Anti-inflammatory activities of Guang-Pheretima extract in lipopolysaccharide-stimulated RAW 264.7 murine macrophages.

BACKGROUND: Guang-Pheretima, which is originated from Pheretima aspergillum, has been documented in academic Chinese herbal studies for nearly 2000 years for its prominent treating effects of various inflammatory diseases such as asthma, cough and fever. However, the anti-inflammatory activity and mechanism of Guang-Pheretima has been rarely reported. Hence, we investigated the inhibitory effect and the underlying mechanism of Guang-Pheretima aqueous extracts on inflammatory response in RAW 264.7 cells. METHOD: RAW 264.7 macrophages were pretreated with various concentrations of Guang-Pheretima decoction (GPD) or protein-free Guang-Pheretima decoction (PF-GPD) and subsequently stimulated with lipopolysaccharide (LPS) to trigger the inflammatory response. Productions of nitric oxide (NO) were determined by Griess reaction, and prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 were measured by enzyme-linked immunosorbent assays (ELISA). The protein expressions and messenger ribonucleic acid (mRNA) amounts of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were analyzed by Western Blot and Real-Time polymerase chain reaction (PCR), respectively. Finally, the translocation of nuclear factor (NF)-κB was observed by Western Blot. RESULTS: GPD of the experimental concentrations showed no anti-inflammatory activity. In contrast, PF-GPD at concentrations of 40-320 µg/mL significantly inhibited NF-κB activation and reduced the production of inflammatory mediators, such as NO, PGE2, TNF-α, as well as the related key synthases including iNOS and COX-2. Moreover, PF-GPD markedly suppressed the release of inflammatory cytokines, such as IL-1ß and IL-6. CONCLUSION: These results demonstrate the excellent anti-inflammatory properties of PF-GPD, and suggest that Guang-Pheretima may be used to treat and prevent certain inflammatory diseases.

Pheretima aspergillum decoction suppresses inflammation and relieves asthma in a mouse model of bronchial asthma by NF-κB inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE AND AIM OF THE STUDY: Guang-Pheretima, the live form of the earthworm Pheretima aspergillum, is a traditional Chinese medicine commonly used for the treatment of asthma, cough, stroke, epilepsy and other diseases due to its anti-inflammatory, anti-asthmatic, anti-seizure, thrombolytic and diuretic properties. Although Guang-Pheretima is effective in the relief of asthma, its pharmacological activity and the underlying molecular mechanisms are not fully understood. Hence, we investigated the effects of a Pheretima aspergillum decoction (PAD) against inflammation in a model of ovalbumin (OVA)-induced asthma in BALB/c mice, as well as the nuclear factor-κB (NF-κB) pathway involved in this process. MATERIALS AND METHODS: OVA was used to sensitize and challenge the airway of the mice, and PAD was administrated by gavage. We measured airway hyperresponsiveness (AHR) in the mice 24h following a final methacholine challenge with whole-body plethysmography. The bronchoalveolar lavage fluid (BALF), serum and pulmonary tissues were collected 48h after the last challenge. The levels of inflammatory factors and the related mRNAs were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR), respectively. The number of differential inflammatory cells in the BALF was counted. Serum total and OVA-specific IgE levels were measured with ELISA. The activation of NF-κB signaling in the lung was detected by western blotting. In addition, the lung tissues were stained with hematoxylin and eosin or periodic acid Schiff stain for histopathological examination. RESULTS: PAD treatment significantly alleviated AHR in the asthmatic mice, decreased the mRNA and protein levels of IL-4, IL-5 and IL-13 and downregulated IgE. In addition, PAD treatment attenuated mucus secretion and infiltration of inflammatory cells in the lung while inhibiting the activation of NF-κB signaling. CONCLUSIONS: PAD effectively inhibited the activation of NF-κB signaling in the lungs of mice with OVA-induced asthma, and mitigated AHR and Th2 type inflammatory reactions. Therefore, PAD may serve as a drug candidate for asthma treatment.