目的 通过网络药理学、分子对接结合动物实验的方法探讨三七总皂苷（PNS）调控自噬改善糖尿病肾病 （DN）小鼠肾脏的作用机制。方法 通过文献检索得到PNS的主要活性成分，利用PubChem、PharmMapper数据 库获取对应靶点；通过 GeneCards、OMIM 数据库收集 DN 疾病相关靶点，取两者交集。通过 Cytoscape 3.9.1软 件构建“中药-活性成分-靶点”网络，STRING 数据库构建蛋白互作（PPI）网络并筛选核心靶点；采用 DAVID 平台对交集靶点进行基因本体论（GO）功能和京都基因与基因组百科全书（KEGG）通路富集分析；使用 AutoDock Vina 软件对PNS主要活性成分和关键靶点进行分子对接。将24只8周龄雄性db/db小鼠随机分成模型 组、PNS组、厄贝沙坦组，每组8只；另取8只同周龄雄性db/m小鼠作为正常组。每天灌胃给药1次，连续8周。 观察各组小鼠一般情况及记录体质量和血糖变化；采用考马斯亮蓝法检测 24 h尿蛋白（24 h-UTP）；果糖胺法 测定糖化血清蛋白（GSP）；GPO-PAP 法测定总胆固醇（TC）和甘油三酯（TG）；肌氨酸氧化酶法检测血肌酐 （Scr）；脲酶法检测尿素氮（BUN）；苏木素-伊红（HE）染色观察小鼠肾脏病理变化；实时荧光定量聚合酶链式 反应（RT-qPCR）检测小鼠肾组织泛素结合蛋白（P62）和微管相关蛋白轻链3（LC3）mRNA表达水平；蛋白免疫印 迹法（Western Blot）检测肾组织P62、LC3及磷脂酰肌醇3激酶（PI3K）、蛋白激酶B（Akt）、哺乳动物雷帕霉素靶 蛋白（mTOR）及磷酸化蛋白（p-Akt、p-PI3K、p-mTOR）表达。结果 PNS的主要活性成分为三七皂苷R1、人参 皂苷 Re、人参皂苷 Rd、人参皂苷 Rb1和人参皂苷 Rg1。网络药理学分析显示，PNS活性成分对应靶点 341个， DN 靶点 1 534 个，PNS 与 DN 交集靶点 113 个。筛选出 PNS 治疗 DN 的关键成分有人参皂苷 Rd、人参皂苷 Re、 人参皂苷Rg1及核心靶点AKT1、ALB、MMP9和mTOR等，相关的信号通路涉及PI3K/Akt、Ras、MAPK、FoxO 等。分子对接显示 PNS 关键成分人参皂苷 Rd、人参皂苷 Re、人参皂苷 Rg1与核心靶点 AKT1、MMP9、mTOR 对接活性良好。动物实验验证结果显示，与正常组比较，模型组小鼠一般状态较差，体质量及血糖、GSP、 TC、TG、Scr、BUN和 24 h-UTP水平均显著升高（P < 0.01）；与模型组比较，PNS组小鼠一般状态改善，体质 量及血糖水平明显下降（P < 0.01），且GSP、TC、TG、Scr、BUN和24 h-UTP水平均显著降低（P < 0.05，P < 0.01）。 HE染色结果显示各治疗组肾脏病理损伤明显改善。RT-qPCR 和 Western Blot 结果显示，与正常组比较，模型 组小鼠肾组织LC3 mRNA及蛋白表达水平明显下降（P < 0.01），而P62 mRNA表达水平升高（P < 0.01），p-PI3K/ PI3K、p-Akt/Akt、p-mTOR/mTOR及P62蛋白表达水平亦明显升高（P < 0.05，P < 0.01）；与模型组比较，PNS组 及厄贝沙坦组 LC3 mRNA 及蛋白表达水平升高（P < 0.05，P < 0.01），P62 mRNA 表达水平明显下降（P < 0.05， P < 0.01），p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR 及 P62 蛋白表达水平均明显下降（P < 0.05，P < 0.01）。 结论 PNS能够明显改善糖尿病肾病小鼠的肾脏损伤，其作用机制可能与抑制PI3K/Akt/mTOR信号通路促进自 噬有关。

Objective To explore the mechanism of Panax notoginseng saponins（PNS）on protecting the kidney of diabetic nephropathy （DN） mice via regulating autophagy through network pharmacology， molecular docking and animal experiments. Methods The main active ingredients of PNS were obtained through literature search， and the corresponding targets were obtained using PubChem and PharmMapper databases. GeneCards and OMIM databases were used to collect DN related targets. After the intersection of active ingredients targets and disease targets was taken， Cytoscape 3.9.1 software was used to construct a“Chinese medicine-active ingredients-targets” network. A proteinprotein interaction（PPI）network was constructed using STRING database and core targets were screened. The DAVID platform was applied to conduct Gene Ontology （GO） functional and Kyoto Encyclopedia of Genes and Genomes （KEGG）pathway enrichment analysis on intersecting targets. AutoDock Vina software was used for molecular docking of the main active ingredients and key targets of PNS. Twenty-four 8-week-old male db/db mice were randomly divided into a model group，PNS group，and irbesartan group，with eight mice in each group. Another eight male db/m mice of the same age were selected as the normal group and orally administered once a day for 8 consecutive weeks. The general condition of mice in each group was observed and changes in body mass and blood glucose were recorded. Coomassie Brilliant Blue method was used to detect 24-hour urinary protein（24 h-UTP），the fructose amine method was applied to measure glycosylated serum protein（GSP）， GPO-PAP method was used to measure total cholesterol（TC）and triglycerides（TG），creatine oxidase method was applied to measure serum creatinine（Scr），urease method was used to measure urea nitrogen（BUN）. Renal pathological changes in mice were investigated using hematoxylin eosin（HE） staining. Real time fluorescence quantitative polymerase chain reaction（RT-qPCR）was used to detect the mRNA expression levels of ubiquitin binding protein（P62）and microtubule associated protein light chain 3（LC3）in the kidney tissues of mice in each group. The protein expressions of P62，LC3，phosphatidylinositol 3 kinase（PI3K）， protein kinase B（Akt），mammalian target of rapamycin（mTOR）and phosphorylated proteins（p-Akt，p-PI3K，pmTOR）in renal tissues were detected by Western Blot. Results The main active ingredients of PNS are notoginsenoside R1， ginsenoside Re， ginsenoside Rd， ginsenoside Rb1， and ginsenoside Rg1. Network pharmacology analysis showed that there were 341 targets corresponding to PNS active ingredients，1 534 targets corresponding to DN，and 113 intersecting targets between PNS and DN. The key components，which were screened from PNS in the treatment of DN， include ginsenosides Rd， Re， Rg1. The core targets are AKT1， ALB， MMP9， and mTOR. The related signaling pathways involve PI3K/Akt，Ras，MAPK，FoxO，etc. Molecular docking showed that the key components of PNS（ginsenosides Rd， Re， and Rg1）exhibited good docking activity with the core targets（AKT1， MMP9， and mTOR）. The animal experiment results showed that compared with the normal group， the mice in model group had generally poorer state and significant increases in body weight，blood glucose，GSP，TC，TG，Scr，BUN，and 24 h-UTP（P < 0.01）. Compared with the model group，the mice in PNS treatment group showed an improvement in the general condition and significant decreases in body weight and blood glucose （P < 0.01）， as well as significant decreases in GSP， TC， TG， Scr， BUN， and 24 h-UTP（P < 0.05， P < 0.01）. HE staining results showed a significant improvement in renal pathological damage of each treatment group. RT-qPCR and Western Blot results showed that compared with the normal group，the mRNA and protein levels of LC3 in the kidney tissues of mice in the model group significantly decreased（P < 0.01），while the mRNA expression of P62 increased（P < 0.01），and the protein expression of p-PI3K/PI3K，p-Akt/Akt，p-mTOR/mTOR and P62 also increased significantly（P < 0.05，P < 0.01）. Compared with the model group，the mRNA and protein levels of LC3 in the PNS group and irbesartan group increased（P < 0.05，P < 0.01），the mRNA expression of P62 significantly decreased（P < 0.05，P < 0.01），and the protein expression of p-PI3K/PI3K，p-Akt/Akt，p-mTOR/mTOR and P62 significantly decreased（P < 0.05，P < 0.01）. Conclusion PNS can significantly improve the renal damage in diabetic nephropathy mice，and its mechanism may be related to the inhibition of PI3K/Akt/mTOR signaling pathway and the promotion of autophagy.

R285.5；R692

国家自然科学基金项目（81973827）；福建省自然科学基金项目（2021J01890）；福建省大学生创新创业训练计划项目（202210393015， S202210393023）