Molecular Mechanism Study Of Icaritin Against Inflammatory Response In Alzheimer's Disease

Alzheimer's disease?AD?is a central nervous system degenerative disease,more and more evidences have shown that microglia-mediated inflammatory responses play an important role in the pathogenesis of AD.Icaritin?ICT?,a derivative of icariin?ICA?,is the most abundant biological active ingredient in Epimedium.ICT is a phytoestrogen and has anti-tumor,anti-inflammatory and neuroprotective effects.Studies have shown that ICT can inhibit A?_25-35-induced apoptosis in rat primary cortical neurons.However,whether it can protect hippocampal neurons and improve learning and memory by inhibiting the inflammatory reaction caused by microglial activation has not been reported yet.Its possible target and molecular mechanism need to be further explored.G protein-coupled estrogen receptor?GPER?is a membranous estrogen receptor.It is mainly located in the cell membrane and endoplasmic reticulum membrane,and is widely expressed in the dentate gyrus,CA1 and CA3 regions of the hippocampus.GPER can be rapidly activated and participate in the non-genomic rapid effects of estrogen.It has been reported that ICT can stimulate the proliferation of breast cancer SKBr3 cells through the GPER-EGFR-MAPK signaling pathway.Our previous study has confirmed that insulin-like growth factor-1 receptor?IGF-1R?signaling pathway is involved in the neuroprotective effects of ICT against MPP⁺-induced toxicity in MES23.5 cells.It is suggested that IGF-1R signaling pathway may be involved in the neuroprotective effects of ICT.Based on the cross-talk between GPER and IGF-1R,we propose whether ICT can exert the neuroprotective effects against AD inflammatory response via GPER and IGF-1R signaling pathways.In the present study,lipopolysaccharide?LPS?was used to established AD inflammatory model.Morris water maze,real time PCR,western blot and immunohistochemistry were used to study the anti-inflammatory effects of ICT and the possible molecular mechanism.The results are as follows:1.Morris water maze results showed that LPS group took longer latency and less quadrant time of training and number of platform crossings compared with the control group?P<0.001?.Pretreatment with different dosages of ICT?5,10,20mg/kg?could significantly reverse LPS-induced behavioral changes in mice?P<0.01,P<0.05?.2.Real time PCR results showed that the gene expressions of inflammatory factor TNF-?,IL-1?and the limited enzyme of proinflammatory cytokine COX-2 and iNOS in hippocampus of LPS group were significantly up-regulated compared with the control group?P<0.001,P<0.05?.Different dosages of ICT?5,10,20mg/kg?pretreatment significantly inhibited the gene expressions of the above inflammatory cytokines?P<0.001,P<0.01,P<0.05?.3.Western blot results showed that pretreatment with different dosages of ICT?5,10,20mg/kg?significantly inhibited LPS-induced protein expressions of COX-2 and iNOS?P<0.001,P<0.01,P<0.05?.In the following experiment,we selected 10mg/kg of ICT for mechanism study.4.Before intragastric administration of ICT,GPER specific antagonist G15 was microinjected into the lateral ventricle to observe its influence on the anti-inflammatory effect of ICT.The results showed that the latency of the water maze test in the G15+ICT group was significantly increased.The quadrant time of training and the number of platform crossings were decreased compared with the ICT group?P<0.05?.Moreover,the gene expressions of TNF-?and IL-1??P<0.01?as well as the gene and protein expressions of COX-2 and iNOS?P<0.001,P<0.01,P<0.05?were significantly increased in hippocampus of G15+ICT group.There was no significant difference between the G15+LPS group and the LPS group.5.Before intragastric administration of ICT,IGF-1R specific antagonist JB-1 was microinjected into the lateral ventricle to observe its influence on the anti-inflammatory effect of ICT.The results showed that JB-1 also blocked the anti-inflammatory effects of ICT on the LPS-induced AD inflammatory model?P<0.001,P<0.05?.There was no significant difference between the JB-1+LPS group and the LPS group.ICT treatment alone also had no effect on the above indicators.6.Compared with the control group,the phosphorylation levels of ERK,JNK and p38 of the MAPKs signaling pathway was significantly increase in LPS group?P<0.001?.ICT treatment significantly decreased phosphorylation levels of the above proteins?P<0.001,P<0.01?.These effects could be blocked by G15?P<0.05?.JB-1 pretreatment could partially block the inhibitory effect of ICT on the above protein phosphorylation.The phosphorylation level of p38 was significantly increased compared with ICT group?P<0.05?.The phosphorylation levels of ERK and JNK were slightly but not significantly increased in JB-1+ICT+LPS group?P>0.05?.There was no significant difference between the G15+LPS or JB-1+LPS group and the LPS group.7.To clarify the activation status of microglia in this experimental model,we used western blot and immunohistochemistry to detect the protein expression of the microglia-specific marker Iba-1 in the hippocampus of mice.Western blot results showed that ICT significantly inhibited LPS-induced Iba-1 protein expression?P<0.001?.These effects could be blocked by G15 or JB-1?P<0.01,P<0.05?.There was no significant difference between the G15+LPS or JB-1+LPS group and the LPS group.These results were consistent with those obtained by immunohistochemistry.8.Nissl staining results showed that ICT could significantly improve the LPS-induced damage of hippocampal CA1 and CA3 neurons?P<0.01,P<0.05?.Both G15 and JB-1 could block the protective effects of ICT?P<0.05?.There was no significant difference between the G15+LPS or JB-1+LPS group and the LPS group.The experimental results indicate that ICT can inhibit the LPS-induced over-activation of microglia and improve learning and memory ability in the AD inflammatory model.GPER and IGF-1R signaling pathways are involved in the anti-inflammatory neuroprotective effects of ICT.This study provides experimental evidence for the discovery of prodrug with potential clinical value.