灯盏乙素抑制糖尿病视网膜病变中 RGC 细胞焦亡的网络药理学机制。

Network pharmacology mechanism of Scutellarin to inhibit RGC pyroptosis in diabetic retinopathy.

机构信息

Department of Anatomy, College of Basic Medicine, Jinzhou Medical University, Jinzhou, 121001, China.

Animal Center, Kunming Medical University, Kunming, 650500, China.

出版信息

Sci Rep. 2023 Apr 20;13(1):6504. doi: 10.1038/s41598-023-33665-3.

To investigate the effect of scutellarin (SCU) in diabetic retinopathy (DR) and explore the associated molecular network mechanism. The animal model of DR was established from diabetic mellitus (DM) rats by intraperitoneally injected streptozotocin (STZ) at dosage 55 mg/kg. Meanwhile, SCU was intraperitoneally administrated to protect retina from cell pyroptosis induced by DM, and cell pyroptosis was detected by using HE, Nissl staining, and immunofluorescence recognition. Moreover, the hub gene involving in pyroptosis in DR was screened by bioinformatics and network pharmacology, designated as Venny intersection screen, GO and KEGG analysis, PPI protein interaction, and molecular docking. Lastly, the expressional change of hub genes were validated with experimental detection. Cell pyroptosis of the DR, specifically in retina ganglion cells (RGC), was induced in DM rats; SCU administration results in significant inhibition in the cell pyroptosis in DR. Mechanically, 4084 genes related to DR were screened from GeneCards and OMIM databases, and 120 SCU therapeutic targets were obtained, by using GeneCards, TCMSP with Swiss Target Prediction databases. Moreover, 357 targets related to pyroptosis were found using GenenCards database, and Drug, disease and phenotypic targets were analyzed online using the Draw Venn Diagram website, and 12 cross targets were obtained. Through GO function and KEGG pathway enrichment analysis, 659 BP related items, 7 CC related items, 30 MF related items, and 70 signal pathways were screened out; Of these, eleven proteins screened from cross-target PPI network were subsequently docked with the SCU, and their expressions including caspase-1, IL-1β, IL-18, GSDMD and NLRP3 in RGC indicated by immunofluorescence, and the mRNA expression for caspase-1 in DR indicated by quantitative PCR, were successfully validated. SCU can effectively protect RGC pyroptosis in DR, and underlying mechanisms are involved in the inhibition of caspase-1, GSDMD, NLRP3, IL-1β and IL-18. Our findings therefore provide crucial evidence to support the clinic practice of SCU for the treatment of DR, and explained the underlying molecular network mechanism.

目的

研究野黄芩苷（SCU）在糖尿病视网膜病变（DR）中的作用，并探讨其相关的分子网络机制。

方法

通过腹腔注射链脲佐菌素（STZ）建立糖尿病大鼠模型（剂量为 55mg/kg），研究 DR 的发病机制。同时，腹腔内给予 SCU 以保护糖尿病诱导的视网膜细胞焦亡，通过 HE、Nissl 染色和免疫荧光识别检测细胞焦亡。此外，通过生物信息学和网络药理学筛选涉及 DR 中焦亡的关键基因，采用 Venny 交集筛选、GO 和 KEGG 分析、PPI 蛋白互作和分子对接。最后，通过实验检测验证关键基因的表达变化。

结果

糖尿病大鼠视网膜神经节细胞（RGC）发生 DR 细胞焦亡，SCU 给药可显著抑制 DR 中的细胞焦亡。机制上，从 GeneCards 和 OMIM 数据库筛选出与 DR 相关的 4084 个基因，利用 GeneCards、TCMSP 与 SwissTargetPrediction 数据库获得 120 个 SCU 治疗靶点。此外，从 GenenCards 数据库中发现 357 个与焦亡相关的靶点，通过在线 Draw Venn Diagram 网站分析药物、疾病和表型靶点，获得 12 个交叉靶点。通过 GO 功能和 KEGG 通路富集分析，筛选出 659 个 BP 相关项目、7 个 CC 相关项目、30 个 MF 相关项目和 70 个信号通路；从交叉靶点 PPI 网络中筛选出 11 个蛋白，用免疫荧光法检测其在 RGC 中的表达，包括半胱氨酸天冬氨酸蛋白酶-1（caspase-1）、白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）、Gasdermin D（GSDMD）和核苷酸结合寡聚化结构域样受体家族包含蛋白 3（NLRP3），用 qPCR 检测 DR 中 caspase-1 的 mRNA 表达，均成功验证。

结论

SCU 可有效保护 DR 中 RGC 的焦亡，其作用机制涉及抑制半胱氨酸天冬氨酸蛋白酶-1（caspase-1）、Gasdermin D（GSDMD）、核苷酸结合寡聚化结构域样受体家族包含蛋白 3（NLRP3）、白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）。本研究为 SCU 治疗 DR 的临床实践提供了重要证据，并解释了其潜在的分子网络机制。