Houguang Zhou

Tissue kallikrein protects cortical neurons against in vitro ischemia-acidosis/reperfusion-induced injury through the ERK1/2 pathway.

Human tissue kallikrein (hTK) gene transfer has been shown to protect neurons against cerebral ischemia/reperfusion (I/R) injury, and exogenous tissue kallikrein (TK) administration can enhance neurogenesis and angiogenesis following focal cortical infarction. Previous studies have reported that acidosis is a common feature of ischemia and plays a critical role in brain injury. However, little is known about the role of TK in ischemia-acidosis-induced injury, which is partially caused by the activation of acid-sensing ion channels (ASICs). Here we report that pretreatment of cultured cortical neurons with TK reduced cell death induced by either acidosis or oxygen and glucose deprivation-acidosis/reoxygenation (OGD-A/R). Immunocytochemical staining revealed that TK largely prevented OGD-A/R-induced neuronal morphological changes. We also observed that TK treatment protected cultured neurons from acidosis and OGD-A/R insults. TK exerted the neuroprotective effects by reducing production of reactive oxygen species (ROS), stabilizing the mitochondrial membrane potential (MMP) and inhibiting caspase-3 activation, and thereby attenuating oxidative stress and apoptosis. In addition, we found that activation of the extracellular signal-regulated kinase1/2 (ERK1/2) signaling cascade but not the PI3K/Akt signaling pathway was required for the survival-promoting effect of TK on neurons exposed to OGD-A/R. Moreover, blockade of ASICs had effects similar to TK administration, suggesting direct or indirect involvement of ASICs in TK protection. In conclusion, TK has antioxidant characteristics and is capable of alleviating ischemia-acidosis/reperfusion-induced injury, inhibiting apoptosis and promoting cell survival in vitro through activating the ERK1/2 signaling pathways. Therefore, TK represents a promising therapeutic strategy for ischemic stroke.

Blockade of bradykinin B2 receptor more effectively reduces postischemic blood-brain barrier disruption and cytokines release than B1 receptor inhibition.

Blood-brain barrier disruption and brain edema are detrimental in ischemic stroke. The kallikrein-kinin system appears to play an important role in the regulation of vascular permeability and is invoked in edema formation. The effects of kinins are mediated by bradykinin receptors B1R and B2R. However, little is known about the exact roles of bradykinin receptors in the early stage of cerebral ischemia. In this study, we demonstrated that ischemia upregulated the level of B1R and B2R at 24h after reperfusion by immunofluorescence assays, mainly expressed in astrocytes and neurons, respectively, in the ischemic penumbra. Moreover, B2R inhibition more effectively reduced neurological severity scores, blood-brain barrier permeability and cytokines release than B1R inhibition did. Additionally, B2R inhibition also significantly suppressed B1R protein level. Therefore, blockade of B2R may be a more effective strategy for the treatment of ischemic brain injury than B1R inhibition within 24h after reperfusion.

A prospective controlled study: minimally invasive stereotactic puncture therapy versus conventional craniotomy in the treatment of acute intracerebral hemorrhage.

BackgroundSpontaneous intracerebral hemorrhage (ICH) is a devastating form of stroke with the high mortality twofold to sixfold higher than that for ischemic stroke. But the treatment of haematomas within the basal ganglia continues to be a matter of debate among neurologists and neurosurgeons. The purpose of this study is to judge the short-term and long-term clinical value of minimally invasive stereotactic puncture therapy (MISPT) on acute ICH.MethodsA prospective controlled study was undertaken. The clinical trial was in compliance with the WMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects. According to the enrollment criterion, there were 168 acute ICH cases analyzed, of which 90 cases were performed by MISPT (MISPT group, MG) and 78 cases by Conventional craniotomy (CC group, CG), by means of compare of short-term outcome such as Glasgow Coma Scale (GCS) score, postoperative complications (PC) and rebleeding incidence (RI), moreover, long-term outcome of 1 year postoperation judged by Glasgow Outcome Scale (GOS), Barthel Index (BI), modified Rankin Scale (mRS) and case fatality (CF).ResultsGCS score of MG patients showed obvious amelioration when compared with that of CG (P = 0.039). The PC incidence of in MG decreased obviously compared with that of CG (P = 0.000). The incidences of rebleeding in MG and CG were 10.0% and 15.4% respectively (P = 0.293). There was no obvious difference between CFs of MG and CG. For three parameters representing long-term outcome,the GOS, BI and mRS in MG were ameliorated significantly than that of CG (P = 0.043, P = 0.011 and P = 0.042 respectively).ConclusionThese data indicated that compared with CC, the advantage of MISPT was not only displayed in short-term outcome such as minute trauma and safety, but also seemed to be feasible and had a trend towards improved long-term outcome such as the GOS,BI and mRS.Trial RegistrationThe Australian New Zealand Clinical Trials Registry (ANZCTR), the registration number:ACTRN12610000945022.

Oxidative Stress and Apoptosis of Human Brain Microvascular Endothelial Cells Induced by Free Fatty Acids

Damage to endothelial cells is a key event in the pathogenesis of atherosclerosis and vascular disease. This study aimed to determine whether free fatty acids (FFAs) induced oxidative stress and apoptosis in human brain microvascular endothelial cells (HBMVECs) in vitro and, if so, which signalling pathway mediated these effects. After culture in different concentrations of FFAs for 24-72 h, cell viability/proliferation was determined using a cell counting kit, apoptosis was detected by measuring caspase-3 activity and by using annexin V-conjugated fluorescein isothiocyanate/propidium iodide staining, and oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). The HBMVECs exposed to FFAs showed significantly decreased cell proliferation, increased apoptosis and ROS levels, and decreased MMP. In conclusion, the results showed that high levels of FFAs induced oxidative stress, which damaged HBMVECs and resulted in apoptosis.

Glutathione Prevents Free Fatty Acids-Induced Oxidative Stress and Apoptosis in Human Brain Vascular Endothelial Cells Through Akt Pathway

The damage of human brain vascular endothelial cells (HBVECs) is the key pathogenesis of diabetes‐associated cerebral vascular complications. The aim of this study was to elucidate the effects of glutathione (GSH) on free fatty acids (FFAs)‐induced HBVECs apoptosis, oxidative stress, and the involved possible signaling pathway.

Tissue kallikrein alleviates glutamate-induced neurotoxicity by activating ERK1.

Glutamate‐induced neurotoxicity consequent to N‐methyl‐D‐aspartic acid (NMDA) and 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl) propionic acid (AMPA) receptor activation underlies the pathogenesis of a wide range of central nervous system disorders, including brain ischemia. Prevention of ischemia/reperfusion (I/R)‐induced neuronal injury has long been regarded as an effective therapeutic strategy for ischemia. Human tissue kallikrein (TK) gene transfer has been shown to protect neurons against cerebral I/R‐induced apoptosis and oxidative stress, via activation of the brandykinin B2 receptor (B2R). However, little is known about the role of TK on glutamate‐induced neurotoxicity. Here we report that pretreatment of cultured cortical neurons with TK largely prevented glutamate‐induced morphological changes and cell death. We found that TK pretreatment alleviated glutamate‐induced oxidative stress by inhibiting neuronal nitric oxide synthase (nNOS) activity, thereby reducing the generation of nitric oxide (NO) and reactive oxygen species (ROS). Blockage of NMDA and AMPA receptors by their specific antagonists MK801 and CNQX had effects similar to those of TK administration. Furthermore, we found that the extracellular signal‐regulated kinase 1/2 cascade (ERK1/2), particularly ERK1, and nuclear factor‐κB (NF‐κB) were involved in TK neuroprotection against glutamate‐induced neurotoxicity. TK pretreatment activated ERK1 and NF‐κB, leading to enhanced expression of brain‐derived neurotrophic factor (BDNF) mRNA and antiapoptotic gene Bcl‐2 protein. Collectively, these findings demonstrate that TK attenuates glutamate‐induced apoptosis through an intracellular signaling pathway including activation of B2R, ERK1/2, and NF‐κB and up‐regulation of BDNF and Bcl‐2 expression. Thus, TK represents a promising therapeutic strategy for ischemic stroke.

Tissue kallikrein protects cortical neurons against hypoxia/reoxygenation injury via the ERK1/2 pathway

Systemic or local delivery of human tissue kallikrein gene (hTK) has been shown to be an effective strategy to alleviate cerebral ischemia/reperfusion (I/R) injury, and tissue kallikrein (TK) administration can suppress glutamate- or acidosis-mediated neurotoxicity in vitro. In the present study, the role of TK in hypoxia/reoxygenation (H/R) induced neuronal cell death was investigated. We found that TK administration could remarkably alleviate H/R-induced neuronal injury by reduction of LDH release and promotion of neuron viability. The protective effects of TK could be counteracted by bradykinin B2 receptor (B2R) antagonist HOE140, which could suppress up-regulation of TK on the ERK signal pathway under H/R condition. These results indicate that TK plays an important role in preventing neurons from H/R damage at least partially through the TK-B2R-ERK1/2 pathway.

Tissue kallikrein protects neurons from hypoxia/reoxygenation-induced cell injury through Homer1b/c.

Previous studies have demonstrated that human tissue kallikrein (TK) gene delivery protects against mouse cerebral ischemia/reperfusion (I/R) injury through bradykinin B2 receptor (B2R) activation. We have also reported that exogenous TK administration can suppress glutamate- or acidosis-induced neurotoxicity through the extracellular signal-regulated kinase1/2 (ERK1/2) pathway. To further explore the neuroprotection mechanisms of TK, in the present study we performed immunoprecipitation analysis and identified a scaffolding protein Homer1b/c using MALDI-TOF MS analysis. Here, we tested the hypothesis that TK reduces cell injury induced by oxygen and glucose deprivation/reoxygenation (OGD/R) through activating Homer1b/c. We found that TK increased the expression of Homer1b/c in a concentration- and time-dependent manner. Moreover, TK facilitated the translocation of Homer1b/c to the plasma membrane under OGD/R condition by confocal microscope assays. We also observed that overexpression of Homer1b/c showed the neuroprotection against OGD/R-induced cell injury by enhancing cell survival, reducing LDH release, caspase-3 activity and cell apoptosis. However, the knockdown of Homer1b/c by small interfering RNA showed the opposite effects, indicating that Homer1b/c had protective effects against OGD/R-induced neuronal injury. More interestingly, TK exerted its much more significantly neuroprotective effects after Homer1b/c overexpression, whereas it exerted its reduced effects after Homer1b/c knockdown. In addition, TK pretreatment increased the phosphorylation of the ERK1/2 and Akt-GSK3β through Homer1b/c activation. The beneficial effects of Homer1b/c were abolished by the ERK1/2 or PI3K antagonist. Therefore, we propose novel signaling mechanisms involved in the anti-hypoxic function of TK through activation of Homer1b/c-ERK1/2 and Homer1b/c-PI3K-Akt signaling pathways.

G-CSF Protects Human Brain Vascular Endothelial Cells Injury Induced by High Glucose, Free Fatty Acids and Hypoxia through MAPK and Akt Signaling

Granulocyte-colony stimulating factor (G-CSF) has been shown to play a neuroprotective role in ischemic stroke by mobilizing bone marrow (BM)-derived endothelial progenitor cells (EPCs), promoting angiogenesis, and inhibiting apoptosis. Impairments in mobilization and function of the BM-derived EPCs have previously been reported in animal and human studies of diabetes where there is both reduction in the levels of the BM-derived EPCs and its ability to promote angiogenesis. This is hypothesized to account for the pathogenesis of diabetic vascular complications such as stroke. Here, we sought to investigate the effects of G-CSF on diabetes-associated cerebral vascular defect. We observed that pretreatment of the cultured human brain vascular endothelial cells (HBVECs) with G-CSF largely prevented cell death induced by the combination stimulus with high glucose, free fatty acids (FFA) and hypoxia by increasing cell viability, decreasing apoptosis and caspase-3 activity. Cell ultrastructure measured by transmission electron microscope (TEM) revealed that G-CSF treatment nicely reduced combination stimulus-induced cell apoptosis. The results from fluorescent probe Fluo-3/AM showed that G-CSF greatly suppressed the levels of intracellular calcium ions under combination stimulus. We also found that G-CSF enhanced the expression of cell cycle proteins such as human cell division cycle protein 14A (hCdc14A), cyclinB and cyclinE, inhibited p53 activity, and facilitated cell cycle progression following combination stimulus. In addition, activation of extracellular signal-regulated kinase1/2 (ERK1/2) and Akt, and deactivation of c-Jun N terminal kinase (JNK) and p38 were proved to be required for the pro-survival effects of G-CSF on HBVECs exposed to combination stimulus. Overall, G-CSF is capable of alleviating HBVECs injury triggered by the combination administration with high glucose, FFA and hypoxia involving the mitogen-activated protein kinases (MAPK) and Akt signaling cascades. G-CSF may represent a promising therapeutic agent for diabetic stroke.

The bradykinin B2 receptor mediates hypoxia/reoxygenation induced neuronal cell apoptosis through the ERK1/2 pathway.

The bradykinin B2 receptor (B2R) mediates many physiological processes such as hypotension, inflammation and blood-vessel permeability. Hypoxia/reoxygenation (H/R) induces neuronal cell apoptosis. It was found that B2R expression was enhanced in primary cultured cortical neurons after H/R treatment. Addition of bradykinin (BK) alleviated the neuronal damage from H/R. This protective effect of BK was inhibited by the B2R antagonist, HOE140, and the ERK1/2 antagonist, PD98059. The phosphorylation of ERK1/2 was increased under H/R, and the addition of BK enhanced this effect. These results indicate that B2R plays an important role in protecting neurons from damage induced by H/R and this effect may function through the ERK1/2 pathway.