5β-hydroxycostic acid from Laggera alata ameliorates sepsis-associated acute kidney injury through its anti-inflammatory and anti-ferroptosis effects via NF-κB and MAPK pathways

Ethnopharmacological relevance: The whole plant of Laggera alata is frequently utilize to remedy inflammatory diseases including nephritis as a traditional Chinese medicine. However, its active ingredients and mechanism of action against sepsis-associated acute kidney injury (SA-AKI) are unknown.

Aim of the study: This study aimed to identify active compounds from L. alata that inhibit renal inflammation and ameliorate SA-AKI, and to elucidate their mechanisms of action.

Materials and methods: The chemical constituents were separated from the ethyl acetate layer of L. alata methanol extract by column chromatography over silica gel, medium-pressure liquid chromatography and semipreparative high-performance liquid chromatography. Extensive spectroscopic techniques were applied to determine the chemical structures. The anti-inflammatory efficiency was measured by analyzing the NO production in RAW 264.7 cells. The levels of IL-6, IL-1β, CCL-2 and CCL-5 mRNA were determined by qRT-PCR. Cecal ligation and puncture (CLP) surgery is a frequently applied method to establish the mouse sepsis model. Sepsis was thus induced in mice via CLP. The effect in the treatment of SA-AKI was evaluated by H&E staining and ELISA detection. Western blotting was used to evaluate the protein levels involved in ferroptosis, NF-κB and MAPK signaling pathways.

Results: Twelve compounds were obtained from L.alata including four unreported sesquiterpenoids (1-4). Compound 5 exhibited the most significant inhibitory effect on NO production with the IC₅₀ value of 6.034 μM and could restrain the mRNA expression of inflammatory factors IL-6, IL-1β, CCL-2 and CCL-5. The in vivo results demonstrated that compound 5 alleviated the renal injury by decreasing the serum IL-6, IL-1β, Cr, and BUN levels, reducing the kidney contents of Cys-C and KIM-1, and regulating the kidney levels of MDA, GSH, ferrous iron, GPX4, FTH1, and SLC7A11. Furthermore, Compound 5 also repressed the NF-κB and MAPK pathways in vitro and in vivo.

Conclusions: This study revealed that compound 5 could ameliorate SA-AKI through exerting its anti-inflammatory and anti-ferroptosis effects via NF-κB and MAPK pathways. The current research supported the traditional use of L.alata in the treatment of renal diseases.