Supplementary file 2_Homotherapy for heteropathy of chronic kidney disease and oligoasthenozoospermia through regulating SIRT1/NF-κB pathway by Shenqi pills.xlsx

BackgroundChronic kidney disease (CKD), defined by a glomerular filtration rate (GFR) below 60 mL/min/1.73 m2 for over 3 months, is a significant global health concern, often progressing to end-stage renal disease (ESRD). Oligoasthenospermia (OA), characterized by reduced sperm count or quality, affects male fertility, contributing to infertility in approximately 15% of couples worldwide. Both conditions share features of yang deficiency, including fatigue, cold intolerance, and weakness. Shenqi Pill (SQP), a Traditional Chinese Medicine (TCM) formula, replenishes kidney yang and demonstrates efficacy in treating yang deficiency-related diseases such as CKD and OA. However, the molecular mechanisms underlying its therapeutic effects remain unclear.
MethodsThis study combined ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), network pharmacology, and machine learning to identify SQP’s active compounds and potential targets. A CKD model was induced in C57BL/6 mice via adenine administration, followed by SQP treatment (0.8 or 1.6 g/kg/day) for 50 days. Renal function, histopathology, and molecular pathways were evaluated. Additionally, in vitro assays were performed to validate SQP’s effects on OA using GC-1spg spermatogonia.
Results41 compounds in SQP were identified. Network pharmacology suggested SQP ameliorates CKD and OA by modulating cellular senescence, with SIRT1, RELA, and NFKB1 as key targets. In vivo, SQP improved renal dysfunction, reduced glomerular atrophy, tubular dilation, and collagen deposition, with higher doses demonstrating superior efficacy. RNA-Seq analysis highlighted SQP’s regulation of the SIRT1/NF-κB pathway and cellular senescence. ELISA, β-galactosidase staining, and Western blotting confirmed reduced senescence-associated secretory phenotype (SASP) release and normalization of SIRT1/NF-κB1 activity. In vitro, SQP-containing serum alleviated cellular senescence in GC-1spg spermatogonia by mitigating SIRT1/NF-κB1 disruptions without cytotoxicity.
ConclusionSQP demonstrates therapeutic potential for CKD and OA by targeting the SIRT1/NF-κB signaling pathway, providing evidence for its clinical application in treating kidney-yang deficiency-related diseases.

背景 慢性肾脏病（chronic kidney disease, CKD）指肾小球滤过率（glomerular filtration rate, GFR）低于60 mL/min/1.73 m²且持续3个月以上的病症，是一项重要的全球公共卫生问题，常进展为终末期肾病（end-stage renal disease, ESRD）。
少弱精子症（oligoasthenospermia, OA）以精子数量减少或质量下降为特征，可损害男性生育能力，全球约15%的不孕夫妇的发病与此相关。上述两种病症均存在阳虚表现，如乏力、畏寒及肢体软弱。参芪丸（Shenqi Pill, SQP）是一种传统中医药（Traditional Chinese Medicine, TCM）方剂，具有温补肾阳的功效，在治疗CKD、OA等阳虚相关病症中展现出良好疗效，但其发挥治疗作用的具体分子机制尚未明确。
方法 本研究联合超高效液相色谱-四极杆飞行时间质谱（ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, UPLC-Q-TOF/MS）、网络药理学与机器学习技术，对SQP的活性成分及潜在作用靶点进行筛选鉴定。通过腺嘌呤灌胃构建C57BL/6小鼠CKD模型，随后给予SQP干预（0.8或1.6 g/kg/天），持续50天。对小鼠的肾功能、组织病理学特征及分子通路进行检测评估。此外，本研究采用GC-1spg精原细胞开展体外实验，以验证SQP对OA的干预效果。
结果 本研究共鉴定出SQP中的41种活性成分。网络药理学分析显示，SQP可通过调控细胞衰老进程改善CKD与OA，其中SIRT1、RELA及NFKB1为核心作用靶点。体内实验结果表明，SQP可显著改善肾功能损伤，减轻肾小球萎缩、肾小管扩张及胶原沉积，且高剂量组的治疗效果更为显著。RNA测序（RNA-Seq）分析显示，SQP可调控SIRT1/NF-κB信号通路及细胞衰老过程。酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）、β-半乳糖苷酶染色及蛋白质印迹实验（Western blotting）证实，SQP可减少衰老相关分泌表型（senescence-associated secretory phenotype, SASP）的释放，使SIRT1/NF-κB1的活性恢复至正常水平。体外实验中，含SQP血清可通过缓解SIRT1/NF-κB1通路紊乱，减轻GC-1spg精原细胞的细胞衰老现象，且无细胞毒性。
结论 本研究证实，SQP可通过靶向SIRT1/NF-κB信号通路发挥治疗CKD与OA的潜力，为其在阳虚相关肾病及生殖病症的临床应用提供了坚实的实验依据。