目的 通过网络药理学、分子动力学模拟及动物实验，探讨二至丸通过抑制异常糖酵解改善肾纤维化的作用机制。方法 从TCMSP、ETCM、BATMAN-TCM数据库获得二至丸的活性成分并预测作用靶点；结合GeneCards、DisGeNET、DrugBank数据库获取肾纤维化交集靶点。构建蛋白质相互作用（PPI）网络筛选核心靶标，并进行基因本体（GO）与京都基因和基因组百科全书（KEGG）通路富集分析。利用AutoDock Vina和Gromacs 2022软件对核心成分与靶标进行分子对接及分子动力学模拟。建立腺嘌呤诱导的大鼠肾纤维化模型，予以二至丸（1.5、3.0 g/kg）及氯沙坦干预。检测大鼠肾功能指标 [血清肌酐（Scr）、尿素氮（BUN）、24 h尿蛋白]；采用HE、Masson染色及免疫组化评估肾组织损伤与纤维化指标[α-平滑肌肌动蛋白（α-SMA）、波形蛋白（Vimentin）]；采用Western blotting检测肾组织磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）磷酸化及糖酵解关键酶[6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3（PFKFB3）、己糖激酶2（HK2）、葡萄糖转运蛋白1（GLUT1）、丙酮酸激酶M2型（PKM2）]蛋白表达。结果 网络药理学筛选获得16个活性成分，与肾纤维化交集靶点297个。PPI网络识别出木犀草素、山柰酚等核心成分，以及Akt1、PFKFB3、低氧诱导因子-1A（HIF-1A）等核心靶点。KEGG分析显示二至丸主要通过调控PI3K/Akt信号通路发挥作用。分子对接及动力学模拟结果显示，木犀草素与糖酵解关键酶PFKFB3结合能最低且在100 ns模拟中结合构象极其稳定。动物实验表明，二至丸能显著降低模型大鼠Scr、BUN、24h UP水平（P＜0.05、0.01），改善肾小管扩张及间质胶原沉积，α-SMA、Vimentin表达显著下调（P＜0.01）。Western blotting结果显示，二至丸组大鼠肾组织中p-PI3K、p-Akt、PFKFB3、HK2、GLUT1、PKM2蛋白表达显著降低（P＜0.05、0.01）。结论 二至丸改善肾纤维化的作用与PI3K/Akt/PFKFB3通路调控异常糖酵解代谢重编程有关。

Objective To investigate the mechanism of Erzhi Pills in ameliorating renal fibrosis by inhibiting abnormal glycolysis, using network pharmacology, molecular dynamics simulations, and animal experiments. Methods Active ingredients of Erzhi Pills were obtained from TCMSP, ETCM and BATMAN-TCM databases, and targets were predicted. Renal fibrosis-related targets were acquired from GeneCards, DisGeNET, and DrugBank databases to identify intersecting targets. A PPI network was constructed to screen core targets, followed by GO and KEGG pathway enrichment analyses. Molecular docking and molecular dynamics simulations of core components and targets were performed using AutoDock Vina and Gromacs 2022 software. A rat model of renal fibrosis was established by adenine administration, and the rats were treated with Erzhi Pill (1.5, 3.0 g/kg) or losartan. Renal function parameters (Scr, BUN, and 24 h UP) were measured. Renal tissue injury and fibrosis (α-SMA and Vimentin) were assessed by HE staining, Masson staining, and immunohistochemistry. Protein expression of phosphorylated PI3K/Akt and key glycolytic enzymes (PFKFB3, HK2, GLUT1, and PKM2) in renal tissues was detected by Western blotting. Results Network pharmacology screening identified 16 active components, and they intersected with 297 target points related to renal fibrosis. PPI network identified core components including luteolin and kaempferol, as well as core targets including Akt1, PFKFB3, and HIF-1A. KEGG analysis revealed that Erzhi Pills primarily exerts its effects through regulating the PI3K/Akt signaling pathway. Molecular docking and dynamics simulations demonstrated that luteolin exhibited the lowest binding energy with the key glycolytic enzyme PFKFB3, and maintained an extremely stable binding conformation during 100 ns simulation. Animal experiments showed that Erzhi Pills significantly reduced Scr, BUN, and 24 h UP levels (P < 0.05, 0.01), ameliorated tubular dilation and interstitial collagen deposition, and markedly downregulated the overexpression of α-SMA and Vimentin (P < 0.01). Western blotting results revealed that Erzhi Pills significantly decreased the protein expression of p-PI3K, p-Akt, PFKFB3, HK2, GLUT1, and PKM2 (P < 0.05, 0.01). Conclusion The ameliorative effect of Erzhi Pills on renal fibrosis is associated with the regulation of aberrant glycolytic metabolic reprogramming via the PI3K/Akt/PFKFB3 pathway.

R285.5;R287.3

昆山市科技专项（KS2350）