Effect of Ginkgo biloba extract on the expression of c-fos and HSP70 following focal cerebral ischemic reperfusion in rats

AIM: To investigate the influence of Ginkgo biloba extract (GBE) on the expression of c-fos, heat shock protein 70 (HSP70) during focal cerebral ischemic reperfusion in rats. METHODS: The middle cerebral artery occlusion (MCAO) model described by Zea longa was used. Healthy Wistar rats were randomized to 4 groups. Immunohistochemistry, in situ hybridization and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were used to detect the expression of c-fos gene, HSP70 and cell apoptosis at different reperfusion time points: 1, 6, 12, 24 hours and 3, 7 days after recirculation. RESULTS: The positive reactions of both c-fos and HSP70 were significantly increased at different reperfusion time in GBE-pretreated ischemia/reperfusion (IR) group than those in ischemia/reperfusion group (P<0.01) and the number of TUNEL-positive cells was reduced in GBE-pretreated IR group. CONCLUSION: The GBE induced the expression of c-fos, HSP70 and contributes to neuroprotective activities after cerebral ischemia.     

Effects of astragalan on neurotransmitters and expression of c-fos mRNA in hippocampus after ischemic brain injury in rats

AIM: To explore the effects of astragalan (AG) on the neurotransmitters,acetylcholine (ACh), norepinephrine (NE) and 5-hydroxytryptamine (5-HT), and the expression of c-fos mRNA in hippocampus after ischemic brain injury in rats. METHODS: Male Wistar rats (180~220 g) were randomly divided into 10 groups (n=10): sham-operated group (SOG), 3 model groups (MG 1 d, 3 d, 7 d) and 3 low- or high-dose AG treatment groups (L/H-AGTG 1 d, 3 d, 7 d), respectively. The middle cerebral artery of the rats in MG group and AGTG group were blocked by operation to induced brain injury. The cerebral blood vessels of the animals were blocked on day 1, day 2 and day 7, respectively, after the L/H-AGTG were treated with AG (5 mg/kg and 15 mg/kg, ip). The content of ACh,5-HT and NE was determined using their respective ELISA kits, and the expression of c-fos mRNA in the hippocampus homogenate was semiquantitative analyzed by RT-PCR after neurologic impairment (NIP) was scored. RESULTS: AG attenuated the injury in hippocampus by cerebral ischemia in a dose-dependent manner. The content of ACh, 5-HT and NE in L-AGTG 7 d,H-AGTG 3 d and 7 d groups was significantly higher than that in MG group, but was lower in SOG group (P<0.05 or P<0.01). The mRNA expression of c-fos in SOG group was lower than that in MG group (P<0.05 or P<0.01), indicating that reinforcement expression of c-fos mRNA by cerebral ischemia and the expression of downstream genes may be beneficial for protecting the neurons. The mRNA expression of c-fos in H-AGTG 3 d/7 d groups was higher than that in MG group (P<0.05). CONCLUSION: AG attenuates the damage of neurons and improves the functions of hippocampus under the condition of cerebral ischemia/reperfusion by increasing the content of ACh, NA and 5-HT, and the mRNA expression of c-fos in hippocampus.     

Construction of recombinant plasmid pEGFP-HSP70 and its expression in rat neural stem cells

AIM：To explore the method of constructing recombinant plasmid pEGFP-HSP70 and its expression in neural stem cells. METHODS：Total RNA was acquired from the fetal liver tissue of SD rat. cDNA complete sequence of heat-shock protein 70 (HSP70) gene was amplified by RT-PCR, and cloned into an eukaryotic expression vector containing the enhanced green fluorescent protein (EGFP) reporter gene, pEGFP-C2. Sequencing analysis was performed to confirm the recombinant plasmid pEGFP-HSP70. The technique of nucleofector transfection was used to transfect the recombinant plasmid pEGFP-HSP70 into neural stem cells. RESULTS：HSP70 cDNA sequence was correctly cloned into the eukaryotic expression vector pEGFP-C2. The recombinant plasmid pEGFP-HSP70 was constructed successfully. Compared with control group, the fluorescence intensities in pEGFP-C2 group and pEGFP-HSP70 group were significantly increased. The fluorescence intensity in pEGFP-HSP70 group after 24 h of transfection was significantly decreased compared with other time points of 7 d, 14 d and 21 d. The expression level of HSP70 significantly increased 1 d, 7 d and 14 d after transfection compared with control group. CONCLUSION：The neural stem cells can be directly used as gene action target cells. The HSP70 expression level in the stem cells is closely related to the time after transfection.     

Changes of cortisol content in plasma and hippocampus after focal cerebral ischemia-reperfusion in rats

AIM: To investigate the relationship between glucocorticoid (Gc) and injury of hippocampus neurons and the effect of Gc on dementia episode after cerebral ischemia-reperfusion. METHODS: The rat model of middle cerebral artery occlusion (MACO) was established. Cortisol contents in hippocampus and plasma of the model rats were examined by means of the radioimmunoassay at 2 h, 6 h, 12 h, 24 h after reperfusion. RESULTS: The levels of cortisol content in model group were significantly higher than those in sham group and normal group both in hippocampus and plasma. The highest cortisol content was observed at 6 hours after reperfusion. HE staining showed that the impairment of hippocampus neurons was aggravated progressively with reperfusion interval elongating. CONCLUSION: The increased cortisol in hippocampus and plasma, after 2 h cerebral ischemia and 24 h reperfusion, could aggravate the injury of hippocampus neurons and lead to dementia post stroke.     

Selective up-regulation of Toll-like receptor 9 in mouse microglia after cerebral infarction

AIM：To observe the expression of Toll-like receptor 9 (TLR9) and the distribution of TLR9 in the neurons and glial cells after cerebral infarction. METHODS：With the intraluminal filament method, middle cerebral artery occlusion (MCAO) model of C57 mice was established, and the filament was removed to begin reperfusion 90 min later. A sham-operated group was set up as a control. At 6 h, 3 d, 7 d or 14 d after reperfusion, the mice were randomly killed to prepare brain coronal cryosections (n=3). The level of TLR9 expression and the distribution of TLR9 in the neurons and glial cells were detected by immunofluorescent staining. RESULTS：The level of TLR9 increased significantly in transitional peri-infarct tissues over time and was constantly higher than that in the contralateral and sham-operated ones. Only a few scattered TLR9 staining was found inside the neurons during the whole process. The percentage of TLR9-positive neurons showed no significant difference among groups and time points. Activated microglia aggregated in transitional peri-infarct tissues, with intracellular TLR9 staining from scattered tiny dots to clustered coarse particles. Meanwhile, the percentage of TLR9-positive microglia increased at the beginning and later decreased with time, and was significantly higher than that in the contralateral and sham-operated ones. No TLR9 expression was found in astrocytes or oligodendrocytes. CONCLUSION：TLR9 is triggered in microglia after cerebral infarction in the CNS, while the neurons maintain inherent expression of TLR9. Besides, there is no evidence to support the expression of TLR9 in the astrocytes and oligodendrocytes.     

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.     

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.     

Effects of new kinds of cannabis preparations O-1602 and cannabidiol on experimental acute pancreatitis

AIM: To investigate the therapeutic effects of O-1602 and cannabidiol (CBD), the new kinds of cannabis preparations, on caerulein (CAE)-induced acute pancreatitis (AP) in mice.METHODS: AP was induced by intraperitoneal injection (ip) of CAE in mice (50 μg/kg hourly with a total of 6 times), and the mice in control group were given normal saline (NS) ip in stead of CAE in the same way. The AP mice were administrated O-1602 or CBD for the therapeutic evaluation by observing the following parameters: pathological changes of pancreatic tissue, plasma activity of amylase and lipase (biochemical methods), the levels of TNF-α and IL-6 in the plasma (ELISA), and the activity of myeloperoxidase (MPO) in the lung (biochemical methods). Meanwhile, real-time PCR and Western blotting were used to evaluate the expression of heat shock protein 60 (HSP60) at mRNA and protein levels, respectively. RESULTS: The pancreatic tissues in AP group appeared obvious edema and neutrophil infiltration, which were significantly improved by treating with AP+O-1602 or AP+CBD. The activity of amylase, lipase and MPO, as well as the levels of TNF-α and IL-6 in AP group significantly increased compared with NS group (P<0.05), while these parameters were significantly lower in AP+O-1602 group and AP+CBD group than those in AP group. Meanwhile, the expression of HSP60 at mRNA and protein levels in pancreas tissues was reduced in AP group (P<0.05), and was improved to some extent after treating with O-1602 or CBD (P<0.05).CONCLUSION: The O-1602 and CBD show anti-inflammatory effects on CAE-induced AP in mice and the mechanisms might be related to the effects of cannabinoids on inhibiting inflammatory mediators and cytokines, and increasing the expression of cytoprotective factor HSP60.     

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.     

Effect of apigenin on expression of VEGF in cerebral ischemia and reperfusion rats

AIM: To investigate the effect of apigenin on the expression of vascular endothelial growth factor (VEGF) in the rats under the condition of cerebral ischemia and reperfusion. METHODS: Ninety-one male SD rats were randomly divided into 13 groups: sham operation group (S), model groups (group M6 h, group M24 h, group M72 h, group M7 d), apigenin treatment groups (group A6 h, group A24 h, group A72 h, group A7 d) and dexamethasone treatment groups (group D6 h, group D24 h, group D72 h, group D7 d). The acute transient focal cerebral ischemia reperfusion model was established by modified method of inserting the nylon thread into middle cerebral artery, staying for 2 h and then withdrawing from the artery. In the experiment groups, the TTC staining of brain slices were performed and the neurological behavior scores were determined. The expression of VEGF by immunohistochemistry (ICH) was semi-quantitatively analyzed by measuring the integrated absorbance(IA). RESULTS: Abnormal neurological behaviors were observed in the animals of M groups, A groups and D groups, but the neurological behaviors of the rats in A7 d group were better than that in the other groups (P<0.05). Typical cortical infarct lesions in M groups, A groups and D groups were found by TTC staining, mainly in cerebral cortex and striatum. The immunnohistochemical results showed that the expression of VEGF was significantly higher in M, A and D groups than that in S group (P<0.05). Moreover,the expression of VEGF in A groups(A24 h and A72 h)was higher than that in M groups (M 24 h and M72 h,respectively)(P<0.01).The expression of VEGF in D72 h group was higher than that in M72 h group (P<0.05), and that in A7 d group was obviously higher than that in D7 d group (P<0.01).CONCLUSION: Apigenin promotes the expression of VEGF in the model of acute transient focal cerebral ischemia-reperfusion injury, improves the process of brain injury and recovers the brain functions in rats.     

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.     

Injured effect of cerebrospinal fluid from experimental subarachnoid hemorrhage after cerebral lymphatic blockage on PC12 cells

AIM: To determine the injured effect of cerebrospinal fluid(CSF) from subarachnoid hemorrhage(SAH) after cerebral lymphatic blockage(CLB) on PC12 cells. METHODS: SAH and CLB models of adult New Zealand rabbits were used. CSF was obtained from experimental animals after 5 d of modeling and was added into cultured PC12 cells. The cells were randomly divided into blank control(F12 Ham’s), normal CSF, SAH CSF, and SAH+CLB CSF groups. At different time points, the survival rate of PC12 cells was measured by MTT assay. LDH leakage was detected. Expression of Bax and heat-shock protein 70(HSP70) was determined by immunohistochemical staining. RESULTS: MTT assay and detection of LDH leakage revealed that the survival rate of PC12 cells was obviously inhibited and the leakage of LDH increased in SAH CSF group and SAH+CLB CSF group. CSF from normal rabbit did not damage the PC12, as compared to blank controls. Above effects were more obvious in SAH+CLB CSF group than those in SAH CSF group. Bax and HSP70 protein expression was found in both SAH CSF group and SAH+CLB CSF group. Expression of Bax protein in SAH+CLB CSF group was stronger than that in SAH CSF group in a time dependent manner. At 0.5 h and 1 h, the expression of HSP70 protein in SAH+CLB CSF group was stronger than that in SAH CSF group, whereas the expression became weaker at 2 h and 4 h in that group. CONCLUSION: Blockage of cerebral lymphatic drainage pathway deteriorates the damage of CSF from SAH on PC12 cells, indicating this pathway may acts as an endogenous protective mechanism in SAH.     

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.     

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.     

Effects of cannabinoids WIN and O-1602 on expression of HSPs in mice with colitis or acute pancreatitis

AIM: To investigate the effects of cannabinoids WIN55, 212-2 (WIN) and O-1602 on the expression of heat-shock proteins (HSPs), such as HSP27, HSP60 and HSP70, in the inflammatory tissues of mice with experimental colitis or acute pancreatitis (AP). METHODS: Mouse colitis was induced by feeding the C57/BL with 4% dextran sulfate sodium (DSS) for 7 d, and AP was induced by intraperitoneal injection of ceruline in the mice (50 μg/kg hourly, with a total of 6 times). The mice were intraperitoneally administered with WIN or O-1602 for the therapeutic evaluation by observing the following parameters: pathological changes of the tissues, plasma activity of amylase, plasma levels of IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), and HSP expression in the colonic and pancreatic tissues. RESULTS: Compared with normal control mice, the colonic tissues from colitis mice and the pancreatic tissues from AP mice appeared obvious signs of inflammation and injury. The plasma levels of IL-6 and CINC-1 significantly increased in colitis mice and AP mice. In DSS group of colitis mice, HSP27 expression increased, but HSP60 and HSP70 were reduced in the colonic tissues. WIN showed anti-inflammatory effects on the pathological changes of colonic tissues and the plasma cytokine levels. WIN also improved the expression of HSP70 (P<0.05). In AP group, the expression of HSP27 and HSP70 in pancreatic tissues increased, and HSP60 decreased. O-1602 also showed some anti-inflammatory effects on the changes of the pathological tissues and the plasma parameters, but had no obvious effects on the HSP expression (P>0.05). CONCLUSION: The experimental colitis and acute pancreatitis in mice were induced by feeding DSS and injection of cerulein, respectively. The changes of HSP expression were different in the inflammatory tissues. WIN and O-1602 show anti-inflammatory effects and increase the expression of several HSPs to some extent.     

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.     

Protective effect of allitridi on hippocampus of rats with cerebral ischemia-reperfusion and P53 expression

AIM: To observe the protective effect of allitridi on hippocampal neuron of rats with cerebral ischemia-reperfusion (I/R) injury and to investigate its effects on P53 expression in hippocampus.METHODS: The global cerebral ischemia-reperfusion models were established by 4-vessel occlusion. Allitridi at doses of 10, 20 or 30 mg/kg was injected through rat’s tail vein, half dose at 30 min before brain ischemia and another half dose at 10 min after reperfusion were injected, respectively. The hippocampus of rat was removed 24 h after reperfusion. Toluidine blue staining was applied to estimate morphologic changes. Flow cytometry was used to evaluate neuronal apoptosis rate of hippocampus. Immunohistochemistry was used to observe the expression of P53 protein.RESULTS: Compared with sham group, survival neuronal density in I/R group was significantly depressed. The rate of neuronal apoptosis and the expression of P53 protein were significantly increased. Allitridi significantly increased the number of survival neurons in hippocampus compared to I/R group. Meanwhile, allitridi remarkably inhibited the rate of neuronal apoptosis and the expression of P53 protein.CONCLUSION: Allitridi has protective role against brain ischemia reperfusion injury. The mechanism may be involved in blocking P53 protein expression in hippocampus of rats with ischemia-reperfusion.     

Expression of hypoxia-inducible factor 1 and neuroglobin in piglet cortex during deep hypothermic circulatory arrest

AIM: To observe the expression of hypoxia-inducible factor 1 (HIF-1) and neuroglobin (NGB) in piglet cortex during deep hypothermic circulatory arrest. METHODS: Wuzhishan piglets were randomly assigned to cardiopulmonary bypass group (CPB group), 40 min of circulatory arrest (CA) at 18 ℃ without cerebral perfusion (DHCA group) or with selective antegrade cerebral perfusion (SACP group). After 180 min of reperfusion, cortical tissue was harvested for determining HIF-1α and NGB expression by HE staining, Western blot and real-time PCR. RESULTS: Severer cerebral injury was observed in DHCA group than that in SACP group. After 180 min of reperfusion, HIF-1α protein and mRNA levels were significantly higher in DHCA group than those in CPB group (P<0.05). Accordingly, SACP animal had higher levels of HIF-1α protein and mRNA than those in DHCA group (P<0.05). Simultaneously, higher NGB protein and mRNA levels were found in DHCA group than those in CPB group after 180 min of reperfusion (P<0.05). The SACP animal had higher levels of NGB protein and mRNA than those in DHCA group (P<0.05). CONCLUSION: Up-regulation of HIF-1 and NGB are involved in the mechanism against cerebral injury resulting from DHCA in the cortex and possibly a part of cerebral protective effect of SACP.     

Expression of CRF and PKC proteins in hippocampus of rats with cerebral ischemia and reperfusion

AIM: To observe the expression of CRF and PKC in rats with cerebral ischemia.METHODS: Using immunohistochemistry technique we measured the expression quantitatively of CRF and PKC proteins in the hippocampus in rats induced by MCAO at 2 h,6 h and 24 h after reperfusion,contrast to CRF antagonist.RESULTS: (1) CRF: there were lots of positive and deeper dyeing neurons in hippocampus in model group and normal saline group rats,while there were a few positive and lighter dyeing neurons in sham group and CRF antagonist group.The positive expression areas of CRF protein in hippocampus in model group and normal saline group were significantly bigger than those in sham group and CRF-antagonist group(P<0.01),respectively.(2) PKC：there were a great number of denser positive granules in hippocampus in model group and normal saline group rats,while there were a few of scattered positive granules in sham group and CRF antagonist group.The positive expression areas of CRF protein in hippocampus in model group and normal saline group were significantly bigger than that in sham group and CRF-antagonist group (P<0.01),respectively.CONCLUSION: The high expression of CRF and PKC induced by cerebral ischemia may be one important factors that resulted in the delayed neuronal death in hippocampus.The CRF protein activated PKC expression,indicating an important pathology mechanism of nerve tissue damage induced by CRF.     

Polydatin downregulates TLR4 expression in lung ischemia reperfusion injury in rabbits

AIM: To explore the influence of polydatin (PD) on Toll-like receptor 4 (TLR4) signal transduction pathway during lung ischemia reperfusion injury in rabbits. METHODS: Rabbit lung model of ischemia reperfusion (IR) injury was constituted in vivo. Thirty rabbits were divided into groups randomly: control (C), IR and PD group, respectively. The concentration of endotoxin (ET) in plasma was analyzed by end-point chromogenic assay. The protein expressions of TLR4, nuclear factor (NF)-κB p65 and heat shock protein 70 (HSP70) were measured by immunohistochemistry. The intracellular adhesion molecule-1 (ICAM-1) mRNA expression was detected by in situ hybridization histochemistry. The ultrastructural changes were observed by electron microscope. RESULTS: No significant difference of ET concentration in plasma between groups (all P>0.05) was observed. The protein expressions of TLR-4, NF-κB p65, HSP70 and ICAM-1mRNA in IR group were significantly increased as compared to C group and PD group, while those expressions in PD group were evidently higher than those in C group (all P<0.01). The lung pathological injuries in PD group were obviously alleviated as compared to IR group under electron microscope. CONCLUSION: It suggests that lung ischemia reperfusion releases endogenous ligands of TLR4 as HSP70, then activates NF-κB, promotes the release of mediators of inflammation such as ICAM-1. PD might have a protective effect on lung ischemia reperfusion injury by regulating TLR4 signal transduction pathway.