Experimental Therapeutic Potential And Mechanisms Of NTR1 Agonist PD149163 On Focal Cerebral Ischemia/Reperfusion-induced Stroke In Rats

Stroke is a kind of acute cerebral vascular disease with high incidence, morbidity and mortality, which is due to the sudden rupture of the blood vessels in the brain or vascular obstruction, causing brain damage resulting from the sharp reduction of blood flow to the brain. It is the leading cause of death and disability in humans worldwide. To date, the usage of tissue plasminogen activator (tPA) is the only approved thrombolytic clinical therapy of acute ischemic stroke treatment. However, due to the narrow therapeutic time, severe adverse reactions and limited therapeutic effects of tPA, its clinical usage is restricted mostly. The pathophysiological mechanisms involved in cerebral ischemic injury are complex. With increasing understanding of the ischaemic cascade reaction and parallel pathways caused by the onset of cerebral ischaemia, researchers are realizing medicines or treatment strategies involved in single mechanism could not be able to affect the whole cascade reaction and to cure ischemic stroke. Therefore, it is of great significance to explore new targets for developing new strategies and medicines of ischemic stroke.Therapeutic hypothermia plays a neuroprotective role by intervention of multiple pathophysiological pathways after ischemic injury, affecting several cell death pathways, including the pathways related to excitotoxicity, apoptosis, free radical, blood brain barrier, and so on. It is a promising treatment strategy of ischemic stroke. Not only physic ways can be used to establish hypothermia therapy, but also the chemical one can, which is targeting on central thermoreceptor. PD149163 is a compound with this kind of effect. Meanwhile, it is an agonist of neurotensin receptor 1, which exhibits BBB permeability. After peripheral administration, it produces a series of central nervous effects, including promoting cognitive, antipsychotic effect, anxiolytic effects and reduced food intake. Furthermore, PD 149163 can effectively induces regulatory hypothermia in rats. Therefore, it is of great significance to further study and exploring the therapeutic effects and mechanisms of PD 149163 on cerebral ischemia for the clinical treatment of cerebral ischemia.Our researches are focusing on neuroprotective effects and mechanisms of PD 149163 on actue phase of focal cerebral ischemia/reperfusion induced neural injury in rats. The results illustrated PD 149163 decreased rat temperature and had neuroprotection through regulating ischemia/reperfusing induced neurovascular unit damage, apoptosis. Its neuroprotective effects may be related to inhibit autophagy via blocking JNK and AMPK/mTOR signaling pathway.AIM:The present study was designed to evaluate the neuroprotective effects and mechanisms of neurotensin receptor 1 (NTR1) agonist PD149163on acute phase of focal cerebral ischemia/reperfusion induced neural injury in rats.METHODS:1. Transient focal cerebral ischemia was established in 2 month-old male Sprague Dawley (SD) rats by intraluminal occlusion of the right MCA for 90min, then restore perfusion. They were all administered PD 149163 (0.5 mg/kg,i.p.) at 1.5 h,4.5 h after reperfusion and were sacrificed to collect specimens at 72 h. Measuring infarct volume by the 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining and evaluating neurological deficits were used to observe the neuroprotective effects of PD 149163 on acute phase of focal cerebral ischemia/reperfusion induced neural injury in rats.2. Intraperitoneally administering PD 149163 at 0 h,4 h,7 h,10 h after intraluminal occlusion was conducted to observe the effect of PD 149163 on body temperature in SD rats within 900 min after ischemia.3. Regional cerebral blood flow in rats was monitored in real time by two dimensional laser speckle imaging techniques and laser Doppler flowmetry in the cerebral ischemia/reperfusion experiments.4. Using Immunohistochemical staining to detect the morphology and number of neurons, astrocytes in different brain regions (ipsilateral cortex and striatum).5. Western blotting assays were applied to detect the levels of protreins in microglia, BBB ingredients, apoptosis and autophagy relating pathways, such as matrix metalloproteinases 9 (MMP-9) and its substrate tight junction protein occludin, apoptosis-related proteins (including apoptotic protein Bax, anti-apoptotic protein Bcl-2 and caspase3), autophagy-related proteins(including LC3, P62 and Beclinl), JNK, p-JNK, AMPK, p-AMPK (Thr172) and mTOR, p-mTOR (Ser 2448).RESULTS:1. PD149163 could lead to the decrease of body temperature in SD rats and reduce focal cerebral ischemia/reperfusion induced acute injury, but it did not significantly influence the local cerebral cerebral blood flow (LCBF) in the lesion area.At 0 h,4 h,7 h,10 h after intraluminal occlusion, intraperitoneally administering PD149163 could cause 2-5℃ reduction in body temperature of SD rats. Using Laser Doppler flowmetry to select rats with ischemic after middle cerebral artery occlusion (MCAO) operation. When administered intraperitoneally 1.5 h,4.5 h after reperfusion, PD 149163 (0.5 mg/kg) could significantly reduce infarct volume and improve neurological dysfunction in MCAO rats. PD 149163 (0.5 mg/kg) could remarkably reduce neuronal injury, inhibit the activation and proliferation of astrocytes and microglia in ipsilateral cortex and striatum of MCAO rats. PD149163 (0.5 mg/kg) did not significantly affect the local cerebral cerebral blood flow (LCBF) in the lesion area. It was prompted preliminarily that PD 149163 had the neuroprotective effects in MCAO rats.2. PD149163 can improve cerebral ischemia/reperfusion injury induced blood-brain barrier disruption.PD 149163 significantly inhibited the tight junction protein occludin degradation through down-regulating MMP-9 expression in ischemic penumbra of rats.3. PD149163 can inhibit the cell apoptosis and autophagy induced by cerebral ischemia/reperfusion injury in rats.PD149163 apparently reduced the expression of Caspase-3 and Bax, increased the expression of Bcl-2 in ischemic penumbra of rats.The ratio of Bax/Bcl-2 in rats treated with PD149163 is lower than that of control.PD149163 significantly inhibited the decrease of P62 protein expression, reduced LC3Ⅱ/LC3Ⅰ ratio and inhibited the expression of Beclinl protein.4. PD149163 can suppress the JNK and AMPK/mTOR signal transduction in cerebral ischemia/reperfusion injury.PD149163 could reduce the ratio of p-JNK/JNK and p-AMPK(Thr172)/AMPK, and increase the ratio of p-mTOR (Ser 2448)/mTOR. Futhermore, it remarkbaly inhibited the phosphorylation of JNK and AMPK on Thr172, and increased the phosphorylation of mTOR on Ser 2448. Thus it would lead to decreasing activity of JNK and AMPK and increasing activity of mTOR.CONCLUSIONS:1. PD 149163 has neuroprotective effects on acute phase of focal cerebral ischemia/reperfusion in rats, and it can alleviate neurovascular unit damage caused by acute phase of focal cerebral ischemia/reperfusion in rats without influencing the focal cerebral blood flow(LCBF) in the lesion area during cerebral ischemia/reperfusion.2.PD149163 blocks apoptosis and inhibits autophagy induced by acute phase of focal cerebral ischemia reperfusion in rats possibly by blocking JNK and AMPK-mTOR autophagy related signal pathway.In summary, the main innovations of this study lie in:1. We discoverde neuroprotective effects of PD149163 on focal cerebral ischemia/reperfusion injury in rats.2. Our results preliminarily clarified the neuroprotective effects of PD 149163 by improving the function of neurovascular unit, inhibiting the autophagy pathway and apoptosis.