Tonifying Kidney Herb Effective Components On The Regulation Of Microglia Polarization And Mechanism Research

Objective:Microglia are resident immune cells in the central nervous system as commonly described for macrophages influencing brain development,maintenance of the neural environment,response to injury and recovery.Microglia played different roles through their different polarization direction.The activation states of microglial can be categorized into "classical activation"(Ml)and"alternative activation"(M2),and thus microglial activation acts as the crucial stuff of immune response and play very important roles in the progress of various CNS diseases.Cornel iridoid glycoside(CIG)is effective component of tonifying kidney herb dogwood.And studies have shown that CIG has multiple pharmacological effects such as anti-inflammatory,immune regulation,neurotrophic effect,etc.BaJiJiaSu is effective component of tonifying kidney herb Morinda officinalis.Previous studies have reported that the medicinal indian mulberry root polysaccharide has improve immunity,antioxidant,etc.Controlling microglial activation has been suggested as an important therapeutic strategy for some neuroinflammatory diseases such as Alzheimer' s disease,Parkinson' s disease and multiple sclerosis.In the present study,the main components from tonifying kidney herb CIG and BaJiJiaSu were selectedand examined the effects on LPS/IFN-r stimulated M1 microglial polarization and then extended the underlying mechanisms.Methods:1.The EAE model was induced by PLP139-151 in female SJL mice.SJL mice were divided into four groups:Ctrl group,EAE group,CIG 100 group and FTY group.After immunization by emulsifier containing PLP139-151 for 9 days,CIG,FTY or vehicle was given to mice intragastrically as treatment or control.Every day the mice nerve function score and record every mice body weight,On the 26st day,the whole brain tissue was removed and evaluated by immunofluorescence staining using microglia markers Iba-1 in the brain.2.LPS(100 ng/ml)and IFN-?(10 ng/ml)combination were used to stimulate BV2 microglial cell line of mice microglia to induce inflammation,M1 polarization.Experiment is divided into control group,the control+CIG(100?g/mL)group,model group(LPS/IFN-?),model+CIG(25,50,100,200 ?g/mL)group.Microglia cells were pretreatment with or without CIG for 2 h,then stimulated with LPS and IFN-? for 24 h to collect samples for testing.Immunofluorescence method is used to measure whether CIG can inhibit the expression of the type of microglial M1 marker CD16/32 and iNOS.Western Blot test was performed to determine the expressions of SOCS1 and iNOS.NO kits was performed to determine whether CIG can inhibit the NO release.ELISA was performed to test whether CIG influence the level of inflammatory factors such as IL-6 in the supernatant.3.By CCK8 method to detect different concentrations of BaJiJiaSu(25,50,100,100,200,400,800 ?M)on the influence of BV2 microglia survival rate.4.LPS(100 ng/ml)and IFN-?(10 ng/ml)were combined to stimulate BV2 cells to induce M1 polarization in vitro model Experiment is divided into control group,the control+BaJiJiaSu(200 ?M)group,model group(LPS/IFN-?),model+ BaJiJiaSu(50,100,100,200,400 ?M)group.Microglia cells were pretreatment with or without BaJiJiaSu for 2 h,then BV2 cells were stimulated with LPS and IFN-? for 24 h to collect samples for testing.Minocycline were served as a positive control drug.Immunofluorescence method is used to eveluate whether CIG can inhibit the expression of the type of M1 marker CD16/32.Western Blot test was performed to determine the expression of COX-2,iNOS,JAK1,p-JAK1,JAK3,p-JAK3,STAT1,p-STAT1(Y701),p-STAT1(S727),STAT3,p-STAT3(Y705),and p-STAT3(S727).Results:1.Female SJL mice could be successfully induced to inflammatory EAE mice model by PLP139-151 peptide fragments.CIG treatment could reduce the numbers of Iba-1+/CD16+ M1 microlglia after EAE.Our results showed that CIG subjects(100 mg/kg)inhibited M1 microglial polarization and the positive control FTY showed a similar effect.2.LPS and IFN-? combination could stimulate M1 polarization,increasing M1 markers such as NO,iNOS,IL-6,IL-1?,CD16/32 expression,and decrease SOCS1 expression.After CIG treatments,the expression of M1 markers NO,iNOS,IL-6,IL-1 ?,CD16/32 decreased significantly,but did not observe CIG have inhibitory effect on level of IL-1 ?.Microscopic photograph shows,CIG treatments can inhibit BV2 microglia cell body change big presented by the activated state and gradually change to the morphology of resting state.3.LPS and IFN-y combination could stimulate M1 polarization,increasing Ml markers such as iNOS,COX-2,CD16/32 expression.After BaJiJiaSu treatments,can reduce the expression of iNOS,COX-2,CD16/32 and was dose dependent,Microscopic photograph shows,BaJiJiaSu not only reduced the expression of CD16/32,and changed the BV2 microglia cell body change big presented by the activated state.LPS and IFN-? compound stimuli can make the JAK/STAT signaling pathway activation,After BaJiJiaSu treatments,can reduce the expression of p-JAK1 and p—JAK3,and BaJiJiaSu of STAT1 and STAT3 phosphorylation of different sites also have obvious inhibitory effect.Conclusion:Both in vivo and in vitro results demonstrated that CIG could inhibit BV2 microglia polarization to M1 induced by LPS and IFN-y pretreatment.BaJiJiaSu also significantly reduced microglia polarization to M1 induced by LPS and IFN-? in BV2,the underlying mechanism may be through through suppressing JAK/STAT signaling pathway.