Antiinflammatory Effects of Sesquiterpene Lactones from Eupatorium lindleyanum DC. in Rodent Models

Summary

This study demonstrates that sesquiterpene lactones from Eupatorium lindleyanum DC. (SLEL) exert significant antiinflammatory effects in xylene-induced mouse ear edema and carrageenan-induced rat paw edema models by alleviating histopathological damage, reducing cytokine release, and inhibiting NF-κB signaling activation.

Abstract

Acute inflammation is a fundamental pathological process involved in many immune-related diseases. Eupatorium lindleyanum DC., a traditional medicinal herb, is rich in sesquiterpene lactones (SLEL), which have been reported to exhibit pharmacological activity; however, their antiinflammatory effects and associated molecular changes remain incompletely characterized. In this study, acute inflammatory responses were induced in a xylene-induced mouse ear edema model and a carrageenan-induced rat paw edema model. Tissue injury and inflammatory infiltration were assessed by hematoxylin and eosin staining. Levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) were quantified in serum by ELISA, and their transcriptional expression in inflamed tissues was analyzed by quantitative PCR (qPCR). Changes in NF-κB pathway–associated proteins, including phosphorylated IκBα and p65, were evaluated by western blotting. SLEL treatment reduced ear and paw edema and alleviated histopathological features of acute inflammation, including tissue edema, inflammatory cell infiltration, and vascular congestion. In parallel, SLEL administration was associated with decreased serum levels and tissue mRNA expression of Tnf-α, Il-6, and Il-1β. Western blot analysis demonstrated reduced phosphorylation of IκBα and p65 in inflamed tissues following SLEL treatment, indicating modulation of NF-κB–related signaling events. Together, these findings demonstrate an experimental workflow for evaluating the anti-inflammatory activity of SLEL in acute inflammation models and suggest that its effects are associated with reduced pro-inflammatory cytokine expression and altered NF-κB pathway–related signaling.

Introduction

Inflammation is a complex defensive response triggered by pathogenic invasion or tissue injury and plays a critical role in limiting infection, eliminating harmful stimuli, and promoting tissue repair¹. While controlled inflammatory responses are essential for maintaining tissue homeostasis, excessive or persistent inflammation can lead to tissue damage, organ dysfunction, and the progression of chronic diseases. Clinically, inflammation is characterized by redness, swelling, heat, and pain, and in severe cases may result in systemic pathological responses. Increasing evidence has linked dysregulated inflammation to the development of cancer, c....

Protocol

The animal study protocol was approved by the Institutional Animal Care and Use Committee of Chongqing Medical University (IACUC-CQMU-2025-10073).

Preparation of sesquiterpene lactone–enriched fraction (SLEL)

Dried medicinal materials of Eupatorium lindleyanum DC. were pulverized and subjected to reflux extraction 2x with 70% (v/v) ethanol under reduced pressure. Each extraction was conducted with a solvent-to-material ratio of 10:1 (v/w) for 2 h. The combined extracts were filtered, concentrated under reduced pressure at ≤60 °C, and subsequently vacuum freeze-dried to o....

Results

Chemical composition in SLEL through UPLC-Q/TOF/MSn

The chemical profile of SLEL was examined using UPLC-Q/TOF-MSn in positive and negative ion modes. Nine sesquiterpene lactones were identified as predominant constituents (Supplemental Figure S1). Molecular formula assignments were derived from observed adduct ions, including [M+H]⁺ and [M+Na]⁺ in positive mode, as well as [M−H]⁻ and [M+HCOO]⁻ in negative mode (Supplemental Table S1).

Discussion

In this study, sesquiterpene lactones from Eupatorium lindleyanum DC. (SLEL) were evaluated using two established acute inflammation models, namely xylene-induced ear edema in mice and carrageenan-induced paw edema in rats. By incorporating histopathological scoring, serum cytokine measurements, tissue-level mRNA quantification, and protein-level signaling readouts, this protocol provides a multi-layer characterization of local and systemic inflammatory responses within a single experimental workflow.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anti GAPDH	ImmunoWay	YN5585
anti IκBα	Cell Signaling Technology	4812
anti P65	Abmart	TA5006
anti p-IκBα	Cell Signaling Technology	2859
anti p-P65	Abmart	TP70621
BCA	Beyotime Biotechnology Co., Ltd	P10010
IL-1β kit	Xiamen Huijia Biotechnology Co., Ltd	EHJ-20537r
IL-6 kit	Xiamen Huijia Biotechnology Co., Ltd	EHJ-20746r
Micropipette	Eppendorf
Microplate reader	Gene Co., Ltd	Odyssey Fc
PVDF	Millipore	IPVH00010
Reverse transcription kit	MedChemExpress	HY-K051A
RIPA buffer	Beyotime Biotechnology Co., Ltd	P0013B
SYBR Green Mastermix	MedChemExpress	HY-K0522
TNF-α kit	Xiamen Huijia Biotechnology Co., Ltd	EHJ-20039r
TRIzol	Thermo Fisher Scientific	15596018CN
Ultra-low temperature freezer	SANYO