Ulinastatin affects focal cerebral ischemia-reperfusion injury via SOCS1-mediated JAK2/STAT3 signalling pathway.

Cerebral ischemia results in loss of cerebral blood flow, which contributes to neuronal damage, neurocognitive impairment, as well as learning and memory difficulties. Although reperfusion is necessary to restore the blood supply to the brain, it also leads to several detrimental effects on the brain. The purpose of this study was to assess the effects of ulinastatin (UTI) on preventing focal cerebral ischemia/reperfusion-induced injury (FCIRI). First, a rat model of FCIRI was established and treated with UTI. The effects of UTI on FCIRI in rats were evaluated using Morris water maze assay, triphenyl tetrazolium chloride staining, TUNEL, western blot assay, and enzyme-linked immunosorbent assay analysis. UTI was found to improve the learning memory ability, reduce infarction area, inhibit apoptosis and decrease inflammation in FCIRI rats. Messenger RNA microarray analysis of hippocampal tissues revealed that suppressor of cytokine signalling-1 (SOCS1) was the downstream target of UTI in FCIRI. SOCS1 depletion impaired the protective effect of UTI on FCIRI in rats. SOCS1 blocked the activation of the JAK2/STAT3 pathway. JAK2 inhibitor caused the JAK2/STAT3 pathway deficit, hence reversing the effect of sh-SOCS1 on FCIRI in rats. Taken together, our results demonstrate that UTI alleviated FCIRI in rats, which was, to some extent, related to SOCS1-mediated JAK2/STAT3 pathway.

Isoastilbin inhibits neuronal apoptosis and oxidative stress in a rat model of ischemia-reperfusion injury in the brain: Involvement of SIRT1/3/6.

BACKGROUND: Isoastilbin (IAB) has been shown to have antioxidative and anti-apoptotic functions. A recent study found that IAB can reduce oxidative stress in Alzheimer's disease. However, whether the antioxidative function of IAB is also protective in other brain diseases remains unknown. OBJECTIVES: To investigate the roles and underlying mechanisms of IAB in middle cerebral artery occlusion-reperfusion (MCAO/R) in rats. MATERIAL AND METHODS: Male Wistar rats were randomly divided into 5 groups: sham group, MCAO/R group, and 3 MCAO/R groups groups administered IAB (20 mg/kg, 40 mg/kg or 80 mg/kg) once a day for 3 days. Infarction size, modified Neurological Severity Score (mNSS), oxidative stress markers, and neuronal apoptosis markers were used to assay the function of IAB. RESULTS: Compared with the MCAO/R group, administration of IAB reduced the infarction size and mNSS scores in MCAO/R rats. Isoastilbin also decreased the level of malondialdehyde (MDA) and enhanced the activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX). Isoastilbin treatment attenuated MCAO/R-induced neuronal apoptosis compared with the MCAO/R group, as indicated by the results of terminal deoxynucleotide transferase-mediated X-dUTP nick end (TUNEL) and western blot assays. Isoastilbin also reversed MCAO/R-induced downregulation of SIRT1/3/6 protein expression. CONCLUSIONS: These observations suggest that IAB protects against oxidative stress and neuronal apoptosis in rats following cerebral ischemia-reperfusion (I/R) injury through the upregulation of SIRT1/3/6, indicating that IAB might be a promising therapeutic agent for cerebral I/R injury.

MicroRNA-193b-3p alleviates focal cerebral ischemia and reperfusion-induced injury in rats by inhibiting 5-lipoxygenase expression.

AIMS: Ischemic stroke has become one of the main causes of death worldwide. MicroRNAs (miRNAs) have been implicated in cerebral ischemia-reperfusion (I/R) injury and could serve as therapeutic targets. 5-Lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of leukotrienes and has been implicated in inflammatory central nerve system disorders. The objective of this study was to explore the neuroprotective effects of miR-193b-3p against focal cerebral I/R injury in rats by regulating 5-LOX expression. METHODS AND MATERIALS: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion injury. The level of miR-193b-3p expression was observed in the rat cortical peri-infarct region after focal cerebral I/R injury. Bioinformatics analysis was used to predict the binding sites of miR-193b-3p, and a dual-luciferase reporter gene assay was applied to verify the potential interaction between 5-LOX mRNA and miR-193b-3p. Then, rats were injected with a miR-193b-3p agomir (modified and enhanced mimic) or antagomir (modified and enhanced inhibitor) in the right lateral ventricle of the brain. Neurological deficit scores, infarct volumes, neuron damage and 5-LOX enzymatic activity and expression were measured. In an in vitro experiment, cultured PC12 cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R). OGD/R-induced cells were treated with a miR-193b-3p mimic or inhibitor and 5-LOX siRNA. Cell viability, lactate dehydrogenase release, apoptosis rate and 5-LOX expression were evaluated. RESULTS: The level of miR-193b-3p expression was increased in the cortical peri-infarct region of rats with cerebral focal I/R injury. The results of the dual-luciferase reporter gene assay showed that a miR-193b-3p binding site was located in the 3' untranslated region (3'UTR) of 5-LOX mRNA. Neurological deficit scores, infarct volumes and neuronal injury were alleviated by miR-193b-3p agomir treatment but aggravated by miR-193b-3p antagomir. Furthermore, leukotriene B4, cysteinyl-leukotrienes and 5-LOX expression in the cortical peri-infarct region of rats with focal cerebral I/R injury were also downregulated by miR-193b-3p agomir treatment but upregulated by miR-193b-3p antagomir. In PC12 cells, miR-193b-3p mimic significantly decreased OGD/R-induced cell death and reduced lactate dehydrogenase release and 5-LOX expression. In contrast, miR-193b-3p inhibitor exacerbated OGD/R-induced injury in PC12 cells. Additionally, the in vitro effects of miR-193b-3p inhibitor on OGD/R-induced cell injury were partially reversed by 5-LOX siRNA treatment. CONCLUSION: MiR-193b-3p has a potentially neuroprotective effect on focal cerebral I/R-induced injury by inhibiting 5-LOX expression.

Conditioned medium obtained from human amniotic mesenchymal stem cells attenuates focal cerebral ischemia/reperfusion injury in rats by targeting mTOR pathway.

Conditioned medium obtained from human amniotic mesenchymal stem cells (hAMSC-CM) was recently shown to have many antioxidant, antiapoptotic and proangiogenic growth factors. The present study was performed to investigate whether protective effects of hAMSC-CM against focal cerebral ischemia/ reperfusion (I/R) injury is associated with modulation of the mammalian target of rapamycin (mTOR) pathway. A rat model of middle cerebral artery occlusion (MCAO) was created and the animals were divided into three groups including sham, MCAO and MCAO + hAMSC-CM. Drug was administrated immediately after cerebral reperfusion (i.v). The expressions of mTOR, p-mTOR and LC3 were measured using Western blotting and real time-PCR, respectively. Apoptosis and neuronal loss were determined using TUNEL and Nissl staining, respectively. Infarct volume and the blood-brain barrier (BBB) damage were evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining and Evans Blue (EB) uptake, respectively. Compared with sham, significant infarct volume, apoptotic cell death, and neuronal loss were found in MCAO rats that reversed by hAMSC-CM (P < 0.05). Likewise, MCAO rats exhibited increased mRNA level of light-chain 3 (LC3) and the LC3II/LC3I ratio as well as decreased expression level of p-mTOR that reversed by hAMSC-CM (P < 0.05). There were no significant differences in the expression of total mTOR among the experimental groups. In summary, our results demonstrate that hAMSC-CM gives rise to neuroprotection following ischemic stroke by restoring mTOR activity and inhibiting autophagy.

Effect of sufentanil postconditioning on the focal cerebral ischemia reperfusion injury in diabetic rats / 中华行为医学与脑科学杂志

Objective To explore the effect of sufentanil postconditioning on the focal cerebral is-chemia reperfusion injury in diabetic rats. Methods An intraperitoneal injection of 50 mg/kg streptozotocin was used to induce diabetes in rats. Meanwhile,the diabetes mellitus model was confirmed by the blood glu-cose level over 16. 7 mmol/L. The diabetes mellitus male SD rats,weighting 250-300 g,were randomly divid-ed into 3 groups:sufentanil postconditioning diabetic group (SP-DM),ischemia reperfusion diabetic group (IR-DM),sham operation diabetic group(sham-DM),with 12 in each group. The non-diabetic rats were randomly divided into 3 groups:sufentanil postconditioning non-diabetic group(SP-NDM), ischemia reperfu-sion non-diabetic group(IR-NDM),sham operation non-diabetic group(sham-NDM),with 12 in each group. All rats in the IR-NDM/DM group and SP-NDM/DM group were exposed to the right middle cerebral artery occlusion for 90 minutes followed by 24 hours reperfusion. The sufentanil 1 μg/kg were injected into the rats in SP-NDM/DM group via tail vein at 5 minutes before reperfusion. Normal saline was injected into the rats in sham-NDM/DM group and IR-NDM/DM group at 5 minutes before reperfusion. At 24 hours after reperfu- sion,the neurological deficit scores( NDS) were assessed,then all the rats were sacrificed. Infarct volume, which was determined by 2,3,5-triph-enyltetrazolium ( TTC) staining,and water content of right hemisphere for brain edema were also measured. Results All rats showed neurological deficit,brain infarction and brain edema after focal cerebral ischemia reperfusion. (1) At 24 hours after reperfusion,the neurological deficit score in IR-DM group(3. 4±0. 4) was significantly higher than that in the IR-NDM group(2. 8± 0. 5) ( t=2. 313,P<0. 05),there was no significant difference in neurological deficit score between the SP-DM group (3. 3±0. 4) and the IR-DM group(t=1. 546,P>0. 05). (2) At 24 hours after reperfusion,the infarct volume in IR-DM group((58. 3±2. 1)%) was significantly higher than that in the IR-NDM group((32. 1±2. 6)%) (t=2. 912, P<0. 05), there was no significant difference in infarct volume between the SP-DM group ((56. 9±2. 1)%) and the IR-DM group(( 58. 3 ± 2. 1)%) ( t=1. 633,P>0. 05). ( 3) At 24 hours after reperfusion,the water content of the right hemisphere in IR-DM group(( 89. 3± 3. 5)%) was significantly higher than that in the IR-NDM group((82. 6±3. 9)%)(t=2. 218,P<0. 05),there was no significant differ-ence in water content of the right hemisphere between the SP-DM group(( 87. 5±3. 4)%) and the IR-DM group(t=1. 730,P>0. 05). Conclusion Sufentanil postconditioning loses neuroprotection against focal cer-ebral ischemia reperfusion injury in diabetic rats.

Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1.

Electroacupuncture preconditioning at acupoint Baihui (GV20) can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 (Drp1) can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 (depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day). Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

Sesamol attenuates oxidative stress, apoptosis and inflammation in focal cerebral ischemia/reperfusion injury.

The aim of the present study was to evaluate the therapeutic potential of sesamol treatment on focal ischemia/reperfusion (I/R) injury in the rat brain. The results demonstrated that pretreatment with sesamol seven days prior to focal cerebral I/R injury had significant positive effects, including improvements in neurological deficits (P<0.05), and a reduction in malondialdehyde content and elevation of antioxidant levels (superoxide dismutase, glutathione and glutatione peroxidase; both P<0.05). Furthermore, levels of B cell lymphoma-2 (Bcl-2)-associated X protein and caspase-3 were significantly downregulated, whereas the level of Bcl-2 was effectively increased. Conversely, the mRNA expression of proinflammatory cytokines were significantly reduced in focal cerebral I/R injury rats upon sesamol intervention. Therefore, the beneficial effects of sesamol on cerebral I/R injury may be due to the reduction of oxidative stress, inhibition of apoptosis and inflammation. The findings of the present study suggest that sesamol supplementation may serve as potent adjuvant in the treatment of focal cerebral ischemia/reperfusion injury due to its neuroprotective effects.

The protective effect of polymerized porcine hemoglobin (pPolyHb) on transient focal cerebral ischemia/reperfusion injury.

Glutaraldehyde-polymerized porcine hemoglobin (pPolyHb) is a hemoglobin-based oxygen carrier currently being developed as a potential red blood cell substitute. We assessed the protective effect of pPolyHb on transient focal cerebral ischemia/reperfusion (I/R) injury. Several outcomes were tested, including infarct size, neurological score, production of inflammatory factors, and markers of oxidative status. Our results show that pPolyHb can not only significantly reduce infarct volume and improve neurological score, but can also inhibit the expression of TNF-α and IL-1ß. Furthermore, the generation of MDA and MPO was reduced by pPolyHb. We conclude that pPolyHb has a positive effect on transient focal cerebral I/R injury.

Senkyunolide I protects rat brain against focal cerebral ischemia-reperfusion injury by up-regulating p-Erk1/2, Nrf2/HO-1 and inhibiting caspase 3.

Oxidative damage and apoptosis are critical factors contributing to neuronal death during a stroke. The aim of the present study was to evaluate the neuroprotective effects of senkyunolide I (SEI) on focal cerebral ischemia-reperfusion (I/R) injury in rats, and investigate the underlying mechanisms. Male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (tMCAO) for 2h, followed by 24h reperfusion, and then randomly assigned into four groups: Sham (sham-operated), Vehicle (tMCAO +normal saline), SEI-L (tMCAO +SEI 36 mg/kg) and SEI-H (tMCAO +SEI 72 mg/kg) groups. SEI was administered intravenously, 15 min after occlusion. Neurological deficit, brain edema and infarct volume were detected after 24h of reperfusion. Histological structures of cortices and hippocampus were observed by hematoxylin and eosin staining. Biochemical indexes in the cortex were assayed by colorimetry. The impact of SEI on the Nrf2-ARE-interaction was assayed using a luciferase reporter gene. Western blotting was performed to analysis the expressions of proteins related to anti-oxidation and apoptosis. SEI administration significantly ameliorated the neurological deficit, reduced the infarct volume and brain edema, reversed the cerebral morphologic damage, decreased the levels of MDA and increased the activities of superoxide dismutase. Furthermore, the high dose SEI could significantly activate the Nrf2/ARE pathway by up-regulating the phosphorylation of Erk1/2 and inducing Nrf2 nuclear translocation with enhanced HO-1 and NQO1 expressions. Additionally, treatment with SEI remarkably promoted the ratio of Bcl-2/Bax and inhibited the expressions of cleaved caspase 3 and caspase 9. These results suggest that the neuroprotective mechanisms of SEI are associated with its anti-oxidation and anti-apoptosis properties.

Protective effect of Sheng-Nao-Kang decoction on focal cerebral ischemia-reperfusion injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Sheng-Nao-Kang decoction (SNK), a modified traditional Chinese medicine (TCM), has been used clinically for the treatment of acute and chronic cerebrovascular related diseases. To evaluate the protective effect of SNK on focal cerebral ischemia-reperfusion (I/R) injury in rats and investigate the underlying mechanisms. MATERIALS AND METHODS: Focal cerebral I/R injury in rats was induced by middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion for 24h. Adult male Sprague-Dawley (SD) rats were randomly divided into six kinds of groups: Sham group; I/R group; SNK-treated groups at doses of 0.7 g/kg, 1.4 g/kg and 2.8 g/kg; and nimodipine (NMP)-treated group. The recoveries of neurological function in rats were estimated by neurological defect scoring and 2,3,5-triphenyltetrazolium chloride (TTC) staining after 24h reperfusion. Various biochemical indexes in serum were assayed by colorimetry, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), inducible nitric oxide synthase (iNOS) and total nitric oxide synthase (TNOS). Histological structures of the brain in rats were observed by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed to detect the caspase-3 protein content in rats. RESULTS: SNK administration significantly reduced the neurological defect scores and lessened the cerebral infarction volume. The treatment of SNK lowered MDA content, up-regulated SOD and GSH-Px levels, down-regulated iNOS and TNOS levels in serum. Furthermore, histological examination indicated that dense neuropil and largely surviving neurons were seen in SNK-treated rats. SNK administration restrained the expression of caspase-3 positive protein significantly. CONCLUSION: The results suggest that SNK demonstrates a strong and ameliorative effect on cerebral I/R damage in rats. The protective mechanisms of SNK are associated with its properties of anti-apoptosis and anti-oxidation as well as regulation of iNOS and TNOS.

Total flavonoid of Litsea coreana leve exerts anti-oxidative effects and alleviates focal cerebral ischemia/reperfusion injury.

In this study, we hypothesized that total flavonoid of Litsea coreana leve (TFLC) protects against focal cerebral ischemia/reperfusion injury. TFLC (25, 50, 100 mg/kg) was administered orally to a rat model of focal ischemia/reperfusion injury, while the free radical scavenging agent, edaravone, was used as a positive control drug. Results of neurological deficit scoring, 2,3,5-triphenyl tetrazolium chloride staining, hematoxylin-eosin staining and biochemical tests showed that TFLC at different doses significantly alleviated cerebral ischemia-induced neurological deficits and histopathological changes, and reduced infarct volume. Moreover, it suppressed the increase in the levels of nitrates plus nitrites, malondialdehyde and lactate dehydrogenase, and it diminished the reduction in gluta-thione, superoxide dismutase and catalase activities induced by cerebral ischemia/reperfusion in-jury. Compared with edaravone, the protective effects of TFLC at low and medium doses (25, 50 mg/kg) against cerebral ischemia/reperfusion injury were weaker, while the protective effects at high dose (100 mg/kg) were similar. Our experimental findings suggest that TFLC exerts neuroprotective effects against focal cerebral ischemia/reperfusion injury in rats, and that the effects may be asso-ciated with its antioxidant activities.

Effects of human urinary kallikrein on cell apoptosis of rats with acute focal cerebral ischemia-reperfusion injury / 中华神经医学杂志

Objective To study the effects of human uriilary kallikrein(HUK)on the number of apoptotic cells and the expressions of Bcl-2 and Bax proteins in rats after focal cerebral ischemia and reperfusion(FCIR) injury. Methods Eighty-four Spmque-Dawley(SD)male rats were randomly divided into sham-operated group(n=12),ischemia-reperfusion group(n=36),and HUK-treated group (n=36). Transient focal cerebml ischemia models were established by middle cerebml artery occlusion.Six rats were chosen from sham-operated group,ischemia-reperfusion group,and HUK-treated group for measuring infarct sizes.The rest were used to evaluate neurologic fhnction impaiment and measure the nunlber of apoptotic cells and Bcl-2 or BaX protein positive cells in cerebral cortex with TUNEL and immunohistochemistry.The latter 2 groups were subdivided into 6,12,24,72,168 h reperfusio groups (each n=6). Results The neurologic function impairmlent score,the infarct sizes,the apoptotic cells and the expression of Bax protein of HUK-treated group at different time points (except 168 h group)significantly decreased compared wilh those of ischemia-reperfsion group (p＜0.05).The expression of Bcl-2 protein of HUK-treated group at different time points(except 168 h group) remarkably increased compared with that of ischemia-reperfusion group(P＜0.05). Conclusions HUK can excrt a protection against FCIR injury, maybe through up-regulating Bcl-2 and down-regulating Bax protein in the initial 3 d of FCIR injury to decrease the number of apoptotic cells

Protective effect of 5-lipoxygenase inhibitor zileuton on focal cerebral ischemia-reperfusion injury in rats / 中国药理学通报

Aim To investigate the protective effect of zileuton,a 5-lipoxygenase inhibitor,on focal cerebral ischemia-reperfusion injury in rats.Methods The right middle cerebral artery of the rat was occluded by inserting a thread through internal carotid artery for 2 h,and then reperfused for 24 h.Zileuton(10,50 mg?kg~(-1)) was orally administered 2 h before ischemia and 0,5,10 h after reperfusion.After 24 h reperfusion,the content of malondialdehyde(MDA) and nitric oxide(NO),the activities of glutathion peroxidase(GSH-PX),myeloperoxidase(MPO) and nitricoxide synthase(NOS) were measured.Results Compared with vehicle group,the infarct size,content of NO and NOS of the brain were significantly reduced in 10 and 50 mg?kg~(-1) zileuton groups;zileuton 50 mg?kg~(-1) also reduced the content of MDA,increased the activity of GSH-PX,and inhibited the increase of MPO in brain tissue.Conclusion Zileuton possesses the neuroprotective effects on focal cerebral ischemia-reperfusion injury in rats.

Protective effects and mechanism of aspirin against cerebral ischemia-reperfusion in rats / 中国药理学通报

Aim To investigate the protective effects and mechanism of aspirin against focal cerebral ischemia-reperfusion in rats. Methods Right middle cerebral artery was occluded by inserting a thread through internal carotid artery for 2 h, and then reperfused for 72 h. 60 mg?kg -1 dose of aspirin was intragastric administrated at 0 h and 6 h after reperfusion. The brain injured area, the mortality, and cerebral edema were estimated. The apoptotic cells of brain tissue were detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) method. Bcl-2 and Bax were detected by immunohistochemical staining method. The activity of calcineurin (CaN) in brain tissue was determined by the inorganic phosphorus method. The content of adenosine 5′-triphosphate (ATP) in brain tissue was separated by capillary electrophoresis. Results By using of aspirin 60 mg?kg -1, all indications were dramatically improved. The injured area of brain [from (10.51?1.12)% to (0.94?0.08)%], the cerebral edema of occluded side [from (82.43?2.0)% to (76.29?0.77)%], and the mortality [from 28% to 0%] were dramatically reduced. In brain tissue of occluded side, 60 mg?kg -1 aspirin helped to reduce the number of apoptotic cells from (26.43?2.0) to (17.53?0.44), increase the ratio of Bcl-2/Bax from (0.61?0.05) to (1.01?0.15), inhibit the activity of CaN from (6.03?1.5) to (3.47?0.96), and improve the ATP level from (10.26?1.02) to (25.65?3.45). Conclusion The neuroprotective effects of aspirin on focal cerebral ischemia-reperfusion injury in rats for 72 h might be attributed to its effects by anti-apoptosis, increasing the ratio of Bcl-2/Bax, inhibiting the activity of CaN, and improving the energy metabolism.