Anti-inflammatory Activity And Regulation On Inflammatiory Signal Transduction Pathway Of Valnemulin

Some antibacterial agents were shown to regulate immune and inflammatory responses, in addition to their antibacterial activity. Valnemulin is a broad-spectrum, pleuromutilin antibiotic; its broad-spectrum activity targets Gram-negative and Gram-positive bacteria, anaerobic bacteria, Mycoplasma and spirochaetes. It is used in veterinary medicine to treat enteric diseases and acute polyarthritis in pigs, and enzootic pneumonia in pigs and poultry. Valnemulin has also been used in human medicine to treat patients with mycoplasma infections that were resistant to conventional treatments. However, valnemulin is widely used only as antibacterial clinic. In order to fully educe clinical value and expand applied scope of valnemulin, we built in vitro inflammatory model by LPS-stimulated murine RAW 264.7 macrophages, and further explored anti-inflammatory molecular mechanism of valnemulin by inflammatory signal transduction pathway. Meanwhile, we studied the effects of valnemulin on LPS-induced murine ALI and endotoxic shock.Macrophages play a crucial role in both the specific and nonspecific inflammatory processes. Therefore, lipopolyssacharide (LPS)-activated macrophages have typically been used to evaluate the anti-inflammatory effects of various materials. LPS-mediated activation of macrophages leads to the production of various cytokines such as tumour necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6 (IL-6) and interleukin-10 (IL-10), and inflammation mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2). The production of these cytokines may result in the systemic inflammatory response syndrome (SIRS), severe tissue damage, and septic shock. So. first, we investigated the effect of different concentrations of valnemulin on cytokine TNF-α. IL-1β, IL-6 and IL-10. inflammatory mediator NO and PGE2 secretions in LPS-stimulated murine RAW 264.7 macrophages. The result showed that valnemulin inhibited TNF-α, IL-6 and IL-1βsecretion and increased IL-10 level in a dose-dependent manner, and valnemulin also inhibited NO and PGE2 synthesis, but had no significant effect on IL-1β.These indicated valnemulin may educe anti-inflammatory effect through regulating the secretion of cytokines and inflammatory mediators in inflammatory process.The mechanism by which LPS induces the production of cytokines and inflammatory mediators has been intensively investigated. Among these. MAPKs and NF-κB pathways may play an essential role. NF-κB and MAPKs are known as important targets for anti-inflammatory molecular mechanism. In order to study anti-infalmmatory molecular mechanism, we further investigated the effect of valnemulin on NF-κB and MAPKs signal transduction pathways in LPS-stimulated murine RAW264.7 macrophages. The result showed that valnemulin inhibited NF-κB activity, ERK, JNK and p38 protein phosphorylation in a dose-dependent manner. It suggested that valnemulin inhibited cytokine TNF-α, IL-6, NO and PGE2 secretion by regulating both NF-κB and ERK, JNK and p38 MAPKs pathways.To provide objective evaluation index on clinical therapeutic of valnemulin, we further studied in vivo ant-inflammatory of valnemulin. In this study, we established a mouse model of LPS-induced inflammatory lung injury and investigated the effect of valnemulin (100 mg/kg) on acute lung injury eight hours after LPS challenge. We prepared bronchoalveolar lavage fluid (BALF) for measuring protein concentrations, cytokine levels and superoxidase dismutase (SOD) activity, and collected lungs for assaying wet-to-dry weight (W/D) ratios, myeloperoxidase (MPO) activity, cytokine mRNA expression and histological change. We found that the preadministration of valnemulin significantly decreases the W/D ratio of lungs, protein concentrations and the number of total cells, neutrophils, macrophages and leukomonocytes, and histologic analysis indicates that valnemulin significantly attenuates tissue injury. Furthermore, valnemulin significantly increases LPS-induced SOD activity in BALF. and decreases lung MPO activity as well, consistent with its effects on neutrophil infiltration. In addition, valnemulin also inhibits the production of TNF-α, L-6, and IL-1β, which is consistent with mRNA expression in lung. The results showed that valnemulin had a protective effect on LPS-induced inflammatory lung injury in mice.We investigated the effect of different concentrations of valnemulin on preventive and therapeutic effect in LPS-induced endotoxic shock mice, and the effect on cytokine TNF-α, IL-1β, IL-6 and IL-10 production in murine serum. The result showed that valnemulin significantly improved murine survival rate and decreased TNF-αand IL-6, increased IL-10 level in serum, but had little effect on IL-1β. It suggested that valnemulin improved murine survival rate through regulating the level of cytokines.The study will provide foundation for reasonable application of valnemulin in clinic and improve clinical value, and it may be of importance as a therapeutics in treatment of excessive inflammatory reaction, SIRS and MODS in macrophages.