Effects and regulating mechanisms of heme oxygenase-1 on brain trauma-induced tissue injury and bacterial infection-induced inflammation of cerebral endothelial cells

• Main Authors: Ruey Horng Shih, 施睿宏
• Other Authors: C. M. Yang
• Format: Others
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/95279053852609258182

博士 === 長庚大學 === 生物醫學研究所 === 99 === Brain trauma, virus/bacteria infection in the brain, and hemorrhagic/ischemic stroke are clinically common conditions that induce brain injury. Oxidative stress and blood-brain barrier (BBB, especially cerebral endothelial cells) disruption contribute to brain injury. Heme oxygenase-1 (HO-1) is a stress-inducible rate-limiting enzyme involves in the oxidative degradation of pro-oxidant heme into potent antioxidants carbon monoxide (CO), free iron, and biliverdin IXα, which confer cytoprotection against oxidative injury and provide a vital function in maintaining tissue homeostasis, for example, in protecting cerebrovascular endothelium against cytotoxic injury. For previous investigation of effects of HO-1 on brain injury, most studies forcused on the effects of HO-1 on stroke. However, the effects of HO-1 on brain trauma-induced brain injury or bacteria infection-induced cerebral vascular inflammation are still unknown. In the present study, we used a cold injury-induced mice brain trauma (CIBT) model, which is a well established model for brain edema and blood-brain barrier (BBB) breakdown, and a murine brain endothelial cells culture (bEnd.3) to investigate the protective effects of HO-1. In the first part of the study (chapter III), we found that HO-1 protein expression and enzymatic activity were up-regulated in the mouse cerebral cortex by the recombinant adenovirus (3.5×107 PFU/ mouse, iv) and cobalt protoporphyrin IX (CoPPIX, 10 mg/kg, iv). Overexpression of HO-1 protected against cold injury-induced secondary damage and behavioral impairment as well as decreased brain edema and neutrophil infiltration. HO-1 expression in the cerebral endothelium was observed by immunofluorescent staining. HO-1 protein was also expressed in bEnd.3 cells. Enhanced HO-1 also protected against cold injury-induced cell loss and damage determined by GAPDH leakage into the cell medium and cytotoxicity assay in bEnd.3 cells. The pharmacological stimulation of HO-1 expression may offer a novel clinical strategy in amelioration of brain trauma. On the other hand, it has been documented that several chemicals, present in cigarette smoke (CS) which leads to oxidant stress, have been reported to induce HO-1 expression. However, the effects of CS extract (CSE) on brain trauma-induced injury are not completely understood. In chapter IV, we found that only pretreatment, but not co-treatment or post-treatment, with CSE (75 mg/kg, 24 h, tail vein injection) reduced cold injury-induced cerebral cortex damage. CSE pretreatment significantly increased HO-1 protein expression in brain cortical tissues. Pretreatment with CSE or CoPPIX (10 mg/kg) significantly reduced the damage area and brain edema as well as rescued the behavioral scores as compared to those of cold injury group. ZnPP (3 mg/kg) effectively reversed the protective effect of CSE on CIBT. The induction of HO-1 by CSE may provide protection against CIBT. The mouse brain endothelial cell culture (bEnd.3) was used to investigate the effect of CSE on HO-1 induction and the mechanisms underlying CSE-induced HO-1 expression in cerebral vessels. In the second part of study (chapter V), we demonstrated that CSE induced HO-1 expression in a time and dose dependent manner and sublethal concentrations of CSE (30 μg/ml) induced HO-1 expression in bEnd.3 cells. NADPH oxidase-dependent ROS generation played a key role in CSE-induced HO-1 expression because ROS scavenger (N-acetyl-L-cysteine) and the inhibitors of NADPH oxidase (diphenyleneiodonium chloride and apocynin) blocked the CSE-induced ROS generation and HO-1 expression. CSE activated NADPH oxidase through c-Src in bEnd.3 cells. CSE-induced HO-1 expression was mediated through PDGF receptor (PDGFR)/JAK2/ STAT3 cascade, which was substantiated by pretreatment with the respective pharmacological inhibitors or transfection with PDGFR shRNA. In chapter VI, we found that CSE-induced HO-1 expression was mediated through phosphatidylcholine phospholipase C (PC-PLC)/PKC/ NADPH oxidase/ROS-dependent PDGFR/PI3K/Akt pathway which was confirmed by pretreatment with their respective pharmacological inhibitors or transfection with dominant negative mutants. HO-1 has also been shown to play an important role in protecting against cerebral vascular inflammation triggered by bacterial infection. Cyclooxygenase-2 ( COX-2) has been suggested to contribute to LPS-induced cellular damage. In the third part of the study (chapter VII), COX-2 protein expression and enzymatic activity induced by lipopolysaccharide (LPS) can be down-regulated by HO-1 induction through pretreatment with CoPPIX or infection with Adv-HO-1 in bEnd.3 cells. The mechanisms underlying HO-1 modulating COX-2 protein expression were shown in bEnd.3 cells. LPS-induced COX-2 expression and PGE2 production were mediated through NF-κB (p65) via activation of TLR4/MyD88. Pretreatment with CORM-2, which can release CO (a byproduct of HO-1), inhibited LPS-induced TLR4/MyD88 complex formation, NF-κB (p65) activation, COX-2 expression and PGE2 production. We show a novel inhibition of HO-1 on LPS-induced COX-2/PGE2 production via inhibitions of TLR4/MyD88 complex and NF-κB (p65) by CO in bEnd.3. Taken together, in the first part of (animal) study, HO-1 overexpression provides neuroprotection against cold-induced brain secondary injury. CSE may play an important role in protection against cold-induced brain injury, at least in part, mediated by HO-1 preconditioning in the animal model. In the second part of (cell culture) study, CSE-induced HO-1 expression was mediated through NADPH oxidase/ROS-dependent PDGFR linking to JAK2/STAT3 and PI3K/Akt cascades in bEnd.3 cells. Finally in the third part of study, HO-1 down-regulated LPS-induced COX-2/PGE2 production suggesting that cerebral endothelium-derived HO-1 as a protector against cerebral vascular inflammation triggered by bacterial infection. Therefore, HO-1 plays a protecting role for brain injury induced by brain trauma or cerebral vascular bacterial infection.