| Neuroinflammation is an important pathological feature of Alzheimer’s disease（AD）.The progressive activation of microglia and astrocytes and the excessive production of pro-inflammatory factors are an important cause ofβ-amyloid peptide（Aβ）deposition and neuronal damage.Therefore,inhibition of the inflammatory response may be an effective way for the treatment of AD.G protein coupled estrogen receptor（GPER）is a seven-transmembrane G protein coupled receptor,which can mediate the rapid non-genomic effects of estrogen.It is widely expressed in many areas of the central nervous system of rodents,especially in the hippocampus and cerebral cortex.It has been reported that GPER mediates the anti-inflammatory effect of estrogen in multiple sclerosis and rat cerebral ischemia models.Although the neuroprotective effect of estrogen has been confirmed,the side effects of estrogen replacement therapy limits its clinical application.Screening estrogen-like active ingredients from traditional Chinese medicine is a hot point in the medicine field.Icariin is the main active ingredient of total flavonoid fraction of Herba Epimedii.Icaritin is a hydrolyzed derivative of icariin,both of them belong to phytoestrogens and have many pharmacological activities such as anti-tumor,anti-inflammatory and neuroprotection.Our previous study showed that icariin and wushanicaritin could inhibit lipopolysaccharide（LPS）-induced inflammatory response in ICR mice,and their anti-inflammatory effects could be blocked by GPER antagonist G15.However,whether GPER can directly bind with icariin and wushanicaritin?Whether GPER can be used as a potential target for the treatment and drug screening of AD?It needs to be further explored.Objective:1.To determine whether GPER was the direct binding target of icariin and wushanicaritin using molecular docking technique.2.To investigate the effect of GPER gene knockout on the anti-AD inflammatory response of icariin and wushanicaritin by using GPER+/+wide type and GPER-/-gene knockout C57BL/6J mice.Methods:1.Online servers such as GPCR-I-TASSER was using to perform modeling of GPER.The targeted binding ability of icariin and wushanicaritin to GPER was analyzed by molecular docking technology.2.GPER gene knockout mice,constructed by CRISPR-Cas9 technology,was used to detect the effect of GPER deletion on the phenotype of mice.3.The inflammatory models of GPER+/+and GPER-/-AD mice were established by stereotactic injection of LPS into the lateral ventricle.Icariin（10 mg/kg）and wushanicaritin（10 mg/kg）were intragastric administration for 12 days.4.The morris water maze was used to detect the spatial learning and memory ability.5.The gene expressions of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,inducible nitric oxide synthase（i NOS）and cyclooxygenase（COX-2）in the hippocampus were detected by real time PCR.6.The protein expressions of i NOS,COX-2,ionized calcium binding adaptor molecule 1（Iba1）and glial fibrillary acidic protein（GFAP）were detected by western blot.Results:1.GPER 3D model was used to simulate the binding ability of icariin and wushanicaritin to GPER.The results showed that the binding energy of icariin and wushanicaritin with GPER were-6.3 kcal/mol and-9.3 kcal/mol,respectively.The binding ability of wushanicaritin with GPER was better than icariin,which was close to the binding energy of GPER specific agonist G1（-9.8 kcal/mol）.2.The results of body weight measurement showed that the body weight of 3-month-old GPER-/-mice was significantly higher than that of GPER+/+mice（P<0.001）.Real time PCR results showed that the gene expressions of i NOS and COX-2 in the hippocampus of GPER-/-mice were significantly up-regulated compared with GPER+/+mice（P<0.05,P<0.01）.While the gene expressions of IL-1βand TNF-αwas slightly but not significantly increased.These results suggested that GPER gene knockout could increase the inflammatory response in the hippocampus of mice.3.The results of morris water maze test showed that there was no significant difference in behavior between GPER+/+and GPER-/-mice.Compared with the control group,the GPER+/+and GPER-/-mice in LPS model groups showed irregular trajectories,significantly prolonged latency,reduction of the target quadrant time ratio and platform crossing times（P<0.05,P<0.01,P<0.001）.Compared with the LPS model group,GPER+/+mice tended to the quadrant where the platform was located and the localization ability was enhanced after the treatment of icariin（10 mg/kg）and wushanicaritin（10mg/kg）.Moreover,icariin and wushanicaritin treatment significantly improved the spatial learning and memory disorder of GPER+/+mice induced by LPS（P<0.01,P<0.001）.The improvement effect of wushanicaritin was slightly stronger than that of icariin.In GPER-/-mice,intragastric administration of icariin and wushanicaritin did not significantly improve the spatial learning and memory disorder induced by LPS,suggesting that GPER gene knockout significantly reduced the improvement of icariin and wushanicaritin on the spatial learning and memory ability.4.Real time PCR results showed that LPS significantly induced the gene expressions of IL-1β,TNF-α,i NOS and COX-2 compared with the control group in GPER+/+mice（P<0.001）.In GPER-/-mice,the gene expressions of IL-1βand TNF-αin the hippocampus were also significantly up-regulated in LPS group（P<0.05,P<0.01,）,while the gene expression of i NOS and COX-2 was slightly but not significantly up-regulated.Compared with the LPS model group,the gene expressions of inflammatory factors in the hippocampus of GPER+/+mice were significantly inhibited by icarrin and wushanicaritin（P<0.05,P<0.01,P<0.001）.In GPER-/-mice,the inhibitory effect of icariin and wushanicaritin on LPS-induced inflammatory response in hippocampus was significantly weakened.Compared with the LPS group,there was no statistical significant.5.Western blot results showed that the protein expressions of i NOS and COX-2 in the hippocampus of GPER+/+and GPER-/-mice were significantly increased in LPS model group compared with control group（P<0.01,P<0.001）.Compared with LPS model group,the protein expressions of pro-inflammatory enzymes in the hippocampus of GPER+/+mice was significantly inhibited by icarrin and wushanicaritin（P<0.01,P<0.001）.After GPER gene knockout,the inhibitory effects of icariin and wushanicaritin on the protein expressions of i NOS and COX-2 were significantly weakened.Compared with the LPS group,there was no statistical significant.6.In order to detect the activation of microglia and astrocyte,we detected the protein expressions of Iba1 and GFAP in hippocampus using western blot.The results showed that LPS significantly up-regulated the protein expressions of Iba1 and GFAP compared with the control group in the hippocampus of GPER+/+and GPER-/-mice（P<0.05,P<0.01,P<0.001）.Compared with the LPS group,icarrin and wushanicaritin significantly inhibited the protein expressions of Iba1 and GFAP in the hippocampus of GPER+/+mice（P<0.05）.But in GPER-/-mice,the inhibitory effects of icarrin and wushanicaritin were not statistically significant.Conclusion:Molecular docking results indicate that icariin and wushanicaritin can bind to GPER,and the binding ability of icariin with GPER is stronger than that of icariin.GPER gene knockout can induce obesity in male mice,promote the inflammatory response in hippocampus,and significantly reduce the anti-AD inflammatory response of icariin and wushanicaritin.These results suggest that GPER may be the direct target of icariin and wushanicaritin against AD inflammatory response. |
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