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, cardiovascular diseases, and autoimmune disorders²˒³.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are currently the most widely used agents for managing inflammation. Despite their effectiveness in symptom relief, long-term NSAID use is frequently associated with adverse effects, including gastrointestinal irritation, hypersensitivity reactions, and hepatotoxicity⁴, which limit their clinical applicability. As a result, there is growing interest in identifying alternative antiinflammatory agents with improved safety profiles. Natural products, including traditional herbal medicines, have been explored as potential sources of bioactive compounds with immunomodulatory properties. However, generalized claims regarding low toxicity or systemic regulatory functions should be interpreted cautiously and require experimental validation in defined disease models⁵.

Eupatorium lindleyanum DC. is a traditional medicinal plant whose characteristic phytochemicals include sesquiterpene lactones, primarily guaianolide- and eudesmanolide-type compounds, along with germacranolides and xanthanolides⁶. Extracts of Eupatorium lindleyanum DC. have been reported to exhibit lipid-lowering, anti-atherosclerotic, antitumor, and antiinflammatory activities in various experimental settings⁶. In particular, ethanol extracts of Eupatorium lindleyanum DC. have shown protective effects in lipopolysaccharide (LPS)-induced acute lung injury models⁷. However, the antiinflammatory activity of SLEL and its effects in classical acute inflammation models remain insufficiently characterized.

To address this gap, we employed two complementary acute inflammation models: xylene-induced ear edema in mice, which captures rapid neurogenic and vascular inflammatory responses, and carrageenan-induced paw edema in rats, a well-established model reflecting time-dependent acute inflammation involving cytokine production and cellular infiltration. Alternative models, such as LPS-induced systemic inflammation, were not selected due to their emphasis on systemic immune activation rather than localized tissue edema and their limited suitability for paired tissue-level histological and molecular analyses⁸.

Using this combined model strategy, inflammatory responses were evaluated through quantitative edema measurements, histopathological assessment by hematoxylin and eosin (H&E) staining, serum cytokine profiling, tissue-level transcriptional analysis, and NF-κB–associated protein changes. This protocol is particularly suited for screening antiinflammatory activity in acute inflammation, where edema formation, cytokine expression, and pathway-associated molecular readouts are of interest. Nonetheless, the limitations of acute models should be recognized, including restricted extrapolation to chronic inflammatory conditions and interpretive boundaries when inferring causality from tissue-level signaling changes.

Collectively, this study highlights a protocol-driven approach that integrates complementary acute inflammation models with paired histological and molecular analyses, providing a reproducible framework for evaluating the antiinflammatory potential of herbal-derived compounds.

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 obtain the crude ethanol extract powder.

The dried extract was fully dissolved in distilled water (final concentration: 100 mg/mL) and sequentially subjected to polyamide resin chromatography, followed by D101 macroporous adsorption resin chromatography to enrich sesquiterpene lactones. After the adsorption equilibrium was reached, the D101 resin was washed with distilled water to remove polar impurities and then eluted with anhydrous ethanol. Following ethanol elution, the collected fraction was concentrated under reduced pressure and subjected to freeze-drying to produce the SLEL fraction, which was stored at −20 ℃ until use.

Separation was achieved on a UPLC platform employing a binary mobile phase system of acetonitrile and 0.1% (v/v) formic acid in water. The gradient program comprised an initial 25 min isocratic hold at low organic content, a short linear ramp to 70% acetonitrile, a high-organic step phase at 90% acetonitrile, and a final equilibration step returning to starting conditions. The system was operated at 0.25 mL·min⁻¹ with a column temperature of 35 °C and an injection volume of 0.1 µL (Supplemental Figure S1).

Animals

A total of 36 male Sprague–Dawley rats (200 ± 20 g) and 36 male BALB/c mice (20 ± 2 g) were used in this study. Animals were housed under standardized conditions (22 ± 2 ℃, 55 ± 5% humidity, 12 h light/dark cycle) with free access to food and water. Animals were acclimated for 7 days prior to experimentation and monitored daily for general health. All procedures were conducted to minimize animal suffering, and anesthesia was applied prior to blood collection and tissue harvesting.

Xylene-induced ear edema in mice

The mouse ear edema model was established as previously described⁹. After acclimatization, mice were randomly assigned into six groups (n = 6 per group): control (CG), model (MG), dexamethasone (DEX, 10 mg/kg), and SLEL low-dose (75 mg/kg), medium-dose (150 mg/kg), and high-dose (300 mg/kg) groups. All treatments were administered by oral gavage once daily for 7 consecutive days (final dosing volume: 10 mL/kg). At 1.5 h after the final administration, inflammation was induced by applying 30 µL of xylene evenly to both surfaces of the right ear, except in the control group. Thirty minutes later, mice were anesthetized, blood samples were collected, and 8 mm circular ear punches were obtained from identical anatomical positions of both ears.

Carrageenan-induced paw edema in rats

The rat paw edema model was established using carrageenan as previously described¹⁰. Rats were randomly assigned to six groups (n = 6 per group): CG, MG, DEX (5 mg/kg), and SLEL low-dose (50 mg/kg), medium-dose (100 mg/kg), and high-dose (200 mg/kg) groups. Treatments were administered orally once daily for 7 days (10 mL/kg). Baseline paw volumes were measured prior to the final dosing. Thirty minutes after the last administration, 0.1 mL of 1% (w/v) carrageenan solution was injected subcutaneously into the plantar surface of the right hind paw. Paw volumes were measured at 1, 2, 3, and 4 h post injection using a plethysmometer. Paw edema was calculated as:

Paw edema (%) = (Vt − V0) / V0 × 100%

Histological examination

Ear and paw tissues were collected immediately after functional assessments. Tissues were fixed in 10% neutral buffered formalin for 72 h at room temperature, embedded in paraffin, sectioned (5 µm), and stained with H&E according to standard protocols¹¹.

Enzyme-linked immunosorbent assay (ELISA)

Serum samples were obtained by centrifugation at 3,000 × g for 10 min at 4 ℃. Levels of TNF-α, IL-6, and IL-1β were quantified using commercially available ELISA kits according to standard protocols.

Western blot analysis

Total protein extracts were prepared from ear and paw tissues using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using a BCA assay, and equal amounts of protein (30 µg per sample) were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight and HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were detected using enhanced chemiluminescence and quantified with ImageJ software.

Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from ear and paw tissues using the RNA extraction reagent listed in the Table of Materials. One microgram of total RNA was reverse-transcribed into cDNA using a commercial reverse transcription kit according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Green chemistry with gene-specific primers (Table 1) in a 10 µL reaction volume under a two-step amplification protocol. Detailed reverse transcription conditions and PCR cycling parameters are provided in the Protocol section. Relative transcript levels were calculated using the 2^⁻ΔΔCT method, with Gapdh used as the internal reference gene.

Data and statistical analysis

Data are expressed as mean ± SD. Statistical comparisons between two groups were performed using an unpaired Student’s t-test, whereas one-way ANOVA was applied for analyses involving more than two groups. Statistical significance was defined as p < 0.05. Significance is indicated as # p < 0.05 and ## p < 0.01 versus the control group (CG), and * p < 0.05 and ** p < 0.01 versus the model group (MG).

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