Huanglian Jiedu decoction ameliorates hypertensive nephropathy by inhibiting the AGEs-RAGE/MAPK/AP-1 axis: An integrated transcriptomics and network pharmacology study.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypertensive Nephropathy (HN) is a severe complication of hypertension characterized by progressive renal fibrosis, which current treatments fail to halt. Huanglian Jiedu Decoction (HLJDD), a classic traditional Chinese medicine (TCM) renowned for its "heat-clearing, detoxifying" and anti-inflammatory properties, is widely used against hypertension and associated target organ damage. Thus, its potential therapeutic mechanisms against HN warrant in-depth investigation.

AIM OF THE STUDY: This study was designed to investigate the renoprotective effects of HLJDD on HN and to determine whether its mechanism is mediated through the modulation of the AGEs-RAGE/MAPK/AP-1 signaling axis.

MATERIALS AND METHODS: UPLC-Q-Exactive Orbitrap-MS was employed to characterize the prototype components in the bloodstream derived from HLJDD-containing serum. Key genes associated with HN were screened from the GSE182517 dataset using Weighted Gene Co-expression Network Analysis (WGCNA). Subsequently, an integrative "component-target-pathway" network was constructed. In vivo, an HN mouse model was established via continuous subcutaneous infusion of Angiotensin II (Ang II; 490 ng/kg/min) for 4 weeks. HLJDD was administered at low, medium, and high doses (2, 4, and 8 g/kg/day). Therapeutic efficacy was assessed by blood pressure monitoring, renal function biochemical indices (Scr, BUN, and UA), and histopathological assessments (HE, Masson, and PAS staining). To establish causal evidence, an in vitro model of Ang II-stimulated HK-2 cells was utilized, with the RAGE antagonist FPS-ZM1 employed to confirm the target-pathway association. The core signaling axis was validated using ELISA, Western blot, qRT-PCR, and immunofluorescence.

RESULTS: Twenty-one prototype components-primarily alkaloids, flavonoids, and iridoids-were identified in HLJDD-containing serum. Integrated network pharmacology and molecular docking analysis indicated that these bioactive components primarily target key molecular nodes, such as MAPK3 (ERK1), JUN, and TGF-β1. In vivo results demonstrated that HLJDD significantly reduced blood pressure and improved renal function in a dose-dependent manner, while effectively attenuating inflammatory infiltration and renal fibrosis. Mechanistically, HLJDD reduced the accumulation of advanced glycation end products (AGEs) and suppressed the expression of their receptor (RAGE). This reduction subsequently inhibited the phosphorylation of p38 MAPK and ERK1/2, blocked the nuclear translocation and activation of the transcription factor AP-1 (c-Jun/c-Fos), and ultimately downregulated pro-fibrotic markers, including TGF-β1 and α-SMA. Notably, in vitro results revealed that HLJDD-containing serum (10%) markedly suppressed Ang II-induced inflammation and MAPK/AP-1 activation in HK-2 cells, exhibiting effects consistent with those of the RAGE antagonist FPS-ZM1.

CONCLUSIONS: HLJDD exerts significant antihypertensive and renoprotective effects in HN mice. The mechanism involves the suppression of renal inflammation and fibrosis, primarily by inhibiting the AGEs-RAGE/MAPK/AP-1 signaling axis, as substantiated by both in vivo findings and in vitro pathway antagonism. These findings provide a scientific basis for the application of HLJDD in treating hypertension and its renal complications.