A New Role Discovered for IGTP: The Protective Effect of IGTP in ICH-Induced Neuronal Apoptosis

Interferon gamma-induced GTPase (IGTP), which is also named Irgm3, has been widely described in regulating host resistance against intracellular pathogens. Previous researches have demonstrated that IGTP exerts beneficial function during coxsackievirus B3 (CVB3) infection. However, little information is available regarding the role of IGTP in central nervous system. Here, our study revealed that IGTP may have an essential role during ICH-induced neuronal apoptosis. We found the expression level of IGTP adjacent to hematoma was strongly increased after ICH, accompanied with the up-regulation of proliferating cell nuclear antigen (PCNA), active-caspase-3, p-GSK-3β, and Bax. IGTP was also observed to be co-localized with PCNA in astrocytes and active-caspase-3 in neurons, indicating its association with astrocyte proliferation and neuronal apoptosis after ICH. Finally, in vitro study, knocking down IGTP with IGTP-specific siRNA promoted active-caspase-3, p-GSK-3β, and Bax expression, and led to more severe neuronal apoptosis after ICH. All these results above suggested that IGTP might play a critical role in protecting neurons from apoptosis after ICH.     

Up-Regulation of TAB3 Is Involved in Neuronal Apoptosis After Intracerebral Hemorrhage

Human transforming growth factor β-activated kinase (TAK1)-binding protein 3 (TAB3) is a regulator of NF-κB which has been mainly found in a variety of cancers. While TAB3 is highly expressed in brain tissue, little is known about the function of TAB3 in central nervous system. Our group established an animal ICH model with autologous whole blood injected into brain, and also a cell ICH model with hemin stimulation. Our Western blot result showed up-regulation of TAB3 during neuronal apoptosis in the model of intracerebral hemorrhage (ICH), which was also approved by immunofluorescence and immunohistochemistry result. Besides, increasing TAB3 level was accompanied by the increased expression of active-caspase-3, active-caspase-8, and decreased expression of Bcl-2. Furthermore, in in vitro study, the level of neuronal apoptosis was decreased by applying TAB3- RNA interference in PC12 cells. All the results above suggested that TAB3 probably participates in the process of neuronal apoptosis following ICH.     

Increased Expression of Ubiquitin-Specific Protease 4 Participates in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats

Ubiquitinating enzymes catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action. Ubiquitin-specific protease 4 (USP4) is a member of the ubiquitin-specific protease (USP) family of DUBs that has a role in spliceosome regulation. In the present study, we demonstrated that USP4 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant up-regulation of USP4 in neurons adjacent to the hematoma following ICH by the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing USP4 level was found to be accompanied by the up-regulation of active caspase-3, γH2AX, Bax, and decreased expression of Bcl-2. In addition, USP4 co-localized well with γH2AX in the nucleus in the ICH model and hemin-induced apoptosis model. Moreover, in vitro study, knocking down USP4 by USP4-specific siRNA in PC12 cells reduced active caspase-3 expression. All these results above suggested that USP4 may be involved in neuronal apoptosis after ICH.     

Up-Regulation of Interferon Regulatory Factor 3 Involves in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats

Interferon regulatory factor 3 (IRF3) is a member of IRF family which plays a significant role in the innate immune response, apoptosis, and oncogenesis. Mounting evidence has demonstrated that IRF3 was involved in central nervous system disease such as cerebral ischemic injury through promoting neuronal apoptosis. However, it remains unclear about the underlying mechanisms of IRF3 upon neuronal apoptosis following intracerebral hemorrhage (ICH). In the present study, we established an adult rat ICH model by injecting autologous whole blood into the right basal ganglia and evaluated their neurological deficits by behavioral tests. IRF3 protein level was up-regulated adjacent to the hematoma following ICH when compared with the sham brain cortex by western blot and immunohistochemistry. Immunofluorescent staining indicated IRF3 was mainly localized in neurons, a few in astrocytes. In addition, we also detected that IRF3 co-localized with active caspase-3 which is a neuronal apoptosis marker. Furthermore, in vitro study, knocking down IRF3 by using IRF3 interference in primary cortical neurons reduced the expression of active caspase-3 and Bax while increased Bcl-2. In conclusion, we speculated that IRF3 might exert pro-apoptotic function in neurons after ICH.     

EP3, Prostaglandin E2 Receptor Subtype 3, Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage

EP3 is prostaglandin E2 receptor subtype 3 and mediates the activation of several signaling pathways, changing in cAMP levels, calcium mobilization, and activation of phospholipase C. Previous studies demonstrated a direct role for EP3 in various neurodegenerative disorders, such as stroke and Alzheimer disease. However, the distribution and function of EP3 in ICH diseases remain unknown. Here, we demonstrate that EP3 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). From the results of Western blot and immunohistochemistry, we obtained a significant up-regulation of EP3 in neurons adjacent to the hematoma following ICH. Up-regulation of EP3 was found to be accompanied by the increased expression of active caspase-3 and pro-apoptotic Bcl-2-associated X protein (Bax) and decreased expression of anti-apoptotic protein B cell lymphoma-2 (Bcl-2) in vivo and vitro studies. Furthermore, the expression of these three proteins reduced active caspase-3 and Bax expression, while increased Bcl-2 were changed after knocking down EP3 by RNA interference in PC12 cells, further confirmed that EP3 might exert its pro-apoptotic function on neuronal apoptosis. Thus, EP3 may play a role in promoting the neuronal apoptosis following ICH.     

Hck Promotes Neuronal Apoptosis Following Intracerebral Hemorrhage

The hematopoietic cell kinase (Hck) is a member of the Src family protein kinases which regulates many signal transduction pathways including cell growth, proliferation, differentiation, migration, and apoptosis. However, the expression and function of Hck after intracerebral hemorrhage (ICH) are unknown. Western blot, immunohistochemistry, and immunofluorescence showed that Hck was obviously up-regulation in neurons adjacent to the hematoma after ICH. In addition, the temporary raise of Hck expression was paralleled with the expression of p53, Bax, and active caspase-3, suggesting that Hck was involved in neuronal apoptosis. Hck siRNA dramatically decrease hemin-induced expression of p53, Bax, and active caspase-3 as well as the amount of apoptotic SH-SY5Y cells in vitro. Furthermore, Hck interacted with p53. Hence, Hck might promote neuronal apoptosis via p53 signaling pathway after ICH.     

IDH1 Associated with Neuronal Apoptosis in Adult Rats Brain Following Intracerebral Hemorrhage

Isocitrate dehydrogenase 1 (IDH1), one member of the IDH family can convert isocitrate to α-ketoglutarate (α-KG) via oxidative decarboxylation. IDH1 and IDH2 mutations have been identified in multiple tumor types and the mutations confer neomorphic activity in the mutant protein, resulting in the conversion of α-KG to the oncometabolite, D-2-hydroxyglutarate (2-HG). The subsequent accumulation of 2-HG results in epigenetic dysregulation via inhibition of α-KG-dependent histone and DNA demethylase. And the glutamate levels are reduced in IDH mutant cells compared to wild-type. We have known that diffuse gliomas contain a high frequency of mutations in the IDH1 gene. However, the expression of IDH1 and its roles in Intracranial hemorrhage (ICH) remain largely unknown. We observed increased expression of IDH1 in neurons after intracerebral hemorrhage. Up-regulation of IDH1 was found to be accompanied by the increased expression of active caspase-3 and pro-apoptotic Bcl-2-associated X protein and decreased expression of anti-apoptotic protein B cell lymphoma-2 in vivo and vitro studies. So we hypothesized that IDH1 was involved in the regulation of neuronal apoptosis. The present research for the first time detected the expression and variation of IDH1 surrounding the hematoma, and all data proved the involvement of IDH1 in neuronal apoptosis following ICH.     

Insulin-like Growth Factor Binding Protein6 Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage in Rats

The insulin-like growth factor (IGF) system is linked to CNS pathological states. The functions of IGFs are modulated by a family of binding proteins termed insulin-like growth factor binding proteins (IGFBPs). Here, we demonstrate that IGFBP-6 may be associated with neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant upregulation of IGFBP-6 in neurons adjacent to the hematoma following ICH with the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing IGFBP-6 level was found to be accompanied by the upregulation of Bax, Bcl-2, and active caspase-3. Besides, IGFBP-6 co-localized well with active caspase-3 in neurons, indicating its potential role in neuronal apoptosis. Knocking down IGFBP-6 by RNA-interference in PC12 cells reduced active caspase-3 expression. Thus, IGFBP-6 may play a role in promoting the brain secondary damage following ICH.     

Expression of somatostatin receptor mRNAs is regulated in vivo by growth hormone, insulin, and insulin-like growth factor-I in rainbow trout (Oncorhynchus mykiss)

Somatostatins are a diverse family of peptide hormones that regulate various aspects of growth, development, and metabolism through interactions with numerous somatostatin receptor subtypes (SSTRs) on target tissues. In this study, we used rainbow trout to evaluate the effects of growth hormone (GH), insulin (INS), and insulin-like growth factor-I (IGF-I) on the expression of SSTR 1A, 1B and 2 mRNAs. GH regulated the expression of SSTRs in a subtype- and tissue-specific manner. GH reduced SSTR 1A, 1B, and 2 expression in optic tectum, reduced SSTR 1A and 1B expression in pancreas, reduced SSTR 1A expression in liver, and increased hepatic SSTR 1B expression. INS also regulated SSTR expression in a subtype- and tissue-specific manner. INS reduced SSTR 1B expression in optic tectum, increased SSTR 2 expression in pancreas, and increased SSTR 1B and 2 expression in liver. IGF-I generally decreased the expression of all SSTRs. These data indicate that GH, INS, and IGF-I modulate the expression of SSTRs and suggest that independent mechanisms may serve to regulate the various receptor subtypes.     

The p35/CDK5 signaling is regulated by p75NTR in neuronal apoptosis after intracerebral hemorrhage

The p75 neurotrophin receptor (p75NTR), a member of tumor necrosis factor receptor superfamily, involves in neuronal apoptosis after intracerebral hemorrhage (ICH). It has been previously demonstrated that phosphorylation of p35 is a crucial factor for fighting against the proapoptotic p25/CDK5 signaling in neuronal apoptosis. Then, in ICH models of rats and primary cortical neurons, we found that the expressions of p75NTR, p-histone H1 (the kinase activity of CDK5), p25, Fas-associated phosphatase-1 (FAP-1), and phosphorylated myocyte enhancer factor 2D (p-MEF2D) were enhanced after ICH, whereas the expression of p35-Thr(138) was attenuated. Coimmunoprecipitation analysis indicated several interactions as follows: p35/p25 and CKD5, p75NTR and p35, as well as p75NTR and FAP-1. After p75NTR or FAP-1 depletion with double-stranded RNA interference in PC12 cells, the levels of p25 and p-histone H1 were attenuated, whereas p35-Thr(138) was elevated. Considering p75NTR has no effect of dephosphorylation, our results suggested that p75NTR might promote the dephosphorylation of p35-Thr(138) via interaction with FAP-1, and the p75NTR/p35 complex upregulated p25/CDK5 signaling to facilitate the neuronal apoptosis following ICH. So, in the study, we aimed to provide a theoretical and experimental basis that p75NTR could be regulated to reduce neuronal apoptosis following ICH for potential clinical treatment.     

Upregulated Expression of Karyopherin α2 is Involved in Neuronal Apoptosis Following Intracerebral Hemorrhage in Adult Rats

Karyopherin α2 (KPNA2) plays a central role in nucleocytoplasmic transport. It is involved in controlling the flow of genetic information and the modulation of diverse cellular activities. Here we explored the KPNA2′s roles during the pathophysiological processes of intracerebral hemorrhage (ICH). An ICH rat model was built and evaluated according to behavioral testing. Using Western blot, immunohistochemistry, and immunofluorescence, significant upregulation of KPNA2 was found in neurons in brain areas surrounding the hematoma following ICH. Increasing KPNA2 level was found to be accompanied by the upregulation of active caspase-3, Bax, and decreased expression of Bcl-2. Besides, KPNA2 co-localized well with active caspase-3 in neurons, indicating its potential role in neuronal apoptosis. What’s more, knocking down KPNA2 by RNA-interference in PC12 cells reduced active caspase-3 expression. Thus, KPNA2 may play a role in promoting the brain secondary damage following ICH.     

MCC950对脑出血大鼠神经损伤的作用*

目的: 研究NLRP3炎性小体抑制剂MCC950对脑出血(ICH)大鼠神经损伤的作用。方法: 72只SD大鼠随机分为3组(n=24):sham组、ICH组和MCC950组。ICH组和MCC950组采用自体非抗凝血注射方法建立大鼠脑出血模型后,给予MCC950组大鼠腹腔注射MCC950 10 mg/kg(2 mg/ml),连续给药3 d。模型建立72 h后,进行前肢放置实验、转角实验和mNSS评分,观察ICH大鼠神经功能情况;新鲜脑组织切片,观察血肿体积变化情况;HE染色,观察脑组织病理学改变情况;干湿比重法,观察脑组织水肿变化情况;FJC染色,观察神经元退化的情况;TUNEL染色,观察神经元凋亡情况;Western blot,观察NLRP3、ASC、caspase-1、IL-1β、IL-18、GSDMD蛋白表达及激活水平的情况。结果: 与sham组比较,ICH组大鼠左前肢放置成功百分比和左侧转身百分比显著下降(P＜0.01,P＜0.05),mNSS评分显著升高(P＜0.01),右侧脑内血肿体积显著增大,血肿周围脑组织中小胶质细胞数量增加,神经元数量减少,神经细胞肿胀,排布不均,部分细胞固缩性坏死,染色加深,右侧基底部含水量显著增多(P＜0.05),血肿周围脑组织中FJC阳性和TUNEL阳性细胞数量显著增加(P＜0.05),NLRP3、ASC、caspase-1、pro-caspase-1、caspase-1/pro-caspase-1比值、GSDMD-N、GSDMD、GSDMD-N/GSDMD比值、IL-1β和IL-18水平显著升高(P＜0.01,P＜0.05)。与ICH组比较,MCC950组大鼠左前肢放置成功百分比和左侧转身百分比显著升高(P＜0.05),mNSS评分显著降低(P＜0.01),右侧脑内血肿体积显著减小,血肿周围脑组织中神经细胞肿胀显著减轻,固缩坏死细胞数量减少,右侧基底含水量显著减少(P＜0.05),血肿周围脑组织中FJC阳性和TUNEL阳性细胞数量显著减少(P＜ 0.05),NLRP3、ASC、caspase-1、pro-caspase-1、caspase-1/pro-caspase-1比值、GSDMD-N、GSDMD、GSDMD-N/GSDMD比值、IL-1β和IL-18水平显著降低(P＜0.05)。结论: MCC950可以通过抑制NLRP3炎性小体介导的炎症反应和细胞焦亡改善ICH后的神经损伤。     

MEKK1 Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage

The JNKs have been implicated in a variety of biological functions in mammalian cells, including apoptosis and the responses to stress. However, the physiological role of these pathways in the intracerebral hemorrhage (ICH) has not been fully elucidated. In this study, we identified a MAPK kinase kinase (MAPKKK), MEKK1, may be involved in neuronal apoptosis in the processes of ICH through the activation of JNKs. From the results of western blot, immunohistochemistry and immunofluorescence, we obtained a significant up-regulation of MEKK1 in neurons adjacent to the hematoma following ICH. Increasing MEKK1 level was found to be accompanied with the up-regulation of p-JNK 3, p53, and c-jun. Besides, MEKK1 co-localized well with p-JNK in neurons, indicating its potential role in neuronal apoptosis. What’s more, our in vitro study, using MEKK1 siRNA interference in PC12 cells, further confirmed that MEKK1 might exert its pro-apoptotic function on neuronal apoptosis through extrinsic pathway. Thus, MEKK1 may play a role in promoting the brain damage following ICH.     

神经活性甾体别孕烯醇酮对脑皮质神经元损伤的保护作用

目的：利用N-甲基-D-天门冬氨酸（NMDA）诱发新生小鼠脑皮质神经元损伤模型,探讨神经活性甾体别孕烯醇酮对脑皮质神经元的保护作用及其机制。方法：应用RT-PCR和Western blot法检测别孕烯醇酮对β2-γ-氨基丁酸受体（β2-GABA-R）表达和对蛋白激酶B（PKB,又称为Akt）磷酸化的影响。应用Western blot和DNA-Ladder方法检测NMDA诱发的神经元凋亡及别孕烯醇酮对NMDA诱发凋亡的影响。结果：Western blot和RT-PCR分析表明0.5×10-6mol/L-5×10-6mol/L别孕烯醇酮使Akt磷酸化增加并促进β2-GABA-R mRNA的表达。1×10-6mol/L别孕烯醇酮预处理小鼠脑皮质神经元有抗凋亡作用,但5×10-6mol/L别孕烯醇酮预处理小鼠脑皮质神经元使NMDA诱发的DNA-Ladder减弱明显,并能有效抵抗NMDA诱发的活化型PRAP、Caspase-3、Caspase-9的增加。结论：别孕烯醇酮可通过促进β2-GABA-R表达和增加Akt磷酸化抵抗NMDA诱发的脑皮质神经元凋亡。     

多巴胺D1和D2受体激动剂和拮抗剂对脑缺血/再灌注损伤的影响

实验应用开阔法、组织病理学方法、原位末端标记(in situ terminal deoxynucleotidyl transferase-metliated de-oxy-UTP mick end labeling,TUNEL)法及免疫组织化学等方法,探讨多巴胺D1、D2受体激动剂和拮抗剂对沙土鼠前脑缺血／再灌注损伤海马CA1区神经元凋亡及凋亡相关基因bcl-2、bax表达的影响。结果显示：前脑缺血5min可引起沙土鼠探索活动增加;再灌注3d,海马CA1区约95％的锥体细胞凋亡;再灌注7d,海马CA1区仅残存约2％—7％的存活锥体细胞;前脑缺血5min可抑制bcl-2的表达并诱导bax表达增高;预先应用D2受体激动剂培高利特可减轻缺血后沙土鼠行为学异常、抑制海马CA1区锥体细胞凋亡、提高锥体细胞存活数、显著诱导bcl-2的表达并抑制bax的表达。预先应用SKF38393、SCH23390及螺哌隆对以上结果无明显影响。实验结果提示,培高利特具有确切的脑保护作用,诱导bcl-2并抑制bax的表达可能是其脑保护作用机制之一。     

Enhanced Neuroprotection of Minimally Invasive Surgery Joint Local Cooling Lavage against ICH-induced Inflammation Injury and Apoptosis in Rats

Hypothermia treatment is one of the neuroprotective strategies that improve neurological outcomes effectively after brain damage. Minimally invasive surgery (MIS) has been an important treatment of intracerebral hemorrhage (ICH). Herein, we evaluated the neuroprotective effect and mechanism of MIS joint local cooling lavage (LCL) treatment on ICH via detecting the inflammatory responses, oxidative injury, and neuronal apoptosis around the hematoma cavity in rats. ICH model was established by type IV collagenase caudatum infusion. The rats were treated with MIS 6 h after injection, and then were lavaged by normothermic (37 °C) and hypothermic (33 °C) normal saline in brain separately. The results indicated that MIS joint LCL treatment showed enhanced therapeutic effects against ICH-induced inflammation injury and apoptosis in rats, as convinced by the decline of TUNEL-positive cells, followed by the decrease of IL-1β and LDH and increase of IL-10 and SOD. This study demonstrated that the strategy of using MIS joint LCL may achieve enhanced neuroprotection against ICH-induced inflammation injury and apoptosis in rats with potential clinic application.     

Protective Effect Against 17β-Estradiol on Neuronal Apoptosis in Hippocampus Tissue Following Transient Ischemia/Recirculation in Mongolian Gerbils via Down-Regulation of Tissue Transglutaminase Activity

We analyzed the protective effect of 17β-estradiol (17β-ED) injection against delayed neuronal death in the hippocampus tissue of the brain in Mongolian gerbils after transient ischemia/recirculation treatment, especially in relation with bcl-2 gene expression and enzymatic activity changes of caspase-3 and tissue transglutaminase (tTGase). Daily intraperitoneal injection of 17β-ED to the animal after the ischemia stimulated the expression of an apoptosis suppressor gene, bcl-2, in the hippocampal tissue for a week. The gradually increasing apoptotic enzyme activity of caspase-3 and increased number of TUNEL positive fragmented neuronal nuclei caused by ischemic attack in the gerbil brain were clearly suppressed by 17β-ED administration. The reduced activity and enzyme protein of tTGase, a neurodegenerative marker of apoptosis in the hippocampus after ischemia, were also restored to nearly normal levels by 17β-ED injection. These results suggest that daily 17β-ED administration to the gerbil after transient ischemic insult with progressing neuronal deteriorative changes in hippocampus tissue can effectively prevent apoptotic changes through a molecular cascade involving gene expression regulation.