Inhibitory Effects And Mechanisms Of Curine,Isochondodendrine And Cycleanine On The Macrophage Hyperactivation

Peripheral inflammation is the reaction of body to defend invasive bacteria,viruses,and parasites.Macrophages,which have a variety of immune functions,are an important part of the immune system.They are the main regulatory cells in the inflammatory response and are closely related to tumor,peripheral inflammatory diseases,metabolic diseases,and other related diseases.The release of NO,TNF-?,IL-1?,and IL-6 was increased when the host received pathogen infection.Then the surface molecules of macrophages were increased,which induce cellular immunity and kill the pathogenic microorganisms.Macrophages play a key role in the effective removal of pathogens.However,hyperactivated macrophages produce a large amount of inflammatory mediators which will cause damage to the surrounding tissues and related to the onset of atherosclerosis of atherosclerosis and other metabolic diseases.The inhibition of macrophage hyperactivation is important for the inhibition of peripheral inflammation.In this study,the inhibitory effects and mechanisms of three dibenzylisoquinoline alkaloids?BBI?,Curine,Cycleanine,and Isochondodendrine,on RAW264.7 macrophage hyperactivation induced by LPS were investigated.First,the mouse RAW264.7 macrophages were cultured in vitro.Cells were treated with three BBI?0.1-30?mol/L?with or without LPS?1?g/ml?,cell viability of macrophages was then evaluated by MTT method to exclude the influences of the drug toxicity.The results showed that the three alkaloids?0.1-30?mol/L?had no significant effect on cell viability of LPS-activated macrophage.In addition,the three alkaloids did not affect the cell viability of macrophages when were used on RAW264.7 cells alone.Then,the inhibitory effects of the three alkaloids on hyperactivated cells were investigated.The inhibitory effects of the three BBI?0.1-30?mol/L?on NO release from LPS-activated macrophages were detected by Griess method.And the effects of the three kinds of BBI were compared.The results showed that the three BBI couldinhibit the release of NO from the activated macrophages on different extent without affecting the release of NO from resting cells.By comparing the half maximal inhibitory concentration?IC50?,the inhibitory effect of Curine on NO release was the most powerful.And the effect was stronger than Tetrandrine?3?mol/L?,the positive control.The effects of three BBI?0.1-10?mol/L?on the release of TNF-?,IL-1?,and IL-6 inflammatory cytokines from LPS-activated macrophages were examined by Enzyme-Linked Immunosorbent Assay?ELISA?.It was showed that the three BBI could significantly inhibit the release of TNF-?,IL-6,and IL-1? by LPS-activated RAW264.7 cells.By comparing the IC50 values,the inhibitory effects of the three BBI on the release of IL-6 and IL-1? were stronger than that of TNF-?.And the inhibitory effect of Curine on the release of inflammatory factors was the most significant among the three BBI.The mechanisms of three alkaloids on macrophage hyperactivation were also investigated by study the influence of the three BBI on the expression of inducible nitric oxide synthase?iNOS?and the intracellular Ca²⁺ level,and the NO and free radicals scavenging ability of the three BBI.Western blot assay was used to study the influence of the three BBI on the expression of iNOS.And it was showed that the three BBI?1-10?mol/L?could significantly inhibit the expression of iNOS protein in LPS-activated macrophages.Then,the effects of the three BBI?10?mol/L?on the level of cytosolic free calcium concentration([Ca²⁺]_i)in LPS-activated macrophages were measured by Fluo-3/AM staining and flow cytometry analysis.The results indicated that the three BBI?10?mol/L?could significantly inhibit the abnormal increasing of [Ca²⁺]_i in LPS-induced macrophages.In order to investigate the ability of three BBI?0.1-30?mol/L?to capture the active nitrogen,the sodium nitroprusside?SNP?was used as the NO donor to detect the direct NO scavenging activity.It was showed that NO2-level in SNP solution was decreased by adding one of the three BBI significantly,indicating that they possess the NO-scavenging activity.In order to further investigate whether the inhibitory effects of three BBI on macrophage activation is related to its anti-oxidative and anti-radiator effect,UV spectrophotometry was used to detect the DPPH scavenging effect of three BBI?0.1-30?mol/L?,·salicylic acid method was used to detect the OHscavenging effect,L-tyrosine method was used to detect the ONOO-scavenging effect,and pyrogallol autoxidation method used to detect its superoxide oxygen anion free radical?·O2-?scavenging effect.The results showed that three BBI?0.1-30?mol/L?had significant clearance effects on DPPH,·OH,ONOO-,and ·O2-.In addition to ONOO-.In summary,the three BBI,Curine,Cycleanine,and Isochondodendrine,can inhibit the NO production from LPS-activated macrophage by down-regulating the expression of iNOS and directly scavenging NO.Moreover,they can inhibit the release of IL-1?,TNF-?,and IL-6 in activated macrophages without the influence in the cell viability.And this effect may be mediated at least in part by inhibiting the[Ca²⁺]_i level and scavenging the free radicals such as DPPH,·OH,ONOO-and ·O2-.Three alkaloids inhibit the activation of macrophages induced by LPS and the scavenging effect of various free radicals were stronger than that of positive control.Comprehensive consideration,Curine was in the best effect of the three alkaloids.Curine,Cycleanine and Isochondodendrine may have certain preventive and therapeutic effects on peripheral inflammation-related diseases caused by macrophage hyperactivation.