Identification of anti-fibrotic compounds from Piper longum L via hollow fiber cell fishing and high-performance liquid chromatography with in vivo and in vitro validation

Traditional Chinese medicine (TCM) is an important source of bioactive compounds, hence enjoying a wide application in clinical treatment, while its pharmacodynamic material basis remains difficult to elucidate as it has a complex chemical composition. Piper longum L is a commonly used herbal medicine in prescriptions for chronic kidney disease (CKD), yet its key active ingredients responsible for anti-renal fibrosis effects remain unclear. This study aimed to establish a novel and efficient strategy for the screening and identification of anti-fibrotic compounds from Piper longum L. HK-2 cells with fibrotic features induced by transforming growth factor-β (TGF-β) were used to develop a hollow fiber cell fishing coupled with high-performance liquid chromatography (HFCF-HPLC) mode. This model was integrated with network pharmacology and molecular biology techniques to screen and validate active anti-fibrotic compounds in Piper longum L. The in vivo efficacy of the identified compounds was further evaluated by a unilateral ischemia-reperfusion injury (uIRI) model with delayed contralateral nephrectomy in C57BL/6 J mice. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were taken into account to assess the renal function, while immunohistochemistry and western blot served for analyzing the fibrosis markers α-SMA and collagen-I. HFCF-HPLC screening identified two key active compounds from Piper longum L: piperlongumine (PIPA) and piperlonguminine (PLG). Among them, PIPA exhibited the strongest inhibitory effect on the expression of fibrosis markers in vitro. In vivo studies demonstrated that PIPA significantly reduced renal fibrosis in the uIRI model, as indicated by lower SCr and BUN levels, improved renal histopathology, and reduced extracellular matrix deposition. A novel HFCF-HPLC model was successfully established to screen active compounds from TCM against renal fibrosis. PIPA was identified as a promising anti-fibrotic agent from Piper longum L, demonstrating significant renoprotective effects in in vitro and in vivo models. This work advances the modernization of herbal medicine research by offering a integrated strategy for identifying bioactive TCM components.