目的：基于转录组学和网络药理学探究清眩润目饮(QRY)治疗干眼(DE)的作用机制，并通过干眼动物模型对QRY的药效和关键靶点进行实验验证。

方法：运用RNA高通量测序(RNA-seq)技术检测干眼小鼠与正常小鼠的差异表达基因(DEGs)，通过数据库筛选QRY有效成分及作用靶点，将二者重叠获取关键靶点后进行GO和KEGG富集分析，构建“药物-成分-靶点-信号通路”网络并分析蛋白质-蛋白质相互作用(PPI)； 自动物实验开始每7 d对小鼠行Schirmer I试验(SⅠt)、泪膜破裂时间检测(BUT)、角膜荧光染色试验(FL)检查； HE染色观察小鼠角膜组织病理变化； ELISA、Western blot、qRT-PCR验证小鼠角膜组织中核心靶点的蛋白和mRNA表达水平。

结果：获得DEGs共2 234个、QRY有效成分233个及相关靶点457个，获得关键靶点64个，GO与KEGG分析结果提示QRY与炎症反应密切相关，通过PPI网络筛选出白介素-6(IL-6)等19个核心靶点； QRY组的SⅠt、BUT、FL结果与模型组相比有差异(均P<0.05)； HE染色结果示模型组角膜上皮细胞分层紊乱且角膜形态发生改变，QRY组可以改善角膜粗糙及分层紊乱的情况，形态接近空白组； ELISA、Western blot、qRT-PCR结果示QRY组的IL-1β、IL-6、肿瘤坏死因子-α(TNF-α)的蛋白和mRNA表达对比模型组都呈现出下降趋势。

结论：清眩润目饮通过多成分、多靶点、多通路联合发挥作用，利用槲皮素等主要成分对IL-6、IL-1β、TNF等靶点进行调控，从而抑制AGE-RAGE/TNF/IL-17等信号通路以实现对干眼的治疗作用。

METHODS:RNA-sequencing(RNA-seq)technology was used to detect differentially expressed genes(DEGs)between mice in the DE group and mice in the normal control group, the active ingredients and potential targets of QRY were screened through database, and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were carried out after overlapping the results and obtaining key targets. Additionally, “drug-component-target signaling pathways” network was built and the protein-protein interaction(PPI)was analyzed. Mice were examined for Schirmer I test(SⅠt), tear film breakup time(BUT), and corneal fluorescein staining(FL)every 7 d from the beginning of the animal experiments. Hematoxylin-eosin staining(HE)was performed to observe pathologic changes in mouse corneal tissues. Enzyme linked immunosorbent assay(ELISA), Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)were performed to verify the mRNA and protein expression levels of the core targets in mouse corneal tissues.

RESULTS:Totally 2 234 DEGs, 233 active ingredients and 457 related targets of QRY were collected, with a total of 64 key targets obtained. GO function and KEGG pathway results showed that QRY was closely related to inflammatory mediators, and 19 core targets such as interleukin-1β(IL-1β)were screened by PPI network construction; SⅠt, BUT and FL results in the QRY group were statistically significantly different compared with the model group(all P<0.05); HE staining showed that corneal epithelial cell stratification was disordered and the corneal morphology was changed in the model group. However, QRY treatment significantly improved corneal morphology and disordered stratification, with a close morphology to the blank group; ELISA, Western blot and qRT-PCR results showed that the protein expression and RNA levels of IL-1β, IL-6, and tumor necrosis factor(TNF-α)in the QRY group showed a decreasing trend compared with the model group.

CONCLUSION: Through the combination of multiple components, multiple targets and multiple pathways, QRY regulated the targets such as IL-6, IL-1β and TNF through quercetin and other main components, thereby inhibiting AGE-RAGE/TNF/IL-17 and other signaling pathways, thus achieving the treatment on DE.