EFFECT OF MILD HYPOTHERMIA ON ACTIVITY NITRIC OXIDE SYNTHASE IN CORTICAL NEURONS AND GLYCEMIA LEVELS OF NEONATAL RATS WITH HYPOXIC ISCHEMIC BRAIN DAMAGE

Through investigating the effect of mild hypothermia on activity of nitric oxide snythase (NOS) in cortical neurons and glycemia levels of neonatal rats with hypoxic ischemic brain damage (HIBD). We studied the mechanism of protecting hypoxic ischemic neurons of mild hypothermia. We established neonatal rat HIBD models, used NOS immunohistochemistry and glycemia determination by micromethod. The number of cortical NOS positive neurons after hypoxic ischemia was significantly decreased as compared with controls. The glycemia levels was significantly increased than that controls. No significant difference was found in number of cortical NOS positive neurons and glycemia levels between 31℃ and 34℃ mild hypothemia. The results imply that hypothermia can decrease overproduction of NO through inhibiting the increase of the activity of NOS, and increase the glycemia levels, thus protect the hypoxic ischemic neurons.

Effects of mild hypothermia combined EPO therapy on cerebral injury, myocardial injury and oxidative stress of neonatal hypoxic ischemic encephalopathy

Objective:To explore the effects of mild hypothermia combined EPO therapy on cerebral injury, myocardial injury and oxidative stress of neonatal hypoxic ischemic encephalopathy. Methods: A total of 72 children with HIE who were diagnosed and treated in the hospital between December 2015 and June 2017 were chosen as the study subjects and divided into control group (n=36) and EPO group (n=36) by random number table method. Control group received mild hypothermia therapy on the basis of conventional therapy, and EPO group received EPO therapy on the basis of the therapy for control group. The differences in serum levels of cerebral injury indexes, myocardial injury indexes and oxidative stress indexes were compared between the two groups before and after treatment.Results: The differences in serum levels of cerebral injury indexes, myocardial injury indexes and oxidative stress indexes were not statistically significant between the two groups before treatment. After the treatment ended, serum cerebral injury indexes VILIP-1, NPY and NSE levels of EPO group were lower than those of control group whereas IGF-1 level was higher than that of control group;myocardial injury indexes CT-1, Myo and cTnⅠ levels were lower than those of control group;oxidative stress indexes GSH-Px and SOD levels were higher than those of control group whereas AOPP and ROS levels were lower than those of control group.Conclusion: Mild hypothermia combined with EPO therapy can improve the cerebral injury, myocardial injury and oxidative stress of neonatal hypoxic ischemic encephalopathy.

Resatorvid protects against hypoxic-ischemic brain damage in neonatal rats

Secondary brain damage caused by hyperactivation of autophagy and inflammatory responses in neurons plays an important role in hypoxic-ischemic brain damage(HIBD).Although previous studies have implicated Toll-like receptor 4(TLR4)and nuclear factor kappa-B(NF-κB)in the neuroinflammatory response elicited by brain injury,the role and mechanisms of the TLR4-mediated autophagy signaling pathway in neonatal HIBD are still unclear.We hypothesized that this pathway can regulate brain damage by modulating neuron autophagy and neuroinflammation in neonatal rats with HIBD.Hence,we established a neonatal HIBD rat model using the Rice-Vannucci method,and injected 0.75,1.5,or 3 mg/kg of the TLR4 inhibitor resatorvid(TAK-242)30 minutes after hypoxic ischemia.Our results indicate that administering TAK-242 to neonatal rats after HIBD could significantly reduce the infarct volume and the extent of cerebral edema,alleviate neuronal damage and neurobehavioral impairment,and decrease the expression levels of TLR4,phospho-NF-κB p65,Beclin-1,microtubule-associated protein l light chain 3,tumor necrosis factor-α,and interleukin-1βin the hippocampus.Thus,TAK-242 appears to exert a neuroprotective effect after HIBD by inhibiting activation of autophagy and the release of inflammatory cytokines via inhibition of the TLR4/NF-κB signaling pathway.This study was approved by the Laboratory Animal Ethics Committee of Affiliated Hospital of Yangzhou University,China(approval No.20180114-15)on January 14,2018.

Hyperbaric oxygen treatment promotes neural stem cell proliferation in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage

Hyperbaric oxygen therapy for the treatment of neonatal hypoxic-ischemic brain damage has been used clinically for many years, but its effectiveness remains controversial. In addition, the mechanism of this potential neuroprotective effect remains unclear. This study aimed to investigate the influence of hyperbaric oxygen on the proliferation of neural stem cells in the subventricular zone of neonatal Sprague-Dawley rats （7 days old） subjected to hypoxic-ischemic brain damage. Six hours after modeling, rats were treated with hyperbaric oxygen once daily for 7 days. Immunohistochemistry revealed that the number of 5-bromo-2＇-deoxyuridine positive and nestin positive cells in the subventricular zone of neonatal rats increased at day 3 after hypoxic-ischemic brain damage and peaked at day 5. After hyperbaric oxygen treatment, the number of 5-bromo-2＇- deoxyuddine positive and nestin positive cells began to increase at day 1, and was significantly higher than that in normal rats and model rats until day 21. Hematoxylin-eosin staining showed that hyperbaric oxygen treatment could attenuate pathological changes to brain tissue in neonatal rats, and reduce the number of degenerating and necrotic nerve cells. Our experimental findings indicate that hyperbaric oxygen treatment enhances the proliferation of neural stem cells in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage, and has therapeutic potential for promoting neurological recovery following brain injury.

Effects of ephedrine on expression of Nogo-A and synaptophysin in neonatal rats following hypoxic-ischemic brain damage

BACKGROUND： Central nervous system axons regenerate poorly following neonatal hypoxic-ischemic brain damage （HIBD）, partly due to inhibitors, such as Nogo-A. Very few studies have addressed the regulation of Nogo-A in neonatal rats following HIBD. However, numerous studies have shown that ephedrine accelerates neuronal remodeling and promotes recovery of neural function in neonatal rats following HIBD. OBJECTIVE： To investigate the effects of ephedrine on expression of Nogo-A and synaptophysin in brain tissues of neonatal rats following HIBD. DESIGN, TIME AND SETTING： A completely randomized, controlled study was performed at the Immunohistochemistry Laboratory of the Research Institute of Pediatrics, Children＇s Hospital of Chongqing Medical University from August 2008 to March 2009. MATERIALS： Ephedrine hydrochloride （Chifeng Pharmaceutical Group, China）, rabbit anti-Nogo-A polyclonal antibody （Abcam, UK）, and rabbit anti-synaptophysin polyclonal antibody （Lab Vision, USA） were used in this study. METHODS： A total of 96 healthy, neonatal, Sprague Dawley rats were randomly assigned to three groups （n = 32）： sham operation, HIBD, and ephedrine. The HIBD model was established by permanent occlusion of the left common carotid artery, followed by 2 hours of hypoxia （8% oxygen and 92% nitrogen）. In the sham operation group, the left common carotid artery was exposed, but was not ligated or subjected to hypoxia. Rats in the ephedrine group were intraperitoneally injected with ephedrine immediately following HIBD, with 1.5 mg/kg each time. Rats in the sham operation and HIBD groups were injected with an equal volume of saline. All neonatal rats were treated once daily for 7 days. MAIN OUTCOME MEASURES： Histopathological damage to the cortex and hippocampus was determined by hematoxylin-eosin staining. Expression of Nogo-A and synaptophysin was detected using immunohistochemical staining. RESULTS： Neuronal degeneration and edema were observed in the hypoxJc-Jschemic cortex and hippocampus by hematoxylin-eosin staining. Compared with the sham operation group, the levels of Nogo-A significantly increased in the HIBD group at various time points （P 〈 0.01）. Nogo-A expression was significantly reduced in the ephedrine group compared with the HIBD group （P 〈 0.01）. Synaptophysin expression was significantly decreased in the hypoxic-ischemJc cortex, compared with the sham operation group （P 〈 0.01）. Synaptophysin levels were significantly increased in the ephedrine group, compared with the HIBD group （P 〈 0.01）. CONCLUSION： Altered Nogo-A expression was associated with inversely altered synaptophysin expression. The use of ephedrine normalized expression levels of Nogo-A and synaptophysin following HIBD.

Estrogen inhibits lipid peroxidation after hypoxic-ischemic brain damage in neonatal rats

Sprague-Dawley neonatal rats within 7 days after birth were used in this study. The left common carotid artery was occluded and rats were housed in an 8% O2 environment for 2 hours to establish a hypoxic-ischemic brain damage model. 17β-estradiol （1 × 10-5 M） was injected into the rat abdominal cavity after the model was successfully established. The left hemisphere was obtained at 12, 24, 48, 72 hours after operation. Results showed that malondialdehyde content in the left brain of neonatal rats gradually increased as modeling time prolonged, while malondialdehyde content of 17β-estrodial-treated rats significantly declined by 24 hours, reached lowest levels at 48 hours, and then peaked at 72 hours after injury. Nicotinamide-adenine dinucleotide phosphate histochemical staining showed the nitric oxide synthase-positive cells and fibers dyed blue/violet and were mainly distributed in the cortex, hippocampus and medial septal nuclei. The number of nitric oxide synthase-positive cells peaked at 48 hours and significantly decreased after 17β-estrodial treatment. Our experimental findings indicate that estrogen plays a protective role following hypoxic-ischemic brain damage by alleviating lipid peroxidation through reducing the expression of nitric oxide synthase and the content of malondialdehyde.

Environmental enrichment promotes neural remodeling in newborn rats with hypoxic-ischemic brain damage

We evaluated the effect of hypoxic-ischemic brain damage and treatment with early environmental enrichment intervention on development of newborn rats, as evaluated by light and electron microscopy and morphometry. Early intervention with environmental enrichment intelligence training attenuated brain edema and neuronal injury, promoted neuronal repair, and increased neuronal plasticity in the frontal lobe cortex of the newborn rats with hypoxic-ischemic brain damage.

Flunarizine and lamotrigine prophylaxis effects on neuron-specific enolase, S-100, and brain-specific creatine kinase in a fetal rat model of hypoxic-ischemic brain damage

BACKGROUND： Calcium antagonists may act as neuroprotectants, diminishing the influx of calcium ions through voltage-sensitive calcium channels. When administered prophylactically, they display neuroprotective effects against hypoxic-ischemic brain damage in newborn rats. OBJECTIVE： To investigate the neuroprotective effects of flunarizine （FNZ）, lamotrigine （LTG） and the combination of both drugs, on hypoxic-ischemic brain damage in fetal rats. DESIGN AND SETTING： This randomized, complete block design was performed at the Department of Pediatrics, Shenzhen Fourth People＇s Hospital, Guangdong Medical College. MATERIALS： Forty pregnant Wistar rats, at gestational day 20, were selected for the experiment and were randomly divided into FNZ, LTG, FNZ ＋ LTG, and model groups, with 10 rats in each group. METHODS： Rats in the FNZ, LTG, and FNZ ＋ LTG groups received intragastric injections of FNZ （0.5 mg/kg/d）, LTG （10 mg/kg/d）, and FNZ （0.5 mg/kg/d） ＋ LTG （10 mg/kg/d）, respectively. Drugs were administered once a day for 3 days prior to induction of hypoxia-ischemia. Rats in the model group were not administered any drugs. Three hours after the final administration, eight pregnant rats from each group underwent model establishment hypoxia-ischemia brain damage to the fetal rats. Cesareans were performed at 6, 12, 24, and 48 hours later; and 5 fetal rats were removed from each mother and kept warm. Two fetuses without model establishment were removed by planned cesarean at the same time and served as controls. A total of 0.3 mL serum was collected from fetal rats at 6, 12, 24, and 48 hours, respectively, following birth. MAIN OUTCOME MEASURES： Serum protein concentrations of neuron-specific enolase and S-100 were measured by ELISA. Serum concentrations of brain-specific creatine kinase were measured using an electrogenerated chemiluminescence method. RESULTS： Serum concentrations of neuron-specific enolase, S-100, and brain-specific creatine kinase were significantly higher in the hypoxic-ischemic fetal rats, compared with the non-hypoxic-ischemic group. Serum concentrations of neuron-specific enolase, S-100, and brain-specific creatine kinase were significantly less in the FNZ, LTG, and FNZ ＋ LTG groups following ischemia, compared with the model group （P 〈 0.01）. However, these values were significantly greater in the FNZ and LTG groups, compared with the FNZ ＋ LTG group, following ischemia （P 〈 0.01）. CONCLUSION： Preventive antenatal use of oral FNZ and LTG has positive neuroprotective effects on intrauterine hypoxic-ischemic brain damage. The combined effect of these two drugs is superior.

660 nm red light-enhanced bone marrow mesenchymal stem cell transplantation for hypoxic-ischemic brain damage treatment

Bone marrow mesenchymal stem cell transplantation is an effective treatment for neonatal hy- poxic-ischemic brain damage. However, the in vivo transplantation effects are poor and their survival, colonization and differentiation efficiencies are relatively low. Red or near-infrared light from 600-1,000 nm promotes cellular migration and prevents apoptosis. Thus, we hypothesized that the combination of red light with bone marrow mesenchymal stem cell transplantation would be effective for the treatment of hypoxic-ischemic brain damage. In this study, the migra- tion and colonization of cultured bone marrow mesenchymal stem cells on primary neurons after oxygen-glucose deprivation were detected using Transwell assay. The results showed that, after a 40-hour irradiation under red light-emitting diodes at 660 nm and 60 mW/cmz, an increasing number of green fluorescence-labeled bone marrow mesenchymal stem cells migrated towards hypoxic-ischemic damaged primary neurons. Meanwhile, neonatal rats with hypoxic-ischemic brain damage were given an intraperitoneal injection of 1 x 106 bone marrow mesenchymal stem cells, followed by irradiation under red light-emitting diodes at 660 nm and 60 mW/cm2 for 7 successive days. Shuttle box test results showed that, after phototherapy and bone marrow mesenchymal stem cell transplantation, the active avoidance response rate of hypoxic-ischemic brain damage rats was significantly increased, which was higher than that after bone marrow mesenchymal stem cell transplantation alone. Experimental findings indicate that 660 nm red light emitting diode irradiation promotes cells, thereby enhancing the contribution ic-ischemic brain damage. the migration of bone marrow mesenchymal stem of cell transplantation in the treatment of hypox-

Negative regulation of miRNA-9 on oligodendrocyte lineage gene 1 during hypoxic-ischemic brain damage

Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair, miRNA-9 is involved in the occurrence of many related neurological disorders. Bioin- formatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups： control group; oxygen-glucose deprivation group （treatment with 8% O2 ＋ 92% N2 and sugar-free medium for 60 minutes）; transfection control group （after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid） and miRNA-9 transfection group （after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid）. From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligoden- drocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.

Neuroprotective effects of ginsenoside Rg1-induced neural stem cell transplantation on hypoxic-ischemic encephalopathy

Ginsenoside Rgl is the major pharmacologically active component of ginseng, and is reported to have various therapeutic actions. To determine whether it induces the differentiation of neural stem cells, and whether neural stem cell transplantation after induction has therapeutic effects on hypoxic-ischemic encephalopathy, we cultured neural stem cells in 10-80 ~tM ginsenoside Rgl. Immunohistochemistry revealed that of the concentrations tested, 20 mM ginsenoside Rgl had the greatest differentiation-inducing effect and was the concentration used for subsequent exper- iments. Whole-cell patch clamp showed that neural stem cells induced by 20 jaM ginsenoside Rgl were more mature than non-induced cells. We then established neonatal rat models of hypox- ic-ischemic encephalopathy using the suture method, and ginsenoside Rgl-induced neural stem cells were transplanted via intracerebroventricular injection. These tests confirmed that neural stem cells induced by ginsenoside had fewer pathological lesions and had a significantly better behavioral capacity than model rats that received saline. Transplanted neural stem cells expressed neuron-specific enolase, and were mainly distributed in the hippocampus and cerebral cortex. The present data suggest that ginsenoside Rgl-induced neural stem cells can promote the partial recovery of complicated brain functions in models of hypoxic-ischemic encephalopathy.

Human insulin-like growth factor 1-transfected umbilical cord blood neural stem cell transplantation improves hypoxic-ischemic brain injury

Human insulin-like growth factor 1-transfected umbilical cord blood neural stem cells were transplanted into a hypoxic-ischemic neonatal rat model via the tail vein. BrdU-positive cells at day 7 post-transplantation, as well as nestin- and neuron specific enolase-positive cells at day 14 were increased compared with those of the single neural stem cell transplantation group. In addition, the proportion of neuronal differentiation was enhanced. The genetically modified cell-transplanted rats exhibited enhanced performance in correctly crossing a Y-maze and climbing an angled slope compared with those of the single neural stem cell transplantation group. These results showed that human insulin-like growth factor 1-transfected neural stem cell transplantation promotes the recovery of the leaming, memory and motor functions in hypoxic-ischemic rats.

水苏碱调节Hippo-YAP信号通路对新生大鼠缺氧缺血性脑损伤的神经保护作用

目的探究水苏碱(stachydrine,STA)对缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)新生大鼠的神经保护作用,并分析其作用机制。方法将新生SD大鼠随机分为假手术组、HIBD组、STA低剂量(5 mg/kg)组、STA中剂量(10 mg/kg)组、STA高剂量(20 mg/kg)组、维替泊芬(10 mg/kg)+STA高剂量(20 mg/kg)组,除假手术组外,其余大鼠构建HIBD大鼠模型。对各组大鼠进行神经功能缺损评分,采用Morris水迷宫实验进行认知功能评价,测定各组大鼠脑含水量和脑指数,HE染色、尼氏染色观察脑组织神经元损伤,TUNEL染色观察脑组织神经元细胞凋亡情况,Western blot法检测YES相关蛋白(YES associated protein,YAP)、p-YAP、哺乳动物STE20样蛋白激酶1(mammalian sterile 20-like kinase 1,MST1)、p-MST1、具有PDZ基序的转录共激活因子(transcriptional coactivator with PDZ-binding motif,TAZ)蛋白表达。结果与假手术组比较,HIBD组海马组织损伤加重,尼氏小体减少(P<0.05),神经功能缺损评分、逃避潜伏期、脑组织含水量、脑指数、神经元细胞凋亡率、p-YAP/YAP比值、p-MST1/MST1比值显著增加(P<0.05),穿越平台次数、TAZ表达显著降低(P<0.05);与HIBD组相比,STA低、中、高剂量组海马组织损伤改善,尼氏小体增加(P<0.05),神经功能缺损评分、逃避潜伏期、脑组织含水量、脑指数、神经元细胞凋亡率、p-YAP/YAP比值、p-MST1/MST1比值显著降低(P<0.05),穿越平台次数、TAZ表达显著增加(P<0.05);与STA高剂量组相比,维替泊芬+STA高剂量组海马组织损伤加重,尼氏小体减少(P<0.05),神经功能缺损评分、逃避潜伏期、脑组织含水量、脑指数、神经元细胞凋亡率、p-YAP/YAP比值、p-MST1/MST1比值显著增加(P<0.05),穿越平台次数、TAZ表达显著降低(P<0.05)。结论STA可能通过调控Hippo-YAP信号通路、减少神经损伤和神经元细胞凋亡、改善神经功能,发挥对HIBD新生大鼠的神经保护作用。

姜黄素改善缺氧缺血性脑损伤新生大鼠远期神经行为缺陷的机制

目的探讨姜黄素减轻缺氧缺血性脑损伤新生大鼠相关神经炎性反应的作用机制并且评估其对缺氧缺血性脑损伤远期神经行为的影响。方法2023年1—6月在武汉大学人民医院中心实验室进行实验。选用7日龄新生SD大鼠66只,采用随机数字表法分为假手术组(n=22)、缺氧缺血脑损伤组(HIBD组,n=22)和缺氧缺血脑损伤+姜黄素组(HIBD+姜黄素组,n=22)。造模后24 h采用Longa评分评估造模是否成功,造模后48 h通过负性趋地反射、翻正反射评估短期神经行为;4周后采用圆筒实验、水迷宫实验、转角实验、爬杆实验评估远期神经行为;采用ELISA试剂盒检测脑组织炎性因子(TNF-α、IL-1β、IL-6)水平;采用Western-blot法检测脑组织蛋白(STAT3、p-STAT3)水平。结果造模24 h后,HIBD组(n=18)与HIBD+姜黄素组(n=15)新生大鼠Longa评分为1~3分提示造模成功。短期行为学评估表明,与HIBD组比较,HIBD+姜黄素组负性趋地反射和翻正反射时间缩短(P<0.05)。远期神经行为学评估表明,与HIBD组比较,HIBD+姜黄素组圆筒实验大鼠左前肢使用率明显增加、水迷宫实验穿越平台潜伏期缩短、穿越平台象限次数增加、转角实验右转得分率减少、爬杆实验中爬杆时间缩短(P<0.01)。ELISA检测显示,与HIBD组比较,HIBD+姜黄素组脑组织中TNF-α、IL-1β和IL-6水平下降(P<0.05)。蛋白免疫印迹技术显示,与HIBD组比较,HIBD+姜黄素组脑组织中p-STAT3/STAT3水平降低(P<0.01)。结论姜黄素可通过抑制STAT3磷酸化介导的神经炎性反应并改善缺氧缺血性脑损伤新生大鼠神经行为学缺陷。

新生儿缺氧缺血性脑损伤程序性细胞死亡及康复治疗干预机制的研究进展

新生儿缺氧缺血性脑损伤(HIBD)是一种脑细胞缺血缺氧性坏死病变,可导致神经发育性残疾,降低儿童生存质量,给患儿家庭及社会带来沉重负担。程序性细胞死亡(PCD)作为一种重要的神经元细胞死亡调控机制,参与HIBD的发生发展。康复治疗是改善HIBD后功能障碍的主要干预方式,临床应用广泛,但其效应机制目前尚未完全阐明。康复治疗可调控PCD,是促进HIBD后功能恢复的关键。因此,深入研究PCD特别是凋亡、自噬、焦亡、铁死亡在HIBD中的作用机制及康复干预机制,可以为疾病的治疗提供新思路。

茶黄素通过抑制细胞过度自噬减轻新生大鼠缺氧缺血性脑损伤

目的基于沉默信息调节因子2相关酶1(SIRT1)/叉头盒蛋白O1(FoxO1)信号通路探讨茶黄素(Theaflavins,TFs)在新生大鼠缺氧缺血性脑损伤(HIBD)中的作用及机制。方法将7日龄Sprague-Dawley(SD)大鼠随机分为假手术组(sham组)、模型组(HIBD组)、茶黄素组(TFs+HIBD组)、SIRT1抑制剂组(TFs+HIBD+Ex-527组),每组40只。假手术组仅游离右侧颈总动脉,不进行缺血缺氧处理;模型组采用改良Rice-Vannucci法,游离并结扎右侧颈总动脉后缺氧制成HIBD模型;茶黄素组和SIRT1抑制剂组在建立HIBD模型前,给予茶黄素灌胃,SIRT1抑制剂组给予Ex-527侧脑室注射。各组于术后6,12,24,48,72 h对新生大鼠进行Longa评分、一般状态、行为学检测及脑组织称重;苏木素-伊红染色(HE)染色观察脑组织病理学改变;蛋白质印迹法(Western blotting)检测SIRT1、FoxO1、Ac-FoxO1、Beclin1、LC3-Ⅱ/Ⅰ在蛋白质水平的表达。结果与sham组比较,HIBD组新生大鼠精神不振,神经功能缺损评分显著升高(P<0.05),体质量增长迟缓(P<0.01),翻正反射时间明显延长(P<0.05),右侧脑组织质量增大(P<0.05),HE染色可见大脑皮层区神经元数目减少,SIRT1、FoxO1蛋白表达明显降低(P<0.01),Ac-FoxO1及自噬相关蛋白Beclin1、LC3Ⅱ/Ⅰ的表达量升高(P<0.01)。与HIBD组比较,TFs+HIBD组新生大鼠精神状态好转,神经功能缺损评分下降(P<0.05),体质量增长较快(P<0.01),翻正反射时间缩短(P<0.05),右侧脑组织质量减轻(P<0.05),HE染色可见脑组织神经元数目增多,SIRT1、FoxO1蛋白表达升高(P<0.01),Ac-FoxO1及自噬相关蛋白的表达量降低(P<0.01)。与TFs+HIBD组比较,TFs+HIBD+Ex-527组新生大鼠神经功能缺损评分升高(P<0.05),体质量增长迟缓(P<0.01),翻正反射时间延长(P<0.05),右侧脑组织质量增大(P<0.05),HE染色可见脑组织神经元细胞减少,SIRT1、FoxO1蛋白表达降低(P<0.01),Ac-FoxO1及自噬相关蛋白的表达量升高(P<0.01)。结论TFs可通过激活SIRT1/FoxO1信号转导通路抑制HIBD引起的过度细胞自噬,从而减轻神经元细胞的损伤。

褪黑素通过PI3K/AKT通路减轻缺氧缺血性脑损伤新生大鼠大脑皮层神经细胞自噬的研究

目的 观察褪黑素(melatonin,Mel)对缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)新生大鼠大脑皮层神经细胞自噬的影响,并从PI3K/AKT信号通路探讨其机制,为Mel的临床应用提供依据。方法 将7日龄Sprague-Dawley新生大鼠随机分为假手术组、HIBD组及Mel组(各组n=9)。采用经典RiceVannucci法构建新生大鼠HIBD模型。采用苏木精-伊红和尼氏染色法观察新生大鼠大脑皮层神经细胞形态;免疫荧光染色和Western blot法检测自噬相关蛋白微管相关蛋白1轻链3 (microtubule-associated protein 1 light chain3,LC3)及Beclin-1蛋白表达水平;免疫组化和Western blot法检测磷酸化磷脂酰肌醇3-激酶(phosphorylated phosphoinositide 3-kinase,p-PI3K)、磷酸化蛋白激酶B (phosphorylated protein kinase B,p-AKT)蛋白表达水平;通过Pearson相关性分析探讨Mel组与HIBD组自噬与PI3K通路的相关性。结果 建模后24h,假手术组新生大鼠大脑皮层神经细胞大小形态正常、排列规则,HIBD组神经细胞胞体肿胀、排列不规则,Mel组神经细胞排列较规整、形态相对正常。假手术组尼氏小体形态规整,HIBD组尼氏小体形态异常,Mel组尼氏小体形态尚规整。HIBD组LC3及Beclin-1平均荧光强度较假手术组增加,Mel组较HIBD组减少(P<0.05)。HIBD组p-PI3K^(+)、pAKT^(+)细胞数较假手术组减少,Mel组较HIBD组增加(P<0.05)。HIBD组LC3、Beclin-1蛋白表达水平较假手术组增加,p-PI3K、p-AKT蛋白表达水平较假手术组减少(P<0.05);Mel组LC3、Beclin-1蛋白表达水平较HIBD组减少,p-PI3K、p-AKT蛋白表达水平较HIBD组增加(P<0.05)。相关性分析显示,Mel组与HIBD组损伤侧大脑皮层LC3、Beclin-1蛋白平均荧光强度差值与p-PI3K^(+)、p-AKT^(+)细胞数差值均呈负相关(P<0.05)。结论 Mel可抑制HIBD新生大鼠大脑皮层神经细胞过度自噬,从而减轻HIBD,其机制与PI3K/AKT通路相关。

天麻素通过CCR5/JAK1/STAT1信号通路抑制缺血缺氧新生小鼠小胶质细胞介导的炎症反应

目的:探究天麻素(GAS)通过C-C趋化因子受体5(CCR5)对新生小鼠缺血缺氧性脑损伤(HIBD)后小胶质细胞JAK1/STAT1信号通路及其介导的炎症反应的影响。方法:选择新出生10 d的C57BL/6J小鼠48只,随机分为假手术(sham)组、HIBD模型组和HIBD与GAS联合处理(HIBD+GAS)组;体外培养BV-2小胶质细胞,分为对照(Con)组、氧糖剥夺(OGD)组、OGD+GAS组、GAS组、Maraviroc(MVC)组、OGD+MVC组和OGD+MVC+GAS组。通过RT-qPCR检测CCL4和CCR5的mRNA表达变化,Western blot检测CCR5、p-JAK1、p-STAT1、肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)蛋白的表达变化,免疫荧光双标染色检测CCR5、p-JAK1和p-STAT1的表达变化。结果:(1)与sham组相比,HIBD组小鼠缺血侧胼胝体区CCL4和CCR5的mRNA水平,以及CCR5、p-JAK1和p-STAT1的蛋白水平明显增高(P<0.05),而HIBD+GAS组中CCL4和CCR5 mRNA水平,以及CCR5、p-JAK1和p-STAT1的蛋白水平显著低于HIBD组(P<0.05)。(2)与Con组相比,OGD组BV-2细胞中CCR5、p-JAK1和p-STAT1蛋白水平明显增高(P<0.05),而OGD+GAS组BV-2细胞中CCR5、p-JAK1和p-STAT1蛋白水平显著低于OGD组(P<0.05)。(3)CCR5拮抗剂MVC在0~80μmol/L范围内不会导致显著的BV-2细胞死亡。与OGD组相比,MVC+OGD组p-JAK1、p-STAT1、TNF-α和IL-1β蛋白水平显著降低(P<0.05),而MVC+OGD组与OGD+MVC+GAS组无明显差异。结论:GAS可通过靶向CCR5抑制小胶质细胞p-JAK1/p-STAT1通路及相关炎症因子的表达,发挥神经保护作用。

藁本内酯抑制缺氧缺血性脑损伤新生大鼠脑组织凋亡相关蛋白的表达

目的:通过检测体内外缺氧缺血条件下NOD样受体蛋白3(NLRP3)、天冬氨酸蛋白水解酶1(Caspase1)、白细胞介素-1β(IL-1β)、GSDMD-N蛋白水平评价藁本内酯(LGSL)对缺氧缺血性脑损伤(HIBD)新生大鼠的神经保护作用。方法:选取7日龄SD雄性大鼠随机分为假手术组(Sham组)、HIBD组和LGSL组各10只,采用左颈总动脉双重结扎构建HIBD模型,体外建立小胶质细胞氧糖剥夺(OGD)模型(OGD/R)。采用CCK-8评估不同浓度LGSL干预24 h对OGD/R小胶质细胞活力的影响;通过免疫印迹评估20μM LGSL对体内外缺氧缺血条件下NLRP3、Caspase1、IL-1β、GSDMD-N表达的影响。结果:在体内外模型中,NLRP3、IL-1β、Caspase1及GSDMD-N表达均上调(P<0.05);20μM LGSL干预24 h后,NLRP3、IL-1β、Caspase1及GSDMD-N表达均下调(P<0.05)。结论:LGSL可以显著抑制新生大鼠体内外因缺氧缺血引起的神经炎症反应。

Neuroprotective effects of autophagy inhibition on hippocampal glutamate receptor subunits after hypoxia-ischemia-induced brain damage in newborn rats

Autophagy has been suggested to participate in the pathology of hypoxic-ischemic brain damage（HIBD）.However,its regulatory role in HIBD remains unclear and was thus examined here using a rat model.To induce HIBD,the left common carotid artery was ligated in neonatal rats,and the rats were subjected to hypoxia for 2 hours.Some of these rats were intraperitoneally pretreated with the autophagy inhibitor 3-methyladenine（10 m M in 10 μL） or the autophagy stimulator rapamycin（1 g/kg） 1 hour before artery ligation.Our findings demonstrated that hypoxia-ischemia-induced hippocampal injury in neonatal rats was accompanied by increased expression levels of the autophagy-related proteins light chain 3 and Beclin-1 as well as of the AMPA receptor subunit GluR 1,but by reduced expression of GluR 2.Pretreatment with the autophagy inhibitor 3-methyladenine blocked hypoxia-ischemia-induced hippocampal injury,whereas pretreatment with the autophagy stimulator rapamycin significantly augmented hippocampal injury.Additionally,3-methyladenine pretreatment blocked the hypoxia-ischemia-induced upregulation of Glu R1 and downregulation of GluR2 in the hippocampus.By contrast,rapamycin further elevated hippocampal Glu R1 levels and exacerbated decreased GluR2 expression levels in neonates with HIBD.Our results indicate that autophagy inhibition favors the prevention of HIBD in neonatal rats,at least in part,through normalizing Glu R1 and GluR2 expression.