Objective To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L.var megalopha Fr.(EEP)on RAW264.7 mouse macrophages.Methods RAW264.7 cells were pretreated with 0–200&...Objective To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L.var megalopha Fr.(EEP)on RAW264.7 mouse macrophages.Methods RAW264.7 cells were pretreated with 0–200µg/mL EEP or vehicle for 2 h prior to exposure to 1µg/mL lipopolysaccharide(LPS)for 24 h.Nitric oxide(NO)and prostaglandin(PGE2)production were determined by Griess reagent and enzyme-linked immunosorbent assay(ELISA),respectively.The mRNA levels of inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2),tumor necrosis factorα(TNF-α),interleukin-1beta(IL-1β),and IL-6 were determined using reverse transcription polymerase chain reaction(RT-PCR).Western blot assay was used to determine the protein expressions of iNOS,COX-2,phosphorylation of extracellular regulated protein kinases(ERK1/2),c-Jun N-terminal kinase(JNK),inhibitory subunit of nuclear factor Kappa B alpha(IκB-α)and p38.Immunofluorescence was used to observe the nuclear expression of nuclear factor-κB p65(NF-κB p65).Additionally,the anti-oxidant potential of EEP was evaluated by reactive oxygen species(ROS)production and the activities of catalase(CAT)and superoxide dismutase(SOD).The 2,2-diphenyl-1-picrylhydrazyl(DPPH),hydroxyl(OH),superoxide anion(O2−)radical and nitrite scavenging activity were also measured.Results The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g.With EEP treatment(100 and 150µg/mL),there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions(P<0.01 or P<0.05).Furthermore,with EEP treatment(150µg/mL),there was a decrease in the mRNA expression levels of TNF-α,IL-1βand IL-6,as well as in the phosphorylation of ERK,JNK and p38 mitogen-activated protein kinase(MAPK,P<0.01 or P<0.05),by blocking the nuclear translocation of NF-κB p65 in LPS-stimulated cells.In addition,EEP(100 and 150µg/mL)led to an increase in the anti-oxidant enzymes activity of SOD and CAT,with a concomitant decrease in ROS production(P<0.01 or P<0.05).EEP also indicated the DPPH,OH,O2−radical and nitrite scavenging activity.Conclusion EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κB pathway and protected against oxidative stress.展开更多
文摘Objective To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L.var megalopha Fr.(EEP)on RAW264.7 mouse macrophages.Methods RAW264.7 cells were pretreated with 0–200µg/mL EEP or vehicle for 2 h prior to exposure to 1µg/mL lipopolysaccharide(LPS)for 24 h.Nitric oxide(NO)and prostaglandin(PGE2)production were determined by Griess reagent and enzyme-linked immunosorbent assay(ELISA),respectively.The mRNA levels of inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2),tumor necrosis factorα(TNF-α),interleukin-1beta(IL-1β),and IL-6 were determined using reverse transcription polymerase chain reaction(RT-PCR).Western blot assay was used to determine the protein expressions of iNOS,COX-2,phosphorylation of extracellular regulated protein kinases(ERK1/2),c-Jun N-terminal kinase(JNK),inhibitory subunit of nuclear factor Kappa B alpha(IκB-α)and p38.Immunofluorescence was used to observe the nuclear expression of nuclear factor-κB p65(NF-κB p65).Additionally,the anti-oxidant potential of EEP was evaluated by reactive oxygen species(ROS)production and the activities of catalase(CAT)and superoxide dismutase(SOD).The 2,2-diphenyl-1-picrylhydrazyl(DPPH),hydroxyl(OH),superoxide anion(O2−)radical and nitrite scavenging activity were also measured.Results The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g.With EEP treatment(100 and 150µg/mL),there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions(P<0.01 or P<0.05).Furthermore,with EEP treatment(150µg/mL),there was a decrease in the mRNA expression levels of TNF-α,IL-1βand IL-6,as well as in the phosphorylation of ERK,JNK and p38 mitogen-activated protein kinase(MAPK,P<0.01 or P<0.05),by blocking the nuclear translocation of NF-κB p65 in LPS-stimulated cells.In addition,EEP(100 and 150µg/mL)led to an increase in the anti-oxidant enzymes activity of SOD and CAT,with a concomitant decrease in ROS production(P<0.01 or P<0.05).EEP also indicated the DPPH,OH,O2−radical and nitrite scavenging activity.Conclusion EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κB pathway and protected against oxidative stress.
