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.

Clematis hexapetala Pall.  /  diuresis  /  renin-angiotensin-aldosterone system  /  AQP/cAMP/CREB pathway  /  clematoside  /  oleanolic acid  /  hederagenin