Effects Of The Active Ingredients Of Atractylodes Macrocephala On Macrophages And Dentritic Cells

Atractylodes macrocephala Koidz as a traditional herb has a diuretic,anti-inflammatory, anti-tumor, anti-aging, pain, immune regulation, lowering bloodsugar and inhibiting metabolic activation enzymes effect. The major bioactivecomponents include Atractylodes macrocephala polysaccharides (AMP) andatractylenolide (ATL) and so on. Some studies have found that AMP and ATL have avariety of pharmacological activities. AMP has immunoregulatory, anti-oxidant,anti-aging, anti-tumor and hypoglycemic effects, whereas ATL has pharmacologicaleffects mainly related to inflammation, gastrointestinal motility, tumor and so on.However, little is known regarding the role of AMP and ATL. Thus it is necessary todo some research to clarify immunoregulatory molecular mechanisms of AMP. Inaddition, it is very important to further elucidate the anti-inflammatory molecularmechanisms of ATL and promote the development of health food and medicaltreatment of a reliable basis.(1) Crude soluble polysaccharides were extracted from Atractylodesmacrocephala Koidz. After graded by freezing and purified by the combineddeprotionization methods of enzyme and sevag, one fraction was obtained: AMP.(2) To study the immunomodulatory effects of AMP in RAW264.7cells, weexamined the effect of AMP on phagocytosis by flow cytometry, TNF-α, IFN-γ andNO release by ELISA and the expression of TLR4, p38activity and the activation ofNF-κB by Western blot. We further investigated the effect of TLR4on phagocytosis,TNF-α and NO release and p38activity. Further, we investigated the effect of p38onTNF-α and NO release and NF-κB activation. Besides, we explored the effect ofNF–κB on TNF-α, IFN-γ and NO release. We found that AMP enhancedphagocytosis, increased TNF-α, IFN-γ and NO release and upregulated the expressionof TLR4, p38and NF-κB activity. Using TLR4antibody, we found that it decreasedphagocytosis, TNF-α and NO release, and p38activity. Using specific inhibitors ofp38(SB203580) and NF-κB (PDTC), we found that they inhibited TNF-α and NOsecretion. And SB203580also inhibited NF-κB activation. In summary, AMP enhanced phagocytosis, the expression of TLR4and TNF-α, IFN-γ and NO release.We further found its signaling pathways such as MAPK and NF-κB have a positiveregulation. Therefore, its immunomodulatory mechanisms may be associated with thetwo signaling pathways, thereby promoting cytokine release.(3) To study the immunomodulatory effects of AMP on DCs, we examined theeffect of AMP on morphology of DCs by microscopy, phagocytosis and surfacemarkers by flow cytometry, IL-12and TNF-α release by ELISA, the expression ofTLR4by Western blot. Further, we examined the effect of TLR4on phagocytosis andIL-12and TNF-α relesse. We found that AMP enhanced the proliferation, induced thesecretion of IL-12and TNF-α in a dose-dependent manner, and increased theexpression of TLR4. Using TLR4antibody, we found that it decreased IL-12andTNF-α release and phagocytosis in a dose-dependent manner. In summary, AMPpromoted the expression of surface markers and TLR4, increased IL-12and TNF-αsecretion and reduced phagocytosis, suggesting AMP can promote the differentiationand maturation of DCs.(4)To study the anti-inflammatory effects of ATL and further clarify themolecular mechanism, we examined the effects of ATL on cell viability by MTT andestablished the inflammatory model by LPS. We first examined the effect ofatractylenolide Ⅰ on TNF-α, and IL-6release by ELISA, the activation of p38,ERK1/2and NF-κB. We found that atractrylode Ⅰ had no effect on RAW264.7cells between1-100μ M. LPS increased TNF-α, IL-6, PGE2and NO release, whereasatractylenolide Ⅰ inhibited the LPS-induced TNF-α and IL-6release. In addition,atractylenolide III suppressed TNF-α, IL-6, PGE2and NO release in a dose-dependentmanner. We further examined anti-inflammatory mechanisms of atractrylode. Wefound that atractylenolide Ⅰ suppressed ERK1/2, p38and NF-κB activity andatractylenolide III inhibited ERK1/2, p38, JNK1/2and NF-κB activity in aconcentration-dependent manner. Thus, atractrylode had anti-inflammatory effectthrough inhibiting the activation of MAPK and NF-κB. Therefore, theanti-inflammatory mechanism may be due to the inhibition of these two pathways, thereby inhibiting the inflammatory cytokines and inflammatory mediators.