Eucalyptol, limonene and pinene enteric capsules attenuate airway inflammation and obstruction in lipopolysaccharide-induced chronic bronchitis rat model via TLR4 signaling inhibition.

BACKGROUND: Chronic bronchitis (CB), a type of chronic obstructive pulmonary disease (COPD), poses a significant global health burden owing to its high morbidity and mortality rates. Eucalyptol, limonene and pinene enteric capsules (ELPs) are clinically used as expectorants to treat various respiratory diseases, including CB, but their acting mechanisms remain unclear. In this study, we investigated the anti-CB effects of ELP in a rat model of lipopolysaccharide (LPS)-induced CB. The molecular mechanisms underlying its inhibitory effects on airway inflammation were further explored in LPS-stimulated Beas-2B cells. METHODS: ELP was characterized using gas chromatography. The production of inflammatory mediators in bronchoalveolar lavage fluid (BALF) was determined using an enzyme-linked immunosorbent assay. The expression of MUC5AC, MUC5B, and p-p65 in the lung tissue was measured using immunohistochemical staining. The gene expression of inflammatory mediators was determined using qRT-PCR. The expression levels of the target proteins were detected by western blotting. Nuclear localization of p65 was determined using an immunofluorescence assay. RESULTS: Compared to the CB model rats, ELP-treated rats showed reduced airway resistance, inflammation, and goblet cell hyperplasia. In BALF, ELP decreased the levels of inflammatory mediators, including TNF-α, IL-6, MIP-1α, and CCL5. ELP also suppressed LPS-induced elevation of MUC5AC, MUC5B, and p-p65 in the lung tissue. The metabolic pathway changes caused by LPS challenge were improved by ELP treatment. In LPS-exposed Beas-2B cells, ELP treatment inhibited the expression of TNFA, IL6, CCL5, MCP1, and MIP2A and decreased the phospho-levels of toll-like receptor 4 (TLR4) signaling-related proteins, including p-p38, p-JNK, p-ERK, p-TBK1, p-IKKα/ß, p-IκB, p-p65, and p-c-Jun. ELP also hindered the nuclear translocation of p65, c-Jun, and IRF3. CONCLUSIONS: This study showed that ELP has a potential therapeutic effect in LPS-induced CB rat model, possibly by suppressing TLR4 signaling. These results justify the clinical use of ELP for the treatment of pulmonary inflammatory diseases.

Xuelian injection ameliorates complete Freund's adjuvant-induced acute arthritis in rats via inhibiting TLR4 signaling.

Background: Xuelian injection (XI), a classic preparation extracted from Saussureae Involucratae Herba, has been clinically used to manage rheumatoid arthritis (RA) for nearly twenty years in China. However, the underlying anti-RA mechanism of XI remains unclear. In this study, complete Freund's adjuvant (CFA)-induced acute arthritic model was used to examine the anti-RA effects of XI in vivo. The molecular mechanisms of this action were further investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods: XI and XI freeze dried powder were characterized by UPLC analysis. CD68 and TLR4 expression in the ankle joints was measured by immunohistochemistry. The secretion of inflammatory mediators was detected by ELISA. The expression levels of TLR4 involved components were measured by Western blotting. The localization of transcription factors was measured by immunofluorescence assay. Results: XI treatment ameliorated arthritic symptoms induced by CFA in the ankle joints of rats. The serum levels of inflammatory mediators, including TNF-α, MCP-1, and Rantes were decreased by XI treatment. The elevation of CD68 and TLR4 levels in ankle joints caused by CFA was suppressed by XI treatment. Moreover, XI treatment inhibited the secretion of nitric oxide and prostaglandin E2 in LPS-treated RAW264.7 macrophages. The expression of their enzymes iNOS and COX-2 was also decreased after XI treatment. The production of inflammatory mediators, including TNF-α, IL-6, IL-1ß, MCP-1, MIP-1α, and Rantes was reduced by XI treatment in LPS-stimulated RAW264.7 cells. The phosphorylation of p38, JNK, ERK, TBK1, IKKα/ß, IκB, p65, c-Jun, and IRF3 was reduced after XI treatment. Additionally, the expression levels of nuclear proteins of p65, c-Jun, and IRF3 were inhibited by XI treatment. Conclusions: Taken together, XI possesses potential anti-RA effect and the underlying mechanism may be closely associated with the inhibition of TLR4 signaling. Our findings provide further pharmacological justifications for the clinical use of XI in RA treatment.