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OBJECTIVE@#To observe the effect of electroacupuncture (EA) pretreatment on inflammatory reaction, apoptosis and expression of Yes-associated protein (YAP) of ischemic penumbra of cerebral cortex in cerebral ischemia reperfusion injury rats, and to explore the possible mechanism of its neuroprotection effect.@*METHODS@#A total of 84 SD rats were randomized into a sham operation group (12 rats), a model group (18 rats), an EA group (18 rats), an EA+YAP virus transfection group (18 rats) and an EA+virus control group (18 rats). Except for the sham operation group, thread embolization method was adopted to establish the middle cerebral artery occlusion (MCAO) model in rats of the other groups. EA was applied at "Baihui" (GV 20) and "Dazhui" (GV 14) for 30 min in the 3 EA intervention groups 2 h before model establishment, disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in intensity. Adenovirus transfection technique was used to induce gene silencing of YAP in the EA+YAP virus transfection group, and adenovirus vectors was injected as negative control in the EA+virus control group 4 d before model establishment. Twenty-four hours after model establishment, neurological function score was evaluated, the relative cerebral infarction area was observed by TTC staining, the apoptosis in the ischemic penumbra of cerebral cortex was detected by TUNEL staining, the levels of inflammatory factors IL-1β, IL-6 and TNF-α in the ischemic penumbra of cerebral cortex was detected by ELISA method, the expression of YAP was detected by Western blot and immunofluorescence.@*RESULTS@#Compared with the sham operation group, the expression of YAP was increased in the model group (@*CONCLUSION@#Electroacupuncture pretreatment can effectively improve the ischemia reperfusion injury, its mechanism may be related to up-regulating the expression of YAP in the ischemic penumbra of cerebral cortex and relieving the apoptosis and inflammatory reaction.
Assuntos
Animais , Ratos , Isquemia Encefálica/terapia , Eletroacupuntura , Infarto da Artéria Cerebral Média , Ratos Sprague-Dawley , Traumatismo por Reperfusão/terapiaRESUMO
AIM: To investigate whether α-ketoglutarate dehydrogenase complex (α-KGDHC) has protective effects on adaptive reperfusion after ischemic stroke in rats, and whether its mechanism is related to inhibition of apoptotic pathways. METHODS: A model of middle cerebral artery occlusion (MCAO) ischemia/reperfusion (I/R) was established, and the volume of cerebral infarction was assessed by TTC staining following computer analysis. The α-KGDHC activity was detected based on chemical reaction, to evaluate the change trend of α-KGDHC activity with cerebral ischemia time, and compare the difference between normal reperfusion group and adaptive reperfusion group; treatment of B35 and SH-SY5Y with CoCl2 to mimic cell hypoxia, and inhibition of α-KGDHC activity with E1K siRNA to detect the expression of apoptosis-related proteins Bcl-2/Bax/Caspase 3 by Western blot. RESULTS: In vivo experiments: In MCAO model, adaptive reperfusion further reduced cerebral infarction volume compared with normal reperfusion (P<0.05), and the expression of Caspase 3 was the lowest; α-KGDHC activity in cerebral cortex and hippocampal brain tissue decreased as ischemic time prolonged (P<0.05), and adaptive reperfusion inhibited the rate of decrease in α-KGDHC activity (P<0.05). In vitro experiments: Inhibiting α-KGDHC activity by interfering E1K expression led to downregulation of Bcl-2 (P<0.05) and upregulation of Bax (P<0.05) and Caspase 3 (P<0.05). CONCLUSION: α-KGDHC is an important factor in the protection of adaptive reperfusion after cerebral ischemia, and it may exert protective effect by inhibiting the activation of apoptotic pathways.
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ObjectiveIn cerebral ischemia, free radicals form in large quantities and activate the inflammasome nod-like recepter protein 3(NLRP3), thereby exacerbating brain damage. Edaravone has the effect of scavenging free radicals, but the relationgship between its neuroprotective effect and inflammasome NLRP3 has not been reported. To study the neuroprotective effects of edaravone on inflammasome NLRP3 and associated protein of the blood-brain barrier against cerebral ischemia injury.Methods60 male Sprague-Dawley rats were randomly divided into Sham group, cerebral embolism group and edaravone group, respectively. Rat thromboembolic MCAO models were established and edaravone (3mg/kg) was intravenous injected immediately after occlusion and 4 hours after occlusion. In the sham group, cervical blood vessels were separated only, and no embolus was injected. At 24 hours after thrombi injected, mNSS score was used to evaluate neurological function deficits. Brain infarct volume was estimated by TTC staining. The expression of inflammasome NLRP3, occludin and ZO-1 was detected by Western blot. Immunohistochemistry was used to measure IgG leakage.ResultsThe mNSS score and infarct volume of cerebral embolism group at 24 hours after stroke were significantly higher than Sham group (P<0.05). The expression of inflammasome NLRP3 in the brain increased (P<0.05), occludin and ZO-1 blood brain barrier associated protein decreased (P<0.05), and IgG leakage increased. But compared with cerebral embolism group, the mNSS score（4.50±2.12 vs 6.50±1.35, P<0.05）and infarct volume （19.29±11.92 vs 29.99±7.56, P<0.05）decreased when treated with edaravone. Edaravone treatment significantly attenuated inflammasome NLRP3 expression in cerebral ischemic area (0.97±0.47 vs 1.58±0.86, P<0.05), degradation of BBB components (occludin, ZO-1) (1.04±0.19 vs 0.53±0.09, 0.66±0.05 vs 0.30±0.04,P<0.05) and IgG leakage decreased.ConclusionInflammasome NLRP3 play an important role in acute cerebral ischemia injury. Edaravone may provide neuroprotection by inhibiting expression of inflammasome NLRP3 and degradation of associated protein of blood brain barrier.
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Objective To investigate the effect of melatonin on mice with ischemia-reperfusion via a silent information regulator 1 (SIRT1) reducing mitochondrial oxidative stress mechanism. Methods A transient middle cerebral artery occlusion ( MCAO) cerebral ischemia-reperfusion ( IR) model in mice was established by the suture-occluded method. One hundred and ninety mice were injected with melatonin intraperitoneally or the SIRT1 inhibitor (EX527) intracerebroventricularly,30 dead and model failure mice were excluded. They were divided into IR,melatonin,melatonin +EX527,and EX527 groups (n =40 in each group ) according to the random number table. The cerebral infarct volume was detected by the triphenyltetrazolium chloride (TTC) method,the brain edema was measured by the wet and dry weight method and the neurological deficit scores were measured. Western blot was used to detect SIRT1,Ac-P53, acetylated-nuclear factorκB (Ac-NF-κB),BCl2,Bax proteins in the mitochondria and cytoplasm,as well as the cytochrome C protein expression. A single factor analysis of variance was used for comparison among the groups. Results ( 1 ) There were significant differences in cerebral infarction volume, neurological dysfunction scores and cerebral edema among the four groups ( F values,16. 452,23. 622,and 18. 786, respectively (all P<0. 05). There were significant differences in the expression levels of SIRT1,Ac-P53, Ac-NF-κB,BCl2, and Bax among the four groups ( F values, 2348. 158, 1434. 841, 7042. 563, 14627. 128,and 691. 475,respectively,all P<0. 05). There were significant differences in mitochondrial membrane potential,mitochondrial reactive oxygen species,and complex I activity in mice among the four groups (F value,28. 454,33. 728 and 29. 716,respectively,all P <0. 05). (2) Compared with the IR group,the infarct volume was reduced (32 ± 5 mm3 vs. 57 ± 5 mm3,P<0. 05),neurological deficit scores were decreased (2. 4 ± 0. 3 vs. 3. 5 ± 0. 3,P<0. 05);brain edema was reduced (80. 2 ± 0. 9% vs. 83. 9 ± 1. 2%,P<0. 05);the expression levels of SIRT1 and anti-apoptosis protein BCL2 were increased in the melatonin group (P<0. 05);the expression levels of pro-apoptotic protein BAX and Ac-P53,Ac-NF-κB were reduced ( P <0. 05 );the mitochondrial membrane potential, mitochondrial complex I activity and cytochrome C level were increased (P<0. 05);and the cytoplasmic reactive oxygen species and cytochrome C level were decreased (P < 0. 05). (3) Compared with the melatonin group,cerebral infarction volume were increased (42 ± 5 mm3 vs. 32 ± 5 mm3,P < 0. 05);nerve dysfunction scores were increased(3. 2 ± 0. 3 vs. 2. 4 ± 0. 3,P<0. 05);cerebral edema was aggravated (83. 4 ± 0. 8% vs. 80. 2 ± 0. 9%, P < 0. 05 );the expression levels of SIRT1 and anti-apoptotic protein BCL2 were reduced (P <0. 05);the pro-apoptotic protein BAX,Ac-P53,and Ac-NF-κB expression levels were increased (P<0. 05);the mitochondrial membrane potential and mitochondrial complex I activity and cytochrome C level were decreased (P<0. 05);and the cytoplasmic reactive oxygen species and cytoplasmic cytochrome C level were increased in the melatonin+EX527 group (P<0. 05). Conclusion In ischemic stroke model mice, melatonin plays a neuroprotective role by activating the SIRT1 signaling pathway and reducing oxidative stress injury and cell death in mitochondria,thus plays a role in cerebral protection.
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Objective To investigate the effect of melatonin on mice with ischemia-reperfusion via a silent information regulator 1 (SIRT1) reducing mitochondrial oxidative stress mechanism. Methods A transient middle cerebral artery occlusion ( MCAO) cerebral ischemia-reperfusion ( IR) model in mice was established by the suture-occluded method. One hundred and ninety mice were injected with melatonin intraperitoneally or the SIRT1 inhibitor (EX527) intracerebroventricularly,30 dead and model failure mice were excluded. They were divided into IR,melatonin,melatonin +EX527,and EX527 groups (n =40 in each group ) according to the random number table. The cerebral infarct volume was detected by the triphenyltetrazolium chloride (TTC) method,the brain edema was measured by the wet and dry weight method and the neurological deficit scores were measured. Western blot was used to detect SIRT1,Ac-P53, acetylated-nuclear factorκB (Ac-NF-κB),BCl2,Bax proteins in the mitochondria and cytoplasm,as well as the cytochrome C protein expression. A single factor analysis of variance was used for comparison among the groups. Results ( 1 ) There were significant differences in cerebral infarction volume, neurological dysfunction scores and cerebral edema among the four groups ( F values,16. 452,23. 622,and 18. 786, respectively (all P<0. 05). There were significant differences in the expression levels of SIRT1,Ac-P53, Ac-NF-κB,BCl2, and Bax among the four groups ( F values, 2348. 158, 1434. 841, 7042. 563, 14627. 128,and 691. 475,respectively,all P<0. 05). There were significant differences in mitochondrial membrane potential,mitochondrial reactive oxygen species,and complex I activity in mice among the four groups (F value,28. 454,33. 728 and 29. 716,respectively,all P <0. 05). (2) Compared with the IR group,the infarct volume was reduced (32 ± 5 mm3 vs. 57 ± 5 mm3,P<0. 05),neurological deficit scores were decreased (2. 4 ± 0. 3 vs. 3. 5 ± 0. 3,P<0. 05);brain edema was reduced (80. 2 ± 0. 9% vs. 83. 9 ± 1. 2%,P<0. 05);the expression levels of SIRT1 and anti-apoptosis protein BCL2 were increased in the melatonin group (P<0. 05);the expression levels of pro-apoptotic protein BAX and Ac-P53,Ac-NF-κB were reduced ( P <0. 05 );the mitochondrial membrane potential, mitochondrial complex I activity and cytochrome C level were increased (P<0. 05);and the cytoplasmic reactive oxygen species and cytochrome C level were decreased (P < 0. 05). (3) Compared with the melatonin group,cerebral infarction volume were increased (42 ± 5 mm3 vs. 32 ± 5 mm3,P < 0. 05);nerve dysfunction scores were increased(3. 2 ± 0. 3 vs. 2. 4 ± 0. 3,P<0. 05);cerebral edema was aggravated (83. 4 ± 0. 8% vs. 80. 2 ± 0. 9%, P < 0. 05 );the expression levels of SIRT1 and anti-apoptotic protein BCL2 were reduced (P <0. 05);the pro-apoptotic protein BAX,Ac-P53,and Ac-NF-κB expression levels were increased (P<0. 05);the mitochondrial membrane potential and mitochondrial complex I activity and cytochrome C level were decreased (P<0. 05);and the cytoplasmic reactive oxygen species and cytoplasmic cytochrome C level were increased in the melatonin+EX527 group (P<0. 05). Conclusion In ischemic stroke model mice, melatonin plays a neuroprotective role by activating the SIRT1 signaling pathway and reducing oxidative stress injury and cell death in mitochondria,thus plays a role in cerebral protection.
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Objective: To investigate the neuroprotective effects and mechanisms of Shennao Fuyuan Decoction (SFD) combined with implantation of human umbilical cord mesenchymal stem cells (hUC-MSCs). Methods: Rats were randomly divided into Sham operation group, model group[cerebral ischemia-reperfusion (I/R) model of rats prepared by middle cerebral artery occlusion, MCAO], SFD group (11.6 g/kg), hUC-MSCs group, combination group (SFD + hUC-MSCs), and these groups were separately divided into other three groups by execution time (days 4,8, and 15). The neurological function of rats was evaluated by neurological severity scores (NSS), the pathological changes of hippocampus were observed by HE staining, and the expression of BDNF and bFGF was detected by immunohistochemical and Werstern blotting method. Results: After cerebral ischemia, the neural necrocytosis of hippocampus in SFD group, hUC-MSCs group, and combination group was alleviated compared with model group; Compared with model group, the NSS was much lower and the expression of BDNF and bFGF was higher in SFD group (days 8 and 15), hUC-MSCs group, and combination group (days 4,8, and 15) (P<0.01 and 0.05); Compared with SFD group and hUC-MSCs group, the NSS was lower and the expression of BDNF and bFGF was higher in combination group (days 8 and 15) (P<0.05). Conclusion: The combination of SFD and hUC-MSCs implantation could significantly improve the restoration of nuerological defects after cerebral ischemia of rats, and this neuroprotective effect is probably due to the promotion of BDNF and bFGF expression.
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Objective:To explore TCM syndrome variation regularity on acute stage of ischemia stroke.Methods:To collect clinical dynamic state information of ischemia stroke from 72h to 14days by using prospective clinical research method and investigate TCM syndrome variation regularity in acute stage of ischemia stroke.Results:Wind,fire and phlegm syndromes were the most frequent syndromes in acute stage of ischemia stroke.They appeared varied regularity.There were forty-four combination patterns about six basic syndromes.The wind syndrome and fire-phlegm syndrome were increasing in acute stage of ischemia stroke.The wind-phlegm syndrome was decreasing in the first three days,the wind-fire syndrome peaked on the third day,the phlegm was decreasing on the fifth day.The wind-fire-phlegm was the most frequent and undulate syndrome on the fifth day.The wind-phlegm-stagnation syndrome was increasing for the first three days.Conclusion:There were fastly morbility,changeable and complicated syndrome change in acute stage of ischemia stroke.It was very necessity and urgency for complementing diagnostic criteria of stroke differentiation.
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A prospective study including 70 patients with acute cerebral infarction at Hospital 108 from July 2004 to Jun 2005. Results: Increasing blood glucose level is very common among acute stroke patients accounting for 54.3%. Blood glucose level due to response during acute stroke is commonly within the range of 7 – 10mmol/l (50%). There are reverse correlations between blood glucose and Glasgow, Herry and Barthel scores. HbA1c is essential to differentiate hyperglycemia due to diabetes or response