Inhibitory Effects And Mechanisms Of Ellagic Acid On Lipopolysaccharide-induced Inflammation

LPS-TLR4-NF-κB pathway has become one of the most important targets of anti-inflammatory drugs. Ellagic acid has shown a variety of biological activity, but it is not clear whether ellagic acid can inhibit LPS-TLR4-NF-κB pathway to act anti-inflammatory effects. In this paper, we explored to solve this problem based on in vivo and in vitro experiments, and the methods and results were shown below.(1)Firstly, the anti-inflammatory effects of ellagic acid were tested in vivo. BALB/c mice were injected LPS to induce endotoxemia, and treated with different concentrations of ellagic acid, serum cytokines(TNF-α, IL-6, IL-1β and IL-10) were detected by ELISA method after LPS injection at different time points. Result: mice serum levels of inflammatory cytokines were increased by LPS injection, peak levels of TNF-α, IL-1β, IL-6 and IL-10 were shown at 6, 1, 12 and 3 h after LPS injection, respectively. Ellagic acid treatment led to significantly decreased levels of TNF-α, IL-6 and IL-1β. Otherwise, IL-10 content was increased by ellagic acid. These results demonstrated ellagic acid could regulate cytokine production to reduce inflammation.(2)Secondly, the anti-inflammatory effects of ellagic acid were tested in cultured RAW264.7 macrophages in vitro to verify the in vivo effects. Cytotoxicity of ellagic acid was determined by the MTT method to provide basis for dose selection. Ellagic acid effect on cytokine(TNF-α、IL-1β、IL-6、IL-10) secretion and m RNA expression were detected by ELISA and PCR method respectively. The results showed LPS significantly stimulated secretion and expression of cytokines of TNF-α 、 IL-1β 、 IL-6 、 IL-10 m RNA, demonstrating the success of inducing inflammation in vitro. Ellagic acid at 2 and 4μg/m L levels significantly reduced the secretion and expression of TNF-α, IL-1β and IL-6 m RNA. However, ellagic acid increased the secretion and m RNA expression of IL-10. The results of ELISA assays were consistent with the expression of cytokine m RNAs. The results demonstrated ellagic acid exhibited antiinflammatory effects in vitro.(3) Thirdly, the molecular antiinflammatory mechanisms of ellagic acid were explored. RAW264.7 macrophages were cultured in vitro, and the effects of ellagic acid on LPS-TLR4-NF-κB pathway were detected. As a result, under the stimulation of LPS, cellular IκB content was decreased obviously, while ellagic acid increased IκB content in a dose-dependent manner(P<0.05). The content of P- IκB was just the opposite. Moreover, ellagic acid could inhibit the expression of NF-κB. For further investigation, TLR4/MD-2 specific antibody was used to block TLR4/MD-2 active site, and the effects of ellagic acid on NF-κB pathway were studied. After blocking TLR4/MD-2 active site, there were no obviously difference of NF-κB pathway proteins were found between LPS and control groups. The NF-κB pathway proteins expression of cells treated with ellagic acid after TLR4/MD-2 specific antibody also showed no obviously difference between groups with or without ellagic acid treatment. The result proved that TLR4/MD-2 was an important target of ellagic acid.Conclusion: Ellagic acid can regulate the expression of cytokines induced by LPS through blocking the LPS-TLR4-NF-κB pathway.