| Objectives:Parkinson’s disease（PD）is a common neurodegenerative disease of the central nervous system,and the main pathological change is the degeneration and death of dopaminergic（DA）neurons in the nigrostriatal pathway.Numerous pieces of evidence have shown that immune inflammation plays an important role in the pathogenesis of PD.Autopsy results show that there are a large number of reactive microglia in the substantia nigra of PD patients.G-protein-coupled estrogen receptor（GPER）is a seven-transmembrane estrogen membrane receptor that mediates the rapid nongenomic effects of estrogen.GPER is widely expressed in neurons and glial cells in the central nervous system.GPER-mediated signal pathways have neuroprotective effects such as anti-inflammatory and anti-apoptosis.Ginsenoside Rg1 is the main pharmacological active component of ginseng,which has the effects of anti-aging,neurotrophy,auxiliary anti-tumor,and so on.In our previous study,we demonstrated that Rg1 can protect against lipopolysaccharide（LPS）-induced inflammation in microglia in vitro through GPER.However,at the animal level,can ginsenoside Rg1 antagonize LPS-induced inflammation in the substantial nigra of mice?Is the mechanism related to GPER?In this study,3-4-month-old and 14-16-month-old GPER wild type(GPER+/+)and GPER gene knockout(GPER-/-)C57BL/6J mice were used to establish PD inflammatory model,to explore the effect of GPER gene knockout on substantia nigra inflammation in mice of different ages,and to determine whether GPER was involved in the anti-inflammatory neuroprotective effect of ginsenoside Rg1.Methods:1.Using stereotaxic techniques,PD inflammatory models were established by microinjection of LPS into the substantia nigra of GPER+/+and GPER-/-mice at 3-4 months and 14-16 months of age,respectively.Rg1（10 mg/kg）was injected intraperitoneally for 14 consecutive days.2.Pole test and rotarod test were used to detect the motor coordination ability of mice.3.The q PCR was used to detect the m RNA expressions of tyrosine hydroxylase（TH）,pro-inflammatory factors tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）,cyclooxygenase-2（COX-2）,inducible nitric oxide synthase（i NOS）and pyroptosis-related factor NOD-like receptor family,pyrin domain containing 3（NLRP3）.4.Western blotting was used to detect the protein expressions of rate-limiting enzyme of dopamine synthesis TH,pro-inflammatory factors COX-2 and i NOS,and pyroptosis-related protein NLRP3.Results:1.In 3-4-month-old mice group,microinjection of LPS into substantia nigra could significantly shorten latency to fall in rotarod test,as well as prolong the time to head turn time（T-turn）and the total time to descend the pole（T-total）in pole test（P<0.01,P<0.001）,which occurs in both GPER+/+and GPER-/-mice.In GPER+/+mice,Rg1 could ameliorate LPS-induced dyskinesia（P<0.05,P<0.001）.However,in GPER-/- mice,the ameliorating effect of Rg1 was significantly weakened（P<0.001）.2.In 3-4-month-old mice group,LPS significantly decreased TH expression in the substantia nigra and striatum of GPER+/+and GPER-/-mice（P<0.001）.In GPER+/+ mice,Rg1 could significantly inhibit the damage induced by LPS（P<0.01,P<0.001）.However,in GPER-/-mice,the neuroprotective effect of Rg1 was abolished（P<0.01,P<0.001）.3.In 3-4-month-old mice,LPS significantly induced the m RNA up-regulation of pro-inflammatory cytokines TNF-α,IL-1β,COX-2 and i NOS as well as the protein up- regulation of COX-2 and i NOS in substantia nigra of both GPER+/+and GPER-/-mice（P<0.01,P<0.001）.Rg1 treatment significantly inhibited the increase of the above pro-inflammatory cytokines（P<0.05,P<0.01,P<0.001）.However,the anti- inflammation effects of Rg1 were significantly attenuated after GPER gene knockout（P<0.01,P<0.001）.In addition,the m RNA expression of TNF-αand IL-1βinduced by LPS was significantly higher in GPER-/-mice than that in GPER+/+mice（P<0.01, P<0.001）,which suggested that GPER gene knockout increased the sensitivity of mice to LPS inflammatory stimulation.4.In 3-4-month-old mice,LPS significantly induced the gene and protein up-regulation of pyroptosis-related factor NLRP3 in the substantia nigra of both GPER+/+and GPER-/-mice（P<0.01,P<0.001）.Rg1 treatment significantly inhibited the above effect（P <0.05,P<0.01）.However,Rg1 could not decrease the up-regulation of NLRP3 in the GPER-/-PD inflammatory model（P<0.001）.5.In 14-16-month-old mice group,LPS could also induce rotarod behavior disorder in GPER+/+and GPER-/-mice,manifested as the residence time of rotarod was shortened（P<0.05,P<0.01）.In GPER+/+mice,Rg1 could ameliorate LPS-induced dyskinesia （P<0.01）.However,in GPER-/-mice,the ameliorative effect of Rg1 was significantly attenuated（P<0.05）.In the pole test,LPS could prolong the head return time（T-turn） of GPER+/+and GPER-/-mice（P<0.05）,and Rg1 still exerted a protective effect in GPER+/+mice（P<0.05）.But in GPER-/-mice,Rg1 treatment showed a slight but not significant protective effect.In the down rod time（T-total）assay,neither GPER+/+nor GPER-/-mice significantly prolonged time after LPS treatment.Compared with the control group of 3-4-month-old mice,the motor coordination ability of 14-16-month- old mice was decreased,manifested as the residence time of rotarod was shortened,and the time of head turn and down rod was prolonged.6.In 14-16-month-old mice group,LPS significantly decreased the expression of TH in the substantia nigra and striatum of GPER+/+and GPER-/-（P<0.01,P<0.001）.In GPER+/+mice,Rg1 could significantly inhibit the LPS-induced decrease of TH expression（P<0.001）.However,in GPER-/-mice,the neuroprotective effect of Rg1 was abolished（P<0.01,P<0.001）.Comparing the gene expression of TH in the substantia nigra of 3-4-month-old mice and 14-16-month-old mice,we found that LPS caused more severe damage to DA neurons in 14-16-month-old mice.7.In 14-16-month-old mice,LPS could up-regulate the m RNA levels of pro-inflammatory cytokines TNF-α,IL-1βand COX-2 as well as the protein up-regulation of COX-2 and i NOS in substantia nigra of GPER+/+and GPER-/-mice（P<0.05,P<0.01,P<0.001）.In GPER+/+mice,Rg1 could significantly inhibit the increase of the above pro- inflammatory factors（P<0.05,P<0.01,P<0.001）,and the anti-inflammatory effect of Rg1 was significantly decreased in GPER gene knockout mice（P<0.05,P<0.01,P<0.001）.8.In 14-16-month-old mice,Rg1 treatment could inhibited the up-regulation of NLRP3 induced by LPS in the substantia nigra of GPER+/+mice（P<0.05,P<0.01）.However, the anti-inflammation effects of Rg1 were significantly attenuated after GPER gene knockout（P<0.01）.Conclusions:1.Rg1 can inhibit LPS-induced inflammatory response in 3-4-month-old and 14-16-month-old PD mice.GPER gene knockout significantly inhibits the anti-inflammatory neuroprotective effect of Rg1.2.GPER gene knockout can increase the sensitivity of DA neurons in the substantia nigra to LPS in 3-4-month-old mice. |
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