Influence of hydrogen sulfide on ileal epithelial cell apoptosis in rats with intestinal ischemia-reperfusion injury

AIM: To investigate the influence of hydrogen sulfide (H₂S) on intestinal epithelial cell mitochondrial morphology and function and the expression of caspase-3, cleaved caspase-3, cytochrome C (Cyt C), Bcl-2 and Bax in rats with intestinal ischemia-reperfusion (I/R) injury. METHODS: Wistar rats (n=24) were randomly divided into 3 groups (8 in each group): sham group, I/R group and I/R+sodium hydrosulfide (NaHS) group. The animal model of intestinal I/R injury was established. The rats in I/R+NaHS group received NaHS (100 μmol/kg bolus +1 mg·kg^-1·h^-1 infusion) 10 min prior to the onset of reperfusion, whereas the rats in I/R group and sham group received equal volume of normal sodium. Ileum epithelial mitochondrial morphology and function were measured. Plasma H₂S was detected by sensitive sulfide electrode. The expression of Bcl-2 and Bax mRNA was studied by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, cytochrome C (Cyt C), Bcl-2 and Bax were tested by Western blot.RESULTS: The area, volume density, maximum diameter, minimum diameter and equivalent diameter of mitochondria, and the expression of cleaved caspase-3, Cyt C and Bax in I/R group were significantly higher than those in I/R+NaHS and sham groups (P<0.01). The mitochondrial count, circumference, specific surface area, area density and population density, plasma H₂S, respiratory control rate (RCR), the ratio of P/O, R₃ , R₄, and the expression of Bcl-2 in I/R group were sharply lower than those in I/R+NaHS and sham groups (P<0.01). H₂S was negatively correlated with caspase-3, cleaved caspase-3, Cyt C and Bax (P<0.01), and was positively correlated with Bcl-2 (P<0.01). CONCLUSION: H₂S has a protective effect on mitochondrial morphology and function in rats with intestinal I/R injury by down-regulating cleaved caspase-3, Cyt C and Bax and up-regulating Bcl-2.     

Preliminary study on pyroptosis involved in cerebral ischemia-reperfusion injury

AIM:To investigate the changes of pyroptosis in hippocampus and cortex at different time points after cerebral ischemia-reperfusion, and to explore its mechanism from NLRP3-mediated classical pyroptosis pathway, and to analyze the role of pyroptosis in different parts of cerebral injury. METHODS:SD rats were randomly divided into sham operation group (sham group) and model group (MCAO/R group). The rats in model group was further divided into cerebral ischemia-reperfusion 6 h group (MCAO/R 6 h group), 12 h group (MCAO/R 12h group)and 24 h group (MCAO/R 24 h group). The rat model was established on rats by middle cerebral artery occlusion and reperfusion (MCAO/R) induced by modified right-side thread method. Neurologic function score, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and morphological observation were used to evaluate the degree of nervous cell injury. TUNEL and caspase-1 immunofluorescence double staining were used to detect pyroptosis. The protein expression of NLRP3, cleaved caspase-1, pro-caspase-1 and interleukin-1β (IL-1β) was determined by Western blot. RESULTS:Neurological damage occurred at different times after cerebral ischemia-reperfusion. TTC staining showed that the volume of cerebral infarction gradually increased with the prolongation of reperfusion time (P<0.05). The hippocampal CA1 area and cortical area showed typical morphological features such as loose tissue structure, interstitial edema, disordered arrangement of nerve cells, deepening of nucleus staining, nuclear fragmentation and decreased cell number. Immunofluorescence double staining showed that there was a phenomenon of pyroptosis at different time after cerebral ischemia-reperfusion. The pyroptosis of hippocampal CA1 and cortical area was most obvious at 12 h and 24 h after reperfusion (P<0.05). Western blot analysis showed that the expression of NLRP3, cleaved caspase-1, pro-caspase-1 and IL-1β in NLRP3-mediated classic pyroptosis pathway was regulated in different degrees after cerebral ischemia-reperfusion. The protein expression of NLRP3 in hippocampus was significantly increased at 12 h and 24 h after reperfusion (P<0.05), and the protein expression of NLRP3 in cortex was significantly increased at 6 h after reperfusion (P<0.05). The protein expression of pro-caspase-1 in hippocampus was significantly increased at each time points of reperfusion (P<0.05), and the protein expression of pro-caspase-1 in the cortex was significantly increased at 24 h after reperfusion (P<0.05). The protein expression of cleaved caspase-1 in the hippocampus was significantly increased at 12 h after reperfusion (P<0.05), and increased in the cortex at 24 h after reperfusion (P<0.05). The protein expression of IL-1β in the hippocampus was significantly increased at 24 h after reperfusion (P<0.05), and increased in the cortex at 6 h after reperfusion (P<0.05). CONCLUSION:Pyroptosis is involved in neuronal injury after cerebral ischemia-reperfusion. The classic pyroptosis pathway plays an important regulatory role in hippocampus and cortex, especially in hippocampus, suggesting that hippocampus is the main part of secondary nerve impairment induced by pyroptosis and inflammation after cerebral ischemia-reperfusion.     

Upregulative effect of CGRP on expression of CREB and phospho-CREB protein during focal cerebral ischemia/reperfusion in rat parietal cortex

AIM: To investigate the effect of calcitonin gene related peptide (CGRP) on the expression of cyclic AMP response element binding protein (CREB) and phosphorylated-CREB in rat parietal cortex during focal cerebral ischemia/reperfusion (I/R).METHODS: Focal cerebral ischemia/reperfusion model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The expressions of CREB and phospho-CREB in the parietal cortex in different groups (sham group, focal cerebral ischemia/reperfusion group and CGRP group) were detected with immunohistochemistry and Western blotting, and the positive products were analyzed by image analysis system.RESULTS: There was definite expression of CREB in right parietal cortex in sham group, while it was lesser in I/R group than that in sham group, but it became more in CGRP group than that in I/R group (P<0.05). Phospho-CREB was barely detected in right parietal cortex in sham group and it became more in I/R group than that in sham group. The expression of phospho-CREB increased in CGRP group than that in I/R group of the right parietal cortex (P<0.05).CONCLUSION: CGRP upregulates the expression of CREB and phospho-CREB in the ischemic neurons of the parietal cortex during focal cerebral ischemia/reperfusion and CREB probably involves in the mechanism of protective role of CGRP to ischemic neurons.     

Effects of Panax quinquefolium saponin on mitochondrial membrane potential and cardiomyocyte apoptosis in ischemia/reperfusion rats

AIM：To investigate whether mitochondrial membrane potential (ΔΨm) and the mitochondrial apoptotic pathway are involved in the protective mechanism of Panax quinquefolium saponin (PQS) against cardiomyocyte apoptosis after ischemia/reperfusion (I/R) injury in rat myocardium. METHODS：Ninety healthy male SD rats were randomly divided into sham group, I/R group, PQS (200 mg·kg-1·d-1) +I/R group, cyclosporine A (CsA) group, CsA (10 mg·kg-1) +I/R group and PQS +CsA +I/R group. The model of myocardial I/R injury in vivo was established by ligating the left anterior descending artery (LAD) for 30 min followed by 120 min of reperfusion in the rats. The serum activity of lactate dehydrogenase (LDH) was measured by automatic chemistry analyzer. The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was detected by in situ TDT-mediated dUTP nick end labeling (TUNEL). The protein levels of Bcl-2, Bax, cleaved caspase-3 and cytosolic cytochrome C were determined by Western blotting. ΔΨm was measured by laser scanning confocal microscopy and fluorescence microplate reader. RESULTS：Compared with I/R group, the serum content of LDH,the infarction size in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group and the myocardial apoptotic index were decreased. Compared with I/R group, the fluorescence intensity of mitochondria after JC-1 staining was enhanced in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, and the relative fluorescence units (RFU) of ΔΨm were improved in those 3 groups. In PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, the protein expression of Bcl-2 was increased, and that of Bax was decreased compared with I/R group. Moreover, in those 3 groups, the protein levels of cleaved-caspase-3 and cytosolic cytochrome C were decreased compared to I/R group, respectively. CONCLUSION：PQS attenuates myocardial injury and cardiomyocyte apoptosis during I/R, and the protective mechanisms of PQS were associated with the modulation of ΔΨm and the inhibition of mitochondrial apoptosis pathway.     

Effects of urocortin-I preconditioning on myocardial mitochondrial respiratory function and enzyme activity in rats

ATM: To investigate the influence of urocortin-I (Ucn I) preconditioning on the myocardial mitochondrial respiratory function and enzyme activity in the rats with ischemia reperfusion, and to observe the changes of ATP content in the myocardial cells. METHODS: (1) The healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal group (Nor group), ischemia reperfusion group (IR group), Ucn I preconditioning group (Ucn I group), 5-hydroxy acid (5-HD)+Ucn I group. Langendorff perfusion was used to establish the in vitro model of cardiac ischemia reperfusion. At the end of the balance (T₁), before ischemia (T₂) and at the end of the reperfusion (T₃) respectively, the myocardial mitochondria was extracted, the mitochondrial respiratory function and respiratory enzyme activity in each group were determined. (2) The method of MPA isolated heart perfusion was used to isolate myocardial cells of the adult rats. After cultured for 24 h, myocardial cells were divided into 4 groups:Nor group, hypoxia/reoxygenation group (I/R group), Ucn I group, 5-HD+Ucn I group. Hypoxia/reoxygenation model of myocardial cells was established. At the end of reoxygenation, the changes of myocardial ATP content were measured by high performance liquid chromatography.RESULTS: (1) Compared with T₁, T₂ time points, the respiratory function (state 3 respiratory rate, respiratory control rate) and NADH oxidase, succinate oxidase and cytochrome C oxidase activities at T₃ time point were significantly decreased (P<0.05) in all groups except Nor group. At T₃ time point, the myocardial mitochondrial respiratory function and respiratory enzyme activity in Ucn I group were superior to 5-HD+Ucn I group and IR group (P<0.05), but was inferior to Nor group (P<0.05). At T₃ time point, the respiratory function of myocardial mitochondria and respiratory enzyme activities (NADH oxidase, succinate oxidase) in 5-HD+Ucn I group were better than those in IR group (P<0.05), but no statistical difference of the cytochrome C oxidase activity between the 2 groups was observed. The respiratory function and 3 kinds of respiratory enzyme activities at T₁, T₂ time points had no statistical change. (2) At the end of the reoxygenation, the myocardial ATP content in Nor group was higher than that in other groups (P<0.01). The myocardial ATP contents in I/R group and 5-HD+Ucn I group were lower than that in Ucn I group (P<0.05). In additon, 5-HD+Ucn I group was higher ATP content compared with I/R group (P<0.05). CONCLUSION: Ucn I preconditioning attenuates the ischemia/reperfusion induced damages of myocardial mitochondrial respiratory function and respiratory enzyme activity, thus ensuring the myocardial ATP contents under the condition of hypoxia/reoxygenation.     

Effects of ischemic preconditioning on hepatocyte apoptosis induced by hepatic ischemia-reperfusion in cirrhotic rats

AIM:To investigate the protective effect of ischemic preconditioning (IPC) on hepatic ischemia-reperfusion(I/R) injury in cirrhotic rats and its possible mechanism. METHODS:Hepatic I/R was induced by Pringle maneuver. The cirrhotic rats were randomized into three groups: Group A: before 30 min of ischemia, a short period of 5 min ischemia and 5 min reperfusion were given; Group B: before 30 min of ischemia, a short period of 10 min ischemia and 10 min of reperfusion were given; Group C: 30 min ischemia only. The serum alanine transferase (ALT), hepatic Fas-mRNA, caspase-3 activity and hepatocyte apoptosis were analyzed. RESULTS:The 7-day survival rate in the group A and B were 100%, respectively. However, it was only 62.5% in the group C. After 6 h of reperfusion, the ALT levels in both group A and B were significantly lower than that of in group C, P＜0.01. The ALT level of group A was also lower than that of group B, P＜0.01. The hepatic Fas-mRNA expression, caspase-3 activity and apoptotic hepatocyte in group A were significantly lower than those of in group C, P＜0.01. CONCLUSIONS:IPC has significant protective effect against hepatic I/R injury. An IPC with 5 min of ischemia and 5 min of reperfusion has the maximal protective effect. The protective mechanism of IPC against hepatic I/R injury is via down-regulation of Fas-mRNA expression, inhibiting caspase-3 activity and subsequently inhibiting hepatocyte apoptosis.     

Influence of high-mobility group box 1 on proliferation of neural stem cell in pre-infarction cortex of focal cerebral ischemia/reperfusion model rats

AIM：To investigate the influence of high-mobility group box 1 (HMGB1) on the proliferation of neural stem cells in peri-infarction cortex of focal cerebral ischemia/reperfusion model rats. METHODS：Male SD rats (n=48) were randomly divided into sham group, ischemia/reperfusion (I/R) group, RNA interference group and negative interference group. The rat middle cerebral artery was blocked to establish focal cerebral I/R model (ischemia for 1 h and reperfusion for 7 d). Lentivirus vector of HMGB1 shRNA was used to suppress the HMGB1 protein expression in the rat brain. The effect of RNA interference was evaluated by the methods of double-immunofluorescence labeling of HMGB1/GFAP and Western blotting. The proliferation of neural stem cells in the peri-infarction cortex was assessed by double labeling of BrdU/nestin. RESULTS：The protein expression of HMGB1 in I/R group was much higher than those in sham group (P<0.05). RNA interference effectively inhibited the HMGB1 expression (P<0.05). Double labeled BrdU/nestin positive cells in I/R group were more than that in sham group (P<0.05). The double labeled BrdU/nestin positive cells were significantly decreased in RNA interference group (P<0.05). CONCLUSION：Focal cerebral ischemia/reperfusion injury promotes the proliferation of neural stem cells in peri-infarction cortex by increasing HMGB1 protein level.     

Effect of tacrolimus on renal ischemia/reperfusion injury in rats

AIM: To investigate the protective effects and mechanism of tacrolimus on renal ischemia/reperfusion(I/R) injury in rats.METHODS: Sixty male Wistar rats were randomly divided into 3 groups: sham-operated group, I/R group and tacrolimus group. After the renal I/R injury model was established, the serum content of creatinine(Cr), tumor necrosis factor-α(TNF-α)and malondialdehyde(MDA) and activity of superoxide dismutase(SOD) were measured at reperfusion time points of 0.5 h, 2 h, 6 h and 24 h. The renal histopathological lesions and the expression of Fas and caspase-3 were observed by the methods of microscopy and immunohistochemistry, respectively.RESULTS: At all the 4 time points, the levels of Cr, TNF-α and MDA in tacrolimus group were lower than those in I/R group(P<0.05). The SOD activity in tacrolimus group was higher than that in I/R group. Compared with I/R group, the renal histopathological lesions were improved, and the levels of Fas and caspase-3 were significantly decreased in tacrolimus group(P<0. 05).CONCLUSION: Tacrolimus inhibits the production of free radical, the expression of TNF-α and apoptosis of renal tubular epithelial cells in renal I/R injury in rats, indicating that tacrolimus has protective function against renal I/R injury.     

Knockdown of Atg5 aggravates cerebral ischemia and reperfusion injury in mice

AIM: To study the role of autophagy-related gene 5 (Atg5) in cerebral ischemia and reperfusion injury in mice. METHODS: BALB/c male mice (weighing 18~22 g) were randomly divided into sham group, ischemia/reperfusion (I/R) group, Atg5 siRNA group and control siRNA group. Focal cerebral ischemia was performed using the method of middle cerebral artery occlusion (MCAO) for 60 min and reperfusion for 24 h. In siRNA group and control group, 5 μL Atg5 siRNA or scrambled siRNA was administered by intracerebroventricular injection 24 h before MCAO. The expression of Atg5 at mRNA and protein levels in ischemic cortex at 24 h after reperfusion was determined by real-time PCR and Western blot. The infarct volume and edema were evaluated by TTC staining, and motor deficits were evaluated by neurological scoring. RESULTS: The expression of Atg5 at mRNA and protein levels was significantly increased 24 h after reperfusion in I/R group compared with sham group. Atg5 siRNA obviously decreased the expression of Atg5 at mRNA and protein levels induced by I/R. Inhibition of Atg5 exacerbated the infarct volume and ameliorated the neurological symptoms. CONCLUSION: Atg5 has neuroprotective effect on focal cerebral ischemia and reperfusion injury.     

Changes of intracellular Ca2+ in living brain slices during focal cerebral ischemia/reperfusion

AIM: The purpose of the present study was to detect intracellular Ca²⁺changes in living brain slices during focal cerebral ischemia/reperfusion (I/R) and reveal the role of intracellular Ca²⁺in the cerebral I/R injury. METHODS: The model of focal cerebral I/R was established in rats by reversible inserting a nylon thread, and dynamic change of intracellular Ca²⁺in brain slices was determined using laser confocal imaging system. RESULTS: ① Ca²⁺gradually enhanced with increase in ischemic time in cortex and striatum. ②At1h ischemia/10min reperfusion, Ca²⁺increased significantly in striatum, but Ca²⁺decreased at 3 h reperfusion compared with10min reperfusion. ③ Ca²⁺markedly enhanced at 6 h ischemia compared with1h ischemia, and after 3 h reperfusion Ca²⁺decreased, but was still higher than that in sham-operation group. ④The striatum is more sensitive than cortex to ischemia/reperfusion. CONCLUSION: Ca²⁺overload in the area of cortex and striatum may play an important role in cerebral ischemia/reperfusion injury in rats.     

Effect of CsA on mitochondria stress after microperfusing glutamate and calcium into tree shrews’ hippocampus

AIM: To observe the changes of glutamate and calcium within the hippocampal microenvironment in mitochondrial stress.METHODS: A lateral hippocampus was microperfused with glutamate and calcium chloride solution by a kind of single-pumped push-pull perfusion system in Tree Shrews. At 24 h, the expression of cytochrome C (Cyt C)was observed by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome C was assessed through Western blotting. The relative amounts of caspase-3 and caspase-9 mRNA were evaluated by real time fluorescence polymerase chain reaction. In the treated group, cyclosporin A (CsA，40 mg/kg) was intravascularly injected at 6 h after perfusion of glutamate-calcium chloride solutions into the hippocampus and inspected the above-mentioned items at 24 h. RESULTS: In the glutamate-calcium group, compared with the control group, cytochrome C immunoreactivity increased and the content of hippocampal mitochondrial cytochrome C decreased. Also, the cytochrome C was detected in cytosol. Cyclosporin A treatment at 6 h after microperfusion, the cytochrome C expression weakened and no Cyt C in cytosol fraction was observed. By real time PCR, in relation to the control group, the caspase-3 and caspase-9 mRNA was higher in the glutamate-calcium group. Cyclosporin A treatment cut down both caspase-3 and caspase-9 mRNA contents. CONCLUSION: The accumulation of glutamate and calcium may promote Cyt C release, caspase cascade activation and the mitochondrial stress. The neuroprotection of CsA may results from uniquely inhibiting the mitochondrial permeability transition pore, and preventing Cyt C release and caspase activation.     

Effects of Le Er Mai on apoptosis of hippocampus neuronal cells in anaphase of cerebral ischemic reperfusion injury in rats

AIM: To investigate the effects and mechanism of Le Er Mai (LEM) on the apoptosis of hippocampus neuronal cells in the anaphase of cerebral ischemic reperfusion injury in rats.METHODS: A rat model of middle cerebral artery occlusion reperfusion (MCAO) was produced with the intraluminal filament. During reperfusion for 30 d after 2 h of ischemia, the TUNEL staining methods were used to detect apoptosis of hippocampus neuronal cells, and immunohistochemical technique were employed to examine the protein expression of Fas, Bax, caspase-3 and caspase-9 in the hippocampial. The gene expressions of fas, bax, caspase-3 and caspase-9 in hippocampial were examined by RT-PCR. RESULTS: After 2 h ischemia and 30 d reperfusion, compared with sham-operated group, TUNEL-positive staining cells and expression levels of Fas, Bax as well as caspase-3 and caspase-9 obviously increased, and the mRNA expressions of fas, bax, caspase-3 and caspase-9 in hippocampial markedly up-regulated in model group. Compared with model group, LEM at dose of 2.00 g/kg or 0.87 g/kg, and flunarizinum significantly reduced apoptosis and decreased the protein expressions of Fas, Bax, caspase-3 and caspase-9 in hippocampial, and down-regulated the mRNA expressions of fas, bax, caspase-3 and caspase-9 (P<0.05), those action of LEM in 0.87 g/kg dosage group was lower than those in 2.00 g/kg dosage group.CONCLUSION: LEM obviously lower the injury of hippocampial in the anaphase of cerebral ischemia reperfusion through inhibiting the apoptosis of hippocampus neuronal cells. The mechanism of LEM may be related to regulate the expression of signal transduction pathway correlated gene of apoptosis in neuronal cells.     

Age-related changes of expression of Bcl-2, Bax and caspase-3 activity after cerebral ischemia/reperfusion in aged rats

AIM: To study age-related changes of expression of Bcl-2, Bax and caspase-3 activity after focal cerebral ischemia/reperfusion (I/R) in aged rats. METHODS: The aged SD rats (20-21 months) and the young animals (4-5 months ) were subjected to 3 h of middle cerebral artery occulsion with the intraluminal filament technique, followed by 3 h, 6 h, 12 h, 24 h and 72 h of referfusion. Expression of Bcl-2, Bax and caspase-3 activity of the young and the aged rats were examined. RESULTS: Cerebral infarct zone increased in the aged at ischemia 3 h and I/R 12 h than that in the young. With I/R time longer, increase in neuron apoptosis showed early and lasted longer in the aged. The Bcl-2 expression increased in the young with I/R time longer, but was not obvious in the aged. Bax expressd obviously and early, and kept on longer in the aged during I/R than that in the young. The enhanced activity of caspase-3 showed early in the aged than that in the young during I/R.　CONCLUSION: The mechanisms of serious cerebral injury and neuron apoptosis might be related to the increase in Bax expression and caspase-3 activity.     

Over-expression of PGC-1α reverses mitochondrial function reduction and apoptosis in OGD/R-induced neurons

AIM: To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was identified by PCR, and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass, reactive oxygen species (ROS) and ATP production, cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining, flow cytometry, ATP metabolic assay kit and TUNEL. RESULTS: Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons (P<0.05), enhanced the ability of ATP synthesis (P<0.01), inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION: PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis, inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.     

Effects of Salvia miltiorrhiza Bunge.f.alba. on mitochondrial damage and apoptosis induced by cerebral ischemia and reperfusion

AIM: To observe the effects of Salvia miltiorrhiza Bunge.f.alba. (Sal) on the mitochondrial ultra-structure, oxidative stress and apoptosis induced by ischemia injury in a rat model of focal cerebral ischemia and reperfusion.METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established by a modified Longa occlusion method. Adult male SD rats were randomly divided into control group, simple ischemia reperfusion group, Sal with ischemia reperfusion group and butylphthalide with ischemia reperfusion group. To study the protective effects of Sal and its mechanism, the intervention of Sal was given and the ultra-structure of mitochondria, functions of mitochondria under oxidative stress and the incidence of apoptosis of brain cells were determined.RESULTS: Many electron dense toxic granulation and vacuolus in mitochondria were observed in the rat brain of focal cerebral ischemia and reperfusion. Under the condition of ischemia and reperfusion, the mitochondria membrane was disaggregative, and the tubular cristae of mitochondrion disappeared. MDA content was obviously increased and the activity of glutathione peroxidase decreased significantly. The apoptosis of brain cells were observed in a great quantity. The changes of ultra-structure of mitochondria and the activity of GSH-Pxase were significantly improved by the treatment of Sal. Furthermore, treatment with Sal delayed the decrease of GSH-Pxase activity, and inhibited the increase in MDA content in brain tissue after ischemia and reperfusion. The incidence of apoptosis of brain cells was also decreased.CONCLUSION: Sal protects the brain tissue from ischemia injury.     

Protective effect of sCR1-SCR15-18 on cerebral ischemia/reperfusion injury in rat via inhibition of complement

AIM: To explore the effect of complement on the cerebral ischemia/reperfusion injury in rat and the protection by sCR1-SCR15-18. METHODS: 75 male SD rats were randomly divided into three groups: sham operation group (SO, n=15), middle cerebral artery occlusion and reperfusion (MCAO) without treatment group (I/R, n=30); MCAO treated with sCR1-SCR15-18 group (sCR1-SCR15-18, n=30). After the MCAO for 2 h, then reperfusion for 24 h, the scores of neural behavioral functional deficits were determined. Infarction area was measured by TTC staining. Activity of MPO in cerebral cortex was detected. C3b deposition and pathological change were observed by immunohistochemial staining and HE staining, respectively. RESULTS: After reperfusion for 24 h, the neurological deficits score, infarction area and activity of MPO in sCR1-SCR15-18 group were decreased compared to I/R group. In sCR1-SCR15-18 group, C3b deposition in ischemic area was decreased and pathological injury was improved compared to I/R group. CONCLUSION: Complement plays a role in cerebral ischemia-reperfusion injury and sCR1-SCR15-18 exerts a protective effect by inhibiting the excessive activation of complement.     

Effect of limb ischemic postconditioning on brain focal ischemia and reperfusion injury in mice

AIM: To establish the mouse model in which the limbic ischemic postconditionning (LIPostC) enhances the tolerance against brain ischemia, and to investigate the effects of LIPostC on the ischemic extent and roles of heat shock protein 70 (HSP70) in ischemia and reperfusion injury. METHODS: The male Kunming mice were used in the study. The brain ischemia reperfusion (I/R) model was made by middle cerebral artery occlusion (MCAO). In the first test, the male mice were randomly divided into 9 groups (n=10): sham group, ischemia/reperfusion (I/R) groups (with ischemia for 0.5 h, 1 h,1.5 h and 2 h) and LIPostC+I/R groups (0.5 h+LIPostC,1 h+LIPostC,1.5 h+LIPostC,2 h+LIPostC). The reperfusion was performed after LIPostC for 24 h. After the neurologic deficit scores were evaluated, the brains were taken out to measure the infarct volume with TTC staining and to observe the pathological changes of cerebral cortex with HE staining. The neuronal apoptosis was determined by TUNEL. In the second test, the male mice were randomized into 4 groups (n=6): sham group, I/R group, LIPostC+I/R group and LIPostC+I/R+quercetin group (2 h ischemia). The neurological deficit scores were evaluated at 24 h after operation. The expression of HSP70 was determined by Western blotting.RESULTS: The duration of brain ischemia was related to the motor behavior and degree of brain injury. The longer the ischemic duration of the brain was performed, the more severe the pathological and behavioral changes were observed. The brain injury in 2 h MCAO mice was more severe than that in 1 h and 1.5 h MCAO mice (P<0.05). Compared to I/R group, each LIPostC group showed lower neurological score, less infarct volume and TUNEL positive neuron. The expression of HSP70 protein was increased and neurological functions were improved significantly in the mice with LIPostC. However, the neuroprotective role of LIPostC was attenuated by treating with quercetin, an inhibitor of HSP70.CONCLUSION: LIPostC promotes the expression of HSP 70, improves the neurological functions and attenuates the ischemia and reperfusion injury in MCAO mice. HSP70 produces a marked effect on the ischemic tolerance induced by LIPostC in MCAO mice.     

Effect of ischemic preconditioning on contents of cytochrome C and mitochondrial calcium in rats after focal cerebral ischemic-reperfusion

AIM: To observe the effect of ischemic preconditioning on contents of cytochrome C and mitochondrial calcium in rats after focal cerebral ischemic-reperfusion. METHODS: Focal cerebral ischemic model was made by occlusion of right middle artery in Wistar rats (ischemia for 2 h and reperfusion for 4 h). Rats were randomly divided into three groups: ischemia pretreatment, model and sham operation. Rats in ischemic pretreatment group were undergone transient ischemic preconditioning (30 min) and reperfusion (72 h). The contents of cytochrome C were measured according to Zhangjuntian's improved methods. The contents of mitochondrial calcium were detected by flame atom absorption. RESULTS: The contents of mitochondrial cytochrome C and calcium in model group were significantly lower than those in sham operation (P<0.05, P<0.01), whereas the contents of plasma cytochrome C were markedly higher (P<0.05, P<0.01). The change in ischemic pretreatment group was obviously difference as compared with the model group. CONCLUSION: Ischemic preconditioning reduces the release of mitochondrial cytochrome C and maintain mitochondrial calcium homeostasis.     

Effects of astragaloside IV on autophagy in rats with cerebral ischemia/reperfusion injury

AIM: To investigate the effects of astragaloside IV (AS-IV) on autophagy in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The focal cerebral ischemia/reperfusion of rat left middle cerebral artery occlusion (MCAO) was induced by suture method. Male SD rats (n=70) were randomly divided into sham operation group, I/R group, solvent control group, AS-IV group, AS-IV+autophagy inhibitor (3-methyladenine, 3-MA) group, 3-MA group and autophagy activator (rapamycin, Rapa) group. Except for sham operation group, the rats in other groups were subjected to ischemia for 2 h and reperfusion for 24 h. The rats with successful modeling were selected according to Zea Longa scoring criteria. The volume of cerebral infarction was measured by TTC staining. The morphological changes of nerve cells in the rats were observed with Nissl staining. The phenomenon of autophagy was observed under transmission electron microscope. The protein expression of beclin-1 and LC3-Ⅱ was determined by Western blot. RESULTS: No neurological deficit in sham operation group was observed, and the cerebral infarction was not found. Compared with sham operation group, obvious cerebral infarction was observed, the Nissl bodies were small in size and number and stained light, typical autophagosomes were observed, and the protein expression of beclin-1 and LC3-Ⅱ was increased in I/R group (P<0.05). Compared with I/R group, the volume of cerebral infarction was decreased obviously, neurological deficit restored significantly, and the number of autophagosomes and the protein expression of beclin-1 and LC3-Ⅱ were increased in AS-IV group and Rapa group (P<0.05). However, no significant difference between solvent control group and I/R group was observed (P>0.05). Compared with AS-IV group, the neurological deficit was serious, the volume of cerebral infarction and the number of autophagosomes were increased, while the expression of beclin-1 and LC3-Ⅱ was decreased in AS-IV+3-MA group and 3-MA group (P<0.05). CONCLUSION: Astragaloside IV may play an important role in atte-nuating cerebral ischemia/reperfusion injury by activating autophagy.     

Quercetin activates mitochondrial autophagy via PINK1/parkin pathway to alleviate cerebral ischemia-reperfusion injury in rats

AIM To analyze the regulatory effect of quercetin （QUE） on PTEN-induced putative kinase 1 （PINK1）/parkin mitochondrial autophagy pathway, and to explore the mechanism of quercetin in relieving cerebral ischemia/reperfusion （I/R） injury. METHODS Sixty SD male rats were randomly divided into sham operation group, model group （I/R group）, QUE group,3-methyladenine （3-MA） group and QUE+3-MA group. Administration started in each group 3 days before modeling, once a day, at 30 min after the last administration,except sham group, the other groups used 4-vessel blockage method to establish the whole brain I/R model. On the day after modeling, the neural function was evaluated by neuropathy disability score （NDS）. The volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride （TTC） staining. The morphological changes of mitochondria in hippocampus were observed by transmission electron microscopy. The contents of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in hippocampus were measured by ELISA. The activity of superoxide dismutase （SOD） and contents of malondialdehyde （MDA） in hippocampus were detected by xanthine oxidase method, thiobarbituric acid condensation method. Western blot was used to detect the proteinex pression of PINK1, parkin and LC3-II in brain tissue. RESULTS Compared with sham group, the hippocampus of the rats in I/R group and QUE+3-MA group showed swelling of mitochondria, destruction or disappearance of internal crista and other pathological damage,also the volume of cerebral infarction, the contents of IL-6, TNF-α and MDA, the protein expression levels of PINK1, parkin and LC3-II were increased （P<0.05）, while NDS score and activity of SOD were decreased （P<0.05）. Compared with I/R group and QUE+3-MA group, the pathological damage degree of hippocampus in QUE group was reduced, the volume of cerebral infarction, the contents of IL-6, TNF-α and MDA were decreased （P<0.05）, the proteinexpression levels of PINK1, parkin and LC3-II, and NDS score and activity of SOD were increased （P<0.05）.The above indexes in 3-MA group were opposite to QUE group. No significant difference in the above indexes between I/R group and QUE+3-MA group was observed （P>0.05）. CONCLUSION Quercetin activates mitochondrial autophagy and reduces cerebral I/R by regulating the expression of PINK1/parkin pathway proteins.