Study On The Molecular Mechanism Of Icariin And Icaritin In Inhibiting The Inflammatory Response Of Primary Microglial

Neuroinflammation is a prominent feature of various neurodegenerative diseases such as Parkinson’s disease(PD)and Alzheimer’s disease(AD).Microglial activation is the principal component of neuroinflammation in the central nervous system(CNS),which provides the first line of defense whenever injury or disease occurs.The molecular and clinical evidences from postmortem analysis and positron emission tomography(PET)imaging have shown an increase of microglia(MG)activation and an increasing accumulated inflammatory mediators during the pathogenesis of PD.The MG appear to be heterogeneous with diverse functional phenotypes that range from pro-inflammatory M1 phenotypes to immunosuppressive M2 phenotypes.M1 phenotypes often setup a vicious cycle between dying neurons and acute inflammation.M2 phenotypes release a variety of anti-inflammatory cytokines,which inhibit the neuroinflammation and promote neuron survival.Therefore,inhibiting the activation of M1 phenotypes and promoting the activation of M2 phenotypes may be a feasible treatment strategy for the treatment of inflammation-related diseases.Icariin(ICA)is the main active ingredient of traditional herb Epimedium.Icaritin(ICT)is the main metabolite of ICA in the body.Studies have shown that both of ICA and ICT have anti-apoptotic,anti-inflammatory and antioxidant effects,ICA and ICT have estrogen-like effects,G protein-coupled estrogen receptor(GPER)-mediated signaling pathways are involved in the proliferation of breast cancer cells induced by ICA and ICT.The preliminary work of our research group proved that ICT could protect against the damage of neurotoxin on dopaminergic neurons through insulin-like growth factor-1receptor(IGF-1R)-mediated signaling pathway.GPER and IGF-1R are involved in the anti-inflammatory neuroprotective effect of ICA and ICT.However,the regulation of MG phenotypes and the target of their anti-inflammatory neuroprotective effect need to be further explored.Objective1.To study the effects of ICA and ICT on the polarization of primary MG;2.To study the target of ICA and ICT in inhibiting the inflammatory response of MG and their related signaling pathways.Methods1.Lipopolysaccharide(LPS)was used to establish the inflammatory model of primary midbrain MG in rats.2.Real-time PCR(RT-PCR)was used to detect the gene expressions of Arg-1,IL-10,IL-1β,TNF-α,i NOS and COX-2.3.Western blot was used to detect the protein expression of i NOS,COX-2,ERK,p38,JNK and IκB.4.Pharmacology blockaded and lentivirus-mediated RNA interference technology were used to interfere the GPER and IGF-1R signal pathways.Results1.ICA(10 μM)and ICT(10 μM)treatment alone could significantly induce the m RNA expressions of M2 phenotypes markers Arg-1 and IL-10(P<0.05,P<0.01,P<0.001).2.LPS(0.5 μg/m L)could significantly induce the m RNA expressions of IL-1β,TNF-α,i NOS and COX-2(P<0.001),ICA and ICT pretreatment could inhibit the expressions of these inflammatory genes(P<0.05,P<0.01,P<0.001),and these effects could blocked by IGF-1R antagonist JB-1(1 μg)and GPER antagonist G15(1 μM)(P<0.05,P<0.01,P<0.001).3.ICA and ICT could inhibit the expressions of i NOS and COX-2 in microglial induced by LPS at the protein level(P<0.001),and these protective effects could be blocked by JB-1 and G15(P<0.05,P<0.01,P<0.001).4.LPS could significantly increase the ptotein phosphorylation levels of ERK,p38,JNK and IκB(P<0.01,P<0.001),ICA and ICT could supress the phosphorylation levels of these proteins(P<0.01,P<0.001).These effects could be blocked by JB-1 and G15(P<0.05,P<0.01,P<0.001).5.Lentivirus-mediated RNA interference technology were used to interfere the GPER gene of BV2.Compared with the Lv-si Con group,the Lv-si GPER group could significantly reduce the GPER gene expression(P<0.01).The knockout rate was about50%.6.At the gene level,the inhibitory effects of ICA and ICT on the dene expressions of IL-1β,TNF-α,i NOS and COX-2 were significantly reduced in the Lv-si GPER group(P<0.01,P<0.001).7.At the protein level,the inhibitory effects of ICA and ICT on the protein expression of i NOS and COX-2 were significantly reduced in the Lv-si GPER group(P<0.05).ConclusionIn summary,ICA and ICT can promote immunosuppressive M2 polarization,and inhibit the M1 polarization induced by LPS.GPER may be the target of the anti-inflammatory effects of ICA and ICT in microglia.IGF-1R is involved in the anti-inflammatory effects of ICA and ICT.