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Objective To investigate synergistic effects of extracellular signal regulated kinase (ERK1/2) and phosphatidylinositol 3 kinase (PI3-K) pathway inhibitors on autophagy in the hippocampus of rats with subarachnoid hemorrhage (SAH),for identification of therapeutic targets in SAH.Methods Totally,200 male SD rats were randomly divided into a sham operated group,SAH group,inhibitor U0126 group,inhibitor LY294002 group,and a U0126+ LY294002 group.Animal models were established by injecting autologous blood twice into the cisterna magna.Morphological changes in the hippocampus nerve cells were detected by HE staining;ERK1/2,PI3-K,beclin-1,and LC3 mRNA expression in the hippocampus were detected by real-time PCR,and phosphorylated ERK 1/2,PI3-K,beclin-1,and LC3-Ⅱ protein expression were detected by immunohistochemistry.Results Neuronal death rate and phosphorylated ERK1/2,PI3-K,beclin-1,and LC3-Ⅱ levels in the hippocampus in the SAH group were higher than in the sham group (all P ＜ 0.05).Neuronal death rate in U0126 or LY294002 group was higher than in SAH group,while ERK1/2,PI3-K,beclin-1,and LC3 mRNA and phosphorylated ERK 1/2,PI3-K,beclin-1,and LC3-Ⅱ protein levels were lower than in SAH group (all P ＜ 0.05).Neuronal death rate in U0126 +LY294002 group was higher than in U0126 or LY294002 group,while ERK1/2,PI3-K,beclin-1,LC3 mRNA and phosphorylated ERK 1/2,PI3-K,beclin-1,and LC3-Ⅱ protein levels in the hippocampus were lower than in U0126 or LY294002 group (all P ＜ 0.05).Conclusion Co-targeting the inhibition of ERK 1/2 and PI3-K pathways can significantly reduce neuronal cell autophagy and aggravate cells loss after SAH.
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Objective To investigate the effect of MAPK activation on autophagy in the hippocampus of rats with subarachnoid hemorrhage.Methods A total of 100 male SD rats were divided into 5 groups randomly:sham operated group,SAH group,inhibitor U0126 group,inhibitor SB203580 group,SP600125 group.The animal model was established by injecting the autologous blood into cisterna magna twice.The morphological changes of hippocampus nerve cells of rat brain were detected with HE.The mRNA levels of ERK1/2,p38MAPK,JNK and LC3 in hippocampus were detected with quantitative real time PCR and the expression of phosphorylated ERK1/2,phosphorylated p38MAPK,phosphorylated JNK and LC3-Ⅱ in hippocampus of rat brain were detected by immunohistochemistry.Results Compared with the Sham group,the survival rate of neurons in SAH group decreased (6 h:(84.982 ± 5.723) ％,24 h:(74.383± 9.860) ％,48 h:(62.860± 10.820) ％,72 h:(52.260± 10.960) ％) (all P＜0.05).The levels of ERK1/2 mRNA,p38MAPK mRNA,JNK mRNA and LC3 mRNA in hippocampus increased (all P＜ 0.05) and the expression of p-ERK1/2,p-p38MAPK,p-JNK,LC3-Ⅱ proteins increased(all P＜0.05).Compared with the SAH group,the survival rate of neurons in U0126 group was decreased (6 h:(71.620±6.542) ％,24 h:(66.221±7.742)％,48 h:(55.208±8.802) ％,72 h:(46.242±7.782) ％),and the ERK1/2 and LC3 in hippocampus decreased both in mRNA level and in protein level(all P＜0.05).Compared with the SAH group,the survival rate of neurons in SB203580 groups was increased (6 h:(89.082±6.602)％,24 h:(85.840±9.726) ％,48 h:(74.96± 10.916) ％,72 h:(69.211 ± 10.745) ％),and the p38MAPK,LC3-Ⅱ in hippocampus decreased at both mRNA and protein levels (all P＜0.05).Compared with the SAH group,the survival rate of neurons in SP600125 groups was increased (6 h:(91.620± 7.542) ％,24 h:(86.221 ± 10.742) ％,48 h:(75.208±11.802) ％,72 h:(70.242± 11.782) ％).The expression of JNK was decreased while the LC3-Ⅱ was increased in hippocampus (P＜0.05).Conclusion MAPK activation is involved in the autophagy of hippocampal neurons after SAH,in which ERK1/2 activation plays a positive role in the regulation of autophagy in hippocampal neurons after SAH,while p38MAPK and JNK activation plays a negative role in autophagy.
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Objective To investigate the relationship of extracellular regulated protein kinases activation and neural cells autophagy in rats after subarachnoid hemorrhage.Methods One hundred twenty male SD rats were randomly divided into sham operated group,SAH group,ERK1/2 inhibitor U0126 group,autophagy inducer rapamycin (Rap) group.The animal models were established by injecting the autologous blood into cisterna magna twice.U0126 (5μ g/μL) and Rap (10nmol/μL) were injected into lateral ventricles in U0126 group and Rap group 30min before SAH.The morphology of hippocampal nerve cells were examined by using light microscopy.The expression levels of phosphorylated ERK 1/2 (p-ERK 1/2),ERK 1/2mRNA and autophagy markers (Beclin-1 and Beclin-1 mRNA、LC3-Ⅱ and LC3mRNA) in the hippocampus were detected by using inmunohistochemistry and real-time fluorescence quantitative PCR.Result Compared with sham group,the rate of dead nerve cells,the mRNA levels of ERK1/2,Beclin-1 and LC3 as well as the levels of the p-ERK1/2,Beclin-1 and LC3-Ⅱ increased in SAH group (P＜0.05).Compared with SAH group,the rate of dead nerve cells increased(P＜0.05),the ERK1/2 mRNA,Beclin-1 mRNA and LC3 mRN A,and p-ERK1/2,Beclin-landLC3-Ⅱ in U0126 group decreased(P＜0.05);the rate of dead nerve cells decreased (P＜0.05),the Beclin-1 mRNA and LC3 mRNA,the Beclin-1and LC3-Ⅱ level increased in Rap group(P＜0.05),but ERK 1/2 mRNA and p-ERK 1/2 remained unchanged (P＞0.05).Conclusion Activation of the ERK1/2 signaling pathway after SAH,can induce nerve cells death by increasing Beclin-1 and LC3-Ⅱ expressions.
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Objective:To study the effect of endothelin receptor antagonist BQ-123 on the nerve function damage after subarachnoid hemorrhage (SAH)in the rats,and to explore the mechanisms.Methods:Total 120 male SD rats were divided into sham group,SAH group,low dose of BQ-123 group (50 μg· kg-1 )and high dose of BQ-123 group (75 μg·kg-1 ).The SAH rat models were established by injecting the autologous blood into cisterna magna twice.The morphological changes of hippocampus nerve cells of rat brain tissue were detected with HE staining, and the expressions of mTOR, Beclin-1 and LC3-Ⅱ in the hippocampus of rats were detected with immunohistochemistry and RT-PCR;the shuttle-box experiment was used to evaluate the abilities of learning and memory,and the holding power evaluation was used to evaluate the forelimb pulling force of the rats in various groups at each time point.Results:Compared with sham group,the morphological damages of neurons of the rats in SAH group were increased,the survival rate of neurons of the rats in SAH group was decreased (P <0.05),the expression levels of mTOR mRNA,Beclin-1 mRNA and LC3-Ⅱ mRNA in hippocampus tissue of the rats were increased (P < 0.05),and the abtilities of learning and memory and the values of holding power were decreased (P <0.05).Compared with SAH group,the morphological damages of neurons of the rats in BQ-123 groups were decreased,the survival rates of neurons of the rats in BQ-123 groups were increased (P < 0.05),the expression levels of mTOR mRNA of rats were decreased (P <0.05),the expression levels of Beclin-1 mRNA and LC3-ⅡmRNA in hippocampus tissue were increased (P <0.05),and the abilities of learning and memory and the values of holding power were increased (P < 0.05 ). The changes were more significant in high dose of BQ-123 group compared with low dose of BQ-123 group (P <0.05).Conclusion:BQ-123 can improve the nerve function damage after SAH in the rats,its mechanism may be related to regulating the mTOR/autophagy signaling pathway.
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Objectives To investigate the treatment effect of endothelin A receptor antagonist BQ-123 on early brain injury of subarachnoid hemorrhage (SAH ) in rats and its mechanism. Methods According to the random number table method,120 SD rats were divided into four groups:a sham operation (sham),a SAH,a high-dose BQ-123 (75 μg/ kg),and a low-dose BQ-123 (50 μg/ kg)(n = 30 in each group). A rat model was induced by using the injection of blood into cisterna magna twice. After establishing models at hours 6,24,72,and 144,the rats were further divided into four subgroups. Light and electron microscopes were used to observe the changes of the morphological structure in hippocampal area. Immunohistochemistry and RT-PCR were used to detect the expression levels of phosphoinositide 3 kina (PI3-K),protein kinase B (PKB/ Akt),and mammal target of rapamycin (mTOR). Results (1)In the process of model making,7 rats died and 1 model did not meet the criteria and was excluded from the SAH group. Six rats died in the high-dose BQ-123 group and the low-dose BQ-123 group respectively. One rat in each group did not meet the criteria and was excluded. The rats were included in the final statistical analysis:30 in the sham group,22 in the SAH group,23 in the high-dose BQ-123 group,and 23 in the low-dose BQ-123 group. (2)Compared with the sham group,the expression levels of PI3-K,AKt and mTOR were increased signifi-cantly (all P < 0. 05). Compared with SAH group,the hippocampal neuronal morphology and structure damage were alleviated in the low-dose BQ-123 group. The number of surviving neurons at each time point was increased ([132 ±18],[110 ±16],[84 ±13],[92 ± 10]cells/ HP,all P < 0. 05). The tensile force values of rats were increased at each time point and the learning and memory function were improved. The expression levels of PI3-K and Akt were further increased (all P < 0. 05). The expression level of mTOR was decreased (all P < 0. 05). (3)Compared with the low-dose BQ-123 group,the morphological and structural damage of hippocampal neurons were alleviated. The number of surviving neurons at each time point ([153 ±20],[131 ± 18],[137 ±19]and [135 ± 17]cells/ HP)was increased (all P < 0. 05). The tensile force values of the rats were increased at each time point. The learning and memory function of the animals were improved. The expression levels of PI3-K (3. 8 ± 0. 8,8. 9 ± 2. 4,8. 6 ± 2. 4,and 6. 2 ± 2. 0)and Akt (4. 86 ± 1. 74, 8. 64 ± 1. 62,7. 94 ± 1. 70,and 6. 48 ± 1. 58)were further increased (all P < 0. 05). The expression levels of mTOR (2. 89 ± 0. 26,2. 14 ± 0. 18,1. 94 ± 0. 17,and 1. 62 ± 0. 12)were decreased (all P < 0. 05). Conclusions BQ-123 has as a good therapeutic effect for early brain injury after SAH. Its mechanism may be associated with the regulation of PI3-K/ Akt signaling pathway.
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Objective To observe the effect of grape seed proanthocyandin extract(GSPE) on aged patients with obstructive sleep apnea-hypopnea syndrome(OSAHS).Methods One hundred and one cases aged patients with OSAHS who were treated in the Affihated Hospital of North China University of Science and Technology from December 2012 to December 2014 were randomly divided into control group,GSPE group A and GSPE group B,36 cases of each group.The apnea hypopnea index (AHI),rapid eye movement (REM) and micro-arousal index(MAI) were observed by polysomnography (PSG);the fatigue,sleepiness of patients were conducted with fatigue severity scale (FSS) and epworth sleepiness scale (ESS);the peripheral blood malondialdehyde(MDA) levels and superoxide dismutase (SOD) level of before and after treatment were observed by enzyme-hnked immunosorbent (ELISA) method.The control group received continuously positive airways pressure (CPAP) treatment,while GSPE group A and GSPE group B received low and high dose of GSPE treatment oral besides CPAP respectively.Results Before the treatment,there were no significant differences in the terms of AHI,REM,MAI,FSS,ESS,MDA and SOD among the three groups(P＞0.05).After treatment,the scores of FSS,ESS,MAI and MDA in GSPE group B were (2.27±0.84)points,(6.20± 1.16)points,(8.42± 3.27) times/h,(69.40 ± 13.70) nmol/L respectively,lower than that of GSPE group A ((3.84 ± 1.20) points,(8.14± 1.26) points,(10.34± 3.48) times/h,(85.38 ± 12.22) nmol/L respectively) and control group((5.02 ± 1.14) points,(9.40 ± 1.14) points,(13.84 ± 4.08) times/h,(97.96 ± 13.24) nmol/L respectively),the differences were significant(P=0.000).The REM in GSPE group B was (18.28±2.54)％,higher than that of GSPE group A ((15.74 ± 4.32) ％) and control group ((12.38 ± 3.77) ％),there were significant differences among the three groups (P =0.000).While there were no significant differences on SOD levels among the three groups(P＞0.05).The rate of effectiveness in control group,GSPE group A and GSPE B were 70.5％,79.4％ and 90.9％ respectively,the rate of effectiveness in GSPE B was significant higher than GSPE group A and control group,and the difference was significant(P＜0.05).Conclusion GSPE can improve the sleep quality and weaken oxidative stress reaction,and has a good clinical effects for aged patients with OSAHS.
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Objective To explore the application of ABCD3 score on stratifying the antithrombotic treatment strategy in patients with capsular warning syndrome (CWS).Methods The clinical features of 15 patients with CWS were analyzed retrospectively,and the risk of stroke were evaluated by ABCD3 score and to guide the treatment of Stratifying antithrombotic therapy.The status of patients hospitalized,discharged and discharged after 90 d were evaluated.Results The frequency of patients with CWS accounted for 2.51％ (15/ 597) of all patients with transient ischemic attack(TIA),and the mean age in patients with CWS was (70.27 ±8.29) years old.The duration of the first onset was (10-30) min,the mean time was (17.33±1.53) min,and ABCD2 score was 5.0-9.0 points,mean score was 7.00±0.26 points,and the total episodes of CWS were 51 times during 24 hours,the mean duration was (18.13 ± 15.36) minutes ((3.0-60.0) min).All 15 cases presented with limb hemiparesis.Of them,9 cases had dysarthria,5 case with ipsilateral facial palsy.All 15 cases CWS patients showed no signs of cortical deficit.The mean NIHSS score at onset was 1.0-6.0 points,mean scores was 3.20±0.31 points.Fourtheen patients were treated with clopidogrel plus aspirin,and 2 cases with administration of the loading dose 300 mg of clopidogrel,1 case was treated with clopidogrel plus aspirin orally followed by intravenous rt-PA thrombolysis.The average hospital periods of all 15 patients was (7.67±0.29) days.The NIHSS score were 0 point at discharge.There was no symptomatic intracranial hemorrhage or death within 90 days follow-up periods.Conclusion CWS is prone to develop a completed stroke.Stratified antithrombotic therapy guiding by ABCD3 score may decrease the risk of ischemic stroke.