Effect And Mechanism Of Curcumin On Microglial Polarization

Background and AimMicroglia,as an innate immune cell in the central nervous system,can regulate neuroinflammation through polarization phenotype changes and play an important role in the development of various cognitive dysfunctions such as Alzheimer’s disease and vascular dementia.Curcumin is a biologically active substance extracted from the roots of traditional Chinese medicine turmeric,and has various pharmacological activities such as anti-inflammatory and anti-oxidation.However,whether curcumin can exert neuroimmune regulation by regulating the polarization state of microglia and its specific mechanism has not been reported.Therefore,in this study,lipopolysaccharide-induced BV2 cells were used as the research object,and curcumin was used as an intervention to observe the regulation of curcumin on microglia polarization in inflammatory response,and to explore its possible mechanism.Methods1.Material: murine BV2 microglia cell lines;2.Construction of cellular inflammatory model: BV2 microglia were treated with 1 μg·m L-1 LPS;3.Cell grouping:(1)Control group: culture medium only;(2)lipopolysaccharide group: culture medium containing 1 μg·m L-1 LPS to induce microglia activation;(3)Curcumin group: culture medium containing 10 μmol·L-1curcumin only;(4)LPS+Cur group: microglia inflammatory cell model(LPS treatment)was superimposed with curcumin treatment,and curcumin treatment group was divided into 5 subgroups according to different concentrations including 1,5,10.(low dose curcumin group)and 20,40 μmol·L-1(high dose curcumin group);4.Inverted phase contrast microscopy to observe the morphological changes of microglia;5.Cell phagocytosis experiments to observe the activation status of microglia;6.CCK-8 method to detect the activity of each group of cells;7.The Griess method detects the release level of Nitric Oxide(NO);8.RT-PCR detection of M1 type pro-inflammatory factors(IL-1β,IL-6 and i NOS)and M2 type anti-inflammatory factors(IL-4,IL-10 and Arg-1)m RNA expression;9.ELISA assay for the expression of M1 proinflammatory cytokines(IL-1β and IL-6)and M2 anti-inflammatory factors(IL-4,IL-10 and Arg-1);10.Flow cytometry was used to detect the expression of M1 microglia markers(CD16/32)and M2 microglia markers(CD206);11.Cellular immunofluorescence technique was used to detect the expression of M1 microglia markers(i NOS)and M2 microglia markers(CD206);12.Western blot analysis of TREM2,TLR4,IκB-α,p-IκB-α,NF-κB p65,pNF-κB p65 protein expression.Results1.Low-dose curcumin group(1,5 and 10 μmol·L-1)had no significant effect on microglia activity;curcumin high-dose group(20 and 40 μmol·L-1)had significant activity of microglia inhibition;2.In the control group,BV2 microglia were in a resting state,the cell body was slender,and the branches of the cell body protruded from the slender protrusions;after LPS treatment,most of the cell bodies became large and round,and some cell surface protrusions became thick and short.It is a typical amoeba;the cell morphology is improved after pretreatment with curcumin;3.After LPS treatment of BV2 microglia,latex phagocytosis experiments showed that the phagocytic activity of cells decreased significantly,and the phagocytic activity increased after curcumin treatment;4.After LPS treatment of BV2 microglia,results of Griess,RT-PCR and ELISA showed increased secretion of NO and pro-inflammatory factors IL-1β,IL-6 and i NOS,and anti-inflammatory factors IL-4,IL-10 and Arg-1 secretion decreased;after treatment with curcumin,NO and pro-inflammatory factors IL-1β,IL-6 and i NOS secretion decreased significantly,and the secretion of anti-inflammatory factors IL-4,IL-10 and Arg-1 increased significantly;5.After LPS treatment of BV2 microglia,flow cytometry and cellular immunofluorescence showed that the expression of M1 microglia markers CD16/32 and i NOS was significantly increased and the expression of M2 microglia marker CD206 was significantly decreased.Pretreatment with curcumin can decrease the expression of M1 microglia markers CD16/32 and i NOS and increase the expression of M2 microglia marker CD206;6.After LPS treatment of BV2 microglia,Western blot analysis showed that TREM2 protein expression decreased,TLR4,p-IκB-α,p-NF-κB p65 protein expression was significantly increased;curcumin pretreatment could induce TREM2 protein expression Increase and decrease the expression of TLR4,p-IκB-α,p-NF-κB p65 protein.ConclusionsCurcumin inhibits LPS-induced microglial proinflammatory cytokine release and regulates microglial phenotypic polarization,which may be through the TREM2/TLR4/NF-κB signaling pathway.