南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (09): 1280-1287.doi: 10.12122/j.issn.1673-4254.2020.09.09

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以caspase-1介导细胞焦亡筛选治疗糖尿病肾病潜在的中药及单体成分

赵为陈，何春远，王法财   

  • 出版日期:2020-09-20 发布日期:2020-09-20

Screening potential Chinese materia medica and their monomers for treatment of diabetic nephropathy based on caspase-1-mediated pyroptosis

  • Online:2020-09-20 Published:2020-09-20

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摘要： 目的 以caspase-1介导细胞焦亡为基础，筛查并探究中药及单体有效成分治疗糖尿病肾病（DN）的分子机制。方法 利用中药系统药理学分析平台（TCMSP）筛选靶向caspase-1的中药以及活性单体成分，并通过GeneCards数据库检索单体成分潜在的作用基因靶点，运用Cytoscape构建单体化合物-基因靶点网络构图。采用基因本体（GO）功能富集分析和京都基因与基因百科全书（KEGG）通路富集分析，预测中药及其单体的分子作用机制。实验SD大鼠随机分为对照组、糖尿病组（DM）和人参皂苷Rh2药物治疗组（Rh2）。DM组和Rh2组通过腹腔注射链脲佐菌素（STZ）（60 mg/kg）构建DN大鼠，对照组注射等量生理盐水。在造模成功后Rh2组大鼠给予Rh2（20 mg·kg-1·d-1）灌胃12周，对照组和DM组大鼠给予等量生理盐水灌胃。HE染色观察大鼠肾脏形态；Western blot检测焦亡标志蛋白caspase-1、GSDMD、IL-1β、IL-18表达；试剂盒检测大鼠血浆IL-1β、IL-18含量以及cathepsin B和cathepsin L活性。结果 人参皂苷Rh2与caspase-1靶点蛋白能够有效分子对接，数据库筛选共涉及14个靶基因，GO功能富集分析共27个条目，其中分子功能（MF）条目4个，细胞组成（CC）条目10个，生物过程（BP）条目13个。KEGG通路富集筛选涉及溶酶体、糖胺聚糖降解、半乳糖代谢和鞘脂代谢通路。DM大鼠造模12周后肾脏出现显著的病理学改变，cleaved-caspase-1、GSDMD-N、IL-1β和IL-18表达显著增加（P<0.05或P<0.01），血浆IL-1β、IL-18含量显著升高（P<0.01），cathepsin B、cathepsin L活性显著降低（P<0.01）。与DM组相比，Rh2组大鼠肾脏病变得到显著改善，焦亡标志蛋白（cleaved-caspase-1、GSDMD-N、IL-1β和IL-18）表达显著下调（P<0.05或P<0.01），血浆IL-1β、IL-18含量显著降低（P<0.01），cathepsin B和cathepsin L活性显著增加（P<0.05）。结论 人参皂苷Rh2可能通过溶酶体途径抑制caspase-1介导的细胞焦亡进而改善DN肾脏损伤。

关键词: 糖尿病肾病, 细胞焦亡, caspase-1, 网络药理学, 人参皂苷Rh2, 溶酶体途径

Abstract: Objective To screen potential traditional Chinese medicine and their active monomer ingredients for treatment of diabetic nephropathy (DN) through the mechanism of caspase-1-mediated pyroptosis. Methods Using the Chinese Medicine System Pharmacology Analysis Platform (TCMSP), we screened traditional Chinese drugs and their active monomer components targeting caspase-1, and searched for the potential gene targets of the monomer components using GeneCards database. Cytoscape was used to construct the monomer compound-gene target network. Gene ontology (GO) functional enrichment analysis and Kyoto Gene and Gene Encyclopedia (KEGG) pathway enrichment analysis were used to predict the molecular mechanism of the screened traditional Chinese medicine and monomers. In SD rat models of diabetic mellitus (DM), we tested the therapeutic effect of ginsenoside Rh2 (daily dose of 20 mg/kg for 12 weeks) by examining renal pathology with HE staining and detecting the expressions of pyroptosis marker proteins caspase-1, GSDMD, IL-1β and IL-18 in the renal tissues using Western blotting, the serum levels of IL-1β and IL-18 and activities of cathepsin B and cathepsin L. Results Ginsenoside Rh2 could effectively dock with caspase-1 molecule. Fourteen targets were identified in ginsenoside Rh2 target network. GO function enrichment analysis revealed 27 GO terms associated with molecular function (4 terms), cell component (10 terms) and biological process (13 terms). KEGG pathyway enrichment analysis identified 4 signaling pathways involving lysosomes, glycosaminoglycan degradation, galactose metabolism, and sphingolipid metabolism pathways. In the animal experiment, treatment with ginsenoside Rh2 significantly alleviated renal pathologies and down-regulated the expressions of pyroptosis marker proteins (cleaved caspase-1, GSDMD-N, IL-1β and IL-18) (P<0.05 or 0.01), lowered serum levels of IL-1β and IL-18 (P<0.01), and enhanced the activities of cathepsin B and cathepsin L in the serum of the diabetic rats. Conclusion Ginsenoside Rh2 may inhibit caspase-1-mediated pyroptosis through the lysosome pathway to improve kidney damages in rat models of DN.

Key words: diabetic nephropathy, pyroptosis, caspase-1, network pharmacology, ginsenoside Rh2, lysosome pathway