Anti-inflammatory Effects And Mechanism Of Flavonoids From Allium Monogolicum Regel

Flavonoids are one of the main bioactive components of Allium mongolicum Regel.The previous studies of our work have shown that flavonoids from Allium mongolicum Regel have functions of antioxidant,antibacterial,promote the proliferation of peripheral blood lymphocytes in sheep,improve the body-specific immunity,improve the flavor of lamb,etc.However,there is a dearth of studies reported on its anti-inflammatory activity.Therefore,this study used in vivo and in vitro inflammation models to systematically study the anti-inflammatory activity and molecular mechanism of Allium mongolicumRegel flavonoids.The main research contents and results are as follows:1.Preliminary evaluation of anti-inflammatory activity of different elutions of Allium mongolicun Regel total flavonoidsIn this study,we used mouse peritoneal macrophages as the research object.The effects of water,35%,55%,and 75%ethanol elutions of Allium mongolicum Regel total flavonoids(AMF I,AMF II,AMF III,AMF IV)on cell viability of mouse peritoneal macrophages were examined by cell counting kit-8 assay,screening out the best concentration of four elution components used in subsequent experiment.Further based on the LPS-induced inflammatory model of mouse peritoneal macrophages,the effects of four kinds of eluting components of total flavonoids on the inflammatory mediators NO,TNF-a,IL-1?,IL-6 and IL-10 in LPS-induced mouse peritoneal macrophages were investigated and compared for the anti-inflammatory activity.The results showed that as compared to the control group.AMF I had no visible cytotoxic effect on mouse peritoneal macrophages when added at concentrations of 50-800 ?g/mL(P>0.05).However,AMF II,AMF III,AMF IV be added within concentrations of 25-100 ?g/mL can significantly up regulate the proliferation activity of mouse peritoneal macrophages(P?0.01).Therefore,in the following experiments the additive concentrations of AMF I were 100.200,400 ?g/mL.and for AMF ?.AMF ? and AMF ? were 25,50.100?g/mL.The four elutions components of total flavonoids from Allium mongolicum Regel can inhibit the excessive secretion of pro-inflammatory mediators such as NO,TNF-a.IL-1?.and IL-6 induced by LPS,and increase the content of inflammatory inhibitory factor cytokine IL-10.The anti-inflammatory effect of AMF III is higher than the other three flavonoids.It indicated that the four elutions components of total flavonoids from Allium mongolicum Regel showed their anti-inflammatory and immunosuppressive properties by inhibiting the secretion of pro-inflammatory mediators in mouse peritoneal macrophages induced by LPS.2.Inhibition of AMF III on inflammatory response and its MechanismThe study tests how AMF III effects the mRNA expression levels of inflammatory mediators such as iNOS,TNF-a,IL-1?,IL-6,and IL-10 in LPS-induced mouse peritoneal macrophages,as well as MyD88-mediated downstream NF-?B and MAPK signaling pathways,and tried to uncover the underlying mechanism.The results indicate that AMF III inhibits the over expression of pro-inflammatory mediators induced by LPS in a dose-dependent manner and alleviates the excessive inflammatory response induced by LPS.The results of the research on the mechanism indicates that AMF III can significantly inhibit the expression of MyD88 at the gene and protein levels in LPS-induced mouse peritoneal macrophages(P<0.05 or P?0.01),and significantly inhibit the phosphorylation of I?B-(P<0.01),reduce the dissociation of NF-B/I?B-a complex and,in this way,inhibits the phosphorylation and nuclear translocation of p65.AMF III also significantly inhibited the phosphorylation levels of MAPK family proteins ERK1/2,JNK,and p38 induced by LPS(P<0.05).3.Protective effect of AMF III on LPS-induced endotoxemia in miceSixty-four male C57BL/6 mice were randomly divided into four groups as:the normal control group,endotoxemic mice model group,low dose AMF III group and high AMF III dose group.Mice in the AMF III low and high dose group were gavaged with 60 and 120 mg/kg AMF III for 7 days.Acute endotoxemic mice model was established by intraperitoneal injection of LPS(10 mg/kg).The activity of ALT and AST,the secretion of inflammatory mediators(iNOS,NO,TNF-a,IL-1?,IL-6,IL-10)and the expression levels of related genes were detected,and the changes of liver tissues were detected by histopathology to investigate the protective effect of AMF III on LPS-induced endotoxemic mice.The results showed that AMF III at a dose of 120 mg/kg significantly reduced the activity of ALT and AST in the serum of endotoxemic mice([<0.001).and significantly inhibited the excessive secretion and expression levels of related genes of proinflammatory mediators iNOS,NO,TNF-? and IL-1?,increased IL-10 content and gene expression level(P<0.05),and significantly improved LPS-induced liver injury in mice.4.The role of TLR4-MyD88-NF-?B/MAPKsignal transduction pathway in AMF III protection endotoxemic miceThe study based on the LPS-induced mouse to establish endotoxemic mice model to reveal the effect of AMF III on MyD88/NF-?B/MAPK signal transduction pathway in the liver of endotoxemic mice,and further explore the protective mechanism of AMF III on LPS-induced endotoxemic mice.The results showed that AMF III significantly inhibited the expression of MyD88 in the liver tissue of mice with endotoxin-induced by LPS(P<0.05),and inhibited the phosphorylation of I?B-a and p65 in a dose-dependent manner(P<0.05).Different doses of AMF ?-protected group could either significantly or highly significantly inhibit the phosphorylation levels of ERK1/2,JNK,and p38 which belong to the MAPK family(P<0.05 or P<0.01).To sum up,all the four kinds of eluting components of Allium mongolicum Regel total flavonoids have anti-inflammatory activity,and the anti-inflammatory effect of AMF III is superior to the other three.AMF III can inhibit the increase of some pro-inflammatory mediators such as iNOS,NO,TNF-a,IL-6,IL-1? induced by LPS,as well as reduce the activity of ALT,AST;increase the secretion of IL-10;and inhibit the inflammatory response and these all do help for improving the liver damage of LPS in mice.Simultaneously,the study also found that this process is achieved by inhibiting the activation of the MyD88-NF-?B/MAPK signal transduction pathway regulated by TLR4.