The Effect Of Progesterone On AQP4in Astrocyte After OGD/R Injury And Its Mechanism

Ischemia brain injury is a devastating neurological injury associated with significant morbidity and mortality. Medical therapies to limit cerebral edema, a cause of increased intracranial hypertension and poor clinical outcome, are largely ineffective, emphasizing the need for novel therapeutic approaches. Progesterone also reversed the induction of aquaporin-4(AQP4), an astrocytic water channel implicated in the development of cellular edema following head trauma. Progesterone is thought can modulate AQP4expression and edema after traumatic brain injury (TBI) in male rats. Progesterone administration facilitates water balance at72h post-TBI by decreasing AQP4expression around the contusion and in the tissue bordering the lateral ventricular area. This raises the question whether progesternone may modulate astrocyte AQP4protein expression and whether this effect may attenuate astrocyte water uptake (water permeability) after ischemia/reperfusion.Objective:The aim of the present study was to determine whether treatment with low or medium concentrations of progesterone (0.03-2μM) might have an effect on AQP4protein and/or mRNA expression in ischemia/reperfusion astrocytes, and whether this influence might be related to its potential positive influence on cell viability (swelling). Moreover, we investigate the classical nuclear progesterone receptor (cPR) and PKC cell signal pathway and try to find the possible mechanism of progesterone effect on AQP4.Content:1. Astrocytes in primary culture.2. We build astrocyte model of cerebral ischemia which is also called oxygen-glucose deprivation (OGD), and study the effect of oxygen-glucose deprivation (OGD) on astrocytes, and make a appropriate choice of OGD time to reperfusion.3. The different reperfusion time influence astrocyte swelling and AQP4protein expression. An optimal reperfusion time points was selected for next research.4. Modulation of progesternone to astrocyte AQP4protein expression and cell edema were studied, and the roles of cPR and protein kinase C (PKC) in this process were investigated.Methods:1. These studies were performed on astrocytes in primary culture from newborn rat cortex.2. Glial fibrillary acidic protein (GFAP) by immunohistochemical staining.3. Build an astrocyte model of cerebral ischemia which is also called oxygen-glucose deprivation (OGD).4. We chose4h for OGD as observed in cell death assays (LDH and MTT)5. AQP4protein expression in the condition of different reperfusion (R) time by Western blotting.6. AQP4protein expression by Western blotting and cell vitality by LDH and MTT to determine the effective dose of progesterone on astrocyte AQP4protein expression in OGD/R injury.7. Progesterone effect on AQP4protein expression of astrocytes during the exposure to the OGD/R by Western blotting and immunofluorescence staining.8. Inhibitors of PR and PKC were employed to find the possible mechanism in progesterone effect on AQP4protein expression.Results:I. Astrocytes in the condiction of OGD.1. Cell activity was significantly decreased4hours after the OGD (P<0.05), and activity decreased by56.4%after4hours.2. Comparison with normal control group, the cell LDH was gradually increased along with the OGD time.Ⅱ. Astrocytes subjected to OGD4hours followed by reperfusion (R).1. AQP4protein expression significantly increased at4hours reperfusion after OGD, and AQP4protein expression levels are still relatively high at8,12,24,48hours.2. Reperfusion8hours, the cell morphology significantly swollen and the cell body lights up. Cell volume reached a peak after24hours reperfusion and lasted for2days,Ⅲ. Different doses of progesterone influence astrocytes during the exposure to OGD4h/R24h.1.1and2μM progesterone can significantly reduce astrocytes LDH release (P<0.05) and increase the MTT value (P<0.05) in OGD4h/R24h injury.2. Compared with the normal control group, Western blotting analysis showed that AQP4protein expression was significantly elevated in Vehicle group, Pg-30nM and Pg-500nM treatment group (P<0.05); Compared with the Vehicle group, Pg-1μM and2uM treatment can significantly down regulate AQP4protein expression induced by OGD4h/R24h (P<0.05).Ⅳ. Progesterone (1μM) effect on AQP4protein expression of OGD4/R24-labeled astrocytes and the cell signal mechanism of it.1. LDH release assay showed that LDH leakage significantly rebounded in Vehicle and Pg+Ro31-8220group (P<0.05).2. Compared with normal control group, Western blotting analysis show that AQP4protein expression was significantly increased in Vehicle and Pg+Ro31-8220group (P<0.05). Compared with Vehicle group, AQP4protein expression was significantly lower in the progesterone treatment group and the RU-486pretreatment group (P<0.05).3. Western blotting analysis show that PKC protein expression has no significance among the groups (P>0.05).4. The morphology observed showed that the cell had a bright body and swollen in Vehicle and Pg+Ro31-8220group.5. Double immunofluorescence staining showed that green fluorescent labeling of AQP4was strong in Vehicle and Pg+Ro31-8220group; Red fluorescent labeling of PKC was no significant difference among the groups.Conclusions:The cell viability decreased about50%and AQP4protein expression was significantly decreased at4hours OGD on astrocytes, but the cell morphology did not change significantly. However, AQP4protein expression increased significantly24hours after reperfusion and the cell body swelled clearly. So OGD/R increases astrocyte swelling, which maybe mediated by an up-regulation of AQP4protein expression. Progesterone at concentrations of1and2μM attenuated the expression of AQP4of astrocyte together with a significant improvement in cell viability following OGD/R, and also progesterone reduced astrocytes swelling induced by OGD/R. Progesterone attenuated the OGD/R-induced aquaporin-4expression through a mechanism that appears to involve, at least in part, the activation of the PKC in an astrocyte model of cerebral ischemia/reperfusion and independent of cPR. As a whole, our data suggest clinically achievable concentrations of progesterone reduce AQP4protein expression and astrocyte swelling following OGD/R, a finding which warrants further investigation.