Experimental Study Of The Neuroprotective Effects Of Ginsenoside Rg1 Against Inflammation-induced Dopaminergic Neuronal Degeneration In Substantia Nigra

Microglia,the resident immune cells of the central nervous system,plays important roles in releasing inflammatory cytokines,antigen presentation and engulfing pathogens.Studies have shown that substantia nigra-striatal system is the main pathological position of Parkinson's disease(PD).Dopaminergic neurons in the substantia nigra pars compacta(SNpc)are very sensitive to the microglia-induced inflammation.Postmortern studies of PD have shown that SNpc contains numerous reactive microglia and astrocyte,indicating neuroinflammation plays an important role in onset and progression of PD.Inhibition of microglia overactivation may be a promising therapeutic strategy for the treatment of PD.Ginsenoside Rg1,the most active components of ginseng,possesses a variety of biological effects,especially on the central nervous system.Rg1 has been reported to exert neurotropic and neuroprotective effects.Moreover,Rg1 has inhibitory effects on microglia-induced inflammation.However,the mechanism that transmits the anti-inflammatory effects of Rg1 is still unclear.Rg1 is a steroid saponin and processes four trans-ring rigid steroid skeleton.Our previous study and the other research group have demonstrated that Rg1 has estrogen-like activity and glucocorticoid-like activity.Studies have shown that Rg1 is a functional ligand of glucocoticoid receptor(GR).GR belongs to the superfamily of nuclear receptors and can be activated by ligand binding.GR activation in the microglia has a determining role in inhibiting microglial activation and reducing inflammation-mediated dopaminergic neuronal degeneration in experimental Parkinsonism.In the first part,we aim to study if GR is involved in the protective effects of Rg1 on lipopolysaccharide(LPS)-induced microglia activation and dopaminergic neuronal degeneration in SN.Estrogen receptor also belongs to the superfamily of nuclear receptor.The genomic effect of estrogen is mediated by classical nuclear receptor ERa and ERb.G protein-coupled estrogen receptor 1(GPER1)mediates the non-genomic effect of estrogen and plays an important role in the anti-inflammatory and anti-apoptotic effects of estrogen.Studies have shown that activation of GPER1 could induce the activation of insulin-like growth factor-I receptor(IGF-IR)and epidermal growth factor-I receptor(EGFR)signaling pathway,which rapidly influence cell physiology or induce the phosphorylation of ERa.Our previous studies have demonstrated that Rg1 is a novel class of potent phytoestrogen.Rg1 could not bind to ERa and ERb,but could activate ERa in a ligand-independent manner.The neuroprotective effects of Rg1 against the neurotoxin 6-OHDA-lesioned rat model and cell model of PD are meditated by the cross-talk of ER and IGF-IR signaling pathway.In vitro study further demonstrates that GPER1 is involved in the anti-inflammatory effect of Rg1 in BV2 microglia cell.The second part of the present study aims to evaluate if GPER1 and IGF-IR are involved in the protective effects of ginsenoside Rg1 on LPS-induced microglia activation and dopaminergic neuronal degeneration in rat SN.G1,the specific agonist of GPER1,is used as a positive control.1.GR is involved in the neuroprotective effect of ginsenoside Rg1 against inflammation-induced dopaminergic neuronal degeneration in substantia nigra(The results of this part has been published in Journal of Steroid Biochemistry & Molecular Biology)In the present study,LPS-induced microglia activation and dopaminergic neuronal degeneration in SN was used as a PD rat model.Rotational behavior was induced by i.p.injection of apomorphine(APO).The contents of DA and its metabolites dihydroxy-phenylacetic acid(DOPAC)and homovanillic(HVA)in the striatum were determined by using high performance liquid chromatography(HPLC).Immunohistochemisty and immunofluorence were used to observe the loss of dopaminergic neuron and microglial over-activation.The production of NO was measured using the Griess reagent.Enzyme-linked immunosorbent assay(ELISA)and real time RT-PCR was used to detect the changes of the content and gene expressions of inflammatory cytokines in SN.The changes of the protein expressions of tyrosine hydroxylase(TH)and GR as well as inflammatory cytokines were determined by western blot.Experimental results are as follows:(1)Administration of APO induced 9.30 turns/min rotations behavior in LPS treatment group(P<0.001).Treatment with Rg1(10mg/kg,i.p.)could ameliorate the apomorphine-induced rotational behavior(4.64 turns/min)compared with LPS-lesioned rats(P<0.01).This effect could be blocked by GR antagonist RU486(P<0.001).Co-treatment with LPS plus RU486 had no effect on the rat's rotation behavior.(2)The HPLC results showed that the DA,DOPAC and HVA levels in the Str of the lesioned side of PD rats were significantly decreased compared with the control rats(P(27)0.001).The decreased percentage is about 72%,64% and 70% separately.Rg1 treatment significantly attenuated LPS-induced decrease of DA,DOPAC and HVA in the Str of the lesioned side(P(27)0.05).This effect could be blocked by GR antagonist RU486(P<0.05).(3)A single intranigral injection of LPS resulted in severe nigrastriatal lesions with a marked loss of approximately 36.7% of TH neurons in the LPS-injected side of SNpc comparing with the un-injected side.TH protein expression was also significantly decreased in the lesioned side of SN compared with that of control group(P<0.001).Rg1 treatment significantly attenuated LPS-induced loss of TH positive neurons in SNpc as well as the TH protein expression(P<0.05).GR antagonist RU486 could abolish the protective effect of Rg1(P<0.05).(4)Immunohistochemisty results showed that Rg1 treatment significantly inhibited the microglial activation induced by LPS in the lesioned side of SNpc.Co-treatment with RU486 significantly blocked the inhibitory effect of Rg1 on LPS-induced over-activation of microglia.(5)Rg1 significantly inhibited LPS-induced microglial activation and production of tumor necrosis factor-alpha(TNF-?),interleukin-1 beta(IL-1?)and nitric oxide(NO)(P<0.01).These effects were abolished by co-treatment with RU486(P<0.01).(6)LPS intranigral injection significantly increased the phosphorylation level of ERK,JNK,p38 and I?B(P<0.01).Rg1 treatment could remarkably attenuate these changes(P<0.05),while co-treatment with RU486 could reverse the protective effects of Rg1(P<0.05).(7)LPS treatment could slightly but not significantly down-regulated the GR expression(P>0.05).Rg1 treatment significantly increased the GR expression in the lesioned side of SN compared to the LPS group(P<0.05).This effect could be completely blocked by RU486(P<0.05).The above data indicates that Rg1 has protective effects on mesencephalic dopaminergic neurons from LPS-induced microglia inflammation.GR signaling pathway might be involved in the anti-inflammatory effect of Rg1.2.GPER1 involved sin the neuroprotective effect of ginsenoside Rg1 against inflammation-induced dopaminergic neuronal degeneration in substantia nigraIn order to determine the roles of GPER1 and IGF-IR in the anti-inflammatory effects of Rg1,the LPS-induced PD rats were treated with Rg1 in the presence or absence of GPER1 antagonist G15 or IGF-IR antagonist JB-1.GPER1 agonist G1 was used as apositive control.Experimental results are as follows:(1)Rg1 or G1 significantly ameliorated APO-induced rotational behavior(P<0.001).The protective effect of Rg1 could be blocked by GPER1 antagonist G15 or IGF-IR antagonist JB-1.(2)Treatment with Rg1 or G1 significantly inhibited the LPS-induced decrease of the contents of DA,DOPAC and HVA in the lesioned side of the Str(P<0.05).Cotreatment with G15 or JB-1 blocked the protective effect of Rg1(P<0.05).(3)Rg1 or G1 treatment significantly attenuated LPS-induced loss of TH positive neurons in SNpc as well as the TH protein expression(P<0.01).These effects could be blocked by G15 or JB-1(P<0.05).(4)Immunofluorence results showed that LPS-induced microglial activation in the lesioned side of SN could be blocked by Rg1 or G1.Cotreatment with G15 or JB-1 significantly inhibited the anti-inflammatory effect of Rg1.Meanwhile,Rg1 or G1 treatment inhibited the LPS-induced the decrease of astrocyte in SNpc.These effects could be blocked by G15 or JB-1.(5)Rg1 or G1 treatment significantly attenuated LPS-induced protein and gene expressions of proinflammatory cytokines i NOS,COX-2,TNF-? and IL-1?(P<0.05).These effects were abolished by G15 or JB-1(P<0.05).(6)Rg1 or G1 treatment significantly inhibited LPS-induced the phosphorylation level of MAPKs and I?B(P<0.05).These effects could be also partly blocked by G15 or JB-1(P<0.05).(7)Immunoprecipitation result showed that Rg1 treatment induced the interaction of GPER1 with IGF-IR.The above data indicates that the inhibitory effect of Rg1 on microglial activation in SN might be mediated by GPER1 and the cross-talk of GPER1 with IGF-IR.Conclusions:Taken together,ginsenoside Rg1 has neuroprotective effects on mesencephalic dopaminergic neurons from LPS-induced microglia inflammation.GR,GPER1 and IGF-IR might be involved in the anti-inflammatory effect of Rg1.