Effect And Mechanism Of Total Alkaloids From The Seed Embryo Of Nelumbo Nucifera On The Anti-neuroinflammation And Neuroprotection

Neuroinflammation and neuronal injury are considered as the main pathophysiological features of Alzheimer's disease.Microglial cells are the main immune cells of the central nervous system and the main source of neuroinflammatory factors.Therefore,inhibition of neuroinflammation and neuronal damage caused by over-activation of microglial cells may be an effective therapeutic strategy for AD.Lotus?Nelumbo nucifera Gaertn.?seed embryo is a medicinal and edible plant approved by the Chinese Ministry of Health,with high function in health care and medicinal value.The total alkaloids from the seed embryo of Nelumbo nucifera?TASENN?is the effective part of alkaloids from Lotus seed embryo,and the main ingredients are neferine,liensinine and isoliensinine.Here we investigated the inhibitory effect of TASENN?The contents of neferine,liensinine and isoliensinine were 44.57,23.01,and 28.02%,respectively;the total content of these three alkaloids was 95.6%?on lipopolysaccharide?LPS?-induced excessive activation of microglial cells and macrophages.Based on the Ca²⁺-Ca M/Ca MK?pathway,we investigated the mechanism of TASENN responsible for inhibition of microglial over-activation.In addition,the inhibitory effect of TASENN on PC12 neuronal injury induced by microglial activation was examined.First,Griess assay was used to detect the effects of TASENN on NO release from LPS-stimulated BV2 microglial cells or RAW264.7 macrophages.The results showed that TASENN inhibited NO release from the activated BV2 microglial cells and RAW264.7 macrophages.MTT assay was used to investigate the effects of TASENN on the survival rate of LPS-activated BV2 microglia and RAW264.7macrophages.The experimental results confirmed that TASENN had no significant effect on the survival rate of the two kinds of activated cells.A microplate reader was used to evaluate the effect of TASENN on NO and DPPH radical scavenging.Results showed that TASENN had NO and DPPH radical scavenging effects in the cell-free reaction systems.Secondly,western blot assay was performed to investigate the effect of TASENN on calmodulin?Ca M?expression,calmodulin-dependent protein kinases??Ca MK??phosphorylation,inhibitor of nuclear factor-?B?I?B??phosphorylation and degradation,and inducible nitric oxide synthase?i NOS?expression in LPS-stimulated BV2 microglial cells.Results indicated that TASENN?2,6?g/m L?inhibited the expression of Ca M and the phosphorylation of Ca MK?,thus contributing to the significant inhibition of intracellular I?B?phosphorylation and degradation and reduction in i NOS expression in LPS-activated BV2 microglial cells.In addition,the effect of TASENN on[Ca²⁺]_iconcentration and reactive oxygen species?ROS?level in LPS-activated BV2 microglial cells were also measured by a fluorescence spectrophotometer.The results showed that TASENN?2,6?g/m L?also inhibited the levels of the intracellular[Ca²⁺]_iand ROS in activated microglial cells.Finally,MTT assay was used to detect the effect of TASENN?2,6?g/m L?on the survival rate of PC12 neuronal cells injured by LPS conditioned medium?L-CM?from BV2 microglial cells.The results showed that TASENN improved survival rate of PC12 cells injured by L-CM.The effect of TASENN?6?g/m L?on the morphological changes of PC12 cells injured by L-CM was detected by HE staining.It was found that TASENN suppressed the morphological changes of PC12 cells damaged by L-CM.The effect of TASENN?2,6?g/m L?on the concentration of[Ca²⁺]_iand the level of ROS in PC12 cells injured by L-CM were measured by fluorescence spectrophotometry.The results showed that TASENN inhibited Ca²⁺overload and ROS production in PC12 cells damaged by conditioned medium.In summary,the total alkaloids from the seed embryo of Nelumbo nucifera?TASENN?inhibited the expression of Ca M,the level of[Ca²⁺]_i,and the activation of Ca MK?;then inhibited the phosphorylation and degradation of intracellular I?B?,the activation of NF-?B,as well as the expression of i NOS,thus contributing to the reduction in NO release in microglial cells.TASENN also inhibited the release of ROS from over-activated BV2 microglial cells.In addition,TASENN inhibited the damage of PC12 neuronal cells by conditioned medium from LPS-activated BV2microglial cells via inhibiting the intracellular Ca²⁺overload and ROS production,increasing cell survival rate,and suppressing cell morphological changes.Taken together,the total alkaloids from the seed embryo of Nelumbo nucifera?TASENN?possessed notable anti-neuroinflammatory effects,and had neuroprotective effects against inflammation-induced neuronal over-damage in vitro,which may exhibit potential preventive and therapeutic effects on neurodegenerative diseases associated with over-activated microglial cells.