Studies On The Role Of Anglotensin â…¡ In Neurotoxic Mechanism Caused By Thrombin After Brain Ischemia-Reperfusion Injury In Rats And Drug Intervention

Objective:It is well known that ischemic cerebrovascular disease (ICVD) can do great harm to human's health. Though many researches have been done in investigating the mechanisms of ICVD, so far effective drugs for the prevention and treatment of ICVD are quite limited and few. It is urgent to find the effective drugs on the foundation of theoretical study. Recent studies have shown that the serine protease thrombin plays an important role in neural degeneration after cerebral ischemia, but there are no reports to clarify the molecular mechanisms of regulating the thrombin. Renin-angiotensin system (RAS) plays an important role in cerebral vascular diseases, of course, angiotensin IⅡ(AngⅡ), the main effector of the renin-angiotensin system, has a hand in occurrence and development of ICVD. The important mechanism of ICVD is that AngⅡactivates oxidative stress caused by NADPH oxidase. Our present study was preformed both in vivo and in vitro to clarify the patho-physiological mechanisms of cerebral ischemia-reperfusion injury, focusing on the interreaction of AngⅡ, NOX2, etc. on regulating thrombin, meanwhile, we observed whether AT1 receptor antagonist losartan and Hydroxysafflor yellow A (HSYA) had neuroprotective effects on brain injury after I/R and implored their probable mechanisms.Methods:1. In vivo experiments:Rats were divided into 8 groups randomly:rats in group 1(sham) only received anesthesia and vessel separation; rats in group 2 received ischemia-reperfusion operation and NS ig; in group 3 and 4 received ischemia-reperfusion and losartan 10mg/kg and 2.5mg/kg ig daily respectively; rats in group 5 received ischemia-reperfusion and NOX inhibitor apocynin 2.5mg/kg; group 6,7 and 8 received ischemia-reperfusion and Hydroxysafflor yellow A (HSYA) 8mg/kg,4mg/kg and 2mg/kg, respectively. Transient focal cerebral ischemia was conducted by the MCAO procedure as described by Zea Longa.Occlusion was done for a period of 2 h. For reperfusion the nylon suture was withdrawn. In order to test their neuroprotective effects, we examined the neurological deficit score of each rat by using a standard scale for a five-point neurological assessment, the number of survival neurons in hippocampus CA1 region and neutrophil infiltration at 24 h after reperfusion. The expression of NOX2, prothrombin, ICAM-1 and TNF-αwere detected by Western blot and RT-PCR. The nuclear translocation of NF-кB p65 subunit and the protein expression of IкBαwere observed by Western blot, the DNA binding activity of NF-кB was observed by electrophoretic mobility shift assay (EMSA). AngiotensinⅡwas determined by radioimmunoassay using type GC-911 radioimmuno-γ-counter.2. In vitro experiments, cultured neonate rat cortical astrocytes were used.The first part:to observe the neuronal injury action of thrombin. Cultured astrocytes were divided into 5 groups:group 1 (control); group 2 (using 5U/ml thrombin); group 3 (thrombin+10-6mol/L losartan); group 4 (thrombin+10-6mol/L PD 123319); group 5 (thrombin+10-6mol/L losartan+ 10-6mol/L PD123319). Cell viability was assessed by methylthiazol tetrazolium (MTT) assay and cell injury by the rate of lactate dehydrogenase (LDH) release. The expression of NF-кB p65 mRNA and protein were examined by RT-PCR and Western Blot.The second part:to explore the molecular mechanisms of angiotensinⅡregulating the expression of thrombin.①After 10-6mol/L AngⅡincubating, the same indexes as vivo experiments and the activity of SOD were observed;②Before 10-6mol/L AngⅡincubating,Ⅰusing NOX inhibitor apocynin;Ⅱobserving the expression of above-mentioned indexes;Ⅲobserving the effects of AT1 receptor specific blocker losartan or plus the AT2 receptor blocker PD123319 or AT2 receptor blocker PD123319 alone on brain injury.Results:1. Compared with sham-group, rats with two hours of ischemia followed by 24 hours of reperfusion exhibited severe neural injury as shown in significant increasing of neurological scores. The levels of the number of neutrophil, TAT, F1+2, prothrombin, NOX2, ICAM-1, TNF-αand NF-кB after cerebral I/R were significantly increased. Plasma and brain angiotensinⅡwere upregulated 24h of reperfusion in model group. While compared with group 2, the levels of above-mentioned indexes were decreased in losartan-treated and HSYA-treated rats significantly.2. In vitro experiments, after 5U/ml thrombin incubating, the relative cell viability was reduced and LDH release from astrocyte was markedly increased. Meanwhile, the mRNA and protein expression of NF-кB p65 were increased. AT1 receptor specific blocker losartan or plus the AT2 receptor blocker PD123319 prevented the cell injury caused by thrombin. After 10-6mol/L Ang II incubating, the above-mentioned indexes were significantly increased as well as vivo experiments. After using NOX inhibitor, the reaction of oxidative stress was significantly inhibited caused by AngⅡ.Prevented with 10-6mol/L AT1 receptor specific blocker losartan inhibited AngⅡ-induced injury. However, prevented with PD123319 alone did not prevent the injury.Conclusions:1. The serine protease thrombin plays an important role in neural degeneration after cerebral ischemia.2. AngⅡcan regulate the expression of thrombin via signal channel of AngⅡ-AT1R-NOX2-thrombin, at least partially.3. Our results demonstrated that losartan could protect ischemia-reperfusion injury through preventing Ang II binding to AT1 receptor, meanwhile inhibit AngⅡ-AT1R-NADPH oxidase (especially NOX2)-ROS signaling pathway.4. HSYA protected focal cerebral I/R injury in rats and the underlying mechanisms might partly be associated with its inhibitory effects on thrombin generation and thrombin-induced inflammatory responses by reducing angiotensinⅡcontent.Significances:Ischemic cerebrovascular disease has become the common harmful disease to elder's health, and the incidence is increasing gradually. But the mechanisms of the disease are not well illustrated, which makes the prevention and treatment very difficult. In the recent researches, thrombin plays an important role in cerebral ischemia. However, the molecular mechanisms of regulating its expression are still unsolved. Oxidative stress triggered by AngⅡthrough stimulating NADPH oxidase (NOX) is an important mechanism, which cause cerebral ischemia-reperfusion injury (CIRI). Therefore, it can be inferred that AngⅡ-NADPH oxidase may play an important regulating role in CIRI caused by thrombin. Our previous research indicated that losartan and hydroxysafflor yellow A played the role in preventing and treating on cerebrovascular disease. However, whether the mechanism is relative to anti-thrombin action has not been reported.This research could take intensive study in molecular mechanism of AngⅡ-NADPH oxidase regulating thrombin and the function of this signal channel in CIRI. We would complete the pathological mechanisms of AngⅡ. Meanwhile, we could provide a new train of thought in prevention and treatment of ischemic cerebral vascular disease via signal channel of AngⅡ-NADPH oxidase-thrombin. And it would provide a new target for medicine development, which has an important academic significance and applied value. We consulted and retrieved the refereces within five years, and did not find the same or similar one with ours.Innovations:1. For the first time, our research reveals the important function of AngⅡin neural toxication induced by thrombin. At the same time, we study the activation of NOX2 in the regulation and effect of it. So we provide an academic basis for further study in mechanisms of neural toxication of thrmbin.2. Losartan and hydroxysafflor yellow A could prevent the expression of thrombin effectively, which could reduce cellular toxication caused by overexpression of thrombin. It provides the academic basis for these two drugs to be the new kinds of prevention and treatment on cerebrovascular disease mediated by thrombin.3. For the first time, it is suggested that we should intervene the signaling pathway of AngⅡ-NOX2-thrombin in order to explore a new strategy to prevent and treat ischemic cerebrovascular disease.