Studies On Anti-inflammatory Effects Of Aqueous Extract Of Acanthopanax Senticosus And Its Mechanisms Of Action

Acanthopanax senticosus (A. senticosus) is known for thousands of years as a medicinal herb and has been clinically used to treat coronary heart disease, angina, ischemic stroke, diabete and hypertension. However, little is known about anti-inflammatory effects and its mechnisms of action. This study aims to investigate the anti-inflammatory effects of aqueous extract of A. senticosus and its mechnisms of action and provide the evidence for the clinical use.In this study, we established the mouse macrophage inflammation model in vitro and mouse endotoxic shock in vivo and investigated the effect and mechanism of aqueous extract of A. senticosus on inflammatory mediators production by mouse macrophages and its protective effect and mechanism against endotoxic shock in mice.1. We examined the effects of aqueous extract of A. senticosus on NO, O₂^-, H₂O₂ and TNF-αproduction in activated RAW 264.7 macrophages and mouse peritoneal macrophages. The contents of NO, O₂^-,H₂O₂ and TNF-αin the medium supematant, respectively, were measured by using Griess Reagent, NBT reduction, phenol red oxidation and ELISA. In vitro exposure of RAW 264.7 macrophages or mouse peritoneal macrophages to aqueous extract of A. senticosus (10～1000μg/mL) significantly suppressed NO production induced by LPS plus IFN-γ, O₂^- production induced by ZYM and H₂O₂ production by PMA. Then, LPS plus IFN-γ-induced TNF-αproduction wasn't affected in RAW 264.7 macropahges and mouse peritoneal macrophages with aqueous extract of A. senticosus incubation. Exposure to aqueous extract of A. senticosus had no effect on cell viability. In vivo administration of aqueous extract of A. senticosus (100 or 200 mg/kg, i.p.) to mice significantly inhibited the production of LPS plus IFN-γ-induced NO, ZYM-induced O₂^- and PMA-induced H₂O₂ by isolated mouse peritoneal macrophages ex vivo. Then, TNF-αproduction wasn't affected in isolated mouse peritoneal macrophages. These results suggest aqueous extract of A. senticosus has an anti-inflammatory effect based on the inhibition of the inflammatory mediators.2. We examined the effects of aqueous extract of A. senticosus on iNOS gene expression and NF-κB signal pathway by LPS plus IFN-γ-stimulated RAW 264.7 macrophages. Real-Time RT-PCR, Western blotting, ELISA and Flow Cytometry, respectively, were used to measure mRNA level, protein level, NF-κB activation and intracellular ROS content. Aqueous extract of A. senticosus inhibited mRNA and protein level in LPS plus IFN-γ-treated RAW264.7 macrophages. Aqueous extract of A. senticosus inhibited the LPS plus IFN-γ, mediated increase in intracellular ROS production, and this inhibition caused the reduction of the activation of NF-κB. This effect was mediated through the suppression of NF-κB p65 nuclear translocation, DNA binding activity of NF-κB and I-κBαdegradation. These results suggest aqueous extract of A. senticosus suppresses iNOS gene expression and NO production through the inhibition of NF-κB activation.3. We examined the protective effect and inflammatory mediators regulatory profiles in mice endotoxic shock, which was induced in mice using an intraperitoneal injection LPS and D-GalN simultaneously. NO concentration, the contents of TNF-αand IL-10 and NF-kB activation, iNOS protein level and histoloty of tissues, respectively, were analyzed by using Griess Reagent, ELISA, Western blotting and HE staining. Intraperitoneal injection of aqueous extract of A. senticosus (100, 200 or 400 mg/kg) prior to LPS/D-GalN challenge significantly improved survival rate of mice compared with that of the LPS/D-GalN alone group. In contrast, treatment of aqueous extract of A. senticosus (200, 400, or 800 mg/kg, i.p.) after LPS/D-GalN didn't reduce the mortality of mice. It is therefore suggested that aqueous extract of A. senticosus exerted prophylactic effect on endotoxic shock in mice. Aqueous extract of A. senticosus preatreament inhibited the production of circulating TNF-αand NO, markedly enhanced that of circulating IL-10. Furthermore, TNF-αand iNOS protein levels in liver were decreased as well as IL-10 level in liver was increased in aqueous extract of A. senticosus preatreated mice compared with the LPS/D-GalN alone group. Aqueous extract of A. senticosus pretreatment inhibited the activation of NF-kB in liver tissue. Inflammatory cells infiltration in heart, liver and lung tissues were alleviated. These results suggest that aqueous extract of A. senticosus protects against LPS/D-GalN induced mice endotoxic shock by a mechanism involving enhancement of IL-10 and inhibition of NF-kB activation, which caused down-regulation of TNF-αand NO, reduction inflammatory cells infiltraion and protection against heart, liver and lung injury.Taken together, our results show the inhibitory effect of aqueous extract of A. senticosus on inflammatory mediators production by mouse macrophages and the prophylactic effect against mice endotoxic shock. Furthermore, its mechanisms of action were elucidated. These data provide the academic evidence for the clinical use and further development of this medicinal herb.