Effect of Hypoxic Preconditioning on Neural Cell Apoptosis and Expression of Bcl-2 and Bax in Cerebral Ischemia-Reperfusion in Rats

In order to investigate the protective effect of hypoxic preconditioning on the cerebral ischemia-reperfusion injury, the expression of Bcl-2 and Bax was detected by using immunohistochemical staining after 3 h cerebral ischemia followed by 1, 6, 12, 24 and 48 h reperfusion respectively in rats treated with or without hypoxic preconditioning before cerebral ischemia. In addition, the apoptosis of neural cells and the behavioral scores for neurological functions recovery were evaluated by TUNEL staining and ＂crawling method＂, respectively. Compared with control group （cerebral ischemia-reperfusion without hypoxic preconditioning）, the expression of Bcl-2 was significantly increased, but that of Bax decreased in the hypoxic preconditioning group （cerebral ischemiareperfusion with hypoxie preconditioning）, both P〈0.05. The pre-treatment with hypoxic preconditioning could reduce the apoptosis of neural cells and promote the neurological function recovery as compared to control group. It was suggested that hypoxic preconditioning may have protective effects on the cerebral ischemia-reperfusion injury by inhibiting the apoptosis of neural cells, increase the expression of Bcl-2 and decrease the expression of Bax.

Hypoxic preconditioning reduces NLRP3 inflammasome expression and protects against cerebral ischemia/reperfusion injury

Hypoxic preconditioning can protect against cerebral ischemia/reperfusion injury. However, the underlying mechanisms that mediate this effect are not completely clear. In this study, mice were pretreated with continuous, intermittent hypoxic preconditioning;1 hour later, cerebral ischemia/reperfusion models were generated by middle cerebral artery occlusion and reperfusion. Compared with control mice, mice with cerebral ischemia/reperfusion injury showed increased Bederson neurological function scores, significantly increased cerebral infarction volume, obvious pathological damage to the hippocampus, significantly increased apoptosis;upregulated interleukin-1β, interleukin-6, and interleukin-8 levels in brain tissue;and increased expression levels of NOD-like receptor family pyrin domain containing 3(NLRP3), NLRP inflammasome-related protein caspase-1, and gasdermin D. However, hypoxic preconditioning significantly inhibited the above phenomena. Taken together, these data suggest that hypoxic preconditioning mitigates cerebral ischemia/reperfusion injury in mice by reducing NLRP3 inflammasome expression. This study was approved by the Medical Ethics Committee of the Fourth Hospital of Baotou, China(approval No. DWLL2019001) in November 2019.

Puerarin inactivates NLRP3-mediated pyroptotic cell death to alleviate cerebral ischemia/reperfusion(I/R)injury through modulating the LncRNA DUXAP8/miR-223-3p axis

NLRP3 inflammasome-mediated cell pyroptosis aggravates the development of cerebral ischemia/reperfusion(I/R)injury,and the aim of this study is to investigate the potential utilization of the Chinese medicine,Puerarin,in treating this disease.Through conducting in vitro and in vivo experiments,the present study illustrated that Puerarin regulated LncRNA double homeobox A pseudogene 8(DUXAP8)/miR-223-3p axis to inactivate NLRP3-mediated pyroptotic cell death,resulting in the attenuation of I/R injury.Specifically,the cerebral I/R injury in rat models and hypoxia/reoxygenation(H/R)in primary hippocampus neuron(PHN)cells were inducted,which were subsequently exposed to Puerarin treatment.As expected,we validated that Puerarin suppressed cell pyroptosis and rescued cell viability in I/R rat hippocampus tissues and H/R PHN cells.Next,through bioinformatics analysis,we noticed that miR-223-3p targeted both LncRNA DUXAP8 and NLRP3 mRNA,and both LncRNA DUXAP8 ablation and miR-223-3p overexpression inactivate NLRP3-mediated cell pyroptosis to rescue cell viability in H/R PHN cells.Interestingly,we evidenced that Puerarin restrained LncRNA DUXAP8 expressions,but upregulated miR-223-3p in I/R rat tissues and H/R PHN cells,and the protective effects of Puerarin on H/R PHN cells were abrogated by overexpressing LncRNA DUXAP8 and silencing miR-223-3p.Collectively,we concluded that Puerarin regulated LncRNA DUXAP8/miR-223-3p/NLRP3 signaling cascade to attenuate I/R injury.

Investigation of Linarinic acid and one of its derivatives against cerebral ischemia in mice

The study aims to investigate the effects of(-)-Linarinic acid(LA) and one of its derivatives(LAd) on brain injury induced by ischemia. Malonaldehyde(MDA) is determined as an index for lipid peroxidation both in vitro and vivo. Mice were pre-treated with LA and LAd for 3 d.Thereafter, they were induced to have incomplete cerebral ischemia with both bilateral carotid artery occlusion and hypotension(BCAOH). In the first part of the in vivo experiment, mice were divided into four groups: sham(control), ischemia, ischemia + LA(200 mg/kg, i.g.) and ischemia + LAd(200 mg/kg, i.g.). In the second part, the dose-response of LAd was investigated at 100, 200 and 400 mg/kg i.g., respectively. A modified neurological severity score was developed for evaluating behavioral deficits of the mice with ischemia. Brains of the mice were excised in order to determinate MDA after ischemia for 6 h. Survival time, survival rate, neurological injury score and MDA level in brains were observed. Results were: 1) The data in vitro showed that both LA and LAd could inhibit the generation of MDA. IC50 values obtained by Probit analysis were 2.9 mM for LAd and 4.88 mM for LA;2) BCAOH could significantly shorten the survival span, reduce the survival rate and cause neurological deficits,which were associated with high level of lipid hydroperoxide production in cerebral tissues;3) LAd decreased lipid peroxidation and improved the neurological outcome more than LA.It is concluded that LAd offers a better neuroprotection than LA against brain damage caused by cerebral ischemia.

Bumetanide promotes neural precursor cell regeneration and dendritic development in the hippocampal dentate gyrus in the chronic stage of cerebral ischemia

Bumetanide has been shown to lessen cerebral edema and reduce the infarct area in the acute stage of cerebral ischemia. Few studies focus on the effects of bumetanide on neuroprotection and neurogenesis in the chronic stage of cerebral ischemia. We established a rat model of cerebral ischemia by injecting endothelin-1 in the left cortical motor area and left corpus striatum. Seven days later, bumetanide 200 μg/kg/day was injected into the lateral ventricle for 21 consecutive days with a mini-osmotic pump. Results demonstrated that the number of neuroblasts cells and the total length of dendrites increased, escape latency reduced, and the number of platform crossings increased in the rat hippocampal dentate gyrus in the chronic stage of cerebral ischemia. These findings suggest that bumetanide promoted neural precursor cell regeneration, dendritic development and the recovery of cognitive function, and protected brain tissue in the chronic stage of ischemia.

杜鹃花总黄酮通过抑制TNF-α/caspase-8/caspase-3信号通路减轻大鼠脑缺血再灌注损伤的作用

目的探索杜鹃花总黄酮(TFR)通过抑制TNF-α/caspase-8/caspase-3信号通路保护脑缺血再灌注(I/R)损伤的机制。方法采用大脑中动脉闭塞术(MCAO)建立大鼠I/R模型,将造模成功大鼠随机分为假手术(Sham)、脑缺血再灌注(MCAO)和脑缺血再灌注术后TFR 200 mg/kg干预(TFR 200 mg/kg)组,制备MCAO大鼠模型,在脑缺血再灌注损伤手术后,TFR 200 mg/kg组连续14 d给予TFR(200 mg/kg)药物溶液。术后14 d,依据大鼠神经功能评分,脑血流图观察脑血流情况,处死大鼠,苏木精-伊红(HE)染色观察脑组织细胞形态学变化,试剂盒检测大鼠血清中乳酸脱氢酶(LDH或LD)和神经特异性烯醇化酶(NSE)两种酶活性;ELISA试剂盒检测白细胞介素-1(IL-1)和IL-6水平,Western blot和免疫组化同时检测大鼠脑组织中裂解型胱天蛋白酶3(cleaved caspase-3)、caspase-8、肿瘤坏死因子(TNF)-α蛋白的表达水平。结果脑缺血再灌注处理后,MCAO导致了大鼠神经功能异常,神经功能评分指数显著升高,脑组织病理形态学和脑血流量变化明显,脑组织中cleaved caspase-3、caspase-8、TNF-α蛋白的表达水平显著增加,血清中LDH、NSE、IL-1、IL-6水平明显升高;TFR 200 mg/kg组大鼠神经功能评分明显降低,脑组织病理损伤显著改善,脑组织中cleaved caspase-3、caspase-8、TNF-α蛋白的表达以及血清中LDH、NSE、IL-1、IL-6水平降低。结论TFR可能通过抑制TNF-α/caspase-8/caspase-3信号通路减轻脑缺血缺氧性损伤。

新生小鼠缺氧缺血性脑病脑组织caspase-3表达的变化

目的观察半胱氨酸天冬氨酸蛋白酶(caspase)-3在新生儿缺氧缺血性脑病(NHIE)小鼠模型脑组织中表达的变化。方法选取7 d CD1新生小鼠30只,按随机数字表法分为假手术组(9只)和NHIE模型组(21只),后者制备NHIE动物模型。用TTC染色法检查2组的脑组织梗死面积;DAPI染色观察脑组织病理变化;原位末端标记技术检测脑组织细胞凋亡;荧光免疫组化法检测脑组织中caspase-3表达水平。结果假手术组小鼠脑组织未见梗死灶,脑组织细胞排列致密整齐,TUNEL阳性细胞数[(18.57±4.98)个]和caspase-3阳性细胞数[(9.17±2.14)个]明显低于NHIE模型组的TUNEL阳性细胞数[(209.57±41.27)个]和caspase-3阳性细胞数[(63.33±16.22)个];与假手术组相比,NHIE模型组小鼠的右侧半球可见梗死灶,脑组织细胞大量坏死脱落、周围组织间隙变大。结论 NHIE模型鼠出现的脑组织损伤可能与caspase-3表达增加、脑组织细胞凋亡增加有关。

低氧预适应对大鼠脑缺血再灌注神经细胞凋亡及Bcl-2、Bax表达的影响

目的 :探讨低氧预适应对大鼠大脑中动脉 (MCA)缺血再灌注神经细胞凋亡 ,Bcl 2、Bax的表达及行为学计分的影响。方法 :成年Wistar大鼠 75只 ,随机分为 3组 :假手术组 ,缺血再灌注组 (对照组 )和低氧预适应组。采用动脉线栓法制作大鼠脑缺血再灌注模型 ,假手术组除不插线外 ,其余同缺血再灌注组 ,低氧预适应组在低氧预适应后 12h制作脑缺血再灌注模型 ,低氧预适应后在大鼠MCA缺血 3h再灌注 1h、6h、12h、2 4h、4 8h不同时间点 ,进行脑切片的TUNEL染色 ,Bcl 2、Bax的免疫组化检测及大鼠的行为学计分。结果 :低氧预适应大鼠的神经细胞凋亡、Bax的表达和行为学计分均较对照大鼠减少 ,Bcl 2的表达较对照组增高 ,差异均有统计学意义 (P <0 .0 5 )。结论 :低氧预适应的脑保护及改善神经功能的作用与其抑制神经细胞的凋亡有关 ;而增加Bcl 2及减少Bax的表达 ,可能是实现保护作用、改善大鼠神经功能的机制之一。

低氧预处理大鼠缺血再灌注脑组织缺氧诱导因子-1α的表达

目的 :探讨低氧预处理对脑缺血再灌注组织缺氧诱导因子 - 1α表达的影响。方法 :SD大鼠 80只随机分为 3组 ,即假手术组 10只 ,缺血再灌注组 35只 ,低氧预处理组 35只。低氧预处理组连续吸入V(O2 ) :V(N2 ) =8:923h作低氧预处理 ,12h后再经插线左大脑中动脉栓塞 (MCAO)作缺血再灌注模型 ,缺血再灌注组不行低氧预处理 ,在大鼠大脑中动脉缺血 3h再灌注后 2h ,6h ,12h ,2 4h ,4 8hTTC法测定脑梗死体积 ,同时采用免疫组织化学方法观察低氧预处理对缺血再灌注大鼠脑组织缺氧诱导因子 - 1α蛋白表达的影响。结果 :与缺血再灌组相比 ,低氧预处理组大鼠的脑梗死体积明显降低 ,缺氧诱导因子 - 1α蛋白的表达增加 ,差异有统计学意义 (P <0 .0 5 )。结论 :低氧预处理对脑缺血再灌注有明显的保护作用 ,缺氧诱导因子 -

低氧预适应对大鼠脑缺血再灌注Bcl-2、Bax mRNA及其蛋白表达的影响

目的 探讨低氧预适应 (8%O2 ,3h)对大鼠大脑中动脉缺血 3h再灌注后不同时间脑梗死体积、神经细胞凋亡及Bcl 2、BaxmRNA及其蛋白表达的影响。方法 低氧预适应后 12h制作脑缺血再灌注模型。在大鼠大脑中动脉缺血 3h再灌注后不同时间进行脑梗死体积测定 (TTC)和原位细胞凋亡检测 (TUNEL) ,通过Northernblot检测脑组织中Bcl 2、BaxmRNA的表达以及免疫组化检测Bcl 2、Bax蛋白的表达。结果 低氧预适应组的脑梗死体积、神经细胞凋亡、Bax的表达较缺血再灌注组明显减少 ;Bcl 2的表达明显增高。结论 低氧预适应可减少大脑中动脉缺血 3h再灌注后梗死体积 ,增强Bcl 2及减少Bax的表达 ,抑制神经细胞的凋亡可能是其脑保护机制之一。

低氧预适应对大鼠脑缺血再灌注神经细胞凋亡和Caspase-3活性的影响

目的探讨低氧预适应(8%O2,3h)对大鼠大脑中动脉缺血2h再灌注后12h神经细胞凋亡及Caspase-3活性的影响。方法低氧预适应后12h制作脑缺血再灌注模型,大鼠大脑中动脉阻塞2h,灌注损伤12h,用TUNEL法和荧光四肽底物法分别检测假手术组、缺血再灌注组和低氧预适应组的凋亡细胞百分率和Caspase-3活性水平。结果与缺血再灌注组比较,低氧预适应组和假手术组的凋亡细胞百分率和Caspase-3活性水平显著降低(P<0.05,P<0.01);低氧预适应组与假手术组比较,细胞凋亡率明显升高(P<0.01),Caspase-3水平反而降低(P<0.01)。结论低氧预适应可减少大脑中动脉缺血2h再灌注后Caspase-3活性,抑制神经细胞的凋亡。

新生大鼠缺氧缺血再灌注后海马神经元凋亡与c-Jun蛋白的表达检测

目的检测新生大鼠缺氧缺血再灌注后即刻早期基因c-jun的表达与海马神经元的凋亡。方法49只7d龄SD大鼠经弹性管穿线阻断右颈总动脉3h,予低氧1h,制备缺氧缺血脑损伤(HIBD)模型,并于再灌注3h、6h、12h、24h、48h、96h、7d处死大鼠,取脑组织。采用免疫组织化学SABC法检测海马CA1、CA3区c-Jun蛋白的表达。采用TUNEL染色法计数海马CA1、CA3区凋亡神经元。8只7d龄SD大鼠仅手术不行HIBD及再灌注(对照组)。结果海马CA1、CA3区c-Jun的表达和凋亡细胞数于HIBD再灌注3h、6h、12h、24h、48h、96h均高于对照组(P<0.05),c-Jun的表达于再灌注6h达高峰,锥体神经元凋亡于再灌注24h达高峰。再灌注7d组c-Jun蛋白的表达及凋亡细胞数与对照组相比差异均无统计学意义。结论c-Jun可能参与轻度HIBD后神经元的凋亡与修复。

缺氧缺血性脑损伤鼠半胱氨酸天冬氨酸蛋白酶-1的表达及其意义

目的 研究新生鼠缺氧缺血性脑损伤 (HIBD)后半胱氨酸天冬氨酸蛋白酶 1(caspase 1)m RNA的表达及其意义。方法 将 112只新生 7d Wistar大鼠随机分为假手术对照组以及 HIBD后 3、8、2 4 h以及 3、6和 14 d组 ,每组 16只。其中 8只应用逆转录聚合酶链反应 (RT- PCR)检测病变侧脑皮质caspase- 1m RNA的表达情况 ;另外 8只进行脑组织苏木素伊红 (HE)染色 ,在光镜下观察脑组织病理变化。结果 假手术对照组中有少量 caspase- 1m RNA表达 ,HIBD 2 4 h组其表达水平开始增加 (与假手术对照组比较 P <0 .0 1) ,6 d达高峰 (与其余各组比较 P均 <0 .0 1) ,14 d时其表达下降 ,但仍高于假手术对照组(P<0 .0 1)。组织病理学检查发现 ,HIBD后 2 4 h～ 6 d神经元大量变性、坏死、丢失 ,同时胶质细胞显著增生。结论 HIBD后 caspase 1m RNA的表达增加 ,其变化规律与光镜观察到的脑损伤进展时间完全吻合 ,提示caspase -1可能参与了新生鼠 HIBD的病理损伤过程。

低氧预适应大鼠皮层缺氧诱导因子-1α的表达及其对细胞凋亡的调控作用

目的探讨低氧预适应对大鼠局灶脑缺血再灌注的脑保护作用及缺氧诱导因子(hypoxic inducible fac-tor,HIF-1)表达对神经细胞凋亡的影响。方法36只大鼠随机分为假手术组、缺血再灌注(I/R)组、低氧预适应(HP+I/R)组。用线栓法建立大鼠局灶脑缺血-再灌注模型,脑缺血前12h将HP+I/R组大鼠放在8%低氧舱中完成低氧预适应。分别用免疫组化、原位末端标记法检测HIF-1α的表达和神经元凋亡。结果与假手术组比较,低氧预适应组和缺血再灌注组的HIF-1α表达水平和凋亡细胞阳性数显著升高(P<0.01,P<0.01);低氧预适应组与缺血再灌注组比较,HIF-1α表达水平明显升高(P<0.01),细胞凋亡阳性细胞降低(P<0.01)。结论低氧预适应可能通过显著增加神经元中HIF-1α表达而抑制神经细胞的凋亡,从而发挥脑保护作用。

Mesenchymal stem cells transplantation attenuates experimentally induced brain injury after neonatal hypoxia by different two routes of administrations

The neonatal hypoxic-ischemic encephalopathy(HIE)is an important cause of neurological morbidity and mortality in neonates.Cell therapy is considered a promising method for treating severe neurological disorders such as this one.Stem cells have the capacity for self-renewal and differentiation into certain cell lineages.The present study was aimed to find out the most beneficial route of bone marrow-derived mesenchymal stem cells(BMSCs)administration for the attenuation of experimentally induced HIE in neonatal rats.Sixty neonatal rats were divided randomly into four groups.Group 1:control group.Group 2:rats were exposed to bilateral ligation of cephalic arteries.Group 3:rats were exposed to bilateral ligation of cephalic arteries and then underwent intravenous(IV)BMSC injection.Group 4:rats were exposed to bilateral ligation of cephalic arteries and then underwent intracerebroventricular(ICV)BMSC injection.The animals were evaluated by(a)neurobehavioral tests;(b)histopathology,i.e.,histological and immuno-histochemical studies;and(3)gene expression studies.The BMSC treated groups(3 and 4)showed improvement in neurobehavioral tests,histopathological studies,and gene expression,as compared to non-injected lesioned rats(Group 2)with better improvement in Group 4(ICV injections)than in Group 3(IV injections).

新生大鼠脑缺氧缺血模型心肌细胞凋亡的研究

目的 研究新生大鼠缺氧缺血性脑损伤 (HIBD)发生后不同时段心肌细胞凋亡情况。方法 结扎新生 7日龄大鼠右颈总动脉后放入 8%低氧舱 2h制成HIBD模型 ,应用AnnexinV/PI双染流式细胞技术检测缺氧缺血 (HI)后 1、4、18、2 4、4 8、72h及 7d心肌细胞凋亡情况。结果 HI后各HI组心肌细胞的凋亡率均明显高于对照组 (P <0 .0 5 ) ,72hHI组凋亡率又显著高于其他组 (P <0 .0 5 ) ,表明HI后 7d内各组大鼠心肌均存在超出正常凋亡 ,尤以 72hHI组凋亡率最高。结论 新生大鼠HIBD后 1h～ 7d内心肌细胞均存在明显凋亡 ,以HI后 72h更明显 ,提示对缺氧缺血性脑病新生儿保护心肌减少心肌凋亡的治疗应至少用至生后

新生鼠缺氧缺血性脑损伤凋亡基因的变化及神经节苷酯的治疗作用

【目的】探讨新生大鼠缺氧缺血性脑损伤(hypoxic-ischemiabraindamage,HIBD)后Bcl-2、Bax基因表达与细胞凋亡的关系及神经节苷酯(gangliosldes,GM1)对其的影响。【方法】建立新生鼠HIBD动物模型,用TUEL法和免疫组化法观察缺氧缺血和GM1l干预后不同时间点细胞凋亡情况及凋亡基因Bcl一2、Bax的变化。【结果】缺氧缺血组随时间延长脑组织中Bcl一2、Bax的表达增加,Bcl一2／Bax的比值下降,同时凋亡细胞数增加,表明Bcl一2、Bax两者比值的降低促进凋亡的发生。GM1干预组Bcl-2表达增加显著,降低了Bax的表达,凋亡细胞减少。【结论】GM1可能通过改变凋亡基因的表达而抑制神经细胞的凋亡过程。

血糖水平对缺氧缺血新生大鼠脑内GLUT3合成的影响

目的探讨血糖水平对缺氧缺血新生大鼠脑内葡萄糖转运蛋白3( glucosetransporter3,GLUT3)合成的影响。方法在成功建立了缺氧缺血合并高、低血糖新生大鼠模型的基础上 ,应用免疫组化方法定量检测新生大鼠脑内海马和皮质部位GLUT3的合成。结果正常情况下GLUT3随日龄增加而合成增加 ;缺氧缺血可引起GLUT3合成在短期内明显增加 ,但在HI后期降至较低的水平 ;HI前低血糖使GLUT3合成在HI后期更进一步降低 ;HI前重度高血糖则可引起各时段GLUT3合成明显增加 ,尤其在HI后期仍保持一定的水平。结论在缺氧缺血前预先补充足量葡萄糖 ,有可能在一定程度上提高脑内应对缺氧缺血的侵袭、改善缺氧缺血程度的能力。

Delta阿片受体参与低氧预处理减轻心肺复苏脑损伤的研究

目的研究delta阿片受体(DOR)是否参与了低氧预处理(HPC)对心肺复苏大鼠脑损伤的保护作用。方法 90只大鼠接受为期7d的HPC后,建立大鼠窒息性心肺复苏脑损伤模型,随机均分为五组:心跳停止(CA)组(A组)、HPC+CA组(B组)、Naltrindole(NTI,DOR特异性拮抗药)+CA组(C组)、HPC+人工脑脊液(ACSF)+CA组(D组)及HPC+NTI+CA组(E组)。A组仅建立心肺复苏模型;C组大鼠在窒息前30min经侧脑室注入含50nmolNTI的ACSF10μl;B、D、E组均接受连续7d的HPC,HPC结束后24h,B组大鼠接受气管插管、8min窒息以及心肺复苏;而D组和E组大鼠则在窒息前30min分别经侧脑室注入含0或50nmolNTI的ACSF10μl。观察复苏成功率并对存活大鼠的神经功能缺损进行评分,观察复苏后海马CA1区神经元损伤及凋亡情况。结果 HPC改善心肺复苏脑损伤后的神经功能缺损,抑制早期海马神经元的凋亡,增加海马CA1区存活神经元的数量,而DOR拮抗药Naltrindole明显消除了HPC的神经保护作用。结论在HPC减轻心肺复苏大鼠脑损伤的过程中,DOR发挥着重要作用。

脑血通颗粒对脑缺血缺氧的实验研究

目的 :探讨脑血通颗粒对脑缺氧的保护作用。方法 :采用断头缺血 ,耐常压缺氧及微循环测验 ,测定脑缺血小鼠呼吸次数 ,维持时间 ,测定脑组织中 L DH,CK,ATP酶含量 ,观察脑缺血时对能量代谢的影响。结果 :脑血通 6 .5 g/kg,13.2g/kg和 2 6 .0 g/kg,ig可明显延长小鼠急性脑缺血、断头喘气时间和喘气次数 ,使小动脉血管口径增大 ,V/A血管口径比减少 ,血流速度明显加快 ,明显提高 L DH和 CK活力 ,2 6 .0 g/kg可延长小鼠耐常压缺氧时间 ,提高脑组织中 ATP酶含量。结论 :脑血通具有改善微循环 ,降低耗氧 ,增加供氧量 ,促进脑缺血后的能量代谢 ,对急性脑缺血缺氧具有较好保护作用。