Studies In Neuroprotective Effects Of Progesterone On Ischemia-induced Cerebral Damage And Underlying Mechanisms

INTRODUCTIONPremenopausal women have lower risks of stroke and better outcomes following stroke relative to men of the same age.Following the menopause,the incidence of stroke in women rapidly increases coincident with decrease in levels of the sex steroid hormones estrogen (E2)and progesterone(P4).The majority of research has focused on E2 as the main source of neuroprotection seen in female and male animals. Although experimental evidence revealed a neuroprotective effect of P4 following cerebral ischemia,the efficacy-time window of P4 -neuroprotection and the underlying molecular mechanisms of the P4-action are not completely understood to date.OBJECTIVEThe objective of this research was to investigate the effects of P4 when administrated before or after cerebral ischemia on the ischemia-induced neuronal damage and determine the efficacy-time window of the P4-neuroprotection.This study further evaluated underlying molecular mechanisms of the P4-neuroprotective effects against ischemia-induced cerebral damage. METHODSMiddle cerebral artery occlusion(MCAO)for 1 hr was performed to induce the model of transient and focal cerebral ischemia.P4(8 mg/kg) was administrated at different time from 96 hr before MCAO to 96 hr after MCAO.Neuronal death and LTP induction in hippocampal CA1 region were examined at the 8th day after MCAO,"Morris" water maze task was observed at 3-8 days after MCAO.ERK1/2 phosphorylation and its nuclear translocation were measured by western blotting analysis. NMDAR-mediated Ca²⁺influx was monitored by calcium imaging.P4 receptor antagonist RU486,ERK1/2 inhibitor U0126,Src inhibitor PP2, PI3K inhibitor LY294002,σ₁R antagonist NE100 and 5α-reductase inhibitor finasteride were used to study the molecular mechanisms underlying the neuroprotection exerted by P4.RESULTS1.P4 when administrated from 48 hr pre-MCAO to 72 hr post-MCAO produced significant protective effects against the ischemia-induced neuronal death,the deficits in spatial cognition and LTP induction.2.We studied the mechanisms underlying the P4-neuroprotection when administrated at 1 hr and 48hr pre-MCAO,termed acute and delayed P4-neuroprotection,respectively.The results revealed that the acute P4-administration could inhibit the enhancing effects ofσ₁R on the NMDAR-mediated Ca²⁺influx.In addition,theσ₁R antagonist NE100 perfectly mimicked the acute P4-neuroprotection.The delayed P4-neuroprotection,but not the acute P4-neuroprotection,was blocked by P4R antagonist RU486,ERK1/2 inhibitor U0126 or Src inhibitor PP2. These findings suggest that the P4-neuroprotection seems to involve two independent processes depending on the administration timing before MCAO:an acute protection by antagonizingσ₁R to inhibit NMDAR-mediated Ca²⁺influx and a delayed one by P4R-mediated Src-ERK signaling pathway.3.We further studied the mechanisms underlying the P4-neuroprotection when administrated at 1 hr and 48hr post-MCAO,termed early and late P4-neuroprotection,respectively.The results revealed that the early P4-neuroprotection was abolished by pre-treatment of the 5α-reductase inhibitor finasteride.The late P4-neuroprotection,but not early one,was blocked by P4R antagonist RU486,ERK1/2 inhibitor U0126,Src inhibitor PP2 or PI3K inhibitor LY294002.These findings suggest that the P4-neuroprotection seems to involve two independent processes depending on the administration timing after MCAO:an early protection through activating GABA_AR to inhibit neuronal excitatory and a late one by P4R-mediated Src-ERK1/2 and PI3K signaling pathways.CONCLUSION1.P4 exerts powerful neuroprotection against ischemic damages with a wide timing-window from 48 hr pre-MCAO to 72 hr post-MCAO, suggesting that P4 is a candidate for preventing and treating stroke.2.The P4 neuroprotection is mediated by two independent processes depending on the administration timing before or after MCAO:when administrated at an acute ischemia and early reperfusion the P4-neuroprotection by antagonizingσ₁R to inhibit NMDAR-Ca²⁺influx and by activating GABA_AR to inhibit neuronal excitatory;when administrated at 48 hr before ischemia or after reperfusion the P4-neuroprotection by P4R-mediated Src-ERK1/2 and PI3K-Akt signaling pathways,both of which eventually protect neurons from ischemic neuronal death and functional impairments.