）的磷酸化水平。治疗后的第14天采用裂隙灯显微镜对角膜混浊程度进行评分，HE染色观察角膜组织结构。结果 治疗后第3天LbGp组小鼠角膜上皮愈合速度快于PBS组（P <0.05）。N组、PBS组及LbGp组的NF-κB-p65、NLRP3、IL-1β及IL-6基因和蛋白相对表达量比较，差异均有统计学意义（P <0.05），PBS组均高于N组（P <0.05），LbGp组均低于PBS组（P <0.05）。LbGp组角膜基质中MPO染色阳性的中性粒细胞的浸润较PBS组减轻。治疗后第14天LbGp组小鼠角膜混浊评分低于PBS组（P <0.05）。结论 LbGp治疗可以抑制小鼠角膜碱烧伤后NF-κB的激活及NLRP3、IL-1β的表达，从而减轻过度的炎症反应，促进小鼠角膜上皮再生及角膜结构恢复，有助于恢复角膜的透明性和完整性。

Abstract:

Objective To investigate the effect and mechanism of lycium barbarum polysaccharide (LbGp) in corneal alkali burn healing.Methods Forty-five healthy SPF-grade male C57BL/6J mice aged 6-8 weeks were randomly divided into normal control group (N), injury control group (PBS) and LbGp treatment group (LbGp), with 15 mice in each group. In the PBS and LbGp groups, mice were subjected to alkali burn injury in the right eye, and then treated with PBS solution or LbGp solution (10 mg/mL) via eye drops and subconjunctival injection, respectively. Three days after treatment, the corneal epithelial repair and corneal tissue structure were observed by slit lamp microscope and HE staining. The expression levels of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), interleukin-1β (IL-1β), interleukin-6 (IL-6), myeloperoxidase (MPO) and the phosphorylation levels of nuclear factor kappa B (NF-κB) in corneal tissues of each group were detected using qRT-PCR, Western blotting, and immunohistochemistry. At 14 days after treatment, the degree of corneal opacity was scored with a slit lamp microscope; the corneal tissue structure was observed by HE staining.Results On the third day after treatment, the corneal epithelial healing rate of LbGp group was significantly faster than that of PBS group. The comparison of genes and proteins relative expression levels of NF-κB-p65, NLRP3, IL-1β, and IL-6 among the N group, PBS group, and LbGp group revealed statistically significant differences (P <0.05). The expression levels were higher in the PBS group than in the N group (P <0.05), while they were lower in the LbGp group compared to the PBS group (P <0.05). Furthermore, the infiltration of MPO-positive neutrophils in the corneal stroma of the LbGp group was lower than that of the PBS group. On day 14 after treatment, the corneal opacity score in the LbGp group was significantly lower than that in the PBS group (P < 0.05).Conclusion LbGp treatment can inhibit the activation of NF-κB and expression of NLRP3 and IL-1β after corneal alkali burn in mice, inhibit excessive inflammatory response, and promote the regeneration of corneal epithelium and the recovery of corneal structure, which is conducive to the restoration of corneal transparency and integrity.