Anti-inflammatory Effect And Molecular Mechanism Of Flavonoids From Radix Tetrastigmae Through TLR4 Mediated Signal Pathway

Objective To observing anti-inflammatory effect offlavonoids from Radix Tetratigmae(RTFs)on lipopolysaccharide(LPS)-induced acute lung injury in Balb/c mice and to explore possible mechanism via TLR4 mediated pathways.From cellular and molecular level,the effect of RTFs on CD14-TLR4-MD-2-MyD88 dependent pathway of macrophages is also discussed.The anti-inflammatory function and mechanism of RTFs are collectively evaluated in vivo and in vitro,which may lay a solid experimental foundation for new drug research and development of RTFs.Methods ?Construction and administration of mouse ALI model All Balb/c mice were divided randomly into 5 groups.LPS 2.5 mg/kg was intratracheally instilled to induce ALI.The experimental group was also administered RTFs 40 mg/kg,80 mg/kg and 160 mg/kg orally everyday and lasted for 3 d.?Collection of bronchoalveolar lavage fluid(BALF)and leukocyte counting Under anesthetization the mouse trachea was cannulated and the lungs were lavaged three times with PBS(0.5 ml/time).The number of leukocytes was enumerated with Wright-Giemsa staining.?Cytokine antibody array and ELISA analysis of cytokines in BALF Select a group of BALF for microarray detection and some cytokines were quantified by ELISA.?Histopathologic examination of lung tissues After paraffin section and hematoxylin eosin staining,histological score was made.?Cell viability assay on RAW264.7 with MTT RTFs(10?160 ?g/ml)and LPS(1 ?g/ml)were used in cell culture for 24 h before cell viability detection.?qRT-PCR of macrophage pro-inflammatory factors and synthetic enzymes Extract cell RNA,quantified real-time RT-PCR detection of iNOS?COX-2?TNF-??IL-1? mRNA expression.?Determination of pro-inflammatory factors secreted by macrophages ELISA of TNF-?,IL-1?,IL-6,IL-12p40 and IL-10 in macrophage supernatant and Griess assay of NO.?Phosphorylated antibody microarray screening of macrophage signaling pathway Extract cell protein,microarray hybridization and data analysis.?Western blot analysis of TLR4,MD-2,NF-?B in mouse lung tissues and NF-?B activity test.?ELISA of MAPKs molecules in mouse lung tissues ?Western blot analysis of macrophage CD 14,TLR4,MD-2,MyD88,NF-?B and NF-?B activity test ? ELISA of macrophage MAPKs molecules ? Effect of NF-?B and JNK blocking agent on NO,IL-6 productonResults ?After 3-day LPS instillation,total leukocytes,neutrophils and macrophages in BALF were significantly increased in all groups exposed to LPS compared with that of the control group.RTFs treatment(40,80 and 160 mg/kg)significantly reduced LPS-induced leukocytes exudation(P<0.05),especially the number of neutrophils and macrophages(P<0.01).?The release of 40 inflammatory cytokines from the BALF of RTFs-treated mice was determined by mouse cytokine antibody array.Compared with the model,the release of B-lymphocyte colony(BLC),granulocyte colony stimulating factor(GCSF),IL-1?,IL-6,IL-12p40/p70,macrophage inflammatory protein-1?(MIP-1?),tissue inhibitor of metalloproteinase-1(TIMP-1)and TNF-? induced by LPS was markedly reduced by RTFs.?LPS treatment induced the release of IL-1?,IL-6,IL-12p40 and TNF-? to a maximum.Compared with the model,production of cytokines in RTFs treated groups was all significantly reduced in a dose-dependent manner.?With the challenge of LPS,the pulmonary function of LPS group was obviously impaired,with various changes,including capillary congestion,hemorrhage,infiltration or aggregation of neutrophils in airspace or vessel wall and thickning of the alveolar wall.As for experimental groups,histopathological changes were obviously abated by RTFs treatment.The lung injury scores soared in LPS-treated groups,but the increase was significantly reduced by RTFs treatment(P<0.01).?Exposure of RAW264.7 cells to 10-160 ?g/ml RTFs or 1 ?g/ml LPS for 24 h showed no significant effect on cell viability.?The quantitative real-time PCR showed that after treatment with LPS and RTFs for 5 h,RTFs significantly inhibited the mRNA expression of TNF-?,IL-1? and iNOS induced by LPS(P<0.05),but had no effect on COX-2 expression(P>0.05).?After treatment for 24 h,the pro-inflammatory factor levels of TNF-?,IL-1?,IL-6,IL-12p40,sTNF-R1 and NO in RAW264.7 cells supernatant were all reduced in a dose-dependent manner(P<0.05),but anti-inflammatory factor IL-10 up-regulated(P<0.05).?Phosphorylation sites modulation of signal pathway subject to four main trends.The trend which is up-regulated with LPS and down-regulated with RTFs comprise of 9 protein and 11 phosphorylated sites,including NF-?B p65(Ser529),c-JUN(Ser73),p38 MAPK(Tyr182),Akt(Thr308/Ser473),FAK(Tyr925),et al.?Western blotting analysis of total protein extract from lung tissues showed that LPS instillation markedly increased the protein levels of TLR4,MD-2,NF-?B p65 and phospho-NF-?B p65 in mouse lungs(P<0.01).However,they were all significantly down-regulated by RTFs(P<0.05).Furthermore,RTFs significantly diminish DNA binding activity of NF-?B in nuclear extract from lung tissues of LPS-induced ALI mice in a dose-dependent manner,as determined by TransAM NF-?B kit(P<0.05).There was no difference in the expression of MyD88 among these groups(P>0.05).?RTFs also significantly reduced the expression and phosphorylation level of p38MAPK and JNK in ALI mice.But ERK and p-ERK exhibited slight difference in the groups.And the difference is of no statistical significance.?In macrophages,the increased expression of CD 14,TLR4,MD-2,MyD88 and NF-?B,as well as NF-?B phosphrylation and activity induced by LPS were all significantly attenuated by RTFs treatment(P<0.05).Besides,flow cytometric analysis used PE-Cy7 conjugated mAb TLR4/MD2 showed that the complex formation was stimulated by LPS but interrupted by RTFs in a concentration-dependent manner.?LPS-induced expression and phosphorylation of JNK was also notably inhibited by RTFs(P<0.05).But RTFs showed no effect on the phosphorylation of ERK and p38MAPK molecules,and only high concentration of TRFs could inhibit the expression of them to some degree.?NF-?B and JNK blocking agent obviously reduced NO and IL-6 produced by LPS-induced macrophages.When they were used together with RTFs respectively,the production of NO and IL-6 was significantly less than RTFs(80 ?g/ml)group.Conclusion Within certain dosage range,RTFs showed anti-inflammatory effect on LPS-induced ALI mice through alleviating leukocytes exudation,inhibiting pro-inflammatory factors secretion and improving pathologic changes of lung tissues.The mechanism may associate with the inhibition effect of RTFs on TLR4,MD-2 mediated JNK and p38MAPK signal pathways.In macrophages,RTFs restrain the pro-inflammatory factors expression and promote anti-inflammatroy factors through CD14-TLR4/MD-2-MyD88 dependent NF-?B and JNK pathways.RTFs effectively suppressed the development of inflammation from multiple targets and in different levels,which may have prospective value in inflammatory disease therapy.