Effects of ischemic postconditioning on hippocampus CA1 area rCBF and astrocyte activation after cerebral ischemia in tree shrews

AIM: To study the effects of ischemic postconditioning (PC) on regional cerebral blood flow (rCBF) and astrocyte (AS) activation in hippocampus CA1 area and to explore the possible mechanism of ischemic PC affecting glial fibrillary acidic protein (GFAP) expression during focal cerebral thrombosis. METHODS: The thrombotic focal cerebral ischemia was induced by photochemical reaction in tree shrews, and ischemic postconditioning was established by cliped ipsilateral carotid of the animal at 4 h after cerebral ischemia. The rCBF and GFAP expressions in hippocampus CA1 area were detected, respectively, by laser-Doppler (LD) fowmeter and immunohistochemistry. RESULTS: The numbers of GFAP positive cells were increased markedly and GFAP expression enhanced (P<0.01). AS oncosis was apparent 24 h after cerebral ischemia. Postconditioning increased hippocampus rCBF from (2.55±0.28) PU to (10.42±3.75) PU (P<0.05) at 24 h and from (9.84±1.22) PU to (18.74±1.60) PU (P<0.05) at 72 h after the cerebral ischemia, and AS oncosis was inhibited markedly. CONCLUSION: Multiple, short, regional carotid occlusions may prolong “time window” of therapeutic cerebral ischemia. The protection mechanism of the ischemic postconditioning may be associated with the increase in rCBF and improvement of hippocampus microenvironment by regulating AS activation.     

Fluorocitrate inhibits regulatory effect of ischemic postconditioning on HIF-1α/iNOS signaling pathway after cerebral ischemia in tree shrews with brain injury

AIM: To investigate the regulatory effect of HIF-1α/iNOS signaling pathway on the neuroprotection of ischemic postconditioning (PC) in tree shrews, and to explore the mechanisms of deteriorated cerebral injury after inhibiting astrocyte (AS) metabolism. METHODS: Thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews. Fluorocitrate (FC) was used to inhibit AS metabolism and the ischemic PC was established at 4 h after ischemia followed by clipped ipsilateral common carotid artery on the ischemia side for 3 times, 5 min/time. A total of 67 male tree shrews were randomly divided into 7 groups:control (n=9), ischemia (4 h and 24 h, n=9 for each group), ischemia with PC (4 h and 24 h, n=9 for each group), and FC pretreatment (4 h and 24 h, n=11 for each group). The cerebral infarction size was detected by TTC staining, and the histological changes of hippocampal neurons were observed under light microscope. The regional cerebral blood flow (rCBF) in ischemic cortex was monitored by laser Doppler brain flowmetry. The protein expression of iNOS in hippocampus was detected both by immunohistochemistry and Western blot. The production of NO detected by spectrophotometer. The level of HIF-1α in hippocampus analyzed by ELISA. RESULTS: The cerebral infarct volume was increased with prolonged duration of ischemia, and the changes of ischemia at 24 h were significant (P<0.05). The cortical rCBF was progressively decreased, and it was decreased at 4 h and 24 h after ischemia (P<0.05). The expression of HIF-1α and iNOS in hippocampus was enhanced, and the production of NO was increased significantly (P<0.05). Ischemic PC restored the cortical rCBF (P<0.05), reduced cerebral infarction volume (P<0.05), down-regulated iNOS expression and reduced NO production in the hippocampus (P<0.05). However, the cortical rCBF in FC pretreatment group was significantly lower than that in ischemic group (P<0.05), the neuronal damage was aggravated, and the infarction volume was increased after pretreatment with FC (P<0.05). CONCLUSION: Ischemic PC may reduce cerebral injury by regulating the expression of HIF-1α and iNOS. Inhibition of AS function may attenuate the protective effect mediated by ischemic PC and aggravate brain injury.     

Mechanism of JAK2-STAT3 signaling pathway in ischemic postconditio-ning neuroprotection after cerebral ischemia in tree shrews

AIM: To investigate the regulatory effect of JAK2-STAT3 signaling pathway on the neuroprotection of ischemic postconditioning (IPoC) in tree shrews, and to explore the mechanisms of cerebral injury deterioration after inhibiting the JAK2-STAT3 pathway. METHODS: The model of thrombotic cerebral ischemia was induced by photochemical reaction in tree shrews and the IPoC was established at 4 h after ischemia followed by clipping ipsilateral common carotid artery on the ischemia side for 5 min (3 times). After IPoC and intracerebroventricular injection of AG490 (JAK2 inhibitor), the changes of cerebral infarction area were detected by TTC staining, and the histological and ultrastructural changes of cortical neurons were observed under light and electron microscopes, respectively. The protein levels of t-STAT3 and p-STAT3 in the cortical tissue were determined by Western blot. RESULTS: The neuronal pycnosis, mitochondrial swelling and vanish of the mitochondrial cristae were found in cortical cortex, and the infarction area was (24.78±3.30)% at 24 h after cerebral ischemia. Meanwhile, the phosphorylation level of STAT3 protein in the cortical tissue was significantly increased (P<0.01). The cortical neuronal damage and mitochondrial swelling were decreased after IPoC, the area of cerebral infarction was significantly reduced to (17.67±1.83)% (P<0.01), and the phosphorylation level of STAT3 protein was further increased (P<0.01). However, the neuronal damage was aggravated, the infarction area was expanded to (23.85±2.77)%(P<0.05) after treatment with AG490, and the phosphorylation level of STAT3 protein was also significantly reduced (P<0.05). CONCLUSION: IPoC may reduce cerebral injury by regulating the phosphorylation of STAT3 protein, and inhibition of JAK2-STAT3 signaling pathway may counteract the cerebral protective effect of IPoC and aggravate brain injury.     

Postconditioning modulates TLR4 expression in hippocampus of tree shrews with thrombotic cerebral ischemia

AIM: To observe the effects of thrombotic cerebral ischemia and postconditioning on the expression of toll-like receptor 4 (TLR4) in hippocampus of tree shrews.METHODS: The model of thrombotic focal cerebral ischemia was established by photochemical reaction.Four hours after the onset of photochemical reaction, ischemic postconditioning was induced by 3 repeated cycles of carotid artery occlusion for 5 min and reperfusion for 5 min. The histological changes of hippocampus (by HE staining), TLR4 protein level (by Western blotting) and TLR4 mRNA expression (by semiquantitative RT-PCR) were observed.RESULTS: The extensive neuronal degeneration in hippocampus was observed from 4 h to 72 h and peaked at 24 h after cerebral ischemia, but was significantly attenuated after postconditioning. Cerebral ischemia caused a progressive increase in the expression of TLR4 protein at 4 h and 24 h (P<0.05), and decreased at 72 h (P<0.05). In contrast to ischemia groups, postconditioning decreased the expression of TLR4 protein at 4 h and 24 h (P<0.05), but an increase in the expression of TLR4 at 72 h (P<0.05) was observed. Simultaneously, the level of TLR4 mRNA in hippocampus showed the tendency of approximate variation in accordance with the protein expression.CONCLUSION: The expression of TLR4 increases by cerebral ischemia. The protection mechanisms of postconditioning may be associated with modulating TLR4 expression.     

Effects of cerebral ischemia and postconditioning on signaling molecules PERK and GRP78 in hippocampus tissue of tree shrew during endoplasmic reticulum stress

AIM: To investigate the effects of cerebral ischemia and postconditioning on protein kinase R-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78) in the hippocampus tissue of tree shrew during endoplasmic reticulum stress and the mechanism of post-conditioning protecting the brain from damage. METHODS: The focal cerebral ischemic model was duplicated by photochemical reaction in tree shrew and the postconditioning was induced by alternatively occluding and opening the carotid artery of ischemic side for 3 cycles (5 min each cycle) at 3.5 h after ischemia. The damage and ultrastructural changes of the hippocampal neurons were observed by HE staining. The expression of PERK and GRP78 at mRNA and protein levels in the hippocampal tissue at different time points after cerebral ischemia and postconditioning was determined by RT-PCR, immunohistochemistry and Western blot. RESULTS: The injuries of hippocampal neurons were aggravated with prolonged cerebral ischemia, which was most severe at 24 h after ischemia while the postconditioning alleviated these damages correspondingly. The expression of PERK at mRNA and protein levels was upregulated at 4 h, 24 h and 72 h after ischemia (P<0.05), while postconditioning downregulated the expressions of PERK at ischemia and postconditioning 4 h (IP4 h) gruop and IP24 h group (P<0.05). The expression of GRP78 at mRNA and protein levels was not changed at 4 h, 24 h and 72 h after ischemia, while postconditioning upregulated the expressions of GRP78 at IP24 h group (P<0.05). CONCLUSION: The focal thrombotic cerebral ischemia activates the endoplasmic reticulum stress in ischemic hippocampus of tree shrews, leading to the changes in mRNA and protein expression of PERK in the PERK/eIF2α signal transduction pathway. The postconditioning treatment alleviates endoplasmic reticulum stress and neuronal damages by downregulating PERK and upregulating GRP78, thereby protecting the brain from injury.     

Effects of hyperglycemia on ionic homeostasis in hippocampal microenvironment and secondary neuronal injury after focal ischemia in cerebral cortex of tree shrews

AIM：To observe the effects of hyperglycemia on the ionic homeostasis in hippocampal microenvironment after thrombotic cortical ischemia in tree shrews, and to explore the action and mechanisms of hyperglycemia in secondary neuronal injury after ischemia. METHODS：High blood glucose in tree shrews was induced by intraperitoneal injection of streptozocin. Focal thrombotic cortical ischemia was induced by photochemical method in tree shrews. At 4, 24 and 72 h after ischemia, the changes of pH, K+, Na+, Ca2+ and Cl- in the ipsilateral ischemic hippocampal microenvironment were tested by a single-pumped push-pull microdialysis system and an ion analyzer. The histopathological changes and hippocampal neuronal density were also examined. RESULTS：After cortical ischemia in tree shrews, the pH and the concentrations of Na+, Ca2+ and Cl- in the hippocampal microenvironment decreased, while the concentration of K+ increased. These differences were the most significant at 4 h, the second at 24 h and insignificant at 72 h. Combination of hyperglycemia and cerebral ischemia worsened the turbulence of ionic homeostasis. Compared with the normoglycemic ischemic animals, the changes of pH, K+ and Ca2+ concentrations at 4 h as well as pH and Na+ at 24 h in the hyperglycemic ischemic animals were more significant (P<0.05). The results of histopathological examination showed that there was ischemic neuronal damage in the exposed cerebral cortex and the ipsilateral hippocampal CA1 region at 4 h after photochemical reaction, and the damage was the most severe at 24 h, subsequently accompanied with glial proliferation at 72 h. The hyperglycemic ischemic animals suffered from greater neuronal injury in the cortex and hippocampus than the normoglycemic ischemic animals, especially at 24 h (P<0.01) and 72 h (P<0.05). CONCLUSION: The disturbance of acid-base equilibrium and ionic homeostasis in hippocampal microenvironment, following the spreading of the microenvironment in ischemic core, may be an important reason for secondary neuronal injury in the hippocampus after thrombotic cortical ischemia in tree shrews. Hyperglycemia aggravates the turbulence of ischemic ionic microenvironment.     

Ischemia postconditioning induces tight junction protein expression and inhibits brain edema after thrombotic cerebral ischemia in tree shrews

AIM: To assess whether the expression of tight junction(TJ) proteins, occludin/zonula occludins(ZO)-1, and regional cerebral blood flow(rCBF) link to brain edema in tree shrews during thrombotic cerebral ischemia and ischemic postconditioning(PC), and to explore how TJ affects brain edema and cerebral infarction. METHODS: Tree shrews were randomly grouped into control, ischemia and cerebral ischemia+PC(n=23), and the remaining 3 animals were used for magnetic resonance imaging(MRI). The local cerebral thrombosis were induced by photochemical reaction in the tree shrews, and ischemic PC was established at 4 h after induction of cerebral ischemia followed by clipped ipsilateral common carotid artery(5 min×3). The changes of the neural ultrastructure were observed under electron microscope. The neuronal apoptosis was analyzed by the method of TUNEL. Laser Doppler brain flowmetry was used to monitor the rCBF. The protein levels of occludin/ZO-1 were determined by immunochemistry and Western blot. The cerebral infarction volume was detected by MRI. The brain water content was measured by dry-wet weight method. RESULTS: Induction of cerebral ischemia led to a significant reduction of the normal neuron numbers in the hippocampal CA1 area, and conversely, the number of neurons with abnormal ultrastructure was increased. The TUNEL positive cells were increased significantly(P<0.01) in ischemia group. Moreover, the rCBF decreased significantly(P<0.01), and occludin/ZO-1 protein expression decreased(P<0.01). The brain water content and cerebral infarction volume were significantly increased(P<0.01). Ischemic PC increased the rCBF and the occludin/ZO-1 expression, but reduced the brain water content, the TUNEL positive cells, and the infarction volume(P<0.01). CONCLUSION: Ischemic PC increases the rCBF but not the local water content, suggesting that reduced cerebral infarction volume after ischemia PC is associated with the attenuation of cerebral edema by the enhancement of occludin/ZO-1 protein expression.     

Inhibitory effect of ischemic postconditioning on autophagy induced by focal cerebral ischemia reperfusion in rats

AIM: To investigate the effect of ischemic postconditioning (IPC) on autophagy induced by focal cerebral ischemia reperfusion (I/R) in rats. METHODS: Healthy male SD rats were assigned randomly into sham-operation (sham) group, I/R group and IPC group with 10 rats in each group. The rats in sham group were only exposed the right common, internal and external carotid artery surgically. The rats in I/R group were subjected to right middle cerebral artery occlusion (MCAO) by the modified Longa suture method for 2 h followed by 24 h of reperfusion. The rats in IPC group were subjected to MCAO for 2 h followed by reperfusion of the ipsilateral common carotid artery occlusion for 10 s for 5 episodes, and then reperfusion for 24 h. Autophagy was obeserved by transmission electron microscopy (TEM). The protein levels of mammalian target of rapamycin (mTOR), p-mTOR and microtubule associated protein light chain 3 (LC3)-II in brain tissue of the rats were determined by Western blot. Pathological changes of brain tissue were observed by HE staining. RESULTS: The protein levels of mTOR and p-mTOR in IPC group were significantly higher than those in I/R group (P<0.05). The expression of LC3-II in IPC group was significantly lower than that in I/R group (P<0.01). The cerebral infarction area and brain water content in IPC group were significantly lower than those in I/R group (P<0.01). HE staining showed that neurons degeneration and necrosis in IPC group were significantly alleviated compared with I/R group. TEM observation showed that IPC revealed fewer autophagosomes, with much less severe cell damage than that in I/R group. CONCLUSION: IPC reduces brain ischemia reperfusion damage by decreasing autophagy of brain cells, which might be related to the activation of mTOR.     

Effect of salidroside on activity of endothelial progenitor cells and phosphoinositide 3-kinase/Akt signaling pathway

AIM：To investigate whether salidroside has influence on the activities of endothelial progenitor cells (EPCs) and its mechanism. METHODS：Mononuclear cells from normal human peripheral blood were cultured in fibronectin coated flasks in endothelial progenitor medium. After 7 d, EPCs were characterized as adherent cells with acLDL-DiI uptaking and lectin binding by direct fluorescent staining. The proliferation and migration of EPCs were analyzed by MTT assay and Transwell chamber assay, respectively. The EPCs adhesion assay was performed by re-plating the cells on fibronectin-coated dishes, and then adherent cells were counted. NO and Akt protein were also detected. RESULTS：Salidroside promoted EPCs proliferative, migratory and adhesive capacities in a concentration dependent manner. Salidroside also increased NO secretion, and the level of phosphorylated Akt protein. However, the effects of salidroside on EPCs were inhibited by phosphoinositide 3-kinase inhibitor LY294002. CONCLUSION：Salidroside regulates the activity of EPCs by phosphoinositide 3-kinase/Akt signaling pathway.     

Effects of ginkgolide B on glial fibrillary acidic protein expression after thrombotic cerebral ischemia in tree shrews

AIM: The present study was designed to examine the changes in glial fibrillary acidic protein (GFAP) expression during cerebral ischemia and the effects of ginkgolide B on GFAP expression. METHODS: The focal thrombotic cerebral ischemia was formed by photochemistry-induced in tree shrews. GFAP stained by ABC immunohistochemistry and absorbance were measured with image analyze system. RESULTS: GFAP expression in astrocytes increased significantly (P<0.01) at 24 h and kept in higher level at 72 h (P<0.01) within penumbra after focal cerebral ischemia. GFAP expression declined when the animals were given GB at 6 h after thrombotic cerebral ischemia. CONCLUSIONS: Neuronal necrosis resulted in GFAP expression in astrocytes after local cerebral ischemia and GB protected neurons by antagonizing PAF receptor and inhibiting GFAP expression.     

Effects of Astragalus injection on neuronal apoptosis and expression of JNK3 in rat hippocampus after cerebral ischemia reperfusion

AIM:To investigate the effects of Astragalus injection on neuronal apoptosis and expression of c-Jun N-terminal kinase 3(JNK3) in the rat hippocampus after cerebral ischemia reperfusion. METHODS:The rat model of cerebral ischemia reperfusion was set up by a four-vessel occlusion method. The SD rats were randomly divided into 4 groups:sham operation group, cerebral ischemia reperfusion group(model group), cerebral ischemia reperfusion+Astragalus injection group(Astragalus injection group) and cerebral ischemia reperfusion+vehicle group(vehicle group). The rats in model group, Astragalus injection group and vehicle group after transient global cerebral ischemia(30 min) were then divided into 7 subgroups according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. The apoptosis of the neuron in the hippocampus was measured by the method of TUNEL staining. The expression of JNK3 at mRNA and protein levels was determined by real-time PCR and Western blotting,respectively. RESULTS:Compared with sham operation group, the number of apoptotic neurons increased in model group(P<0.05). Compared with model group, the number of apoptotic neurons decreased obviously in Astragalus injection group(P<0.05). Compared with sham operation group, the expression of JNK3 at mRNA and protein levels in the hippocampus increased obviously in model group at all time points except 120 h(P<0.05). Compared with model group, the expression of JNK3 at mRNA and protein levels in the hippocampus decreased obviously in Astragalus injection group at all time points except 120 h(P<0.05). CONCLUSION:Astragalus injection decreases neuronal apoptosis in rat hippocampus after cerebral ischemia reperfusion by inhibiting the expression of JNK3 at mRNA and protein levels.     

Effects of propofol on lung injury and PI3K/Akt pathway in rats after liver ischemia and reperfusion

AIM：To study the effect of propofol on phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway in the model of rat lung injury after hepatic ischemia and reperfusion (IR). METHODS：Sixty-six SD rats were randomly divided into 4 groups：sham operation group (n=6), IR group (n=24), propofol group (n=24) and propofol+wortmannin group (n=12). The rats in IR group and propofol group were further divided into 4 subgroups according to the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The rats in propofol+wortmannin group were also divided into 2 subgroups according to the time points of 3 h and 6 h after reperfusion. The rats in sham group were only dissected porta without ligation. The ligation of hepatic pedicle in the rats in IR group was performed to induce liver ischemia for 30 min and then reperfusion was conduced. The rats in propofol group were given slow injection of propofol (20 mg/kg) from the caudal vein 10 min before ischemia, and then propofol was continuously pumped at dose of 20 mg·kg-1·h-1 until rats' death. Other procedures were the same as the rats in IR group. The rats in propofol+wortmannin group were injected with wortmannin, a blocker of PI3K, at dose of 15 μg/kg before ischemia, and also given the injection of propofol as the rats in propofol group. The lung tissues of the rats were collected at the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The protein levels of total Akt (t-Akt), phosphorylated Akt (p-Akt) and Bcl-2 in the lung tissues were detected by Western blotting. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. RESULTS：Compared with sham group, the protein levels of p-Akt and Bcl-2, and the cell apoptotic rate in the lung tissues in IR group, propofol group and propofol+wortmannin group increased. Compared with IR group, the protein levels of p-Akt and Bcl-2 increased, and cell apoptotic rate of the lung tissues decreased in propofol group. Compared with propofol group, the protein levels of p-Akt and Bcl-2 decreased, and cell apoptotic rate in the lung tissues increased in propofol+wortmannin group. CONCLUSION：Propofol reduces the lung injury in rats induced by liver ischemia and reperfusion, and its mechanism may be involved in PI3K/Akt signaling pathway.     

Effects of adrenal gland on the expression of bax and bcl-2 in hippocampus after cerebral ischemia

AIM: To observe the effects of adrenal gland on the hippocampus responses to cerebral ischemia. METHODS: 36 Wistar rats were randomly divided into three groups: sham-operated control group (sham), unilateral adrenalectomy were performed in ADX and GC group, and GC group were injected with 5 mg/per rat of dexamethasone before cerebral ischemia. Fourteen days after the first operation, all animals were performed occlusion of bilateral carotid artery for 15 min, and then reperfusion. 3 rats of each group were sacrificed at 1 h, 4 h, 8 h and 24 h after reperfusion and hippocampus were dissected. The total RNA was rapidly extracted from hippocampus tissue. The expressions of c-fos, bcl-2 and bax gene were quantified with the method of semiquantitive RT-PCR. RESULTS: The expressions of c-fos and bax in three groups showed no statistical differences (P>0.05). The expression of bcl-2 in sham group was significantly higher than that in GC and ADX groups (P<0.05). However, no differences of bcl-2 expression between GC and ADX group (P>0.05) was observed. The ratio of bax to bcl-2 in sham group was significantly lower than that in GC and ADX groups (P<0.05), no significant differences of the ratio displayed between ADX and GC group. CONCLUSION: The expression of c-fos and bax in hippocampus after cerebral ischemia is not affected by adrenal gland. The excision of unilateral adrenal gland downregulates bcl-2 expression and raises the ratio of bax to bcl-2 in rat hippocampus after cerebral ischemia. Dexamethasone treatment does not alter the expression of bcl-2 in ADX and GC groups. The results indicate that the adrenal gland can counteract cell apoptosis in hippocampus tissue induced by cerebral ischemia. Adrenal steroids are not sufficient to enable the compensatory increase in bcl-2 expression in steroid-deficient animal, some other mechanism may exist.     

Astragalus injection inhibits expression of Apaf-1 in rat hippocampus after cerebral ischemia and reperfusion

AIM: To investigate the effect of Astragalus injection on the expression of apoptotic protease-activating factor 1 (Apaf-1) in the hippocampus of global cerebral ische-mia-reperfusion rats. METHODS: Male SD rats were randomly divided into 4 groups with 30 each: sham operation group, cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group, and cerebral ischemia-reperfusion+vehicle group. The global cerebral ischemia-reperfusion model of the rats was established by 4-vessel occlusion. The rats in cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group and cerebral ischemia-reperfusion+vehicle group were further divided into 7 subsets, according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. After reperfusion, the brains were removed at the corresponding time points. The protein expression of Apaf-1 in hippocampal neurons was detected by immunohistochemistry and Western blotting. The mRNA expression of Apaf-1 was observed by RT-PCR. RESULTS: Compared with sham operation group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h increased obviously in cerebral ischemia-reperfusion group (P<0.05). Compared with cerebral ischemia-reperfusion group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h decreased obviously in cerebral ischemia-reperfusion+Astragalus injection group (P<0.05). However, those in cerebral ischemia-reperfusion+vehicle group had no obvious change (P>0.05). CONCLUSION: Astragalus injection inhibits the expression of Apaf-1 at mRNA and protein levels in hippocampus of global cerebral ischemia-reperfusion rats, thus inhibiting the apoptosis of hippocampal neurons.     

Roles of PI3K/Akt and JAK2/STAT3 signaling pathways in protection of SO2 against limb ischemia/reperfusion-induced acute lung injury in rats

AIM: To investigate the role of PI3K/Akt and JAK2/STAT3 pathways in the protection of sulfur dioxide (SO₂) against limb ischemia/reperfusion (I/R)-induced acute lung injury (ALI) in rats. METHODS: ALI was induced by limb I/R in the SD rats. Na₂SO₃(0.54 mmol/kg, ip)/NaHSO₃ (0.18 mmol/kg, ip) as SO₂ donor was injected at 20 min before reperfusion. The inhibitors of JAK2/STAT3 and PI3K/Akt pathways, Stattic (3 mg/kg, iv) and LY294002(40 mg/kg, iv), respectively, were injected at 1 h before reperfusion. Peripheral blood and lung tissues were collected for determining the contents of the cytokines, the protein levels of the molecules related to the signaling pathways, apoptosis and histopathologic changes by ELISA, TUNEL and Western blot. RESULTS: Compared with control group, the content of MDA, the activity of MPO, lung coefficient, apoptotic index, cytokine expression, and the protein levels of p-Akt and p-STAT3 in I/R group all increased significantly, and administration of Na₂SO₃/NaHSO₃ attenuated the damage in the lung. Besides, the results of Western blot showed that the rat lung tissues expressed p-STAT3 protein and p-Akt protein. After I/R, the protein levels of p-STAT3 and p-Akt were increased. After using Na₂SO₃/NaHSO₃, p-Akt was increased, but p-STAT3 was decreased (P<0.05). CONCLUSION: Both JAK2/STAT3 and PI3K/Akt pathways are likely involved in the protective effect of SO₂ against limb I/R-induced ALI in rats. The activation of JAK2/STAT3 signaling pathway increases I/R injury. Reversely, the activation of PI3K/Akt signaling pathway reduces I/R injury. Besides, JAK2/STAT3 and PI3K/Akt signaling pathways may have crosstalk during I/R-induced ALI and JAK2/STAT3 pathway may have an impact on the P13K/Akt pathway.     

Effect of naoluo xintong on expression of Fas,Fas L in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats

AIM: To investigate the effets of naoluo xintong on the expression of Fas, FasL protein in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats. METHODS: The local cerebral ischemia /reperfusion model was established by intraluminal thread occlusion of the middle cerebral arteries (MCAO), the middle cerebral arteries of rats were occluded for 2 hours and reperfused for 1, 3 and 7 days. The animals were divided into pseudo surgery group(sham group), model group, Yiqi group, Huoxue group and naoluo xintong group. Using the techniques of immuno-histochemical staining and in situ hybridization, the expression of Fas and FasL was observed in hippocampus CA1 area, the expression of Fas mRNA was also observed in the cortex of frontal and parietal lobe. RESULTS: A value of Fas and FasL protein expression or A value and positive unit of Fas mRNA expression in control group were higher than those in sham in hippocampus CA1 area, the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats (P<0.01). A value and/or positive unit of their expression in naoluo xintong group were lower than those in control group (P<0.05 or P<0.01). A value and/or positive unit of their expression in Yiqi and Huoxue groups were higher than those in naoluo xintong group for 3 and/or 7 days (P<0.05 or P<0.01). CONCLUSION: naoluo xintong could resist neuron apoptosis, alleviate pathologic injury after local cerebral ischemia/reperfusion in MCAO rats by inhibiting the expression of Fas, FasL protein and Fas mRNA.     

Effect of aminoguanidine on TNF-α,IL-1β and neuronal apoptosis after focal cerebral ischemic injury in rats

AIM: To evaluate the effect of aminoguanidine (AG) on inflammatory factors and neuronal apoptosis after focal cerebral ischemic injury in rats and the possible mechanism of protective effect of AG against cerebral ischemic injury.METHODS: Thirty male SD rats (weighing 250 g-280 g) were randomly divided into three groups: (1) sham operated group (SH group,n=10),(2) ischemic groups (IS group,n=10),(3) AG group (n=10).In AG group,AG at dose of 100 mg·kg-1 was given intraperitoneally twice a day for 3 consecutive days.In IS group,normal saline was given instead of AG.Focal cerebral ischemia was produced by middle cerebral artery occlusion (MCAO) for 12 h.A nylon thread with rounded tip was inserted into left internal carotid artery cranially until resistance was felt.The distance from bifurcation of common carotid artery to the tip of the thread was about 18-19 mm.Focal cerebral ischemia was confirmed by left Horners syndrome and right side hemiplegia.In SH group,the carotid artery was exposed but no thread was inserted.The expression of tumor necrosis factor-α(TNF-α) was determined by immunochemistry and the content of interleulin-1β(IL-1β) was measured by radioimmunoassay.The expressions of Bcl-2 and Bax protein were detected by flow cytometry.RESULTS: The expression of TNF-α and the content of IL-1β were markedly increased after MCAO.Significantly increased DNA fragmentation,the indication of apoptosis,was detected after MCAO.The expression of TNF-α and the content of IL-1β were significantly lower in AG group than those in IS group.The percentage of apoptosis cells and expression of Bax protein were markedly lower in AG group than those in IS group but still significantly higher than those in SH group.The expression of Bcl-2 protein was markedly higher in AG group than that in IS group.No significant difference in the expression of Bcl-2 protein between IS and SH group was observed.CONCLUSION: AG inhibits the increase in the expression of TNF-α and the content of IL-1β,and protects neurons from apoptosis induced by focal cerebral ischemia through increasing the Bcl-2 protein expression and inhibiting the Bax protein expression.     

Effects of soybean isflavones on mitochondrial damage and neuronal apoptosis induced by cerebral ischemia/reperfusion

AIM: To study the effects of soybean isoflavones on mitochondrial ultrastructure, neuronal apoptosis and expression of cytochrome C, caspase-9 and caspase-3 in the rats with cerebral ischemia/reperfusion.METHODS: Adult healthy SD rats (n=60) were randomly divided into 3 groups: sham group, ischemia/reperfusion injury (I/R) group and soybean isoflavone (SI) pretreatment group. Soybean isoflavones (120 mg·kg-1·d-1) were fed by gastric lavage for 21 d. The global ischemia/reperfusion model of the rats was established by blocking 3 vessels, and then reperfused for 1 h after 1 h of ischemia. The morphological change of the cerebral cortex cells was observed under light microscope. The mitochondrial ultrastructure of the cerebral cortex cells was determined by transmission electron microscope. The apoptotic rate of the cerebral cortex cells was detected by flow cytometry. The expression of cytochrome C, caspase-9 and caspase-3 in the cerebral cortex cells was determined by semi-quantitative RT-PCR and immunohistochemical techniques.RESULTS: Disintegration of mitochondria membrane and disappearance of the mitochondrial cristae were seen in I/R group. Compared with I/R group, the change of ultrastructure of mitochondria was significantly improved by soybean isoflavone pretreatment, and the neuronal apoptotic rate was also significantly decreased (P<0.01). The mRNA expression and protein content of cytochrome C, caspase-9 and caspase-3 in I/R group were obviously higher than those in sham group (P<0.01). Compared with I/R group, the mRNA expression and protein content of cytochrome C, caspase-9 and caspase-3 in SI group were significantly decreased (P<0.01).CONCLUSION: Soybean isoflavones attenuate cerebral ischemia/reperfusion injury by stabilizing the structure of mitochondria, preventing cytochrome C release to the cytoplasm, inhibiting the activation of caspase-9 and caspase-3 and decreasing cell apoptosis．