| According to the World Health Organisation, over 15 million people a year, equating to one in every 400 people, suffer a stroke worldwide. Stroke is a leading cause of mortality after heart disease, equating to 9% of total deaths each year. It is the most common cause of long-term disability, with up to 40% of stroke patients not expected to recover independence. Ischaemic stroke accounts for approximately 80–85% of all cases, with approximately 30% of the former undergoing haemorrhagic transformation.Cerebral ischemia/reperfusion injury is a complex pathophysiologic process which is not been clarified now. Recent studies found that the injuries caused by cerebral blood flow cessation and reperfusion are a rapid cascade reaction which includes disfunction of ATP production, overproduction of reactive oxygen species, intracellular acidosis, increased release of excitatory amion acids, destabilization of intracellular calcium and apoptosis-related genes activation and so on. These pathophysiologic processes overlap and intercommunicate then form a vicious cycle which results in cell apoptosis or necrosis. It is a focus in neuroscience that looking for the ideal neuroprotective agents that can block ischemic cascade reaction.Ursolic acid (UA), one of pentacyclic triterpene acids, is ubiquitous in the plant kingdom and is found in fruits, vegetables, and medicinal herbs. Its molecular formula is C15H24N2O. Although the molecular targets of this molecule are not completely characterized, the best known effect of UA is anti-bacterial, antiviral, antioxidant, anti-cancer, anti-inflammatory, anti-HIV and immunomodulatory propertiesICR mice and Nrf2-deficient or control mice were subjected to transient focal cerebral ischemia by right MCA occlusion. UA was used to activate Nrf2. This study evaluated the time course expression regularity of Nrf2, HO-1, ROS, TLR4, NF-κB, NF-M, NF-H andα-Tub, and UAs role in cerebral ischemia and it's potential mechanism.Partâ… Anti-oxidative Effect of UA on focal cerebral ischemia/reperfusion injury in mice and its mechanism of signal transduction pathwayObjective:This study is to evaluate the time course expression regularity of Nrf2, HO-1, ROS, and estimate the anti-oxidative effects of UA on focal cerebral ischemia/reperfusion injury in mice and explore its possible mechanisms and related signal transduction pathways.Methods:CD-1 mice and Nrf2-deficient or control mice were subjected to transient focal cerebral ischemia/reperfusion model in rats was established by middle cerebral artery occlusion using modified suture occlusion technique. Experiment 1: The mice were randomly divided into Sham group(n=30), MCAO group(n=90), The time course expression of Nrf2 and HO-1 in the brain tissue after MCAO. Two groups were studied, including Sham group, MCAO group. The last two group included 3 h, 6 h, 12 h, 24 h, 48 h and 72 h sub-groups. Experiment 2: UA's neuroprotection against damage from cerebral ischemia/reperfusion. UA was injected intraperitoneally 15 minutes after MCAO. mice were reanesthetized and killed at 24 h after MCAO. In this part, mice were individed into 4 groups randomly. Group 1: Sham-operated group (Sham) that received equal volume PBS including 1% DMSO; group 2: Vehicle controls (Vehicle) that received equal volume PBS including 1% DMSO after MCAO; group 3: UA low dose (L-UA) rats that received UA at 68 mg/kg after MCAO; group 4: UA high dose (H-UA) mice that received UA at 130 mg/kg after MCAO. Experiment 3: Nrf2–/–, Nrf2+/+mice were randomly divided into Sham-operated group (Sham) that received equal volume PBS including 1% DMSO; Vehicle controls (Vehicle) that received equal volume PBS including 1% DMSO after MCAO; UA high dose (H-UA) mice that received UA at 130 mg/kg after MCAO. Infarct volume was measured by TTC staining and morphologic changes were observed by H.E. we measured Malondialdehyde (MDA) of cerebral tissue to evaluate antioxidation activitis of UA. RT-qPCR, Western blot, and confocal microscope were used to analyse the expression of Nrf2 and HO-1.Results:Compared with Sham group, Nrf2 and HO-1 were upregulated at gene and protein level in ischemia/reperfusion brain, beginning at 6 h and peaking at 24 h after MCAO (P<0.05). UA high dose (130 mg/kg) upregulated Nrf2 and HO-1 in MCAO-affected brain tissue, The Nrf2 RQ of Vehicle group, MCAO group, L-UA group, H-UA group were (0.22±0.27, 0.85±0.10, 0.70±0.05, 0.53±0.06). UA reduced infarct volume (0.30±0.02 vs. 0.48±0.04) (P<0.05), and behavioral deficits(1.78±0.30 vs. 2.47±0.46) (P<0.05) caused by MCAO. And also, UA could decrease the level of MDA. Consistent with our prediction, Nrf2 deficiency significantly worsened the neurologic deficit caused by MCAO and also obliterated the anti-oxidative effect of UA. We next examined the location of Nrf2 expression at 24 h after MCAO using confocal microscope. The nuclear translocation of Nrf2 showed a significant increase in H-UA treated animals as compared to MCAO group.Conclusions:Nrf2, HO-1 and MDA were induced at the early stage after MCAO. UA reduce cerebral infarct volume and improve neurologic impairment and protected the brain from damage caused by MCAO, this effect may be through upregulation the transcription factor Nrf2 expression. We further verified the importance of Nrf2 in oxidative stress with Nrf2–/– mice. Consistent with our prediction, Nrf2 deficiency significantly worsened the neurologic deficit caused by MCAO and also obliterated the anti-oxidative effect of UA. The neuroprotection of UA was accomplished by antioxidation. It was one of important neuroprotective mechanisms of UA that regulating the anti-oxidative effect by Nrf2/ARE pathway.Partâ…¡Anti-inflammatory Effect of UA on focal cerebral ischemia/reperfusion injury in mice and its mechanism of signal transduction pathwayObjective:This study is to evaluate the time course expression regularity of TLR4,NF-κB, and estimate the anti-inflammatory effects of UA on focal cerebral ischemia/reperfusion injury in mice and explore its possible mechanisms and related signal transduction pathways.Methods:CD-1 mice and Nrf2-deficient or control mice were subjected to transient focal cerebral ischemia/reperfusion model in rats was established by middle cerebral artery occlusion using modified suture occlusion technique. Experiment 1: The mice were randomly divided into Sham group(n=30), MCAO group(n=90), The time course expression of Nrf2 and HO-1 in the brain tissue after MCAO. Two groups were studied, including Sham group, MCAO group. The last two group included 3 h, 6 h, 12 h, 24 h, 48 h and 72 h sub-groups. Experiment 2: UA's neuroprotection against damage from cerebral ischemia/reperfusion. UA was injected intraperitoneally 15 minutes after MCAO. mice were reanesthetized and killed at 24 h after MCAO. In this part, mice were individed into 4 groups randomly. Group 1: Sham-operated group (Sham) that received equal volume PBS including 1% DMSO; group 2: Vehicle controls (Vehicle) that received equal volume PBS including 1% DMSO after MCAO; group 3: UA low dose (L-UA) rats that received UA at 68 mg/kg after MCAO; group 4: UA high dose (H-UA) mice that received UA at 130 mg/kg after MCAO. Experiment 3: Nrf2–/–, Nrf2+/+mice were randomly divided into Sham-operated group (Sham) that received equal volume PBS including 1% DMSO; Vehicle controls (Vehicle) that received equal volume PBS including 1% DMSO after MCAO; UA high dose (H-UA) mice that received UA at 130 mg/kg after MCAO. RT-qPCR, Western blot were used to analyse the expression of TLR4 and NF-κB.Results : Compared with Sham group, TLR4 and NF-κB were upregulated at gene and protein level in ischemia/reperfusion brain, beginning at 6 h and peaking at 24 h after MCAO (P<0.05). UA high dose (130 mg/kg) upregulated TLR4 and NF-κB in MCAO-affected brain tissue(P<0.05), The TLR4/ NF-κB RQ of Vehicle group, MCAO group, L-UA group, H-UA group were (TLR4 0.40±0.31, 1.69±0.09,1.20±0.13, 1.00±0.26; NF-κB 0.50±0.04, 1.52±0.25, 1.30±0.09, 0.96±0.40). Nrf2 deficiency significantly worsened the neurologic deficit caused by MCAO and also obliterated the anti-inflammatory effect of UA. Conclusions:TLR4 and NF-κB were increased at the early stage after MCAO. UA reduce the expression of TLR4 and NF-κB, and protected the brain from damage caused by MCAO. The tendency of anti-imflammtory of UA is coincidence with the alter of Nrf2 in partâ… . Then we presume this effect of anti-inflammtory may be through upregulation the transcription factor Nrf2 expression. We further verified the importance of Nrf2 in inflammtory reaction with Nrf2–/– mice. Consistent with our prediction, Nrf2 deficiency significantly worsened the neurologic deficit caused by MCAO and also obliterated the anti-inflammtory effect of UA. The neuroprotection of UA was accomplished by antioxidation and anti-inflammtion. It was one of important neuroprotective mechanisms of UA that regulating the anti-oxidative and anti-inflammtory effect by Nrf2/ARE pathway.Partâ…¢Promotion neuronal differentiated Effect of UA on focal cerebral ischemia/reperfusion injury in mice and its mechanism of signal transduction pathwayObjective:This study is to evaluate the time course expression regularity of NF-M, NF-H andα-Tub, and estimate the Promotion neuronal differentiated effect of UA on focal cerebral ischemia/reperfusion injury in mice and explore its possible mechanisms and related signal transduction pathways.Methods:CD-1 mice and Nrf2-deficient or control mice were subjected to transient focal cerebral ischemia/reperfusion model in rats was established by middle cerebral artery occlusion using modified suture occlusion technique. Experiment 1: The mice were randomly divided into Sham group(n=30), MCAO group(n=90), The time course expression of Nrf2 and HO-1 in the brain tissue after MCAO. Two groups were studied, including Sham group, MCAO group. The last two group included 3 h, 6 h, 12 h, 24 h, 48 h and 72 h sub-groups. Experiment 2: UA's neuroprotection against damage from cerebral ischemia/reperfusion. UA was injected intraperitoneally 15 minutes after MCAO. mice were reanesthetized and killed at 24 h after MCAO. In this part, mice were individed into 4 groups randomly. Group 1: Sham-operated group (Sham) that received equal volume PBS including 1% DMSO; group 2: Vehicle controls (Vehicle) that received equal volume PBS including 1% DMSO after MCAO; group 3: UA low dose (L-UA) rats that received UA at 68 mg/kg after MCAO; group 4: UA high dose (H-UA) mice that received UA at 130 mg/kg after MCAO. Experiment 3: Nrf2–/–, Nrf2+/+mice were randomly divided into Sham-operated group (Sham) that received equal volume PBS including 1% DMSO; Vehicle controls (Vehicle) that received equal volume PBS including 1% DMSO after MCAO; UA high dose (H-UA) mice that received UA at 130 mg/kg after MCAO. RT-qPCR, Western blot were used to analyse the expression of NF-M, NF-H andα-Tub.Results:Compared with Sham group, NF-M, NF-H andα-Tub were decreased at gene and protein level in ischemia/reperfusion brain, beginning at 6 h and at a low ebb at 24 h after MCAO (P<0.05). UA high dose (130 mg/kg) upregulated NF-M in MCAO-affected brain tissue(P<0.05), but have no effect on NF-H andα-Tub. The NF-M, NF-H andα-Tub RQ of Vehicle group, MCAO group, L-UA group, H-UA group were (NF-M 1.80±0.53, 0.76±0.10,0.85±0.03, 1.02±0.26; NF-H 1.50±0.14, 0.41±0.05, 0.42±0.19, 0.48±0.05;α-Tub 1.50±0.14, 0.58±0.05, 0.52±0.03, 0.56±0.04). Nrf2 deficiency significantly worsened the neurologic deficit caused by MCAO and also obliterated the Promotion neuronal differentiated effect of UA.Conclusions:NF-M, NF-H andα-Tub were decreased at the early stage after MCAO. UA reduce the expression of NF-M, and protected the brain from damage caused by MCAO. The tendency of Promotion neuronal differentiationt of UA is coincidence with the alter of Nrf2 in part 1. Then we presume this effect of Promotion neuronal differentiation may be through upregulation the transcription factor Nrf2 expression. We further verified the importance of Nrf2 in promotion neuronal differentiation with Nrf2–/– mice. Consistent with our prediction, Nrf2 deficiency significantly worsened the neurologic deficit caused by MCAO and also obliterated the Promotion neuronal differentiated effect of UA. The neuroprotection of UA was accomplished by antioxidation, anti-inflammtory and promotion neuronal differentiation. It was one of important neuroprotective mechanisms of UA that regulating the anti-oxidative, anti-inflammtory and promotion neuronal differentiation effect by Nrf2/ARE pathway.
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