Inhibitory Effects And Mechanisms Of Liensinine,Neferine And Isoliensinine On The Macrophage Hyperactivation Based On The Ca²⁺-CaM/NF-κB Pathway

Macrophages are important members of human innate immunity and participate in many kinds of immune processes.Macrophages play an important regulatory role in the inflammatory responses.Moderately-activated macrophages release NO,interleukin-1β（IL-1β）,interleukin-8（IL-8）,tumor necrosis factor-α（TNF-α）,reactive oxygen species（ROS）and so on to induce cellimmunity and kill pathogenic microbes.However,if the the proinflammatory responses induced by hyperactivated macrophages can not be terminated in time,it will damage the tissues and cells of the body and endanger the health.The first part of the thesis is the pharmacodynamic studies,in which the inhibitory effects of three alkaloids from Plumula Nelumbinis（Liensinine,Neferine,and Isoliensinine）on LPS-stimulated macrophages were investigated.First,the mouse RAW264.7 macrophages were cultured.The effects of three alkaloids（10μM）on the migratory ability of LPS-activated macrophages were investigated by the wound healing assay and transwell assay.The results showed that three alkaloids could significantly inhibit the migratiory ability of hyperactivated macrophages induced by LPS.Secondly,the effects of three alkaloids of Plumula Nelumbinis on the release of inflammatory cytokines IL-8 from LPS-activated macrophages were examined by Enzyme-Linked Immunosorbent Assay（ELISA）.The results indicated that liensinine,neferine and isoliensinine（0.1,0.3,1,3 and 10μM）could significantly suppress the LPS-stimulated release of IL-8 by macrophages in a concentration-dependent manner,with the IC₅₀0 values of 0.67,0.14 and 0.33μM,respectively.The second part of the thesis is the mechanism studies,in which the effects of three alkaloids of Plumula Nelumbinis（Liensinine,Neferine,and Isoliensinine）on the phosphorylation and degradation of IκBα,the intracellular free Ca²⁺level,and the expression of calmodulin（CaM）in hyperactivated macrophages induced by LPS were examined.Western blot assay was used to measure the phosphorylation and degradation of IκBαin LPS-activated macrophages treated with Liensinine,Neferine,or Isoliensininefor.The results showed that three alkaloids（1,3 and 10μM）could inhibit the phosphorylation and degradation of IκBαin LPS-stimulated macrophages.In order to investigate the effects of three alkaloids（10μM）on the intracellular free calcium concentration([Ca²⁺]_i)in the macrophages activated by LPS,the level of[Ca²⁺]_i was detected by Fluo-3/AM staining and flow cytometry analysis.It was showed that three alkaloids of Plumula Nelumbinis could significantly inhibit the excessive-increase of[Ca²⁺]_i level in macrophages.Moreover,the level of[Ca²⁺]_i in the macrophages was also detected by Fura-2/AM staining and fluorescence spectrophotometer analysis.The results showed that three alkaloids（10μM）could inhibit the increase of[Ca²⁺]_i level in LPS-activated macrophages.The binding capacity of three alkaloids to calmodulin was evaluated by molecular docking method.The results showed that three alkaloids could combine with calmodulin in different extent,and the binding ability of the three compounds to the site one of calmodulin is better than that of the site two.Furthermore,the effects of three alkaloids on the calmodulin expression in LPS-activated macrophages were investigated by western blot assay.The results indicated that three alkaloids of Plumula Nelumbinis（1,3 and10μM）could significantly inhibit the enhanced expression of calmodulin in LPS-stimulated macrophages after the treatment with Liensinine,Neferine,or Isoliensinine.In summary,these findings demonstrate that three alkaloids of Plumula Nelumbinis（Liensinine,Neferine,and Isoliensinine）act to inhibit LPS-induced increase of IL-8 in macrophages by suppressing LPS-induced phosphorylation and degradation of IκBα,which is required for NF-κB activation.And this effect is mediated by inhibiting the binding of Ca²⁺with CaM resulted from the binding of Liensinine,Neferine,or Isoliensinine with CaM,the inhibition of the expression of CaM and the cytosolic free Ca²⁺level in hyperactivated macrophages.Besides,three alkaloids could inhibit the excessive migration by inhibitng[Ca²⁺]_i level in hyperactivated macrophages induced by LPS.All these results suggested that Liensinine,Neferine,and Isoliensinine have the anti-neuroinflammatory activity which might exert potential in the treatment of diseases accompanied by macrophages hyperactivation.