Experimental Study Of The Anti-inflammatory Effect Of Ginsenoside Rg1 Via G Protein-coupled Estrogen Receptor In BV2 Microglial Cells

Many studies have shown that microglial cells plays a major role in the pathogenesis of neurodegenerative diseases. Microglial activation induce neuronal death by producing proinflammatory enzymes and cytokines. Ginsenoside Rg1 is the major pharmacological active ingredient in traditional Chinese medicine ginseng. Studies have shown the anti-inflammatory effects of ginsenoside Rg1, but the molecular mechanism is unclear till now. Our previous studies have demonstrated that ginsenoside Rg1 is a phytoestrogen and possess estrogen-like activities. Rg1 can protect dopaminergic neurons from the neurotoxin-induced neuronal death throgh the esgrogen receptor pathway. G protein-coupled estrogen receptor(GPER) is a novel class of membrane estrogen receptor reported to mediate non-genomic estrogen responses. There are no current data to suggest that the anti-inflammatory effects of Rg1 is related to GPER. In the present study,lipopolysaccharide(LPS) was used to induce inflammatory reponse in BV2 microglial cells. By using MTT assay, immunofluorescence, western blot, real-time quantitative RT-PCR and lentivirus-mediated si RNA interference, the present study was designed to observe the molecular mechanism of the anti-inflammatory effect of ginsenoside Rg1 via GPER in BV2 microglial cell. Experimental results are as follows:1. LPS(1μg / m L) had no effects on cell viability and proliferation in BV2 microglial cells(P> 0.05).2. LPS could significantly increase the m RNA expression i NOS, COX2, IL-10, IL-1βand TNFα in BV2 microglial cells. Ginsenoside Rg1(1-50μM) could inhibit LPS-induced inflammation, and the most effective dosage is 10μM(P <0.05).3. GPER agonist G1(1μM) could inhibit LPS-induced m RNA expression of i NOS,COX2, IL-10, IL-1β and TNFα in BV2 microglial cells(P <0.05).4. GPER antagonist G15(1μM) could block the anti-inflammatory effect of ginsenoside Rg1 and G1 at the m RNA level(P <0.05).5. Pre-treatment with ginsenoside Rg1 and G1 could inhibit LPS-induced protein expression of i NOS and COX2 in BV2 microglial cells, and this effect could be blocked by G15(P <0.05).6. LPS could significantly increase the phosphorylation level of MAPKs(ERK, JNK,p38) and IκB. Pre-treatment with ginsenosides Rg1 and G1 could protect against the protein phosphorylation induced by LPS, and this effect could be blocked by G15(P<0.05).7. Lentivirus mediated GPER si RNA(Lv-si GPER) knocked down the GPER expression in BV2 microglial cells, which leaded to the significant decrease of the inhibitory effect of ginsenoside Rg1 on LPS-induced m RNA expression of i NOS, IL-10 and TNFα(P <0.05) and the inhibitory effect of G1 on LPS-induced m RNA expression of i NOS, COX2, IL-10 and TNFα(P <0.05).8.The knockdown of GPER by Lv-si GPER resulted in a significant decrease of the inhibitory effect of ginsenosides Rg1 on LPS-induced protein expression of i NOS(P<0.01) and the inhibitory effect of G1 on LPS-induced protein expression of i NOS and COX2 in BV2 microglial cells(P <0.01).These results indicate that ginsenoside Rg1 significantly inhibits LPS-induced inflammation in BV2 microglial cells. GPER is involved by suppressing the LPS-induced activation of MAPKs and IκB signaling pathways. This study provides not only the experimental evidence of the molecular mechanisms of anti-inflammative effect of ginsenoside Rg1, but also a new target for PD therapy.