The Anti-Inflammatory Effect And Potential Mechanism Of Betulinic Acid On ?-Carrageenan Induced Paw Edema In Mice

Betulinic acid?BA?,a naturally occurring pentacyclic triterpenoid extracted from white birch bark,has been demonstrated to have a variety of biological activities,including immunomodulatory,anti-oxidative stress,anti-inflammatory,antitumor,anti-malarial,anti-HIV.In recent studies,BA has been shown to have an inhibitory effect on the Nuclear factor-kappa B?NF-?B?mediated Cyclooxygenase-2?COX-2?signaling pathway up-regulated by different stimuli in different cell lines.In addition to the NF-?B signaling pathway,a number of MAPKs can affect the expression of COX-2 and prostaglandin E2?PGE₂?.These results suggested a possible relationship between BA and MAPK mediated release of inflammatory mediator.Thus far,few studies have assessed the effects of BA on MAPK mediated release of COX-2 and PGE₂ expressions in vivo,which deserves further investigation.Therefore,in this project,a well-documented model to induce acute paw edema inflammation was established in mice by intraplantar injection of?-carrageenan?Carr?.This study was designed to investigate the anti-inflammatory effect of BA on Carr-induced acute paw edema inflammatory in mice and to elucidate the underlying mechanism of anti-inflammatory effect via MAPK/COX-2/PGE₂ signaling pathway.Objective:To investigate the anti-inflammatory effect and its molecular mechanism of BA on?-carrageenan induced acute paw edema inflammation in mice by regulating MAPK/COX-2/PGE₂ signaling pathway which provides a scientific basis for further research and development of BA.Methods:A total of 40 male Kunming mice were randomly divided into 5 groups:the control group,the Carr group,the low,medium and high dose of BA?2.5,10 and 40 mg/kg?with Carr groups.Mice in the control and the Carr groups were orally administered 1%starch solution,and the other groups were orally administered different doses of BA for 7days.Mice were given 25?l of 1%Carr intraplantar injection to set up paw edema inflammatory model,30 min after the last administration of BA,while the control group received an equal volume of sterile saline.After the thickness of the right paw edema was continuously monitored for 6 h,the mice were sacrificed,and serum,liver,spleen,thymus and right paw were collected for analysis.The activities of alanine aminotransferase?ALT?and aspartate aminotransferase?AST?,the content of albumin?ALB?,and the levels of calcium ions(Ca²⁺),Magnesium ions(Mg²⁺)in serum were determined by using the automated blood analyzer.The levels of 23 cytokines and PGE₂ in serum were measured by reagent kits.The activities of superoxide dismutase?SOD?,Catalase?CAT?,Glutathione peroxidase?GSH-Px?and the contents of malondialdehyde?MDA?and glutathione?GSH?in liver were determined by reagent kits.The histological change and inflammatory cell infiltration in paws were measured by H&E stain.The protein expressions and phosphorylation of JNK,P38,ERK1/2 and COX-2 in paw were detected by Western blot.Results:1)BA pretreatment significantly reduced?-carrageenan induced paw edema inflammatory response in mice,especially 4h after intraplantar injection of Carr.2)BA pretreatment reduced the activities of ALT and AST,while increased the level of Mg²⁺,and no obvious changes of the ALB and Ca²⁺levels in serum of mice induced by Carr.3)BA pretreatment significantly reduced the levels of pro-inflammatory cytokines?IL-1?,IL-1?,IL-5,IL-6,GM-CSF,KC and MCP-1?and PGE₂,while increased the levels of anti-inflammatory cytokines?IL-12?in serum of mice treated with Carr.4)BA pretreatment enhanced the SOD,CAT,GSH-Px activities and GSH content,and reduced MDA content in liver of mice induced by Carr.5)BA pretreatment reduced the infiltration of neutrophils in paw subcutaneous and paw muscle of mice induced by Carr.6).BA pretreatment decreased protein expression of COX-2 and reduced the phosphorylation of JNK,P38,ERK in Carr-induced paw edema inflammatory in mice.Conclusions:BA has a protective effect on Carr-induced acute paw edema inflammatory by improving anti-oxidation capacity,inhibiting pro-inflammatory cytokine secretion and reducing infiltration of inflammatory cells which mainly regulated by MAPK-COX-2-PGE₂ signaling pathway.