【摘要】 目的：通过对比不同剂量二至丸对东莨菪碱诱导的小鼠干眼模型干眼症状及炎症状态的治疗效果，从而探讨二至丸在干眼中的疗效及作用机制。方法：选取30只C57/BL6小鼠，将小鼠分为5组，每组6只，其中24只采用皮下注射东莨菪碱的方法诱导干眼模型，最终分为：空白对照组（不建立干眼模型，口服0.9%氯化钠溶液1 m L），模型组（建立干眼模型后，口服0.9%氯化钠溶液1 mL），高剂量组（建立干眼模型后，口服高剂量二至丸12 g/kg），中剂量组（建立干眼模型后，口服中剂量二至丸6 g/kg），低剂量组（建立干眼模型后，口服低剂量二至丸3 g/kg），分别在造模前1天，造模后隔日检测各组小鼠泪液分泌量以及角膜荧光素染色评分的变化，并于14 d后处死小鼠，收集角结膜组织，检测角结膜组织中炎症因子肿瘤坏死因子-α (TNF-α)、白细胞介素-1β (IL-1β)、IL-4表达，采用实时定量PCR技术检测RNA水平改变，流式细胞仪的液相蛋白定量技术（CBA）检测蛋白水平的改变，收集泪腺组织，采用Protein Simple公司的Wes全自动蛋白质印迹定量分析系统对泪腺的核因子κB (NF-κB)通路核心蛋白P65以及磷酸化P65进行胞浆和胞核定性定量的检测验证。结果：用药14 d后，在对干眼症状的改善方面：对比模型组，二至丸各个剂量组的泪液分泌量及角膜荧光素染色评分均有不同程度改善（P<0.05），其中低剂量组与中剂量组的治疗效果相近，高剂量组的治疗效果最佳。在对眼表炎症的改善方面：(1)mRNA水平，与模型组相比，高剂量组的TNF-α、IL-1β、IL-4的mRNA水平均明显下降，差异有统计学意义（P<0.05）。高中低3个剂量组的IL-4的mRNA表达水平均低于模型组，差异有统计学意义（P<0.05）。说明不同剂量的二至丸对眼表炎症因子IL-4的mRNA表达均具有抑制作用，但高剂量组同时可以抑制TNF-α、IL-1β的mRNA表达。(2)蛋白水平：高中低3个剂量组的TNF-α、IL-1β蛋白表达量均低于模型组，差异有统计学意义（P<0.05）。各实验组IL-4蛋白表达均小于模型组，但差异无统计学意义（P>0.05）。提示各剂量二至丸对TNF-α、IL-1β的蛋白表达均有抑制作用，但对IL-4的蛋白表达抑制作用不明显。在对泪腺炎症相关通路影响方面：高中低3个剂量组中二至丸均可抑制细胞浆内P65的磷酸化及磷酸化的P65向核内转运，从而抑制NF-κB通路的激活。结论：二至丸不仅可以有效改善干眼小鼠的泪液分泌和角膜荧光色素染色评分，同时也可以改善干眼小鼠眼表的炎症状态，其对干眼小鼠的炎症抑制作用可能是通过抑制NF-κB通路来实现。更多还原

【Abstract】 Objective:To compare the therapeutic effects of different doses of Erzhi Pills on dry eye symptoms and inflammatory status induced by scopolamine in the mouse dry eye model, in order to explore the therapeutic effect and mechanism of Erzhi Pills in dry eyes. Methods:Thirty C57/BL6 mice were selected and divided into 5 groups,with 6 mice in each group. Among them,24 mice were induced into dry eye models by subcutaneous injection of scopolamine. They were ultimately divided into the blank control group(no dry eye model was established, oral administration of 1 mL of 0.9% sodium chloride solution),the model group(after establishing a dry eye model,oral administration of 1 mL of 0.9% sodium chloride solution),and the high-dose group(after establishing a dry eye model,oral administration of 12 g/kg of high-dose Erzhi Pills),the medium-dose group(after establishing a dry eye model,oral administration of medium-dose Erzhi Pills 6 g/kg) and the low-dose group(after establishing a dry eye model, oral administration of low-dose Erzhi Pills 3 g/kg) were tested for changes in tear secretion and corneal fluorescein staining scores in each group on the first day before and the next day after modeling. The mice were euthanized 14 days later,and the corneal conjunctiva tissue was collected to detect the inflammatory factor tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-4 expression in the corneal conjunctiva tissue was detected using real-time quantitative PCR technology to detect changes in RNA levels. Flow cytometry was used to detect changes in protein levels using Cytometric Bead Array(CBA). Tear gland tissue was collected,and nuclear factors-κB(NF-κB) in the lacrimal gland were analyzed using Protein Simple’s Wes fully automated protein blotting quantitative analysis system. Quantitative detection and validation of cytoplasmic and cytoplasmic validation were performed on pathway core protein P65 and phosphorylated P65. Results: After 14 days of medication, in terms of improving dry eye symptoms,compared with the model group,the tear secretion and corneal fluorescence staining scores of each dose group of Erzhi Pills were improved to varying degrees(P<0.05). The treatment effect of the low-dose group and the medium dose-group was similar,while the treatment effect of the high-dose group was the best.In terms of improving ocular surface inflammation:(1)mRNA levels,compared with the model group,the mRNA levels of TNF-α, IL-1β, IL-4 in the high-dose group were significantly decreased, with statistically significant differences(P<0.05). The mRNA expression levels of IL-4 in the high-,medium-,and low-dose groups were lower than those in the model group,with statistically significant differences(P<0.05). It indicates that different doses of Erzhi Pills have inhibitory effects on the mRNA expression of ocular surface inflammatory factor IL-4, but the high-dose group can also inhibit mRNA expression of TNF-α,IL-1β.(2)Protein level:the protein expression levels of TNF-α, IL-1β in high-,medium-,and low-dose groups were lower than that of the model group,and the difference was statistically significant(P<0.05). The protein expression of IL-4 in each experimental group was lower than that in the model group,but the difference was not statistically significant(P>0.05). Reminder of the effects of each dose of Erzhi Pills on the protein expression of TNF-α,IL-1β is inhibited,but the inhibitory effect on IL-4 protein expression was not significant. In terms of its impact on the lacrimal gland inflammation related pathways,Erzhi Pills in the high-,medium-,and low dose groups can inhibit the phosphorylation of intracellular P65and the transport of phosphorylated P65 to the nucleus, thereby inhibiting activation of the NF-κB pathway. Conclusion: Erzhi Pills can not only effectively improve tear secretion and corneal fluorescent pigment staining scores in dry eye mice,but also improve the inflammatory state of the ocular surface in dry eye mice. Its anti-inflammatory inhibitory effect on dry eye mice may be achieved by inhibiting NF-κB channel.更多还原