The Acidic Fraction of Isatidis Radix Regulates Inflammatory Response in LPS-Stimulated RAW264.7 Macrophages through MAPKs and NF-κB Pathway

Isatidis Radix, the dried root of Isatidis indigotica Fort, is a traditional heat-clearing and detoxicating herb, which has the antiviral and anti-inflammatory activity and immune regulation. It has been widely used to treat cold, fever, sore throat, mumps, and tonsillitis in clinics. A previous study demonstrated that the acidic fraction of Isatidis Radix (RIAF) had strong anti-inflammatory activity, but the mechanism of action was not well elucidated. Lipopolysaccharide- (LPS-) induced RAW 264.7 cells were employed to observe the anti-inflammatory activity of RIAF. The level of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), prostaglandin E-2 (PGE(2)), and interleukin-6 (IL-6) was determined by enzyme-linked immunosorbent assay kits. Western blot was performed to quantify the expression of extracellular signal-regulated kinase (ERK) 1/2, c-jun NH2-termianl kinase (JNK), p38, inducible NO synthetase (iNOS), cyclooxygenase (COX)-2, andnudear factor-kappa B (NF-kappa B). Immunofluorescence assay and electrophoretic mobility shift assay (EMSA) were used to quantify the translocation and the binding-DNA activity of NF-kappa B. RIAF could inhibit the secretion of inflammatory cytokines (PGE(2), IL-6, IL-1 beta, and NO, other than TNF-alpha) in a dose-dependent manner. Further investigation showed that the expression of iNOS and COX-2 induced by LPS were downregulated by treatment with RIAF. Meanwhile, data from the signal pathway exhibited that RIAF significantly suppressed the phosphorylation of ERK1/2, JNK, and p38 and reduced the translocation of NF-kappa B from the cytoplasm to nucleus, as well as the binding-DNA activity. The anti-inflammatory mechanism of action of RI AF was to reduce inflammation-associated gene expression (iNOS, COX-2, IL-1 beta, IL-6) by regulating the phosphorylation of the mitogen-activated protein kinases (MAP K) pathway and interventing the activation of the NF-kappa B pathway, which partly illustrated the basis of treatment of Isatidis Radix on cold, fever, sore throat, mumps, and tonsillitis in clinics.