ABSTRACT
OBJECTIVE To investigate the role of PDE4 inhibition in astrocyte swelling caused by cerebral ischemic/reperfusion(I/R)injury and the molecular mech-anisms.METHODS SD rats were subjected to 2 h of focal cerebral ischemia induced by middle cerebral artery occlusion/reperfusion(MCAO/R).Roflumilast(Roflu)was intraperitoneally injected 2 h after MCAO.At 24 h after reperfusion,a high-resolution MRI was performed and using the wet-dry weighting method to measure the water content.The oxygen-glucose deprivation/reoxygenation(OGD/R)model was established in primary astrocytes for 2 h.After 24 h of reoxygenation,CellMask? plasma membrane stain was used to label the plasma membrane to calculate cell volume.The protein expressions insides astrocytes and penumbra were detected by Western blot-ting.To investigate the role of Akt/FoxO3a in mediating the effect of Roflu on the expression of AQP4.The astro-cytes were treated with an Akt inhibitor MK2206 before treatment with Roflu and the activation of Akt,the expres-sion of AQP4 and cell volume were determined as described above.In addition,an IL-1β-stimulated cell model was established in astrocytes,the expression of AQP4 and the activation of Akt/FoxO3a were detected by Western blotting.The change of AQP4 expression inside astrocytes and penumbra were visualized by immunofluo-rescence staining.RESULTS Roflu reduced MCAO/R-induced water contents,the expression of AQP4 and the phsophorylation of Akt and FoxO3a in the brains of MCAO/R rats.Inhibition of PDE4 decreased the cell volume and the expression of AQP4 in primary astro-cytes subjected to OGD/R.PDE4 inhibition activated Akt/FoxO3a,and inhibition of Akt by MK2206 blocked the protective effect of Roflu against OGD/R induced astro-cyte swelling.PDE4B knocking down reduced the expres-sion of AQP4,while PDE4B overexpression reversed the effect of PDE4B siRNA in astrocytes.Roflu exert-ed similar protective effect in IL-1β-cultured astrocytes,and importantly overexpression of FoxO3a remarkably increased the expression of AQP4 in IL-1β-stimulated astrocytes.CONCLUSION Our findings indicate that PDE4 inhibition limits I/R-induced brain edema and astro-cyte swelling via the Akt/FoxO3a/AQP4 pathway.PDE4 inhibition is a promising strategy for the treatment of brain edema after I/R injury.
ABSTRACT
During cerebral ischemia-reperfusion injury(CIRI),endoplasmic reticulum stress(ERS)leads to the development and progression of a series of deleterious physiological responses such as oxidative stress,dis-turbed calcium ion homeostasis,inflammation,apoptosis and autophagy.The unfolded protein response(UPR)is the main pathway activated by ERS,which regulates the expression of related factors within the endoplasmic reticu-lum(ER)and reduces protein translation levels.Prolonged and intense ERS may lead to cell death.Excessive ERS induces apoptosis mediated by C/EBP homologous pro-tein(CHOP),caspase-12 and c-Jun N-terminal kinase(JNK),thereby exacerbating brain damage.The thresh-old for the transition from adaptive mechanisms to apop-totic mechanisms during ERS depends on multiple fac-tors,including the cell status and environment,signaling pathway activity status,cumulative cascade,and the dose and time of ERS inducers.Further research is needed to completely elucidate the mechanism of ERS.Although the factors associated with the PERK and ATF6 path-ways are less extensively studied,their regulators still exist.Deficiency of protein tyrosine phosphatase 1B(PTP1B)leads to increased phosphorylation of PERK-eIF2α,while regulation of the proteasome and regulation of the XBP1 target gene WFS1 may also affect ATF6 sta-bility.In addition,differences in the structure,gene expres-sion,and metabolism of different types of neurons,as well as in their internal environment,may lead to differ-ences in their response to and impact on ERS.Differenc-es in UPR signaling pathways occur in hippocampal neurons and medial thalamic cells,and Purkinje cells and pyramidal cells may be more sensitive to ERS than other types of neurons.Our group's previous study found that ERS induced apoptosis in neurons after the onset of CIRI by regulating proteins such as GRP78,CHOP and caspase-12,but the effects of UPR activation on different cells need to be further investigated.
ABSTRACT
OBJECTIVE:To study the effects of anisodine hydrobromide on cell apoptosis and extracellular signal-regulated pro-tein kinase 1/2 (ERK1/2) phosphorylation (p-ERK1/2) level in brain tissue of model rats with acute cerebral ischemia-reperfusion injury. METHODS:Rats were randomly divided into sham operation group,model group,positive control group(nimodipine 1.0 mg/kg),anisodine hydrobromide high-dose,medium-dose,low-dose,extreme low-dose groups(1.2,0.6,0.3,0.15 mg/kg),8 in each group. Suture method was used to establish the rat models with acute cerebral ischemia-reperfusion injury. Rats were intrave-nously injected once in tail at 2nd of ischemia and 6th of reperfusion. Then adenosine triphosphate (ATP) enzyme activity,Ca2+content,cell apoptosis in brain tissue,p-ERK1/2 protein expression in brain tissue,and p-ERK1/2/total ERK1/2 (t-ERK1/2) pro-portion in brain tissue of rats were detected after 22 h of reperfusion. RESULTS:Compared with sham operation group,ATP en-zyme activity in brain tissue of rats in model group was obviously decreased,Ca2+ content was obviously increased,density of cell apoptosis in brain tissue was obviously increased,with statistical significances(P<0.01). Compared with model group,density of cell apoptosis in brain tissue was obviously decreased in each administration group;Ca2+ contents in brain tissue of rats in positive control group,anisodine hydrobromide high-dose,low-dose groups were obviously decreased;and p-ERK1/2/t-ERK1/2 proportion in brain tissue of rats in anisodine hydrobromide high-dose,low-dose,extreme low-dose groups were obviously increased,with sta-tistical significances(P<0.05 or P<0.01);the other differences were not statistically significant(P>0.05). CONCLUSIONS:An-isodine hydrobromide can inhibit the cell apoptosis in brain tissue of model rats with acute cerebral ischemia-reperfusion injury,andthe mechanism may be related with activating ERK1/2 signal pathway and regulating ATP enzyme activity to decrease the Ca2+content in the brain tissue.
ABSTRACT
OBJECTIVE:To study the effects of anisodine hydrobromide on cell apoptosis and extracellular signal-regulated pro-tein kinase 1/2 (ERK1/2) phosphorylation (p-ERK1/2) level in brain tissue of model rats with acute cerebral ischemia-reperfusion injury. METHODS:Rats were randomly divided into sham operation group,model group,positive control group(nimodipine 1.0 mg/kg),anisodine hydrobromide high-dose,medium-dose,low-dose,extreme low-dose groups(1.2,0.6,0.3,0.15 mg/kg),8 in each group. Suture method was used to establish the rat models with acute cerebral ischemia-reperfusion injury. Rats were intrave-nously injected once in tail at 2nd of ischemia and 6th of reperfusion. Then adenosine triphosphate (ATP) enzyme activity,Ca2+content,cell apoptosis in brain tissue,p-ERK1/2 protein expression in brain tissue,and p-ERK1/2/total ERK1/2 (t-ERK1/2) pro-portion in brain tissue of rats were detected after 22 h of reperfusion. RESULTS:Compared with sham operation group,ATP en-zyme activity in brain tissue of rats in model group was obviously decreased,Ca2+ content was obviously increased,density of cell apoptosis in brain tissue was obviously increased,with statistical significances(P<0.01). Compared with model group,density of cell apoptosis in brain tissue was obviously decreased in each administration group;Ca2+ contents in brain tissue of rats in positive control group,anisodine hydrobromide high-dose,low-dose groups were obviously decreased;and p-ERK1/2/t-ERK1/2 proportion in brain tissue of rats in anisodine hydrobromide high-dose,low-dose,extreme low-dose groups were obviously increased,with sta-tistical significances(P<0.05 or P<0.01);the other differences were not statistically significant(P>0.05). CONCLUSIONS:An-isodine hydrobromide can inhibit the cell apoptosis in brain tissue of model rats with acute cerebral ischemia-reperfusion injury,andthe mechanism may be related with activating ERK1/2 signal pathway and regulating ATP enzyme activity to decrease the Ca2+content in the brain tissue.
