Anti-inflammatory Effects of Eucommia ulmoides Oliv. Male Flower Extract on Lipopolysaccharide-Induced Inflammation
Eucommia ulmoides Oliv. is a traditional medicinal plant native to China, known for its various pharmacological effects, including anti-inflammatory, analgesic, and immune-regulatory properties. While the bark of Eucommia ulmoides has been extensively studied, the male flowers of the plant have recently gained attention due to their availability and potential health benefits. This study investigates the anti-inflammatory effects of a 70% ethanol extract of Eucommia ulmoides male flowers (EF) in both in vitro and in vivo models of lipopolysaccharide (LPS)-induced inflammation.
Introduction
Inflammation is a critical biological response to injury or infection, but excessive or prolonged inflammation can lead to tissue damage and chronic diseases. Macrophages play a central role in the inflammatory response by releasing pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, is a potent inducer of inflammation, triggering the release of these cytokines and activating the nuclear factor-kappa B (NF-κB) signaling pathway.
Eucommia ulmoides male flowers, unlike the bark, are abundant and easy to harvest, making them a promising source of natural anti-inflammatory agents. Previous studies have shown that extracts from Eucommia ulmoides male flowers possess anti-inflammatory, analgesic, and immune-regulatory effects. This study aims to explore the anti-inflammatory potential of EF in LPS-stimulated RAW 264.7 macrophages and in an LPS-induced acute inflammation mouse model.
Methods
Plant Material and Extraction
Eucommia ulmoides male flowers were collected from Zhangjiajie City, Hunan Province, China. The flowers were washed, sliced, dried, and extracted twice with 70% ethanol at 60°C for three days each. The extract was then concentrated under vacuum, yielding a 20.47% extract with a total flavonoid content of 1.72%.
Cell Culture and Viability Assay
The mouse macrophage cell line RAW 264.7 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. RAW 264.7 cells were seeded in 96-well plates and treated with various concentrations of EF (10, 20, 30, 60, 120, and 250 µg/mL) for 24 hours. The absorbance was measured at 450 nm to determine cell viability.
Nitric Oxide (NO) Production
RAW 264.7 cells were treated with EF (10, 20, and 30 µg/mL) for 4 hours, followed by stimulation with 1 µg/mL LPS for 48 hours. The concentration of NO in the culture supernatant was measured using the Griess reagent, and the absorbance was read at 550 nm.
Cytokine Secretion and mRNA Expression
RAW 264.7 cells were treated with 30 µg/mL EF for 4 hours, followed by LPS stimulation for 24 hours. The levels of TNF-α, IL-1β, and IL-6 in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA) kits. The mRNA expression levels of IL-17, IL-23, and IL-10 were determined using quantitative real-time polymerase chain reaction (qPCR).
NF-κB Pathway Activation
RAW 264.7 cells were treated with EF (10, 20, and 30 µg/mL) for 4 hours, followed by LPS stimulation for 20 minutes. The phosphorylation levels of NF-κB p65, inhibitor of kappa B (IκBα), and IκB kinase (IKKα/β) were analyzed using Western blotting.
In Vivo Anti-inflammatory Effects
Female and male ICR mice were randomly divided into four groups: control, LPS only, LPS + 10 mg/kg EF, and LPS + 20 mg/kg EF. The EF groups were orally administered EF for seven days, followed by intraperitoneal injection of 10 mg/kg LPS on day 7. Twenty-four hours after LPS injection, the mice were euthanized, and serum and lung tissues were collected. Serum levels of TNF-α and IL-6 were measured using ELISA, and myeloperoxidase (MPO) activity in lung tissue was determined. Lung tissue sections were stained with hematoxylin and eosin (H&E) for histopathological analysis.
Results
Cell Viability and Proliferation
EF showed no significant cytotoxicity at concentrations ranging from 10 to 60 µg/mL (cell viability > 80%). However, higher concentrations of EF (60, 120, and 250 µg/mL) significantly inhibited RAW 264.7 cell proliferation compared to the negative control.
Inhibition of NO Production
EF significantly reduced NO production in LPS-stimulated RAW 264.7 cells at concentrations of 20 and 30 µg/mL. The inhibitory effect of EF on NO production was concentration-dependent.
Reduction of Pro-inflammatory Cytokines
EF significantly suppressed the secretion of TNF-α, IL-1β, and IL-6 in LPS-stimulated RAW 264.7 cells. Additionally, EF downregulated the mRNA expression of IL-17, IL-23, and IL-10 in LPS-stimulated cells.
Inhibition of NF-κB Pathway Activation
EF inhibited the LPS-induced phosphorylation of NF-κB p65, IκBα, and IKKα/β in RAW 264.7 cells. The inhibition of these key components of the NF-κB pathway suggests that EF exerts its anti-inflammatory effects by modulating this signaling cascade.
In Vivo Anti-inflammatory Effects
EF significantly reduced lung inflammation in LPS-administered mice, as evidenced by decreased inflammatory cell infiltration and granuloma formation in lung tissue sections. EF also suppressed serum levels of TNF-α and IL-6 and reduced MPO activity in lung tissue, indicating inhibition of neutrophil activation.
Discussion
The findings of this study demonstrate that the 70% ethanol extract of Eucommia ulmoides male flowers (EF) possesses significant anti-inflammatory properties in both in vitro and in vivo models of LPS-induced inflammation. EF effectively inhibited the production of pro-inflammatory cytokines, downregulated the expression of inflammatory genes, and suppressed the activation of the NF-κB signaling pathway in LPS-stimulated RAW 264.7 macrophages. In the LPS-induced acute inflammation mouse model, EF reduced lung inflammation, decreased serum levels of TNF-α and IL-6, and inhibited neutrophil activation.
The anti-inflammatory effects of EF are likely mediated through the inhibition of the NF-κB pathway, which plays a central role in regulating the expression of pro-inflammatory cytokines. By suppressing the phosphorylation of NF-κB p65, IκBα, and IKKα/β, EF prevents the nuclear translocation of NF-κB and the subsequent transcription of inflammatory genes. Additionally, EF’s ability to reduce NO production and inhibit the secretion of TNF-α, IL-1β, and IL-6 further supports its potential as a natural anti-inflammatory agent.
The in vivo results indicate that EF can effectively mitigate acute inflammation by reducing inflammatory cell infiltration and suppressing the overactivation of neutrophils. The reduction in MPO activity in lung tissue suggests that EF may protect against tissue damage caused by excessive inflammation. These findings highlight the therapeutic potential of EF in the treatment of inflammatory diseases.
Conclusion
This study provides comprehensive evidence that the 70% ethanol extract of Eucommia ulmoides male flowers (EF) exhibits potent anti-inflammatory effects in both in vitro and in vivo models of LPS-induced inflammation. EF inhibits the production of pro-inflammatory cytokines, downregulates inflammatory gene expression, and suppresses the activation of the NF-κB signaling pathway. In vivo, EF reduces lung inflammation, decreases serum levels of TNF-α and IL-6, and inhibits neutrophil activation. These findings suggest that EF has significant potential as a natural anti-inflammatory agent and warrants further investigation for its therapeutic applications.
doi.org/10.1097/CM9.0000000000000066
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