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Free PDF Download European Review for Medical and Pharmacological Sciences 2019; 23 (3 Suppl): 126-134 Promethazine inhibits neuronal apoptosis via PI3K/Akt signaling pathway in rats with cerebral infarction X.-D. PAN1, X.-L. CHEN2, S.-F. DING1, D. KOU3,4, H.-L. HU5, L. LI6 1Department of Critical Care Medicine, Qilu Hospital of Shandong University, Jinan,China 2Department of Laboratory, Zhangqiu District People’s Hospital, Jinan, China 3Department of Neurology, Weifang Hanting District People’s Hospital, Weifang, China 4Department of Economic Management, Medical Research Department, PLA Rocket Force Characteristic Medical Center, Beijing, China 5Department of Radiation Sickness and Hematology, PLA Rocket Force Characteristic Medical Center, Beijing, China 6Department of Pharmacy, Sunshine Union Hospital, Weifang, Shandong, China Abstract. – OBJECTIVE: To study the effect with that in the model group (p<0.05). The of promethazine on neuronal apoptosis in rats escape latency was significantly prolonged with cerebral infarction (CI) through the phos- and the times of crossing platform were sig- phatidylinositol 3-hydroxy kinase/protein kinase nificantly reduced in the model group and B (PI3K/Akt) signaling pathway. promethazine group compared with those in MATERIALS AND METHODS: A total of 36 the sham group (p<0.05), while the escape Sprague-Dawley rats were randomly divided in- latency was significantly shortened and the to the sham group (n=12), model group (n=12), times of crossing platform were significant- and promethazine group (n=12). The external ly increased in the promethazine group com- carotid artery was only exposed in the model pared with those in the model group (p<0.05). group, and the ischemia-reperfusion model af- Compared with those in the sham group, the ter CI was established using the suture method positive expression of Bax was significant- in the other two groups. After modeling, the nor- ly increased, while the positive expression of mal saline was intraperitoneally injected in the Bcl-2 was remarkably decreased in the model sham group and model group, while prometh- group and promethazine group (p<0.05). Com- azine was intraperitoneally injected in the pro- pared with those in the model group, the pos- methazine group. The rats were sampled after itive expression of Bax was significantly de- 1 week of intervention. The neurological defi- creased, while the positive expression of Bcl- cits of rats were evaluated using the Zea-Lon- 2 was remarkably increased in the prometha- ga score, and the cognitive function, the spatial zine group (p<0.05). Besides, the model group learning, and memory of rats were detected via and promethazine group had evidently higher the water maze test. Moreover, the expressions relative protein expressions of PI3K p85, PI3K of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associat- p110, and p-Akt than the sham group (p<0.05), ed X protein (Bax) in brain tissues were detect- while the promethazine group also had evi- ed via immunohistochemistry, and the relative dently higher relative protein expressions of protein expressions of PI3K p85, PI3K p110, and PI3K p85, PI3K p110, and p-Akt than the mod- p-Akt were detected via Western blotting. The el group (p<0.05). Compared with the sham mRNA expressions of Bax and Bcl-2 were de- group, model group, and promethazine group tected via quantitative Polymerase Chain Reac- had remarkably increased relative mRNA ex- tion (qPCR), and the apoptosis was detected via pression of Bax, and remarkably decreased terminal deoxynucleotidyl transferase-mediated relative mRNA expression of Bcl-2 (p<0.05). dUTP nick end labeling (TUNEL) assay. Compared with those in the model group, the RESULTS: The Zea-Longa score was signifi- relative mRNA expression of Bax was remark- cantly increased in the model group and pro- ably decreased, while the relative mRNA ex- methazine group compared with that in the sh- pression of Bcl-2 was remarkably increased am group (p<0.05), while it significantly de- in the promethazine group (p<0.05). Finally, clinedRETRACTED in the promethazine group compared the apoptosis rate was significantly higher in 126 Corresponding Author: Shifang Ding, MD; e-mail: [email protected] Promethazine in neuronal apoptosis in rats the model group and promethazine group than is an important cell signal transduction path- that in the sham group (p<0.05), while it was way, which plays an important regulatory role significantly lower in the promethazine group in such processes as cell proliferation, apoptosis, than that in the model group (p<0.05). CONCLUSIONS: and necrosis, and has an important influence Promethazine inhibits neu- on the repair and reconstruction of neurological ronal apoptosis in CI rats by upregulating the 7,8 PI3K/Akt signaling pathway, thereby exerting a injury . It is now thought that the PI3K/Akt protective effect. signaling pathway, as a classical anti-apoptotic pathway, can effectively reduce the degree of Key Words: apoptosis after injury. In particular, the PI3K/Akt Promethazine, Cerebral infarction, PI3K/Akt signal- ing pathway, Apoptosis. signaling pathway in brain neurons is activated after CI, which can reduce the degree of neuronal apoptosis to play a protective role9. As a com- monly used sedative drug in clinic, promethazine functions as the H1 receptor blocker, exerting a Introduction certain protective effect on neuronal apoptosis, but its mechanism of action remains unclear. In Cerebral infarction (CI), as a clinically common this experiment, therefore, the protective mech- acute cerebrovascular disease, has become one of anism of promethazine in neuronal apoptosis in the diseases seriously threatening human life and CI rats through the PI3K/Akt signaling pathway health. It is currently believed that the damage to was explored. the neurological function caused by CI severely affects the limb motor function of patients in mild cases and leads to the death of patients in severe Materials and Methods cases, so the disability and mortality rates of CI are extremely high1,2. According to epidemiolog- Laboratory Animals ical statistics, the morbidity rate of CI has been A total of 36 specific pathogen-free increasing with the aging of the population in the Sprague-Dawley (SD) rats aged 1 month old world and changes in people’s lifestyles, and ap- were purchased from Shanghai SLAC Laboratory proximately 75% of patients suffer from sequelae Animal Co., Ltd. [license No. SCXK (Shang- caused by neurological deficits after CI. In other hai, China) 2014-0003], and they were fed with words, CI is characterized by high morbidity, normal feed and sterile filtered water every day disability, and mortality rates and high incidence in the Laboratory Animal Center under 12/12 h of sequelae, making it a major disease for clinical light-dark cycle, room temperature, and regular medical workers and researchers. Therefore, how humidity. This research was approved by the An- to effectively prevent, treat CI, and reduce the se- imal Ethics Committee of Shandong University quelae and death caused by neurological deficits Animal Center. after CI is of great significance3,4. Currently, it is believed that the patholog- Laboratory Reagents and Instruments ical responses after CI are very complicated, The main reagents and instruments used including a series of cascade reactions, such as were: promethazine hydrochloride injection inflammation, lipid peroxidation, and release of (KingYork, Tianjin, China), anti-PI3K p110 anti- free radicals, all of which can lead to neuronal body (Abcam, Cambridge, MA, USA), anti-PI3K apoptosis, necrosis, and other pathological re- p85 antibody (Abcam, Cambridge, MA, USA), sults, thereby affecting neurological repair and anti-p-Akt antibody (Abcam, Cambridge, MA, resulting in neurological deficits5,6. In particular, USA), anti-B-cell lymphoma-2 (Bcl-2) antibody the degree of the neuronal apoptosis, as one of (Abcam, Cambridge, MA, USA) and anti-Bcl-2 the most important pathological responses after associated X protein (Bax) antibody (Abcam, CI, determines the degree and scope of CI injury Cambridge, MA, USA), secondary antibodies and the degree of neurological deficits in patients. (Abcam, Cambridge, MA, USA), immunohisto- Therefore, early effective anti-neuronal apoptosis chemistry kit and AceQ quantitative Polymerase is considered as one of the key steps in the treat- Chain Reaction (qPCR) SYBR Green Master ment of CI. Mix Kit (Vazyme, Nanjing, China), HiScript II The phosphatidylinositol 3-hydroxy kinase/ Q RT SμperMix for qPCR (+gDNA wiper) kit proteinRETRACTED kinase B (PI3K/Akt) signaling pathway (Vazyme, Nanjing, China), optical microscope 127 X.-D. Pan, X.-L. Chen, S.-F. Ding, D. Kou, H.-L. Hu, L. Li (Leica DMI 4000B/DFC425C, Wetzlar, Ger- Sampling many), and fluorescence qPCR instrument (ABI After successful anesthesia, the brain tissues 7500, Foster City, CA, USA). were directly taken from 6 rats in each group, washed with normal saline and stored in the Animal Grouping and Treatment Eppendorf (EP; Eppendorf, Hamburg, Germany) The above 36 SD rats were randomly divided tube at -80°C for later use. The samples were into the sham group (n=12), model group (n=12), taken via perfusion fixation from the remaining 6 and promethazine group (n=12) using a random rats in each group: the skull was cut open to ex- number table. The rats were adaptively fed in the pose the brain, and 400 mL of 4% paraformalde- Laboratory Animal Center for 7 d before exper- hyde was perfused. Then, the brain tissues were iments. taken and fixed in 4% paraformaldehyde. The common carotid artery, external carotid artery, and internal carotid artery were exposed Zea-Longa Score and Morris only in the sham group, and the CI model was Water Maze Test established in the model group and promethazine After intervention for 2 weeks, the neurologi- group. After the operation, the promethazine cal deficits were evaluated using the Zea-Longa hydrochloride injection was intraperitoneally in- score according to the symptoms and behaviors jected (7.5 mg/kg) every day in the promethazine of rats (Table I).
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