方法：通过500mOsm/L浓度渗透压作用于HCECs制造干眼细胞模型。空白组：正常条件培养的角膜上皮细胞； 模型组：模型细胞加入10%空白血清； 西药组：模型细胞加入0.3%玻璃酸钠滴眼液； 杞参方高剂量组：模型细胞加入15%杞参方高剂量含药血清； 杞参方中剂量组：模型细胞加入15%杞参方中剂量含药血清：杞参方低剂量组：模型细胞加入15%杞参方低剂量含药血清。CCK-8法检测不同药物干预对HCECs存活率的影响； ELISA法检测各组细胞外液炎性因子TNF-α、IL-6的表达； 免疫细胞化学法检测各组HCECs凋亡因子caspase-1与AQP5的蛋白表达变化； Western blotting法检测各组HCECs的JNK1、p-JNK1、AQP5表达情况。

结果：与空白组相比，各组HCECs存活率均显著减少(均P<0.01)，各组细胞外液炎症因子TNF-α、IL-6及HCECs中caspase-1、p-JNK1、AQP5蛋白表达水平均显著增高(均P<0.01)； 与模型组相比，各用药组HCECs存活率均显著增加(均P<0.01)，各用药组的细胞外液中TNF-α、IL-6及HCECs中AQP5、p-JNK1蛋白表达水平及西药组及杞参方高中剂量组caspase-1、AQP5蛋白表达均减少(均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)，杞参方各剂量组的TNF-α、IL-6表达水平均减少(均P<0.05)，杞参方高剂量组caspase-1及杞参方高、中剂量组的AQP5蛋白表达水平减少(均P<0.05)。Western blotting法检测HCECs的JNK1蛋白表达各组比较无差异(P>0.05)。

结论：杞参方颗粒可降低高渗诱导HCECs的JNK1的磷酸化和AQP5的蛋白表达，降低细胞外液炎症因子TNF -α、IL-6和HCECs凋亡因子caspase-1的表达，最终有效抑制炎症反应和细胞凋亡。

METHODS: Human corneal epithelial cells(HCECs)model was created by osmotic pressure at a concentration of 500mOsm/L for 24h. Serum of rats containing drugs in the blank group, model group, Western medicine group, and Qishen recipe low-dose, medium-dose and high-dose groups were treated on the modeled DE HCECs, and the effects of different drug stimulation on the survival rate of HCECs were tested by CCK-8 method. The expressions of inflammatory factors TNF-α, IL-6 in extracellular fluid were explored by ELISA. The expression of apoptosis factors caspase 1 and AQP5 were detected by immunocytochemistry(ICC). The expressions of AQP5, JNK1, p-JNK1 of HCECs after intervention with different drug concentrations were found by Western blotting.

RESULTS: Compared with the blank group, the survival rate of HCECs in each group was significantly reduced(P<0.01). The extracellular fluid inflammatory factors TNF-α, IL-6 and caspase-1, p-JNK1, AQP5 protein expression levels increased significantly in each group(all P<0.01); In comparison to the model group, the survival rate of HCECs in each medication group increased significantly(all P<0.01). The expression levels of TNF-α, IL-6 in the extracellular fluid of each drug group, AQP 5 and p-JNK1 protein expression in HCECs, and the expression of caspase-1 and AQP5 protein in the western medicine group and the Qishen recipe high and medium dose group were all reduced(all P<0.05). Compared with the western medicine group, the survival rate of HCECs in the Qishen prescription high-dose group was significantly increased(P<0.01). The expression levels of TNF-α and IL-6 in each dose group of Qishen recipe were reduced(all P<0.05), while the expression levels of caspase-1 in the high-dose Qishen recipe group and the AQP5 protein expression levels of the high and medium-dose Qishen recipe group saw a decrease(all P<0.05). However, there was no statistically significant difference in the JNK1 protein expression of HCECs of all the groups detected by Western blotting method(P>0.05).

CONCLUSION: Qishen recipe can not only reduce the JNK1 phosphorylation and AQP5 protein expression of HCECs induced by hypertonicity, but also reduce the expression of inflammatory factors TNF-α, IL-6 and the apoptotic factor caspase-1 of HCECs in the extracellular fluid, thus effectively Inhibit inflammation and apoptosis.