| Ischemic stroke is the most common type of stroke. All over the world, evaluation system of disease burden, injury and risk factors display that cerebral infarction is the second most common cause for the death, the third most common cause for the disabled. The incidence, mortality, disability rate and recurrence rate of cerebral infarction are increasing year by year. Ischemic stroke brings a heavy burden to the social and family because of its long recovery time and unsatisfactory prognosis. After cerebral infarction, in the infarct core area, obvious oxygen content reduces and nerve tissue is necrosis rapidly. However in the infarct surrounding area, oxygen content and blood perfusion moderately reduce. This damage could be saved. Because there is a risk of infarction in this area, but injure has not been formed yet. As a result, the ischemic penumbra is the key point of vascular recanalization and neuroprotective therapy. Intravenous thrombolysis is considered to the most effective treatment for acute cerebral infarction, but due to the strict treatment time window and bleeding complications, intravenous thrombolysis rate is not high. Therefore, it is very important to find new drug targets and provide a reliable basis for the treatment of cerebral infarction.Blood brain barrier(BBB) is a special barrier between the brain blood circulation and nerve tissue, the main function of BBB is maintaining brain tissue homeostasis, regulating the balance within the substance exchange in brain and protect brain tissue against injure. BBB is constituted by brain capillary endothelial cells, tight junction protein between endothelial cell, capillary basement membrane, the surrounding astrocyte and pericytes, each of them plays the indispensable role in maintaining the integrity of the BBB. When a type of cell is damaged, the barrier is broken, a series of serious consequences such as neuroinflammation and neurodegeneration will come up. In the pathological and physiological process of ischemic stroke, BBB function disorder and cell primary and secondary destruction, death. Because BBB plays a crucial role in the pathogenesis of cerebrovascular disease, BBB is considered to be a new target for the treatment of cerebral infarction, and the researches about blood brain barrier is the basis for new clinical drug studies.Nuclear transcription factor Nrf-2(factor NFE2-related 2, Nrf-2), which is involved in the process of transcription by binding to cell pressure signals, regulates many kinds of detoxifying enzymes and antioxidant enzymes expression. Nrf-2/HO-1(heme oxygenase 1, HO-1) signaling pathway is a most important cellular defense mechanisms, Nrf-2 as a brain protective factor of cerebral infarction, plays the vital role of regulating inflammatory reaction and oxidative stress response. After cerebral infarction, there were a large number of reactive oxygen species(ROS). ROS is involved in the oxidative stress reaction and the release of toxic substances. However, after cerebral infarction, using drugs to activate the Nrf-2/HO-1 signaling pathway, will trigger the intracellular antioxidant stress response, so the Nrf-2 signaling pathway plays an role in cell protection. In the experimental brain injury model, the Nrf-2 signal pathway was activated by sulforaphane, a Nrf-2 agonist, the integrity and stability of the blood brain barrier were improved. Therefore, the Nrf-2 signaling pathway and its downstream protective factor are the key targets for the treatment of cerebral infarction.Leonurine(LEO, C14H21N3O5), as the effective components of Herba Leonuri, has a variety of biological effects such as antioxidant, antiplatelet aggregation, promoting uterine contraction, activating blood and dissolving. LEO inhibits vascular smooth muscle contraction by blocking calcium influx. In the rat middle cerebral artery model, leonurine protected the brain tissue though maintaining mitochondrial function. In vitro experiments, leonurine played a protective role in myocardial ischemia by antioxidant reactions. However, it is poorly understood whether leonurine can stabilize the BBB function and maintain BBB integrity after stroke. In this study, we explored the potential beneficial effects of leonurine on BBB and gained insight into the underlying mechanisms after stroke.Male, healthy CD-1 mice and Nrf-2 gene mice were used and subjected to modified permanent middle cerebral artery occlusion(p MCAO), as described by Longa previously. In the mice experimental cerebral infarction model induced by permanent middle cerebral artery occlusion using leonurine, observed the protective effect of leonurine on mice experimental cerebral infarction by evaluating cerebral infarction volume, brain tissue water content and neurological function assessment; evaluated leonurine protective effect on mice experimental cerebral infarction BBB through observation Evans blue(EB) exudation, tight junction proteins related factors matrix metalloproteinases-9(MMP-9) and claudin-5 expression, vascular endothelial growth factor(VEGF) expression, astrocytes activation marker glial fibrillary acidic protein(GFAP) expression and blood brain barrier ultrastructure; determinated the mechanism of leonurine protective effect on mice experimental cerebral infarction blood brain barrier, by detecting Nrf-2/HO1 signal pathway related factors expression. PartⅠ The neuroprotective effect of leonurine on the mice of focal cerebral ischemiaObjective: The aim was to study the neuroprotective effects of leonurine by evaluating the neurological deficit scores, brain water content and infarct volume.Methods: Male, healthy CD-1 mice were used and subjected to modified permanent middle cerebral artery occlusion(p MCAO). CD-1 Mice were divided into 6 groups randomly: Sham group: mice received sham operation intraperitoneal injection with equal volume of normal saline on 2h after surgery; Vehicle group: mice received p MCAO intraperitoneal injection with equal volume of normal saline on 2h after surgery; LEO25 group: mice received p MCAO intraperitoneal injection with leonurine 2.5mg/kg on 2h after surgery;LEO50 group: mice received p MCAO intraperitoneal injection with leonurine 5.0mg/kg on 2h after surgery; LEO100 group: mice received p MCAO intraperitoneal injection with leonurine 10.0mg/kg on 2h after surgery; LEO150 group: mice received p MCAO intraperitoneal injection with leonurine 15.0mg/kg on 2h after surgery; Neurological behavior was evaluated by neurological deficit scores, brain water content was measured by wet-dry method and infarct volume was analyzed with 2, 3, 5- triphenyltetrazolium chloride(TTC) staining. Neurological deficits, brain water content and infarct volume were evaluated at 24 h after p MCAO to estimate leonurine’s neuroprotective effect.Results:1 Mice in Sham group had no neurologic defect and a neurological deficit score of zero; Vehicle group was more serious in the neurological deficit scores at 24 h. Compared with Vehicle group, LEO25 group was not significantly reduced in the neurological deficit scores at 24 h, the difference was not statistically significant(P>0.05). By contrast, compared with vehicle group, LEO50, LEO100 and LEO150 group was significantly reduced in the neurological deficit scores at 24 h, the difference was statistically significant(P< 0.05)2 Compared with vehicle group, there was no significant effect in brain water content in LEO25 and LEO50 group at 24 h after p MCAO, the difference was not statistically significant(P>0.05). However, there was significantly lower effect in brain water content in LEO100 and LEO150 group at 24 h after p MCAO, the difference was statistically significant(P< 0.05)3 Compared with vehicle group, there was no significant amelioration about infarct volume in LEO25 group(P>0.05) but totally significant amelioration in LEO50, LEO100 and LEO150 group(P<0.05).Conclusions: After leonurine administration, we observed ameliorated neurological infarct volume, infarct volume, brain edema. Part Ⅱ Effect of leonurine on protection of blood brain barrier in experimental ischemic strokeObjective: Evaluated effect of leonurine on protection of blood brain barrier in experimental ischemic stroke, through the evans blue exudation, MMP-9, claudin-5, VEGF and GFAP expression, blood brain barrier ultrastructure changes.Methods: Male, healthy CD-1 mice were used and subjected to modified permanent middle cerebral artery occlusion(p MCAO). CD-1 Mice were divided into 6 groups randomly: Sham group: mice received sham operation intraperitoneal injection with equal volume of normal saline on 2h after surgery; Vehicle group: mice received p MCAO intraperitoneal injection with equal volume of normal saline on 2h after surgery; LEO25 group: mice received p MCAO intraperitoneal injection with leonurine 2.5mg/kg on 2h after surgery;LEO50 group: mice received p MCAO intraperitoneal injection with leonurine 5.0mg/kg on 2h after surgery; LEO100 group: mice received p MCAO intraperitoneal injection with leonurine 10.0mg/kg on 2h after surgery; LEO150 group: mice received p MCAO intraperitoneal injection with leonurine 15.0mg/kg on 2h after surgery; neurological behavior was evaluated by neurological deficit scores. Observed the evans blue exudation at 24 h after p MCAO. In subsequent experiments, CD-1 Mice were divided into 3 groups randomly: Sham group: mice received sham operation intraperitoneal injection with equal volume of normal saline on 2h after surgery; Vehicle group: mice received p MCAO intraperitoneal injection with equal volume of normal saline on 2h after surgery; LEO100 group: mice received p MCAO intraperitoneal injection with leonurine 10.0mg/kg on 2h after surgery; Neurological behavior was evaluated by neurological deficit scores, western blot, reverse transcription-polymerase chain reaction and immunehistochemical staining were employed to detect the expression of MMP-9, claudin-5, VEGF and GFAP at 24 h after p MCAO. The ultrastructure changes of blood brain barrier were observed by electron microscope at 24 h after p MCAO.Results: Ischemic brain tissue was observed blood brain barrier perm- eability increased, MMP-9 expression increased, claudin-5, VEGF and GFAP expression decreased, endothelial cells edema and pinocytotic vesicles increased at 24 h after cerebral infarction. However, leonurine relieved BBB permeability, decreased MMP-9, improved tight junction claudin-5, enhanced the factor of angiogenesis VEGF, advanced the astrocyte activation marker GFAP, relieved endothelial cells edema, reduced the pinocytotic vesicles.1 There were no evans blue leakage in sham group; there were evans blue leakage very serious in vehicle group at 24 h after p MCAO; Compared with vehicle group, there was a statistically significant decreased of the permeability of BBB in LEO25, LEO50, LEO100 and LEO150 group(P<0.05). Accordingly, we observed from neurological deficit score, cerebral edema, cerebral infarction volume and Evans blue extravasation, and found that leonurine can play a beneficial therapeutic effect in high dose(15.0mg/kg) and middle dose(10.0mg/kg).Therefore, we focused on the leonurine treatment at 10.0 mg/kg for the subsequent study.2 In immunohistochemical staining, compared with Vehicle group, the number of MMP-9, claudin-5, VEGF and GFAP positive cells were changed in LEO100 group at 24 h after cerebral ischemia.3 In western blot analysis, compared with Vehicle group, the MMP-9, claudin-5, VEGF and GFAP expression levels in the right cortex were significantly changed in LEO100 group at 24 h after cerebral ischemia(P< 0.05).4 In RT- q PCR analysis, compared with Vehicle group, the MMP-9, claudin-5, VEGF and GFAP expression levels in the right cortex were significantly changed in LEO100 group at 24 h after cerebral ischemia(P< 0.05).5 There are normal structure and just little pinocytosis vesicles in natural blood brain barrier endothelial cells. Normal structure and little pinocytosis vesicles are found in sham group. Swollen endothelial cell and plenty of pinocytosis vesicles are found in Vehicle group at 24 h after cerebral ischemia. Compared with Vehicle group, mild swollen endothelial cell and little pinocytosis vesicles are found in LEO100 group at 24 h after cerebral ischemia.Conclusions: Ischemic brain tissue was observed blood brain barrier permeability increased, leonurine relieved BBB permeability. Leonurine decreased MMP-9 expression, improved tight junction claudin-5 expression, enhanced the expression of VEGF and advanced the astrocyte activation marker GFAP expression. Leonurine relieved endothelial cells edema, reduced the pinocytotic vesicles. Leonurine can inhibit the degradation of the tight junction protein, reduced the tight junction protein redistribution, start the angiogenesis after infarction, stable endothelial cells, activation of glial cells, maintain the blood brain barrier function, increase the stability of the blood brain barrier. While the function of leonurine maintain blood brain barrier is consistent with protective effect in experimental cerebral infarction. So we speculated that leonurine play a protective effect on the brain through maintaining the function of BBB. Part Ⅲ The related mechanism of leonurine protecting blood brainbarrier in experimental ischemic strokeObjective: Though observing the expression of Nrf-2/HO-1 signaling pathway related factors after experimental cerebral infarction and the expression of blood brain barrier related factor after cerebral infarction in Nrf-2 gene mice, studied the related mechanism of leonurine protecting blood brain barrier in experimental ischemic stroke.Methods: Male, healthy CD-1 mice and Nrf-2 gene mice were used and subjected to modified permanent middle cerebral artery occlusion(p MCAO). CD-1 Mice were divided into 3 groups randomly: Sham group: mice received sham operation intraperitoneal injection with equal volume of normal saline on 2h after surgery; Vehicle group: mice received p MCAO intraperitoneal injection with equal volume of normal saline on 2h after surgery; LEO100 group: mice received p MCAO intraperitoneal injection with leonurine 10.0mg/kg on 2h after surgery. Neurological behavior was evaluated by neurological deficit scores, western blot, reverse transcription-polymerase chain reaction and immunehistochemical staining were employed to detect the expression of Nrf-2 and HO-1 at 24 h after p MCAO. Nrf-2 gene mice were divided into 4 groups randomly: Nrf-2+/+Vehicle group: mice received p MCAO intraperitoneal injection with equal volume of normal saline on 2h after surgery; Nrf-2+/+LEO100 group: mice received p MCAO intraperitoneal injection with leonurine 10.0mg/kg on 2h after surgery. Nrf-2-/-Vehicle group: mice received p MCAO intraperitoneal injection with equal volume of normal saline on 2h after surgery; Nrf-2-/-LEO100 group: mice received p MCAO intraperitoneal injection with leonurine 10.0mg/kg on 2h after surgery. Western blot and reverse transcription-polymerase chain reaction were employed to detect the expression of MMP-9, claudin-5, VEGF and GFAP at 24 h after p MCAO.Results:Ischemic brain tissue was observed Nrf-2 and HO-1 expression decreased at 24 h after cerebral infarction. However, leonurine improved the expression of Nrf-2 and HO-1 in CD-1 mice. Leonurine could not decrease the expression of MMP-9, improve the expression of claudin-5, VEGF and GFAP in Nrf-2 gene mice.1 In immunohistochemical staining, compared with Vehicle group, the number of Nrf-2 and HO-1 positive cells were increased in LEO100 group at 24 h after cerebral ischemia.2 In western blot analysis, compared with Vehicle group, the Nrf-2 and HO-1 expression were increased in LEO100 group at 24 h after cerebral ischemia(P<0.05).3 In RT- q PCR analysis, compared with Vehicle group, the Nrf-2 and HO-1 expression were increased in LEO100 group at 24 h after cerebral ischemia(P<0.05).4 In western blot analysis, compared with Nrf-2-/-Vehicle group, the MMP-9, claudin-5,VEGF and GFAP expression levels in the right cortex were not significantly changed in Nrf-2-/-LEO100 group at 24 h after cerebral ischemia(P>0.05).5 In RT-q PCR analysis, compared with Nrf-2-/-Vehicle group, the MMP-9, claudin-5,VEGF and GFAP expression levels in the right cortex were not significantly changed in Nrf-2-/-LEO100 group at 24 h after cerebral ischemia(P>0.05).Conclusions: Leonurine can activate Nrf-2/HO-1 signaling in ischemic brain tissue pathway after experimental cerebral infarction. The role of leonurine protect blood brain barrier is inhibited in Nrf-2 knockout mice. This suggests that leonurine may play a beneficial role of blood brain barrier function through activation Nrf-2/HO-1 signaling pathways, so as to protect brain tissue. |
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