Mechanism Study Of The Involvement Of GPER In The Anti-inflammatory Response Of Icariin And Icaritin In Parkinson’s Disease

G protein-coupled estrogen receptor（GPER）belongs to the family of seven-transmembrane G protein-coupled receptors.It is widely expressed in the brain such as hippocampus,striatum,hypothalamus and substantia nigra.Many studies have confirmed the neuroprotective effect of estrogen via GPER.Icariin is the main active ingredient of the Herbal Epimedium.The content of icariin was the highest in the total flavonoids of Epimedium.Icaritin is one of the major metabolites of icariin and belongs to the flavonols of phytoestrogen.Our previous study confirmed that icariin and icaritin could inhibit the inflammatory response of rat ventral mesencephalic microglia and astrocytes.However,the anti-inflammatory target of icariin and icaritin remain unclear.Aim:1.GPER 3D model was used to determine whether icariin and icariin could directly bind to GPER;2.GPER^+/+wide type and GPER^-/-gene knockout C57BL/6J mice were used to investigate the role of GPER in the anti-inflammatory effect of icariin and icaritin in Parkinson’s disease（PD）mice.Methods:1.Online servers such as GPCR-I-TASSER were used to perform modeling of GPER.The software autodock vina1.1.2 was used to perform molecular docking analysis of GPER with icariin and icaritin.Moreover,the molecular dynamics stimulation of icaritin/GPER complex was carried out.2.The PD inflammatory model was established by stereotactic injection of LPS in the substantia nigra of GPER^+/+and GPER^-/-C57BL/6J mice.Icariin（10 mg/kg）and icariin（10 mg/kg）were administered intragastrically for 14 days.3.Motor coordination was detected using rotarod test and pole test.4.Real-time PCR was used to detect the gene expressions of inducible nitric oxide synthase（i NOS）,cyclooxygenase-2（COX-2）,tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）.5.Western blot was used to detect the protein expressions of i NOS,COX-2,tyrosine hydroxylase（TH）,microglia specific antibody（ionized calcium binding adapter molecule1,Iba1）and glial fibrillary acidic protein（GFAP）.6.Immunohistochemistry was used to detect the numbers of TH-positive neurons.Result:1.The results of molecular docking showed that the binding energy between G1 and GPER is-9.8 kcal/mol.Icaritin could bind to the hydrophobic core of the N terminal of GPER and the binding energy is-9.3 kcal/mol which was closed to the value of G1.The binding energy between icariin and GPER is-6.3 kcal/mol,suggesting that the binding ability of icariin is weaker than that of icaritin.2.The results of molecular dynamics simulation showed that the overall binding free energy of icaritin/GPER complex is-30kcal/mol,further confirmed that icaritin could combine well with GPER.3.The results of behavioral experiment showed that LPS induced motor dysfunctions in GPER^+/+and GPER^-/-mouse（P<0.05,P<0.01,P<0.001）.Icaritin could improve LPS-induced motor dysfunctions in GPER^+/+mouse（P<0.05,P<0.001）.The protective effect of icaritin is not obvious in GPER^-/-mouse.It suggested that the neuroprotective effect of icaritin was interfered by GPER gene knockout.Icariin inhibited the LPS-induced motor dysfunctions of GPER^+/+mouse in pole-test（P<0.05,P<0.01）,but not in rotarod test.In pole test of GPER^-/-mouse,icariin had no effect on extension of the return time（T-turn）induced by LPS,but improved the total time of descend（T-total）（P<0.01）.4.Western blot results showed that LPS could reduce the TH protein level in the substantia nigra of GPER^+/+and GPER^-/-mice（P<0.01,P<0.001）.Icaritin could significantly inhibit the inhibitory effect of LPS on the TH protein expression in GPER^+/+mouse（P<0.01）and the neuroprotective effect of icaritin was significantly reduced in GPER^-/-mice.Icariin has no obvious effect on LPS-induced decrease of TH protein expression both in GPER^+/+and GPER^-/-mice.5.Immunohistochemical results showed that LPS could induce the decrease of the number of TH-positive neurons in the substantia nigra in GPER^+/+and GPER^-/-mice（P<0.01）.Icaritin ameliorated the decrease of TH positive neurons in substantia nigra induced by LPS in GPER^+/+mice（P<0.01）.Icaritin could not exert neuroprotective effect in GPER^-/-mice.Icariin could not inhibit the decrease of TH positive neurons induced by LPS both in GPER^+/+and GPER^-/-mice.6.Real time PCR results showed that LPS induced the up-regulation of m RNA of inflammatory cytokins i NOS,COX-2,TNF-αand IL-1βin the substantia nigra of GPER^+/+and GPER^-/-mice（P<0.05,P<0.01,P<0.001）.LPS-induced inflammatory response was suppressed by icaritin in GPER^+/+mouse（P<0.01,P<0.001）,while the anti-inflammatory effect of icaritin was significantly decreased in GPER^-/-mouse（P<0.05,P<0.01,P<0.001）.Icariin could reduce the LPS-induced upregulation of m RNA levels of TNF-αand i NOS in GPER^+/+mice（P<0.01）.While compared with LPS group,the m RNA levels of IL-1βand COX-2 only could slightly but not significantly decrease.In GPER^-/-mice,icariin could inhibit the LPS-induced upregulation of m RNA levels of IL-1β,i NOS and COX-2（P<0.05）.These resutls suggested that GPER gene knockout had no significant effect on the anti-inflammatory activity of icariin at m RNA level.7.Western blot results showed that LPS could induce the increase of COX-2 and i NOS protein expressions in substantia nigra of GPER^+/+and GPER^-/-mouse（P<0.01,P<0.001）.Icaritin significantly inhibited the LPS-induced inflammation in GPER^+/+mouse（P<0.01）.However,the anti-inflammatory effect of icaritin was significantly weakened in GPER^-/-mouse（P<0.05,P<0.01）.Icariin had a tendency to inhibit the increase of COX-2 and i NOS expressions induced by LPS,but it is not statistically significant in GPER^+/+mouse.Icariin could not inhibit the increase of COX-2 and i NOS protein expression induced by LPS in GPER^-/-mice.8.Western blot results showed that LPS could increase the protein expressions of Iba1 and GFAP in the substantia nigra of GPER^+/+and GPER^-/-mice（P<0.01,P<0.001）.Icaritin could inhibited the increase of Iba1 and GFAP protein levels induced by LPS in GPER^+/+mice（P<0.001,P<0.05）.In GPER^-/-mice,the anti-inflammatory effect of icaritin was significantly reduced（P<0.01）.Icariin inhibited the increase of Iba1 protein level,but not GFAP protein level,induced by LPS in GPER^+/+mice（P<0.001）.Icariin could not inhibit the LPS-induced up-regulation of Iba1 and GFAP protein levels in GPER^-/-mice.Conclusion:1.The binding energy of icariin with GPER is-9.3 kcal/mol,showing good binding ability.The binding energy of icariin with GPER is-6.3kcal/mol and the binding ability is relative weak.2.Icaritin can inhibit the inflammatory response induced by LPS in the substantia nigra of mice of GPER^+/+mice,but cannot exert the anti-inflammatory effect in GPER^-/-mice.It is suggested that GPER mediates the anti-inflammatory effect of icaritin in PD,and GPER may be the direct receptor for icaritin.3.Icariin has a certain inhibitory effect on the inflammatory effect induced by LPS in the substantia nigra of mice,but GPER gene knockout cannot completely block the anti-inflammatory effect of icariin.Combining with molecular docking result,we speculate that there may be other signaling pathway involved in the anti-inflammatory effect of icariin.