目的 探讨棉团铁线莲Clematis hexapetala对盐水负荷大鼠模型的急性利尿和长期利尿作用及其利尿机制。方法 设置模型组、呋塞米（10 mg/kg）组、川木通（20 g/kg）组和棉团铁线莲低、中、高剂量（5、10、20 g/kg）组，每组8只。给予药物急性及长期干预后，测定大鼠尿量、电解质浓度；测定血清中Na⁺-K⁺-三磷酸腺苷酶（adenosine triphosphatase，ATPase）活性及血管紧张素II（angiotensin II，Ang II）、抗利尿激素（antidiuretic hormone，ADH）、醛固酮（aldosterone，ALD）、心房肽素（atrial natriuretic peptide，ANP）水平；采用苏木素-伊红（hematoxylin-eosin，HE）染色观察肾脏组织病理变化。采用网络药理学筛选棉团铁线莲活性成分和利尿靶点，构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络，对核心靶点进行基因本体（gene ontology，GO）功能及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析。对核心成分和核心靶点进行分子对接验证，采用qRT-PCR、Western blotting检测肾脏组织水通道蛋白（aquaporin，AQP）/环磷酸腺苷（cyclic adenosine monophosphate，cAMP）/环磷腺苷效应元件结合蛋白（cAMP-response element binding protein，CREB）通路相关基因及蛋白表达。结果 从TCMSP数据库中鉴定出棉团铁线莲7种活性成分，354个靶点。从GeneCards、OMIM数据库中筛选出了1 531个疾病相关靶标，其中交集靶点133个。PPI分析揭示前5个核心靶标分别为前列腺素内过氧化物合酶2（prostaglandin-endoperoxide synthase 2，PTGS2）、肾上腺素能受体β1（adrenergic receptor β1，ADRB1）、过氧化物酶体增殖物激活受体α（peroxisome proliferator-activated receptor α，PPARA）、血管紧张素II 1型受体（angiotensin II receptor type 1，AGTR1）和PPARG。KEGG通路富集分析发现cAMP通路在棉团铁线莲利尿过程中发挥重要的作用。动物实验结果显示，棉团铁线莲显著增加大鼠尿量和尿液中Na⁺、Cl⁻、K⁺水平（P＜0.05、0.01），显著降低Ca²⁺水平（P＜0.05），从而增加了NaCl的排泄；棉团铁线莲显著降低大鼠血清中ADH、Ang II和ALD水平（P＜0.05、0.01），显著提高Na⁺-K⁺-ATPase活性及ANP水平（P＜0.05、0.01），从而抑制肾素-血管紧张素-醛固酮系统（renin-angiotensin-aldosterone system，RAAS）的激活；棉团铁线莲显著抑制AQP/cAMP/CREB通路相关基因和蛋白表达（P＜0.05、0.01、0.001）。结论 棉团铁线莲可能通过抑制盐水负荷大鼠AQP/cAMP/CREB通路，发挥急性和长期利尿作用。

Objective To investigate the acute and chronic diuretic effects and diuretic mechanisms of Clematis hexapetala in a saline-loaded rat model. Methods Model group, furosemide (10 mg/kg) group, C. armandii (20 g/kg) group, C. hexapetala low-, medium- and high-dose (5, 10, 20 g/kg) groups, with eight rats in each group. Urine volume and electrolyte concentration in rats were measured after acute and long-term drug intervention. The activity of Na⁺-K⁺-triphosphatase (ATPase) and levels of angiotensin II (Ang II), antidiuretic hormone (ADH), aldosterone (ALD), atrial natriuretic peptide (ANP) in serum were measured. Hematoxylin-eosin (HE) staining was used to observe pathological changes in renal tissue. Network pharmacology was used to screen the active ingredients and diuretic targets of C. hexapetala, a protein-protein interaction (PPI) network was constructed to perform gene ontology (GO) functional and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis on core targets. Molecular docking validation was performed on the core components and targets, qRT-PCR and Western blotting were used to detect the expressions of genes and proteins related to aquaporin (AQP)/cyclic adenosine monophosphate (cAMP)/cAMP response element binding protein (CREB) pathway in renal tissue. Results A total of seven active compounds and 354 potential drug targets were identified for C. hexapetala from TCMSP database. A total of 1 531 disease-related targets were screened from GeneCards and OMIM databases, including 133 intersecting targets. PPI analysis revealed that the top 5 core targets were prostaglandin-endoperoxide synthase 2 (PTGS2), adrenergic receptor β1 (ADRB1), peroxisome proliferator-activated receptor α (PPARA), angiotensin II receptor type 1 (AGTR1) and PPARG. KEGG pathway enrichment analysis revealed that cAMP pathway played an important role in the process of diuresis in C. hexapetala. Animal experiment results showed that C. hexapetala significantly increased urine output and Na⁺, Cl⁻, K⁺ levels in urine of rats (P < 0.05, 0.01), significantly reduced Ca²⁺ level (P < 0.05), thereby increasing NaCl excretion. C. hexapetala significantly reduced the levels of ADH, Ang II and ALD in serum of rats (P < 0.05, 0.01), significantly increased Na⁺-K⁺-ATPase activity and ANP level (P < 0.05, 0.01), thereby inhibiting the activation of renin-angiotensin-aldosterone system (RAAS). C. hexapetala significantly inhibited the expressions of genes and proteins related to AQP/cAMP/CREB pathway (P < 0.05, 0.01, 0.001). Conclusion C. hexapetala may exert acute and long-term diuretic effects by inhibiting AQP/cAMP/CREB pathway in saline loaded rats.

R285.5

第七批全国老中医药专家学术经验继承工作项目[国中医药人教函(2022)76号]; 基于古今品种差异研究威灵仙功效性能的变化(330023153); 中医药高层次人才培育工程-第四届十大名中医张廷模(600008241005)