托珠单抗调控角膜碱烧伤后修复的研究OA
Study on the regulation of tocilizumab on corneal repair after alkali burns
目的:评估外用白介素(Interleukin,IL)-6特异性抑制剂托珠单抗滴眼液在调控角膜碱烧伤后修复的安全性和有效性.方法:6只角膜假烧伤小鼠局部使用托珠单抗滴眼(2.5 mg/mL)和6只角膜假烧伤小鼠局部使用生理盐水滴眼,分别作为实验组和空白组以评估托珠单抗滴眼液的安全性.30只碱烧伤小鼠,按照1∶1随机分配到治疗组和对照组,治疗组使用托珠单抗滴眼液滴眼,对照组使用生理盐水,每日6次,连续用14 d.通过前段光学相干断层扫描观察虹膜前粘连、角膜后弹力层脱离及角膜水肿,在体视显微镜下检查角膜瘢痕形成及上皮伤口愈合.在角膜切片上评估IL-6定位、肌成纤维细胞、免疫细胞浸润和角膜上皮化生.在角膜铺片上评估角膜新生血管和新生淋巴管面积.通过实时荧光定量聚合酶链式反应(Quantitative Real-time Polymerase Chain Reaction,qRT-PCR)方法检测小鼠角膜 IL-6的表达水平.结果:对未进行碱烧伤的角膜使用托珠单抗治疗未观察到明显的角膜结构的损伤.角膜碱烧伤后可见角膜结构的破坏,角膜瘢痕形成并伴有角膜上皮伤口的延迟愈合.使用托珠单抗治疗后,虹膜前粘连的发生率从86.67%下降至20%(P<0.01),角膜后弹力层脱离的发生率从93.33%下降至53.33%(P<0.05),角膜厚度小于对照组[(100.03±15.73)μm vs.(207.02±56.30)μm,P＜0.001],角膜混浊评分从对照组的3.76±0.44下降到治疗组的1.94±0.83(P<0.001),治疗组在第5天(P<0.05)、第10天(P<0.001)和第14天(P<0.001)的上皮愈合率高于对照组.角膜碱烧伤后可见IL-6大量分布于角膜全层,且可见大量肌成纤维细胞形成及免疫细胞浸润,托珠单抗治疗后抑制了IL-6的表达(下降77.5%,P<0.05),肌成纤维细胞数量从每视野(91.44±65.60)个减少至(12.89±10.51)个(P<0.01),免疫细胞的数量从每视野(60.30±28.71)个细胞减少至每视野(6.80±3.82)个细胞(P<0.001).此外,托珠单抗还减少角膜切片中每视野的杯状细胞数目,由(11.30±5.29)个减少至(2.00±1.90个)(P<0.01),并减少角膜新生血管和新生淋巴管的形成(分别减少了76.86%和71.16%,均P<0.001).结论:局部使用托珠单抗抑制IL-6未见明显角膜毒性,且可以调控角膜碱烧伤后的修复.
Objective:To evaluate the safety and effect of topical IL-6 inhibitor tocilizumab eye drops in regulating corneal alkali burn repair.Methods:Six mice without corneal burns were locally treated with tocilizumab eye drops(2.5 mg/mL)and six mice with corneal pseudo burn were treated with saline,respectively,as experimental and blank groups to evaluate the safety of tocilizumab eye drops.30 alkali burned mice were randomly divided into a treatment group and a control group in a 1:1 ratio.The treatment group received tocilizumab eye drops,while the control group received physiological saline solution 6 times per day for 14 days.Observe the anterior adhesion of the iris,detachment of the Descemet membrane,and corneal edema through anterior segment optical coherence tomography(AS-OCT),and examine corneal scarring and epithelial wound healing under a stereomicroscope.Evaluate IL-6 localization,myofibroblasts,immune cell infiltration,and corneal epithelial metaplasia on corneal sections.Evaluate corneal neovascularization and neovascularization area by whole-mount cornea staining.Detect the expression level of IL-6 in mouse cornea by qRT-PCR.Results:No significant damage to the corneal structure was observed in the treatment of unburned corneas with tocilizumab.After corneal alkali burns,the corneal structure was damaged,corneal scarring was formed,and delayed healing of corneal epithelial wounds was observed.After treatment with tocilizumab,the incidence of anterior synechia of the iris significantly decreased from 86.67%to 20%(P<0.01),the incidence of Descemet membrane detachment decreased from 93.33%to 53.33%(P<0.05),the corneal thickness was significantly less than that of the control group(100.03±15.73)μm vs.(207.02±56.30 μm)(P<0.001),the corneal opacity score decreased from 3.76±0.44 in the control group to 1.94±0.83 in the treatment group(P<0.001),and the epithelial healing rate in the treatment group was significantly higher than that in the control group on day 5(P<0.05),day 10(P<0.001),and day 14(P<0.001).After corneal alkali burns,IL-6 was distributed throughout the corneal layer,and a large number of myofibroblasts and immune cells were observed.After treatment with tocilizumab,the expression of IL-6 was inhibited(decreased by 77.5%,P<0.05),the number of myofibroblasts decreased from(91.44±65.60)per field to(12.89±10.51)per field(P<0.01),and the number of immune cells decreased from(60.30±28.71 cells)per field to 6.80±3.82 cells per field(P<0.001).In addition,tocilizumab also reduced the number of goblet cells per field in corneal sections(from 11.30±5.29 to 2.00±1.90)(P<0.01),and reduced the formation of corneal neovascularization and neovascular lymphatic vessels(by 76.86%and 71.16%,respectively,both P<0.001).Conclusion:Topical use of tocilizumab to inhibit IL-6 showed no significant corneal toxicity and can regulate the repair of cornea after alkali burns.
张祺恩;王佳琦;何嘉瑜;卓晴晴;袁钊辉
中山大学中山眼科中心,眼病防治全国重点实验室,广东省眼科视觉科学重点实验室,广州 510060
临床医学
角膜碱烧伤白介素-6托珠单抗
corneal alkali burninterleukin-6tocilizumab
《眼科学报》 2024 (010)
489-500 / 12
广州市科技项目(202102080039).This work was supported by grants from the Science and Technology Project of Guangzhou Municipal(202102080039).
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