Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells

ObjectiveTo investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages.MethodsRAW264.7 cells were pretreated with 0-200 & mu;g/mL EEP or vehicle for 2 h prior to exposure to 1 & mu;g/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE(2)) 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 factor & alpha; (TNF-& alpha;), interleukin-1beta (IL-1 & beta;), 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 (I & kappa; B-& alpha;) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-& kappa; B p65 (NF-& kappa; B 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 (O-2(-)) radical and nitrite scavenging activity were also measured.ResultsThe total polyphenol and flavonoid contents of EEP were 23.50 & PLUSMN;2.16 mg gallic acid equivalent/100 g and 43.78 & PLUSMN;3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 & mu;g/mL), there was a notable decrease in NO and PGE(2) production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (PP<0.05). Furthermore, with EEP treatment (150 & mu;g/mL), there was a decrease in the mRNA expression levels of TNF-& alpha;, IL-1 & beta; and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, PP<0.05), by blocking the nuclear translocation of NF-& kappa; B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 & mu;g/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (PP<0.05). EEP also indicated the DPPH, OH, O-2(-) radical and nitrite scavenging activity.ConclusionEEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-& kappa; B pathway and protected against oxidative stress.