Experimental Study Of The Anti-inflammatory Action Of Icariin In Primary Astrocytes

The traditional Chinese medicine Epimedium has the functions of enhancing immunity,strengthing the body,tonifying kidney,anti-tumor and anti-neuritis.Icariin(ICA)is the main active ingredient of traditional Chinese medicine Epimedium.It has been found that neuroinflammation is associated with the overactivation of microglia and astrocytes.Our previous study has demonstrated the anti-inflammatory effects of ICA in lipopolysaccharide(LPS)-induced neuroinflammation in animal model and primary cultured microglia.So whether ICA can exert the anti-inflammatory action in the primary astrocytes and the molecular mechanism is not very clear.ICA was known as an estrogenic compound.Recently,G protein-coupled estrogen receptor(GPER)has been demonstrated to be a membrane estrogen receptor.Studies have shown that estrogen exerts neuroprotective effect via the cross-talk between the GPER and insulin-like growth factor-1 receptor(IGF-1R).Our previous study has demonstrated that the ICA derivative icaritin can protect against neurotoxin MPP~+-induced dopaminergic neuronal damage via IGF-1Rsignaling pathway in MES23.5 cells.Therefore,we propose whether GPER and IGF-1Rare involved in the anti-inflammatory effect of ICA in primary astrocytes.The experiment was divided into two parts.In the first part,LPS was used to induce the inflammatory reaction of primary astrocytes.The role of GPER and IGF-1R in the anti-inflammatory effect of ICA was investigated.In the second part,the effect of ICA on astrocyte function was evaluated.The experimental results are as follows:1.Experimental study of the effect of ICA against LPS-induced inflammation in astrocytes(1)The purity of cultured primary astrocytes was measured by immunofluorescence technique.The astrocyte-specific marker glial fibrillary acidic protein(GFAP)was detected.The results showed that the purity of primary astrocytes was above 95%.(2)LPS(1μg/mL)significantly increased the m RNA expressions of inflammatory cytokines of TNF-αand IL-1βin astrocytes(P<0.001).The mRNA expression of GFAP was also significantly increased(P<0.01).Pretreatment with different dosages of ICA(0.1、1、10 and 20μM)for 1h,the above gene expressions were apparently inhibited(P<0.05).10μM ICA exerted significantly anti-inflammatory effects which was chosen as the optimal drug concentration.(3)GPER antagonist G15(1μM)could significantly block the anti-inflammatory effects of ICA on LPS-induced gene expressions of COX-2 and IL-1βin astrocyte(P<0.05). While,G15 could not block the inhibitory effect of ICA on LPS-induced gene expressions of i NOS and TNF-α.(4)G15 could not block the inhibitory effect of ICA on the protein expressions of COX-2 and iNOS.(5)IGF-IR antagonist JB-1(1μM)could block the inhibitory effects of ICA on LPS-induced gene expressions of COX-2,IL-1β,iNOS and TNF-α(P<0.05).(6)JB-1 could block the inhibitory effect of ICA on iNOS protein expression(P<0.05),but could not block the protein expression of COX-2.2.Experimental study of the regulatory effect of ICA on astrocyte function(1)The astrocytes were treated with ICA for different time(0h,1h,2h,4h,6h).Them RNA expression of c-fos was significantly increased after 1h treatment(P<0.05).While the gene expressions of GLAST and GLT-1 were significantlyincreases after 2h treatment(P<0.05).(2)Both G15 and JB-1 were able to block ICA-induced gene expressions of c-fos,GLAST,and GLT-1.(3)After 0.5h treatment of ICA on astrocytes,the protein phosphorylation levels of ERK,AKT and CREB were significantly increased.These effects could be blocked by JB-1,but not by G15.Conclusions:These results indicate that ICA can inhibit LPS-induced inflammation in astrocytes,which may be related to GPER and IGF-1R signaling pathways.But the other signaling pathway might be also taken part in this process.The present study provide new experimental data on the anti-inflammatory effect of ICA in astrocytes.