摘要： 目的 基于ERK1/p38MAPK通路观察杞参方对高渗诱导的角膜上皮细胞（HCECs）的保护作用。方法 通过500 mOsm/L浓度渗透压作用于HCECs制造干眼细胞模型，将空白血清、玻璃酸钠滴眼液、杞参方颗粒高、中、低剂量含药血清作用于干眼模型HCECs。采用CCK-8法检测HCECs的存活率，ELISA法检测细胞外液炎性细胞因子白细胞介素（IL）-1β、IL-8的表达，ICC法检测凋亡因子caspase-3的蛋白表达，Western blotting法检测HCECs的ERK1、p38MAPK及磷酸化蛋白表达情况。结果 与空白组相比，各组HCECs存活率均显著降低（P<0.01），各组细胞外液炎性细胞因子IL-1β、IL-8及HCECs中caspase-3、p-ERK1、p-p38MAPK蛋白表达水平显著增高（P<0.01）；与模型组相比，各用药组HCECs存活率显著增加（P<0.01），各用药组IL-1β和IL-8、西药组和杞参方高、中剂量组caspase-3和p-ERK1及杞参方高、中剂量组p-p38MAPK蛋白表达减少（P<0.05）；与西药组相比，杞参方高剂量组HCECs存活率显著增加（P<0.01），杞参方高剂量组的IL-1β、IL-8、caspase-3、p-ERK1、p-p38MAPK与杞参方中剂量组的IL-1β、p-ERK1、p-p38MAPK及杞参方低剂量组的IL-8蛋白表达减少（P<0.05）。结论 杞参方颗粒可降低高渗诱导HCECs的ERK1、p38MAPK的磷酸化蛋白表达，降低细胞外液炎性细胞因子IL-1β、IL-8和HCECs凋亡因子caspase-3的表达，最终有效抑制炎症反应和细胞凋亡。

杞参方对高渗诱导的干眼模型角膜上皮细胞保护机制的研究

赵磊^1,2, 杨笑薇¹, 左韬², 姜艳华³, 周慧敏¹

1. 辽宁中医药大学研究生学院, 沈阳 110032;
2. 辽宁中医药大学附属第二医院眼科, 沈阳 110034;
3. 沈阳市第四人民医院眼科, 沈阳 110031

收稿日期:2021-08-25出版日期:2022-01-30发布日期:2021-12-30
通讯作者:左韬E-mail:ykzt208@163.com
作者简介:赵磊(1988-),男,主治医师,博士.
基金资助:国家自然科学基金（82104714）；辽宁省自然科学基金（20170540557）


关键词: 干眼, 杞参方, 角膜上皮细胞, 细胞外调节蛋白激酶1, p38丝裂原活化蛋白激酶
Abstract: Objective To observe the protective effects of different formulas composed of varying Qishen prescriptions on human corneal epithelial cells (HCECs) treated with hypertonic fluid. Methods The osmotic pressure of 500 mOsm/L was applied to HCECs to create a dry eye cell model. Blank serum (control group), sodium hyaluronate eye drops (Western medicine group), and a Qishen prescription granule of medicated serum were each applied separately to the dry eye model HCECs, with each ingredient constituting its own group in the study. In other words, three separate groups tested the protective effects blank serum, sodium hyaluronate eye drops, and a Qishenfang granule of medicated serum had on HCECs. The Qishenfang granule of medicated serum was applied in high, medium, and low doses, constituting three additional subcategories that became their own groups in the study. The CCK-8 method was used to detect the effects that the different drug stimulations had on the survival rate of the HCECs. Using ELISA, the expressions of inflammatory factors IL-1β and IL-8 in extracellular fluid were explored. Similarly, the expression of apoptosis factor, caspase 3, was detected using immunocytochemistry. Finally, the expressions of p38MAPK, p-p38MAPK, ERK1, and p-ERK1 of HCECs were found using western blotting. Results Compared to the group using only blank serum, the survival rates of HCECs in all other groups were significantly reduced (P<0.01). In every group, the extracellular fluid inflammatory factors of IL-1β, IL-8, and caspase-3 increased significantly. Moreover, the protein expression levels of p-ERK1, and p-p38MAPK also significantly increased in each group. (P<0.01). In comparison to the control group, the survival rate of HCECs in each medication group increased significantly (P<0.01). Regarding the prescriptions containing medium- and high-dose levels of Qishen granule, the expression levels of both IL-1β and IL-8 were reduced in the extracellular fluid. The caspase-3, p-ERK1, and p-p38MAPK protein expression in HCECs in the medium- and high-dose Qishen granule groups were also all reduced. Likewise, the expression of caspase-3 and the p-ERK1 protein in the Western medicine group were reduced (P<0.05, 0.01). Compared to the Western medicine group, the survival rate of HCECs in the high-dose Qishen granule group was significantly increased (P<0.01). The expression levels of IL-1β, IL-8, caspase-3, p-ERK1, and p-p38MAPK in the high-dose Qishen granule group were reduced; similarly, the IL-1β, p-ERK1 and p-p38MAPK in the medium-dose Qishen granule group was reduced. Meanwhile, the IL-8 in the low-dose Qishen granule group was reduced (P<0.05, 0.01). However, there were no statistically significant differences in the expression of the ERK1 and p38MAPK proteins in HCECs as detected by western blotting (P>0.05). Conclusion A prescription containing any level of Qishen granule of medicated serum reduces not only the ERK1 and p38MAPK phosphorylation protein expression of HCECs induced by hypertonicity, but also the expression of inflammatory factors IL-1β and IL-8 as well as the apoptotic factor caspase-3 of HCECs in the extracellular fluid. Therefore, this effectively blocks inflammation and apoptosis.
Key words: dry eye, Qishen prescription, corneal epithelial cells, extracellular regulated protein kinase 1, p38 mitogen activated protein kinase
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