| Background: Cardiovascular disease is one of the most common causes of death in worldwide.Inflammation is a common physiological and pathological response of the body.It has been recognized that cardiovascular diseases are closely related to inflammation.Many cardiovascular diseases are accompanied with inflammation.And in these inflammatory reactions,mononuclear-macrophages play an important role.They can trigger the activation of signaling pathway by releasing a variety of inflammatory mediators,and finally participate in the occurrence and development of cardiovascular diseases.Astragalus is a traditional Chinese medicine for tonifying kidney.Its main active ingredient,flavonoids(astragalus complanatus flavonoids),has various pharmacological effects such as antihypertensive,lipid-lowering,anti-oxidation and anti-inflammatory.However,the anti-inflammatory effects and mechanism of astragalus complanatus flavonoids have not been reported yet.Objective: By using in vivo acute inflammation injury model of genetically modified zebrafish and in vitro LPS-induced RAW264.7 macrophage inflammation response model,this study explored the inhibitory effect and mechanism of astragalus complanatus flavonoids on inflammation.To provided a new idea for the study of Chinese medicine with definite curative effect and high safety.Methods: After extracting of astragalus complanatus flavonoids,we analysis the quality control of astragalus complanatus flavonoids by HPLC;To observe the anti-inflammatory effect of astragalus complanatus flavonoids on acute inflammation injury in zebrafish Tg(Cora1a: EGFP / Lyz: Ds RED),we construct Tg(Cora1a: EGFP / Lyz: Ds RED)zebrafish acute inflammation injury model,add astragalus complanatus flavonoids to intervene treatment,by observing the migration of fluorescent labeled macrophages and neutrophils,detecting the production of NO in zebrafish by micro-method,and detecting the gene changes of inflammatory factors,such as i NOS,TNF-?,IL-1? and IL-10 by RT-PCR.The LPS-induced RAW264.7 macrophage inflammation model was constructed.After the intervention of astragalus complanatus flavonoids,the cell activity was detected by CCK-8,the production of NO was detected by Griess reagent,and the production of TNF-? was detected by TNF-? Elisa kit.The expression of i NOS gene was detected by RT-PCR,and the protein expression of i NOS,NF-?B,I-?B,P38,JNK and ERK1/2 was detected by Western Blot.Through these data,we investigate the signaling pathway and molecular mechanism of astragalus complanatus flavonoids in anti-inflammatory response.Results: The main components of astragalus complanatus flavonoids are Astragaloside A and Astragaloside B;In the acute inflammatory injury model of zebrafish Tg(Cora1a:EGFP / Lyz: Ds RED),astragalus complanatus flavonoids have no toxic effect on zebrafish in the concentration range of 15-30 ?g/m L.We find that astragalus complanatus flavonoids can significantly inhibit the migration of macrophages and neutrophils,the production of NO and the expression of inflammatory genes i NOS,but have no effect on inflammatory factors TNF-?,IL-1? and anti-inflammatory factor IL-10;In the LPS-induced RAW264.7 macrophage inflammation model,the astragalus complanatus flavonoids had no toxic effect on macrophages in the concentration range of 12.5-400 ?g/m L.Our research found that astragalus complanatus flavonoids can significantly inhibited the production of NO and i NOS,but have no effect on TNF-?.The results of Western Blot showed that astragalus complanatus flavonoids significantly inhibited the expression of NF-?B and I-?B,and this reaction could be blocked by PDTC.Conclusion: Astragalus complanatus flavonoids have an inhibitory effect on the acute inflammatory response of zebrafish and LPS-induced RAW264.7 macrophage inflammatory response.The mechanism may be related to the regulation of NO production and inhibition of NF-?B/i NOS signaling pathway. |
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