Possible pathophysiological roles of mitogen-activated protein kinases (MAPKs) in endometriosis

PROBLEM: Endometriosis accompanies local inflammatory reactions in the peritoneal cavity. We examined the phosphorylation of mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and c-Jun N-terminal kinase (JNK) in endometriotic stromal cells, and their possible pathophysiological roles in endometriosis in relation to proinflammatory substances. METHOD OF STUDY: Endometriotic stromal cells were isolated from endometriomas and were cultured for the experiments. Phosphorylation of MAPKs in endometriotic stromal cells treated with interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and H(2)O(2) were examined by Western blot analysis. Effects of PD98059, SB202190 and SP600125 (inhibitors of ERK, p38 and JNK, respectively) on IL-1beta-induced secretion of IL-6 and IL-8, and on IL-1beta-induced expression of cyclo-oxygenase-2 (COX-2) in endometriotic cells were studied. In addition, eutopic endometrial tissues were collected, and the phosphorylation rate of p38 in eutopic endometrial tissues and endometriotic tissues were determined. RESULTS: IL-1beta, TNFalpha and H(2)O(2) stimulated the phosphorylation of ERK, p38 and JNK, while the total amounts of proteins of the respective MAPKs were virtually the same compared with those in the unstimulated controls. Both SB202190 and SP600125 suppressed IL-1beta-induced secretion of IL-6 and IL-8, and PD98059 suppressed IL-1beta-induced secretion of IL-8. Both SB202190 and PD98059 suppressed IL-1beta-induced expression of COX-2 in endometriotic cells. The p38 phosphorylation rates in the endometriotic tissues were significantly higher than those in the eutopic endometrial tissues of the same patients. CONCLUSIONS: Given the current theory that inflammatory changes are involved in the progression of endometriosis, MAPKs could play as pivotal intracellular signal transducers in endometriotic cells, and thus have a pathophysiological role in the disease.     

Roles of peripheral mitogen-activated protein kinases in melittin-induced nociception and hyperalgesia

Recently, we have reported that melittin, a major toxic peptide of the whole bee venom, plays a central role in production of local inflammation, nociception and hyperalgesia following the experimental honeybee’s sting. However, the exact peripheral mechanisms underlying melittin-induced multiple pain-related behaviors are still less characterized. In the present study, we sought to investigate the potential roles of peripheral mitogen-activated protein kinases (MAPKs) in melittin-induced nociception and hyperalgesia by pre- and post-administration of three MAPK inhibitors, namely U0126 (1 μg, 10 μg) for extracellular signal-regulated kinase (ERK), SP600125 (10 μg, 100 μg) for c-Jun N-terminal kinase (JNK) and SB239063 (10 μg, 100 μg) for p38 MAPK, into the local inflamed area of one hind paw of rats. Both pre- and post-treatment with three drugs significantly suppressed the occurrence and maintenance of melittin-evoked persistent spontaneous nociception (PSN) and primary heat hyperalgesia, with little antinociceptive effect on mechanical hyperalgesia. In vehicle-treated group, ipsilateral injection of melittin produced no impact on thermal and mechanical sensitivity of the other hind paw, suggesting no occurrence of contralateral heat and mechanical hyperalgesia in the melittin test. In addition, local administration of each inhibitor into the contralateral hind paw exerted no significant influence on either PSN or heat/mechanical hyperalgesia tested in the primary injured hind paw, excluding the systemically pharmacological effects of the three drugs. Furthermore, local administration of the three compounds in naïve animals, respectively, did not change the basal pain sensitivity to either thermal or mechanical stimuli, suggesting lack of peripherally functional roles of the three MAPK subfamily members in normal pain sensitivity under the physiological state. Taken together, we conclude that activation of peripheral MAPKs, including ERK, JNK and p38, might contribute to the induction and maintenance of persistent ongoing pain and primary heat hyperalgesia in the melittin test. However, they are not likely to be involved in the processing of melittin-induced primary mechanical hyperalgesia, implicating a mechanistic separation between mechanical and thermal hyperalgesia in the periphery.     

Noxious cold stimulation induces mitogen-activated protein kinase activation in transient receptor potential (TRP) channels TRPA1- and TRPM8-containing small sensory neurons

Two cold-sensitive transient receptor potential (TRP) channels, TRPA1 and TRPM8, have been identified and considered interesting because of their possible roles in thermosensation, nociception and other functions. Recently, we have reported that the phosphorylation of extracellular signal-regulated protein kinase and p38 mitogen-activated protein kinase occurred in primary afferent neurons in response to noxious heat stimulation of the peripheral tissue, i.e. activity-dependent activation of extracellular signal-regulated protein kinase and p38 in dorsal root ganglion neurons. In the present study, we investigated the phosphorylation of extracellular signal-regulated protein kinase, p38, and c-Jun N-terminal kinase in the rat dorsal root ganglion by cold stimulation using immunohistochemistry. Cold stimuli (28-4 °C) were applied by immersion of the hind paw into a water bath (six times of 10 s stimulation and 10 s interval, total 2 min). Noxious cold stimulation induced phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38, but not phosphorylated-c-Jun N-terminal kinase, in small to medium diameter sensory neurons with a peak at 2 min after stimulation. We found that a cold stimulation at 4 °C showed a marked increase in the number of activated neurons. Furthermore, double staining for phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38 showed no colocalization in the dorsal root ganglion neurons. We then performed double-labeling experiments for TRPA1 and TRPM8 mRNA and phosphorylation of mitogen-activated protein kinase. The majority of phosphorylated-extracellular signal-regulated protein kinase-positive neurons also expressed TRPM8 mRNA, whereas phosphorylated-p38 heavily colocalized with TRPA1 mRNA after noxious cold stimulation. Our data suggest that the noxious, but not innocuous, cold stimulation in vivo induced differential activation of extracellular signal-regulated protein kinase and p38 pathways in each subpopulation containing TRPA1 or TRPM8 in dorsal root ganglion.     

ApoE肽段预处理通过调节c-jun氨基端激酶阻抑小鼠脑缺血再灌后神经细胞的凋亡

目的:为探讨外源性载脂蛋白E(apoE)肽段对局灶性脑缺血再灌(I/R)的保护机制,观察apoE-1410拟肽对I/R小鼠脑组织磷酸化c-jun氨基端激酶(JNK)表达变化的影响。方法:实验选用160只雄性ICR小鼠,随机分为3组:假手术对照组(sham组),模型组(I/R组),apoE治疗组(apoE组),采用可逆性大脑中动脉栓塞(MCAO)模型,术后行神经功能评分。I/R3、6、12、24、48h行HE染色观察皮质区神经细胞的形态变化,并计算存活神经元的数目。利用WesternBlot法、免疫组织化学法检测p-c-jun的表达,行TUNEL法检测凋亡细胞数。结果:与对照组比较,模型组小鼠神经功能评分均降低,I/R12h后存活神经元数目明显减少;p-c-jun阳性反应术后3、6、12、24h显著增高(P0.05);随缺血时间延长,凋亡细胞增多,并于48h达高峰(P0.05)。与模型组比较,治疗组小鼠神经功能评分增加,各时相点p-c-jun和TUNEL表达均不同程度下调(P0.05)。I/R3h至48h皮质区p-c-jun表达与TUNEL呈正相关(r=0.716,P0.01)。结论:缺血侧皮质区p-c-jun表达的增强可诱导I/R后神经细胞凋亡;拟apoE-1410肽对I/R具有一定的治疗作用,其机制之一可能是通过抑制JNK活化实现的。     

Long-term study of dendritic spines from hippocampal CA1 pyramidal cells, after neuroprotective melatonin treatment following global cerebral ischemia in rats

Melatonin reduces pyramidal neuronal death in the hippocampus and prevents the impairment of place learning and memory in the Morris water maze, otherwise occurring following global cerebral ischemia. The cytoarchitectonic characteristics of the hippocampal CA1 remaining pyramidal neurons in brains of rats submitted 120 days earlier to acute global cerebral ischemia (15-min four vessel occlusion, and melatonin 10mg/(kg h 6h), i.v. or vehicle administration) were compared to those of intact control rats in order to gain information concerning the neural substrate underlying preservation of hippocampal functioning. Hippocampi were processed according to a modification of the Golgi method. Dendritic bifurcations from pyramidal neurons in both the oriens-alveus and the striatum radiatum; as well as spine density and proportions of thin, stubby, mushroom-shaped, wide, ramified, and double spines in a 50 microm length segment of an oblique dendrite branching from the apical dendrite of the hippocampal CA1 remaining pyramidal neurons were evaluated. No impregnated CA1 pyramidal neurons were found in the ischemic-vehicle-treated rats. CA1 pyramidal neurons from ischemic-melatonin-treated rats showed stick-like and less ramified dendrites than those seen in intact control neurons. In addition, lesser density of spines, lower proportional density of thin spines, and higher proportional density of mushroom spines were counted in ischemic-melatonin-treated animals than those in the sinuously branched dendrites of the intact control group. These cytoarchitectural arrangements seem to be compatible with place learning and memory functions long after ischemia and melatonin neuroprotection.     

巴曲酶对脑缺血再灌注大鼠海马CA1区的影响

目的 通过观察大鼠局灶性脑缺血再灌注不同时段，巴曲酶对海马CAl神经元及星形胶质细胞数目、形态等方面的影响，从而探讨巴曲酶对局灶性脑缺血再灌注损伤的保护作用。方法 采用改良的线栓法制备大脑中动脉阻塞(MACO)2h、不同再灌注时间段(3h、6h、12h、24h、48h、72h、7d)的大鼠短暂局灶性脑缺血(transient focal cerebral isehemia)模型，随机设立巴曲酶组(Bat)、生理盐水对照组(N．S)、假手术组(sham-operated)，通过HE染色及胶质原纤维酸性蛋白(GFAP)和神经元特异核抗原(NeuN)的免疫组化染色，观测CAl区神经元和星形胶质细胞的形态、数目的动态变化。结果巴曲酶能显提高再灌注早期(6～24h)CAl区GFAP阳性细胞的数目，再灌注7d组存活锥体细胞的数量较盐水对照组有明显提高，提示局灶性脑缺血后早期反应性星形胶质细胞的增多对维持神经元的存活有积极意义，巴曲酶对短暂局灶性脑缺血再灌注引起的海马CAl区延迟性神经元坏死(DND)有一定的抑制作用。     

Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death

Summary The ultrastructural changes in the pyramidal neurons of the CA1 region of the hippocampus were studied 6 h, 24 h, 48 h, and 72 h following a transient 10 min period of cerebral ischemia induced by common carotid occlusion combined with hypotension. The pyramidal neurons showed delayed neuronal death (DND), i.e. at 24 h and 48 h postischemia few structural alterations were noted in the light microscope, while at 72 h extensive neuronal degeneration was apparent. The most prominent early ultrastructural changes were polysome disaggregation, and the appearance of electron-dense fluffy dark material associated with tubular saccules. Mitochondria and nuclear elements appeared intact until frank neuronal degeneration. The dark material accumulated with extended periods of recirculation in soma and in the main trunks of proximal dendrites, often beneath the plasma membrane, less frequently in the distal dendrites and seldom in spines. Protein synthesis inhibitors (anisomycin, cycloheximide) and an RNA synthesis inhibitor (actinomycin D), administered by intrahippocampal injections or subcutanously, did not mitigate neuronal damage. Therefore, DND is probably not apoptosis or a form of programmed cell death. We propose that the dark material accumulating in the postischemic period represents protein complexes, possibly aggregates of proteins or internalized plasma membrane fragments, which may disrupt vital cellular structure and functions, leading to cell death.     

Role of apoptosis inducing factor (AIF) for hippocampal neuronal cell death following global cerebral ischemia in mice

The molecular mechanisms of neuronal cell death following circulatory arrest are still not fully understood. In the current study we investigated the role of apoptosis-inducing factor (AIF), a major caspase-independent mitochondrial cell death protein, for neuronal cell death following global cerebral ischemia (GCI). C57/Bl6 or low AIF expressing Harlequin mutant mice (AIF^low) and their wild-type littermates were subjected to 10 min of GCI. DNA damage, nuclear pathology, and localization of AIF were investigated 6, 24, and 72 h after GCI by TUNEL and DAPI staining, and immunohistochemistry, respectively. Cell death of hippocampal CA1 neurons following GCI was associated with nuclear translocation of AIF, nuclear pyknosis, and DNA fragmentation, i.e. ∼80% of all TUNEL-positive neurons had nuclear AIF staining. In AIF^low mice neuronal cell loss was reduced by 60% (p < 0.02). The current experiments suggest that AIF-mediated signaling represents a novel mechanism of neuronal cell death following GCI.     

自噬标志性分子LC3B在大鼠全脑缺血复灌损伤中海马CA1区的表达及意义

目的　探讨自噬标志分子LC3B在大鼠全脑缺血复灌不同时间的表达以及对海马神经元损伤的影响。 方法　采用四血管法(4-vessel-occlusion,4-VO)法制作大鼠全脑缺血模型,随机将33只　SD大鼠分成假手术组和缺血再灌注组。在大鼠全脑缺血20 min后,分别恢复血流灌注30 min、1、2、4、6、8、12、24、48、72 h,用免疫组织化学法检测海马CA1区神经元LC3B的表达。 结果　LC3B在大鼠全脑缺血20 min复灌2 h开始表达,在复灌12 h表达到达高峰,之后逐渐减弱。 结论　自噬的激活介导了大鼠全脑缺血再灌注海马CA1区神经元的损伤死亡,长时间脑缺血再灌注损伤中自噬激活的时间更早及介导神经元的损伤更严重。     

亚低温后适应对树鼩局部脑缺血海马CA1区神经元的保护机制

目的： 观察亚低温后处理(HPC)对树鼩局部脑缺血后不同时间海马CA1区血管内皮生长因子（VEGF）表达及神经元缺失的影响,探讨亚低温后适应保护脑缺血后海马CA1区神经元的可能机制。方法：建立光化学诱导树鼩脑缺血模型,于缺血后6h采用局部恒温控温装置使脑温降低并维持亚低温(31-32 ℃)状态1 h。用免疫组化法及图像分析仪测定海马CA1区VEGF表达的改变;计数海马神经元并观察其超微结构变化。结果：与常温组相比,亚低温后适应组海马CA1区VEGF表达在24 h时增加而72 h时明显下降（P＜0.01）;24 h时神经元坏死减少,72 h时坏死细胞增多,缺血侧超微结构呈现相同变化。结论：在脑缺血后早期VEGF的表达可能与其直接发挥对神经元细胞的保护作用有关,亚低温后适应对脑缺血的保护作用在脑缺血的早期有明显意义,而在缺血的晚期则可能加重脑缺血的损伤,低温后适应脑保护的意义在于延长脑缺血治疗时间窗。     

黄芪注射液对脑缺血再灌注大鼠海马神经元凋亡及JNK3表达的影响

目的:观察黄芪注射液对脑缺血再灌注大鼠海马神经元凋亡及c-Jun N末端激酶3(JNK3)表达的影响。方法:四血管阻断法制备脑缺血再灌注大鼠模型。设假手术组、脑缺血再灌注模型组(模型组)、脑缺血再灌注模型+黄芪注射液组(黄芪注射液组)和脑缺血再灌注模型+黄芪注射液溶剂对照组(溶剂对照组)。除假手术组外其余3组根据再灌注时间不同又分为0 h、0.5 h、2 h、6 h、24 h、72 h和120 h 7个亚组。采用TUNEL法检测海马神经元凋亡,Western blotting法检测海马组织JNK3蛋白变化,real-time PCR法检测海马组织JNK3 mRNA 的表达变化。结果:与假手术组比,模型组大鼠各个时点凋亡细胞数均增多(P<0.05);与模型组比,黄芪注射液组各个时点的细胞凋亡数明显减少(P<0.05),而黄芪注射液溶剂对照组各个时点的细胞凋亡数无明显变化(P>0.05)。除120 h外,模型组各时点海马组织JNK3蛋白及mRNA表达均较假手术组增加(P<0.05);与模型组相比,黄芪注射液可减弱除120 h之外的各时点JNK3 蛋白及mRNA的表达(P<0.05),而黄芪注射液溶剂对照组则无明显变化(P>0.05)。结论:黄芪注射液可抑制脑缺血再灌注大鼠海马神经元凋亡,其抗凋亡机制可能与下调JNK3 mRNA及蛋白表达有关。     

脑缺血再灌注损伤的海马神经元对Bax、Bcl-2和Caspase-3的表达

目的：凋亡调控基因在脑缺血再灌后海马神经元的表达。方法：采用免疫组织化学的方法，观察 昆明小鼠双侧颈总动脉结扎7min后不同再灌时间组(24h组、48h组、72h组、7d组、14d组)海马CAl区神经元Bax、Bcl-2和Caspase-3的活性形式CM1的免疫反应活性。结果：Bax和CM1阳性神经元数在48h组最多，与其他各组相比差异有显著性(P＜0．01)，72h组明显下降，14d组完全消失；而Bcl-2阳性神经元数在48h组增多(与24h组相比，P＜0．01)，72h组下降，7d组再次上升(与72h组相比，P＜0．01)，14d组最多(与48h组相比，P＜0．01)。在24h、48h、72h、7d组，Bax阳性神经元多于Bcl-2阳性神经元(P＜0．05)，14d组则相反。结论：Bax和caspase-3在脑缺血再灌早期表达增强，然后下降以至消失，Bcl-2于再灌后期表达增强。Bax表达上调可能与Caspase-3激活相关。     

行为学训练对缺血性脑损伤大鼠脑组织X盒结合蛋白1表达的影响

目的:研究大鼠脑缺血再灌注后对细胞核转录因子X盒结合蛋白1(XBP1)表达的影响,探讨穿梭箱行为学训练对脑缺血再灌注大鼠脑组织XBP1的调节作用及机制.方法:应用线栓法制作大鼠局灶性脑缺血再灌注模型,训练组于造模1d后开始给予穿梭箱训练,采用免疫组织化学检测不同时间点大脑皮质内XBP1的表达.结果:正常对照组大鼠大脑皮质内XBP1少量表达,缺血再灌注后高于正常对照组,训练组大脑皮质内XBP1阳性表达较缺血再灌注组明显增多.结论:穿梭箱训练上调脑缺血诱导的XBP1表达,对脑缺血再灌注大鼠皮质神经元的具有保护作用.     

Neuroprotection against ischemic brain injury by a small peptide inhibitor of c-Jun N-terminal kinase (JNK) via nuclear and non-nuclear pathways

Our previous studies and the others have strongly suggested that c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Here we reported that Tat-JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1), a smaller 11-mer peptide corresponding to residues 153-163 of murine JIP-1 conjugated to Tat peptide, perturbed the assembly of JIP-1-JNK3 complexes, thus inhibiting the activation of JNK3 induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. As a result, Tat-JBD diminished the increased phosphorylation of c-Jun (a nuclear substrate of JNK) and the increased expression of Fas ligand induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and the release of Bax from Bcl-2/Bax dimers, Tat-JBD attenuated Bax translocation to mitochondria and the release of cytochrome c induced by ischemia/reperfusion. Furthermore, the activation of caspase3 and hydrolyzation of poly-ADP-ribose-polymerase induced by brain ischemia/reperfusion were also significantly suppressed by preinfusion of the peptide Tat-JBD. Importantly, Tat-JBD showed neuroprotective effects on ischemic brain damage in vivo, and administration of the peptide after ischemia also achieved the same effects as preinfusion of the peptide did. Thus, our findings imply that Tat-JBD induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via inhibiting nuclear and non-nuclear pathways of JNK signaling. Taken together, these results indicate that Tat-JBD peptide provides a promising therapeutic approach for ischemic brain injury.     

Neuroprotection of hypoxic postconditioning against global cerebral ischemia through influencing posttranslational regulations of heat shock protein 27 in adult rats

We previously reported that hypoxic postconditioning (HPC) ameliorated hippocampal neuronal death induced by transient global cerebral ischemia (tGCI) in adult rats. However, the mechanism of HPC‐induced neuroprotection is still elusive. Notably, heat shock protein 27 (Hsp27) has recently emerged as a potent neuroprotectant in cerebral ischemia. Although its robust protective effect on stroke has been recognized, the mechanism of Hsp27‐mediated neuroprotection is largely unknown. Here, we investigated the potential molecular mechanism by which HPC modulates the posttranslational regulations of Hsp27 after tGCI. We found that HPC increased expression of Hsp27 in CA1 subregion after tGCI. Inhibition of Hsp27 expression with lentivirus‐mediated short hairpin RNA (shRNA) abolished the neuroprotection induced by HPC in vivo. Furthermore, pretreatment with cycloheximide, a protein synthesis inhibitor, resulted in a significant decrease in the degradation rate of Hsp27 protein in postconditioned rats, suggesting that the increase in the expression of Hsp27 after HPC might result from its decreased degradation. Next, pretreatment with leupeptin, a lysosomal inhibitor, resulted in an accumulation of Hsp27 after tGCI, indicating that autophagic pathway may be responsible for the degradation of Hsp27. We further showed that the formation of LC3‐II and autophagosomes increased after tGCI. Meanwhile, the degradation of Hsp27 was suppressed and neuronal damage was reduced when blocking autophagy with 3‐Methyladenine, whereas activating autophagy with rapamycin showed an opposite tendency. Lastly, we confirmed that HPC increased the expression of phosphorylated MAPKAP kinase 2 (MK2) and Hsp27 after tGCI. Also, administration of SB203580, a p38 mitogen‐activated protein kinase inhibitor, decreased the expressions of phosphorylated MK2 and Hsp27. Our results suggested that inhibition of Hsp27 degradation mediated by down‐regulation of autophagy may induce ischemic tolerance after HPC. Additionally, phosphorylation of Hsp27 induced by MK2 might be associated with the neuroprotection of HPC.     

Neuroprotection of ischemic preconditioning is mediated by thioredoxin 2 in the hippocampal CA1 region following a subsequent transient cerebral ischemia

Preconditioning by brief ischemic episode induces tolerance to a subsequent lethal ischemic insult, and it has been suggested that reactive oxygen species are involved in this phenomenon. Thioredoxin 2 (Trx2), a small protein with redox‐regulating function, shows cytoprotective roles against oxidative stress. Here, we had focused on the role of Trx2 in ischemic preconditioning (IPC)‐mediated neuroprotection against oxidative stress followed by a subsequent lethal transient cerebral ischemia. Animals used in this study were randomly assigned to six groups; sham‐operated group, ischemia‐operated group, IPC plus (+) sham‐operated group, IPC + ischemia‐operated group, IPC + auranofin (a TrxR2 inhibitor) + sham‐operated group and IPC + auranofin + ischemia‐operated group. IPC was subjected to a 2 minutes of sublethal transient ischemia 1 day prior to a 5 minutes of lethal transient ischemia. A significant loss of neurons was found in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia‐operated‐group 5 days after ischemia‐reperfusion; in the IPC + ischemia‐operated‐group, pyramidal neurons in the SP were well protected. In the IPC + ischemia‐operated‐group, Trx2 and TrxR2 immunoreactivities in the SP and its protein level in the CA1 were not significantly changed compared with those in the sham‐operated‐group after ischemia‐reperfusion. In addition, superoxide dismutase 2 (SOD2) expression, superoxide anion radical ( ) production, denatured cytochrome c expression and TUNEL‐positive cells in the IPC + ischemia‐operated‐group were similar to those in the sham‐operated‐group. Conversely, the treatment of auranofin to the IPC + ischemia‐operated‐group significantly increased cell damage/death and abolished the IPC‐induced effect on Trx2 and TrxR2 expressions. Furthermore, the inhibition of Trx2R nearly cancelled the beneficial effects of IPC on SOD2 expression, production, denatured cytochrome c expression and TUNEL‐positive cells. In brief, this study shows that IPC conferred neuroprotection against ischemic injury by maintaining Trx2 and suggests that the maintenance or enhancement of Trx2 expression by IPC may be a legitimate strategy for therapeutic intervention of cerebral ischemia.     

Neanthes japonica (Iznka) fibrinolytic enzyme reduced cerebral infarction,cerebral edema and increased antioxidation in rat models of focal cerebral ischemia

Thrombolytic agent is increasingly being used in treating acute ischemic stroke. A novel protease with strong thrombolytic activity, Neanthes japonica (Iznka) fibrinolytic enzyme (NJF) discovered in our laboratory has been reported with characteristics of direct hydrolyzing fibrin and fibrinogen. The neuroprotective effect of NJF and urokinase (UK) was tested in rat models of middle cerebral artery occlusion (MCAO). The model was successfully produced by introducing an intraluminal suture into the left middle cerebral artery (MCA). NJF (0.25, 0.5, 1 mg/kg) was injected intravenously 1 h after the onset of reperfusion. Compared with vehicle group, MCAO animals treated with NJF showed dose dependent reduction in cerebral infarction with improved neurological outcome. Meanwhile, ischemia induced cerebral edema was reduced in a dose dependent manner. Treatment with NJF at 0.5 mg/kg was almost equivalent to UK at 15,000 U/kg dosage in the reduction of cerebral infarction and cerebral edema. Biomedical assay showed that NJF treatment suppressed lipid peroxidation and restored superoxide dismutase (SOD) activities in brain tissue. These results suggest that NJF posses neuroprotective potential in rat MCAO and reperfusion model. Neuroprotection shown by NJF may be attributed to inhibition of lipid peroxidation, increase in endogenous antioxidant defense enzymes.     

降钙素基因相关肽对局灶性脑缺血再灌注大鼠海马CREB和磷酸化CREB的上调作用

目的:探讨外源性降钙素基因相关肽(CGRP)对局灶性脑缺血再灌注大鼠海马CREB和磷酸化CREB(p- CREB)表达的影响。方法:制作局灶性脑缺血再灌注模型实验组并给予CGRP,应用免疫组织化学、Western印迹和图像分析方法检测大鼠海马CA1区CREB和p-CREB表达。结果:缺血再灌注组CA1区CREB表达较假手术组减少,CGRP组CA1区的CREB表达高于缺血再灌注组。缺血再灌注组CA1区p-CREB表达高于假手术组,CGRP组p-CREB表达多于缺血再灌注组。结论:CGRP上调局灶性脑缺血再灌注大鼠海马CREB和p-CREB的表达,CGRP对缺血神经元的保护作用可能是通过上调神经元内CREB和p-CREB来实现的。     

Convergence of stress granules and protein aggregates in hippocampal cornu ammonis 1 at later reperfusion following global brain ischemia

The delayed and selective vulnerability of post-ischemic hippocampal cornu ammonis (CA) 1 pyramidal neurons correlates with a lack of recovery of normal protein synthesis. Recent evidence implicates sequestration of translational machinery into protein aggregates and stress granules as factors underlying persistent translation arrest in CA1 neurons. However, the relationship between protein aggregates and stress granules during brain reperfusion is unknown. Here we investigated the colocalization of protein aggregates and stress granules using immunofluorescence microscopy and pair-wise double labeling for ubiquitin/T cell internal antigen (TIA-1), ubiquitin/small ribosomal subunit protein 6 (S6), and TIA-1/S6. We evaluated the rat dorsal hippocampus at 1, 2 or 3 days of reperfusion following a 10 min global brain ischemic insult. At 1 day of reperfusion, ubiquitin-containing aggregates (ubi-protein clusters) occurred in neurons but did not colocalize with stress granules. At 2 days' reperfusion, only in CA1, cytoplasmic protein aggregates colocalized with stress granules, and ubiquitin-containing inclusions accumulated in the nuclei of CA1 pyramidal neurons. Functionally, a convergence of stress granules and protein aggregates would be expected to sustain translation arrest and inhibit clearance of ubiquitinated proteins, both factors expected to contribute to CA1 pyramidal neuron vulnerability.     

Antioxidant N-acetylcysteine inhibits the activation of JNK3 mediated by the GluR6–PSD95–MLK3 signaling module during cerebral ischemia in rat hippocampus

Cerebral ischemia induces kainate receptor glutamate receptor 6 (GluR6) binding to the postsynaptic density protein 95 (PSD95), which in turn anchors mixed lineage kinase 3 (MLK3) via SH3 domain in rat brain. MLK3 subsequently activates c-Jun NH₂-terminal kinase (JNK) via MAP kinase kinases (MKKs). In this study, we investigated the association of PSD95 with GluR6 and MLK3, the autophosphorylation of MLK3, the combination of MLK3 with JNK3, and the phosphorylation of JNK3 during cerebral ischemia in rat hippocampus CA1. Our results indicate that the GluR6–PSD95–MLK3 complex quickly enhanced at 5 min of ischemia and peaked at 10 min of ischemia, and then gradually reduced with the prolonged time of ischemia. Interestingly, the combination of MLK3 and JNK3 gradually increased from 5 min to 30 min of ischemia. JNK3 phosphorylation first increased and then attenuated in cytosol, suggesting the translocation of activated JNK3 to nucleus during ischemia. To further investigate the possible mechanism of JNK3 activation, antioxidant N-acetylcysteine (NAC) was given to the rats 20 min prior to ischemia. Results indicate that NAC distinctly inhibited the association of PSD95 with GluR6 and MLK3, the autophosphorylation of MLK3, the combination of MLK3 with JNK3 and JNK3 activation. Taken together, these finding indicate that ischemic stimulation results in JNK3 activation through the GluR6–PSD95–MLK3 signaling module, and that the activation of JNK3 is closely related to oxidative stress.