Inhibitory Effects Of Three Alkaloids Of Plumula Nelambinis On Lipopolysaccharide And Manganese Chloride Induced Microglial Activation

Manganese is an essential trace element in the human body and participates in many important synthesis and metabolism in the body.The lack of manganese in the body can lead to growth retardation.Excessive exposure to manganese can cause brain diseases such as nervous system disorders.Manganese is widely present in air,water and soil.Medium,but the content of soil in the north and south is different.Occupational personnel such as manganese ore mining and smelting and welding operations are susceptible to damage from high concentrations of manganese to cause damage to the nervous system.Many environmental pollution conditions and occupational environments can cause different degrees of damage to the nervous system.Microglia are immune resident cells of the central nervous system,which are important for maintaining the steady state health of the nervous system.Microglia will be overactivated and then release a large amount of nitric oxide?NO?,reactive oxygen species?ROS?,and interleukin-8?IL-8?,which may be closely related to the occurrence of neurodegenerative diseases.This study investigated the effects of various exogenous substances on microglia:the excessive activation of microglia induced by lipopolysaccharide?LPS?and manganese chloride,and the inhibitory effects of bisbenzylisoquinoline alkaloids?Liensinine,Neline,and Isoliensinine?on microglial hyperactivation.The first part of these pharmacodynamic studies,in which the inhibitory effects of three alkaloids from Plumula Nelumbinis?Liensinine,Neferine,and Isoliensinine?on LPS-stimulated microglia were investigated.First,the migration ability of microglia was examined by healing assay.The results showed that the three lotus alkaloids?10?M?alone did not change the basal migration ability of microglia,while three alkaloids could significantly inhibit the migratioryability of hyperactivated microgliainduced by LPS.Secondly,the phagocytic ability of microglia was investigated by phagocytosis of chicken red blood cells.The results showed that microglia showed phagocytic ability after incubation of chicken red blood cells for about 2 hours,and LPS significantly enhanced the phagocytic ability of microglia.After the combined action of three kinds of alkaloids,the phagocytic ability was inhibited.Finally,the effects of three alkaloids of Plumula Nelumbinis on the release of inflammatory cytokines IL-8 from LPS-activated microglia were examined by Enzyme-Linked Immunosorbent Assay?ELISA?.The results indicated that the three alkaloids of lotus plumule can inhibit the excessive release of IL-8 by LPS-activated microglia.The second part of the experiment examined the ability of three alkaloids of lotus plumule to inhibit LPS-induced microglial hyperactivation.Firstly,DCFH-DA was used as a fluorescent probe to detect the level of reactive oxygen species?ROS?in microglia by fluorescence spectrophotometer.The results showed that the ROS level of microglia was significantly increased after LPS induction.1,3 and 10?M of alkaloids can inhibit the excessive release of ROS,and this inhibition has a concentration effect relationship.Secondly,using Fura-2/AM as a fluorescent probe,the[Ca²⁺]_i level in microglia was detected by fluorescence spectrophotometer.The results showed that LPS can enhance the[Ca²⁺]_i level in microglia.It was showed that three alkaloids of Plumula Nelumbinis could significantly inhibit the excessive increase of[Ca²⁺]_i level in microglia.Finally,the expression of calmodulin?CaM?in microglia was examined by Western Blot.The results showed that all three lotus alkaloids could inhibit the expression of CaM in microglia activated by LPS.The third part experimentally studied the effect of manganese chloride on the activation of microglia and the inhibition of three kinds of three alkaloids from Plumula Nelumbinis on the LPS-induced microglia.Firstly,the microglia was induced by different concentrations of manganese chloride and LPS,the survival rate of microglia was examined by MTT assay.The results showed that manganese chloride alone or in combination with LPS can kill microglia when the concentration of manganese chloride is greater than 100?M.Thus,the concentration of manganese chloride should not exceed 30?M in further experiments.Secondly,the ability of microglia released NO was tested by Griess assay.The results showed that manganese chloride alone had no significant effect on the release of NO by microglia,but it could significantly increase the NO release of LPS-induced microglia.Next,the morphological changes of microglia were examined by HE staining.The results showed that LPS can make the microglia synaptic lengthening and exhibit an activated form,which further enhances the activation effect after combined with manganese chloride.All three kinds of alkaloids can inhibit the abnormal changes of morphology of microglia induced by manganese chloride and LPS.Finally,the migration of microglia after LPS combined with manganese chloride was examined by healing assay.The experimental results showed that under the combined action of LPS and manganese chloride,the migration ability of microglia is significantly enhanced,and the three alkaloids inhibit the excessive migration of microglia.In summary,these findings demonstrate that three lotus alkaloids can inhibit the phagocytosis and migration of microglia induced by LPS,and the ability of the cells to release inflammatory factor IL-8,which may related to the inhibition of intracellular ROS level,[Ca²⁺]_i level and CaM expression.Manganese chloride can enhance the activation and flammatory response of LPS-induced microglia.Three alkaloids of lotus plumule can inhibit the morphological changes and excessive migration of microglia induced by manganese chloride and LPS.However,the detail mechanisms need further study.This study can preliminarily elucidate the role of manganese chloride in the development of neuroinflammation,and clarify the role of alkaloidsoflotusplumuleinthepreventionandtreatmentof neuroinflammation-related diseases caused by manganese chloride pollution.