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Objective To investigate the effect of expressions of endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) in the ischemic cortex on ischemic cerebral injury in rats with diabetes mellitus.Methods A total of 36 healthy male Sprague-Dawley rats were divided into 3 groups:a shamoperation group,a cerebral ischemic group,and a diabetic cerebral ischemic group according to the random number table method.A diabetes model was induced by injection of streptozocin,and then,a permanent focal cerebral ischemic model was induced by the suture method.At 24 h after ischemia,the neurological deficit scores were conducted.The triphenyl tetrazolium chloride staining was used to measure the infarct volume.TUNEL was used to detect the apoptotic cells.Real-time fluorescence quantitative polymerase chain reaction was used to detect the expression levels of VEGF and VEGFR2 mRNAs.Western blot was used to detect the expression levels of VEGF and VEGFR2 proteins.Results In the sham operation group,there were no neurological deficit and infarcts,and there were only a few apoptotic cells and a few expressions of VEGF,VEGFR2 mRNAs and protein.The neurological function score (4.25 ±0.54 vs.2.86 ±0.73);t =5.303,P＜0.001),infarct volume (51.69 ±2.26 mm3 vs.30.15 ±2.08 mm3;t =23.166,P＜0.001),and the number of apoptotic cells (24.22 ± 1.34/HP vs.13.28 ±0.37/HP;t =27.261,P＜0.001) in the diabetic cerebral ischernia group were significantly increased than those in the cerebral ischemic group,while VEGF,VEGFR2 mRNA,and protein expression level were significantly decerased (VEGF mRNA:4.74 ± 0.54 vs.6.71 ± 0.91,P ＜ 0.001;VEGFR2 mRNA:4.06 ± 0.60 vs.6.16 ± 0.96,P ＜ 0.001,VEGF protein:0.99 ± 0.13 vs.1.55 ± 0.23,P ＜ 0.001;VEGFR2 protein:4.12 ± 0.74 vs.6.23 ± 0.76,P ＜ 0.001) compare with the cerebral ischemic group.Conclusions VEGF/VEGFR2 signal pathway participates in diabetes aggravating ischemic cerebral injury.The downregulating of VEGF/VEGFR2 may be one of the mechanisms of diabetes aggravating ischemic cerebral injury.
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Glucagon and its analogues are a intestinal stimulating insulin screened by the small intestinal L cells. It can smoothly penetrate the blood-brain barrier into the brain tissue and play a neuropro-tective role. Liraglutide and glucagon and its analogues have higher homology. After entering the brain tissue, it is able to bind with the related receptors and activates Nrf2/HO-1 signaling pathway and thus reducing the production of oxidative stress products, increases the glutathione, heme oxygen synthase, superoxide dismutase and other phase Ⅱ detoxification enzymes, promotes angiogenesis, and protects the nerve cells of diabetes combined with cerebral ischemia injury.
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Objective To investigate the protective effect of salvianolic acid A (SAA) on permanent focal cerebral ischemia in rats and its possible mechanisms. Methods Fifty-four adult male Sprague-Daw ley rats w ere randomly divided into a sham operation group, a cerebral ischemia group, and a SAA group ( n =18 in each group). A model of permanent middle cerebral artery occlusion w as induced by the intraluminal suture method.At 0 h and 6 h after modeling, the rats of the SAA groups w ere intraperitonealy injected SAA (3 mg/kg). The other groups w ere injected equal volume of saline. At 24 h after modeling, the neurological deficit scores w ere performed. 2,3,5-Triphenyl tetrazolium chloride (TTC) staining w as used to detect cerebral infarction volume. TUNEL staining w as used to detect cel apoptosis. Both immunohistochemical staining and Western blotting w ere used to detect the expressions of Wnt3a, β-catenin, and phosphor-glycogen synthase-kinase-3β(p-GSK-3β) in the ischemic cortex. Results The neurological deficit scores show ed that no neurological deficits w ere observed in the sham operation group (score 0). The neurological deficit score in the SAA group (median and interquartile range) w as significantly low er than that in the cerebral ischemia group (3 [2-3] vs.4 [3-5]; Z = -2.679, P =0.007). No infarcts w ere observed in the sham operation group. The infarct volume in the SAA group w as reduced significantly compared w ith the cerebral ischemia group (79.038 ±10.665 mm 3 vs.212.702 ±8.029 mm 3; t = 24.525, P < 0.001). Very few positive cels w ere observed in the sham operation group. The numbers of TUNEL -positive cels in the SAA group and the cerebral ischemia group w ere 29.667 ±1.366/HP and 63.333 ±0.894/HP, respectively. The former w as significantly less than the latter ( t = 14.115, P < 0.001). Immunohistochemical staining show ed that the number of Wnt3a positive cels in the sham operation group, the cerebral ischemia group, and the SAA group w ere 35.500 ±2.572/HP, 18.056 ±3.765/HP, and 29.000 ±2.376/HP, respectively. There w ere significant differences among the 3 groups ( F = 115.972, P < 0.001), and those in the SAA group w ere significantly more than the cerebral ischemia group ( P < 0.01). The numbers of p-GSK-3βpositive cels in the sham operation group, the model group, and the SAA group w ere 7.944 ±2.127/HP, 37.444 ±3.434/HP, and 11.222 ±1.734/HP, respectively. There w ere significant differences among the three groups (F =730.580, P < 0.001), and those in the SAA group w ere significantly less than the cerebral ischemia group ( P < 0.01). The numbers of β-catenin positive cels in the sham operation group, the cerebral ischemia group, and the SAA group w ere 26.722 ±26.722/HP, 16.556 ±1.854/HP, and 21.333 ± 1.940/HP, respectively. There w ere also significant differences among the 3 groups ( F < 33.385, P <0.01), and those in the SAA group w ere significantly more than the cerebral ischemia group ( P < 0.01). Western blot analysis show ed that Wnt3a expression levels in the sham operated group, the cerebral ischemia group, and the SAA group w ere 1.000 ±0.190, 0.800 ±0.185, and 1.198 ±0.262, respectively. There w ere significant differences among 3 groups ( F = 9.621, P < 0.001), and those in the SAA group w ere significantly higher than the cerebral ischemia group ( P < 0.01). The p-GSK-3βexpression levels in the sham operation group, the cerebral ischemia group, and the SAA group w ere 0.650 ±0.150, 1.290 ± 0.250, and 1.190 ±0.250, respectively. There w ere also significant differences among the 3 groups ( F =19.668, P < 0.001), and those in the SAA group w ere significantly higher than the cerebral ischemia group (P <0.01). The β-catenin expression levels in the sham operation group, the cerebral ischemia group, and the SAA group w ere 1.200 ±0.210, 0.500 ±0.120, and 1.100 ±0.220, respectively. There w ere significant differences among the 3 groups ( F = 33.385, P < 0.001), and those in the SAA group w ere significantly higher than the cerebral ischemia group ( P < 0.01). Conclusions SAA has certain protective effect on permanent cerebral ischemia injury in rats. Its mechanism may be associated w ith the up -regulation of the expression of Wnt3a and β-catenin and the dow n-regulation of the expression of p-GSK-3β.
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ObjectiveToinvestigatetheprotectiveeffectandmechanismofscutelarincombinedwith paeoniflorin after permanent cerebral ischemia in rats. Methods Forty-eight adult male SD rats w ere randomly divided into four groups: sham-operation, cerebral ischemia, scutelarin+ paeoniflorin, and cyclopamine (n=12 in each group). A model of permanent middle cerebral artery occlusion w as induced by suture method. The intraperitoneal injection of cyclopamine 6 mg/kg, a specific inhibitor of sonic hedgehog (SHH) pathw ay, at 15 min before ischemia in the cyclopamine group, w hile other groups w ere intraperitoneal y injected an equal volume of saline. At 0 hour and 3 hours after ischemia, the scutel arin+paeoniflorin group and cyclopamine group w ere intraperitoneal y injected scutel arin ( 20 mg/kg ) and paeoniflorin (30 mg/kg), while other groups were intraperitonealy injected an equal volume of saline. Neurological deficit scores w ere performed at 24 hours after ischemia, and then the rats w ere decapitated. The cerebral infarct volume w as measured by using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Real-time fluorescent quantitative polymerase chain reaction and Western blotting w ere used respectively to detect the expression levels of SHH, Patched-1, Gli-1 mRNAs and proteins in the ischemic cortex. Results The neurological deficit scores in the cerebral ischemia group, scutel arin+paeoniflorin group, and cyclopamine group w ere 3.33 ±0.52, 1.50 ±0.55, and 3.67 ±0.52, respectively. The neurological deficit score in the scutel arin+paeoniflorin group w as significantly low er than that in the cerebral ischemia group ( P<0.05), and the neurological deficit score in the cyclopamine group w as significantly higher than that in the scutelarin+paeoniflorin group ( P<0.05). The infarct volume percentage in the cerebral ischemia group, scutelarin+paeoniflorin group, and cyclopamine group were 31.77%±1.19%, 22.94%±2.65%, and 35.53%±0.20%, respectively. The infarct volume in the scutel arin+paeoniflorin group w as significantly less than that in the cerebral ischemia group ( P<0.05), and the infarct volume in the cyclopamine group was significantly larger than that of the scutelarin+paeoniflorin group (P<0.05). The expression levels of SHH, Patched-1, Gli-1 mRNAs and proteins in the cerebral ischemia group, scutelarin+paeoniflorin group, and cyclopamine group w ere significantly higher than those in the sham -operation group (al P<0.05). The expression levels of SHH, Patched-1, Gli-1 mRNAs and proteins in the scutelarin+paeoniflorin group were significantly higher than those in the in the cerebral ischemia group (al P<0.05), and the expression levels of Gli-1 mRNA and protein in the cyclopamine group were significantly lower than those in the scutelarin+paeoniflorin group ( al P<0.05 ). Conclusions The scutel arin combined w ith paeoniflorin has certain protective effect on focal cerebral ischemia injury in rats. Its mechanism is associated w ith the activation of SHH signaling pathw ay.
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Objective To investigate the roles of tumor necrosisfactor-α(TNF-α) and nuclear factor-κB (NF-κB) in cerebral ischemia-reperfusion injury in rats w ith diabetes mel itus. Methods Thirty-six healthy male Sprague-Daw ley rats w ere divided into a euglycemic sham operation group, a euglycemic isc hemia-reperfusion group, and a diabetes ischemia-reperfusion group (n=12 in each group) according to a random number table. A diabetes model w as induced by intraperitoneal injection of streptozotocin, and then a focal cerebral ischemia-reperfusion model w as induced by the suture method. The neurological deficit score was performed at 24 h after reperfusion. 2,3,5 triphenyl tetrazolium staining was used to measure the cerebral infarction area. Western blotting w as used to detect the expression levels of NF-κB and TNF-αon the ischemic sides. Results The neurological function scores w ere 0.00 ±0.00, 2.50 ±1.08, and 3.20 ± 1.03, respectively in the euglycemic sham operation, euglycemic cerebral ischemia-reperfusion and diabetes cerebral ischemia-reperfusion groups, and there w ere significant differences (F=38.015, P<0.001). The neurological deficit scores of the diabetes cerebral ischemia-reperfusion group w ere significantly aggravated compared with the euglycemic cerebral ischemia-reperfusion group (P<0.05). The infarct areas of the euglycemic sham operation, euglycemic cerebral ischemia-reperfusion and diabetes cerebral ischemia-reperfusion groups w ere 0.00% ±0.00%, 33.09% ±5.17%, and 55.45% ±9.29%, respectively, and there w ere significant differences among the groups (F=206.614, P<0.001), in w hich the infarct area in the diabetes cerebral ischemia-reperfusion group w as enlarged significantly compared w ith the euglycemic cerebral ischemia-reperfusion group ( P< 0.05 ). At 24 h after reperfusion, there w ere no significant differences in the expression levels of the cortical NF-κB (F=29.993, P<0.001) and TNF-α(F=28.722, P<0.001) on the ischemic sides in each group, in w hich the expression levels of NF-κB and TNF-αin the diabetes cerebral ischemia-reperfusion group w ere increased significantly compared w ith the euglycemic cerebral ischemia-reperfusion group (al P<0.05). Conclusions Diabetes may aggravate cerebral ischemia reperfusion injury. The upregulated expression of TNF-αand NF-κB may be one of the mechanisms of diabetes aggravating cerebral ischemia-reperfusion injury.
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AIM:To evaluate the effect of microRNA-155(miRNA-155) on the regulation of angiogenesis in diabetic rats with cerebral ischemic injury .METHODS: Adult male Sprague-Dawley rats were randomly divided into 5 groups:sham group, cerebral ischemia group , diabetic cerebral ischemia group , diabetic cerebral ischemia +miRNA-155 inhibitors group and diabetic cerebral ischemia +scramble group .Diabetes model was made by injection of streptozocin and permanent cerebral ischemic model was developed by suture-occluded method .The scores of neurological deficit and infarct volume were estimated at 24 h after cerebral ischemia .miRNA-155 level was detected by real-time polymerase chain reaction.The expression of platelet endothelial cell adhesion molecule-1 ( PECAM-1/CD31 ) and vascular endothelial growth factor ( VEGF) was detected by Western blotting .RESULTS:miRNA-155 inhibitor significantly reduced miRNA-155 levels in the ischemic cortex (P<0.05), improved the scores of neurological deficit , reduced infarction size and up-regulated the levels of CD31 and VEGF (P<0.05).CONCLUSION:miRNA-155 has a critical role in the regulation of angiogenesis in diabetic rats with cerebral ischemia .Down-regulation of miRNA-155 using miRNA-155 inhibitor attenuates brain infarct injury in diabetic rats .
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Objective To investigate the protective effect of lipoxin A4 on diabetic rats with focal cerebral ischemia-reperfusion and its mechanisms.Methods Thirty-six adult male Sprague-Dawley rats were randomly divided into a sham operation group,a cerebral ischemia-reperfusion group,and a lipoxin A4 group (n=12 in each group).Diabetes was induced by repeated intraperitoneal injection of low-dose streptozotocin.A model of middle cerebral artery occlusion and reperfusion was induced by the intraluminal suture method.Five minutes after cerebral ischemia,lipoxin A4 0.03 nmol/5 μ1 was injected via intracerebroventricular in the lipoxin A4 group.The other groups were injected equal volume of saline.Two hours after ischemia,the suture was pulled out and reperfusion was achieved.Neurological deficit scores were performed at 24 hours.Then the rats were decapitated and their brains were taken out.2,3,5-triphenyl tetrazolium chloride (TTC) staining was used to detect infarct size.Western blotting was used to detect the expression of cortical tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB).Results The neurological deficit score showed that no neurological deficit was observed in the sham operation group (score 0).The neurological deficit score in the lipoxin A4 group was significantly lower than that in the cerebral ischemia-reperfusion group (2.20 ± 1.03 vs.3.20 ± 1.03; P ＜0.05).TTC staining showed that no infarct was observed in the sham operation group.The infarct size in the lipoxin A4 group was significantly lower than that in the cerebral ischemia-reperfusion group (27.52％ ± 5.71％ vs.55.45％ ± 9.29％ ; P ＜0.05).Western blotting showed that the expression levels of TNF-α in the sham operation,cerebral ischemiareperfusion,and lipoxin A4 groups were 0.64 ± 0.16,1.85 ± 0.52,and 1.40 ± 0.34,respectively.There were significant differences among the 3 groups (F =18.868,P ＜0.001).The expression level of TNF-α in the lipoxin A4 group was significantly lower than that in the cerebral ischemia-reperfusion group (P ＜0.05).The expression levels of NF-κB in the sham operation,cerebral ischemia-reperfusion and lipoxin A4 groups were 0.79 ±0.24,2.09 ± 0.47,and 1.27 ± 0.35,respectively.There were significant differences among the 3 groups (F =16.736,P ＜ 0.001).The expression level of NF-κB in the lipoxin A4 group was significantly lower than that in the cerebral ischemia-reperfusion group (P ＜0.05).Conclusions Lipoxin A4 has certain protective effect on focal cerebral ischemia-reperfusion injury in diabetic rats,its mechanism may be associated with the inhibition of the expression of TNF-α and NF-κB.
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Lipoxin A4 is an arachidonic acid metabolite,it is an important endogenous anti-inflammatory mediators in the body,which is known as an inflammatory braking signal. Inflammatory response is an important factor for causing cerebral ischemia-reperfusion injury.Lipoxin A4 can exert neuroprotective effects by inhibiting inflammatory response.In addition,lipoxin A4 can also reduce blood-brain barrier permeability,reduce cerebral edema,and promote recovery of neurological function.This article reviews the neuroprotective roles and mechanisms of lipoxin A4 in cerebral ischemia-reperfusion.
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Objective To investigate the relationship between the expression changes of p-Akt (Ser473),p-Bad (Ser136) and the cell apoptosis in peri-infarction tissue of permanent middle cerebral artery occlusion (MCAO) in rats.Methods Sixty male Sprague-Dawley rats were randomly allocated into sham operation,MCAO 3 h,MCAO 12 h,LY294002 intervention MCAO 3 h,and LY294002 intervention MCAO 12 h groups (n =12 in each group).A permanent MCAO model was induced by the modified suture method.At 15 minutes before modeling,the rats in the LY294002 intervention MCAO groups were administered via lateral ventricle.The neurological function score was scored by using Zea Longa method.2,3,5-triphenyltetrazolium chloride (TTC) staining was used to detect infarct volume.Immunohistochemical staining was use to detect pAkt (Ser473) and p-Bad (Ser136) expressions in peri-infaretion tissue.TUNEL assay was used to detect apoptotic cells in peri-infarction tissue.Results Three hours after modeling all the experimental rats awoke from anesthesia.The neurological deficit score in the sham operation group was 0,and the scores of the MCAO 3 h,MCAO 12 h,LY294002 intervention MCAO 3 h and LY294002 intervention MCAO 12 h groups were 2.25 ± 0.45,2.92 ± 0.99,3.00 ± 0.95,and 3.02 ± 0.36,respectively.There were significant differences among all groups (F =26.520,P =0.000).The score of the LY294002 intervention MCAO 3 h group was significantly higher than that of the MCAO 3 h group (P =0.009).There was no significant difference between the LY294002 intervention MCAO 12 h group and the MCAO 12 h group (P =0.354).TTC staining showed that no infarct was observed in the sham operation group.The infarct volumes of the MCAO 3 h,MCAO 12 h,LY294002 intervention MCAO 3 h and LY294002 intervention MCAO 12 h groups were 23.4 ± 1.4,40.3 ± 1.1,31.9 ±6.0 and 44.4 ±3.8 mm3,respectively.There were significant differences among the groups (F =30.440,P =0.000).The score of the LY294002 intervention MCAO 3 h group was significantly greater than that of the MCAO 3 h group (P =0.002).There was no significant difference between the LY294002 intervention MCAO 12 h group and the MCAO 12 h group (P=0.113).Compared with the sham operation group,the p-Akt (Ser473) expression in peri-infarction tissue in the MCAO 3 h group was significantly high,and it was significantly decreased in the MCAO 12 h group; the expression level of p-Bad (Ser136) showed a progressive decline with the passage of MCAO time,at the same time,the number of apoptotic cells increased progressively.After the LY294002 intervention,the expression levels of p-Akt (Ser473) and p-Bad (Ser136) in peri-infarction tissue decreased significantly at 3 h after MCAO,and the number of apoptotic cells increased significantly (P ＜0.05),but there was no significant effect on each index at 12 h after MCAO.Conclusions The activation of the PI3K/Akt signal transduction pathway in early cerebral infarction and the stress elevation of the key protein p-Akt (Ser473) of this pathway have brain protection,while the failure of this pathway activity and the drastical decrease of its key protein in the late cerebral infarction are associated with brain injury.
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microRNAs (miRNAs) are a class of small non-coding RNAs (length 21 to 25 nt).They can complement and bind to 3' non-coding region of their target genes,directly degrade target genes or inhibit the translation of encoding proteins,and extensively participate in development,metabolism,apoptosis,and a variety of disease processes.Studies have shown that miRNAs play important roles in the occurrence and development of ischemic stroke,and participate in post-stroke protection and regulation of repair mechanisms.
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Apoptosis is one of the important forms during cerebral ischemia.Phosphoinositide-3 kinase (PI3K)/serine/threonine kinase (Akt) is the important cell survival signaling pathway,while c-jun N-terminal kinase (JNK) is the important pro-apoptotic signaling pathway.The dynamic equilibrium of the two signal transduction pathways maintains cell survival and apoptosis under the physiological state.Stimulation during cerebral ischemia breaks this physiological balance and results in the apoptosis of massive neurons.A variety of proved neuroprotective factors are associated with the amplification of enhancement of cell survival signal or inhibition of apoptosis signal,and thrus maintain the balance between the two signal pathways.