Effects of flavonoids isolated from Scutellaria stem and leaf on expression of NMDAR and VEGF in chronic cerebral ischemia rats

AIM: To study the effects of flavonoids isolated from Scutellaria stem and leaf (SSF) on the expression of N-methyl-D-aspartate receptor (NMDAR) and vascular endothelial growth factor (VEGF) in chronic cerebral ischemia rats. METHODS: The model of chronic cerebral ischemia was established by bilateral carotid artery occlusion for 2 months in female SD rats. The effects of SSF on mRNA expression of NMDAR in hippocampus and VEGF in cerebral cortex were evaluated by the method of RT-PCR. RESULTS: Compared with the sham group, the expression of NMDAR1, NMDAR2A and NMDAR2B in hippocampus and VEGF in cerebral cortex were significantly increased (P<0.01). However, the cerebral ischemia rats daily and orally administered with SSF at doses of 17.5 mg·kg^-1·d^-1, 35 mg·kg^-1·d^-1 and 70 mg·kg^-1·d^-1 for 38 days appeared that the mRNA expression of NMDAR1, NMDAR2A and NMDAR2B in hippocampus was obviously reduced (P<0.05), and the mRNA content of VEGF in the cortex (P<0.05) was increased. CONCLUSION: SSF decreases the expression of NMDAR in hippocampus, increases the expression of VEGF in cerebral cortex of cerebral ischemia rats, suggesting that the neuroprotective effect of SSF may be exerted by influencing the production of NMDAR and VEGF in the brain.     

Effect of scutellarin on VEGF expression in human retinal pigment epithelial cells and retinas of diabetic rats

AIM: To evaluate the influence of scutellarin on the expression of vascular endothelial growth factor (VEGF) in high glucose-treated human retinal pigment epithelial cell line ARPE-19 and to observe the effects of scutellarin on the protein expression of VEGF, p-ERK and VEGFR2 in the retinas of type II diabetic rats. METHODS: Cultured ARPE-19 cells were divided into normal control group, scutellarin group, high glucose group and high glucose+scutellarin group. The protein levels of VEGF, p-ERK and VEGFR2 were measured by Western blot. The VEGF release in ARPE-19 cells was detected by ELISA. Normal rats were randomly divided into normal control group and scutellarin group. Diabetic rat model was established by feeding with high-fat diet and injecting with streptozocin, and randomly divided into diabetes group and diabetes treated with scutellarin group. After 16 weeks, the eyes were removed. The morphological changes of the retinas were observed under light microscope with HE staining, and histopathological score was recorded. The expression of VEGF in the retinas was observed by the method of immunohistochemistry. RESULTS: Compared with normal control group, the protein levels of VEGF, p-ERK and VEGFR2 in the ARPE-19 cells decreased in scutellarin group, but increased in high glucose group. The histopathological score of the retinas showed significant difference among diabetes group, diabetes treated with scutellarin group and normal control group, and no significant difference between normal control group and scutellarin group was observed. The expression of VEGF increased in diabetic group and was significantly higher than that in scutellarin treatment group (P<0.05). CONCLUSION: Scutellarin inhibits the increased protein le-vels of VEGF, p-ERK and VEGFR2 in ARPE-19 cells, and decreases the expression of VEGF in the retinas of diabetic rats. The suppression of the diabetic retinopathy development by scutellarin may be partly involved in the ERK/MAPK pathway.     

Effect of ginsenoside Rg1 on activity and protein expression of NOS in rats with cerebral ischemic reperfusion injury

AIM: To observe the neuroprotective effects of ginsenoside Rg1 on focal cerebral ischemia reperfusion (I/R) injury in rats. METHODS: SD rats were applied to right middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h of reperfusion. The rats were randomly divided into sham-operation group, I/R group and ginsenoside Rg1 pretreatment groups. The rats in ginsenoside Rg1 pretreatment groups were pretreated with ginsenoside Rg1 at doses of 10, 20 or 40 mg/kg once a day for 7 days and then subject to MCAO. The neurological deficit score was measured by Longa's method. The neurons were observed with Nissel staining. The nitric oxide (NO) content, the activity of nitric oxide synthase (NOS) and inducible NOS (iNOS) in the brain tissues were determined. The expression of neuronal NOS(nNOS) and iNOS was detected by Western blotting. RESULTS: Compared with sham-operation group, ginsenoside Rg1 significantly reduced the neurological deficit score and increased the neuron number in the hippocampus. The activity of NOS and iNOS, and NO content were decreased. Ginsenoside Rg1 also down-regulated the expression of nNOS and iNOS. CONCLUSION: Ginsenoside Rg1 has protective effect on the brain during cerebral I/R injury in rats. The mechanism may be related to reducing the content of NO and the activiy of NOS dose-dependently.     

Effect of erigeron on intercellular adhesion molecule-1 and its mRNA expression during cerebral ischemia and reperfusion in rats

AIM: To investigate the effect of erigeron on intercellular adhesion molecule-1 (ICAM-1) and mRNA expression during cerebral ischemia/reperfusion. METHODS: The rat models of middle cerebral artery (MCA) focal cerebral ischemic reperfusion were established with the suture method in the study. The ICAM-1 mRNA and protein expression were measured by RT-PCR and immunohistochemistry techniques, respectively. RESULTS: By down-regulating the expression of ICAM-1 protein and mRNA and alleviating inflammation in cerebral ischemic region, erigeron exerted a protective effect in cerebral ischemia and reperfusion. CONCLUSION: The results suggest that erigeron protects the brain against cerebral ischemia and reperfusion injury via inhibiting ICAM-1 expressino.     

Effect of cholecystokinin-octapeptide on focal cerebral ischemia/reperfusion injury in rats

AIM:To examine the effect of cholecystokinin-octapeptide (CCK-8) on focal cerebral ischemia/reperfusion injury and its underlying mechanisms.METHODS:By using the suture model of focal cerebral ischemia and reperfusion, the effects of intracerebroventricular (icv) injection of CCK-8 and proglumide, nonselective CCK receptors antagonist, on the infarct size, regional cerebral blood flow (rCBF), and the levels of nitric oxide (NO), malondialdehyde (MDA) were observed in different brain regions of rats subjected to 1 h focal cerebral ischemia followed by 24 h reperfusion.RESULTS:(1) pretreatment with different doses of CCK-8 (0.3 μg,1.0 μg,2.0 μg or 4.0 μg) could attenuate the infarct size, but the statistically significant effects of CCK-8 were obtained only at the doses of 1.0 μg and 2.0 μg(P＜0.05). The neuroprotective effects of CCK-8 were blocked by pretreatment with proglumide. Administration of proglumide alone could worsen the ischemia/reperfusion injury. (2) CCK-8 (1.0 μg) inhibited the increase in NO, MDA levels in the ischemic core, and also inhibited the increase in NO level in the ischemic penumbra. The rCBF in the CCK-8 group was significantly higher than the normal value at 24 h after reperfusion (P＜0.05).CONCLUSIONS:These results suggest that both endogenous and exogenous CCK-8 alleviate focal cerebral ischemia/reperfusion injury. Such an action may be associated with inhibition of free radical-induced injuries and the improvement in rCBF.     

Effects of bFGF on neuronal apoptosis and fractalkine expression in cerebral ischemia/reperfusion rats

AIM: To study the effects of basic fibroblast growth factor (bFGF) on neuronal apoptosis and fractalkine expression in ischemic penumbra after cerebral ischemia/reperfusion in rats.METHODS: Thirty-six rats were randomly divided into 3 groups: sham operation group, ischemia/reperfusion group and bFGF group. The model of middle cerebral artery occlusion was established by the method of intraluminal filament blockage. The middle cerebral arteries were blocked for 1 h and then reperfused for 24 h. Neurological performances of all rats were scored with Bederson's standard. The brain tissues of the rats were stained and the average infarct volume was calculated. TUNEL method was used to determine the number of apoptotic neurons, and the expression of fractalkine was detected by the method of immunohistochemistry.RESULTS: The score of neurological performances in bFGF group was 2.23±0.59, lower than that in ischemia/reperfusion group (3.18±0.65). The number of apoptotic neurons in bFGF group (13.22±1.35) was lower than that in ischemia/reperfusion group (17.28±1.01, P<0.05), which was the lowest in sham operation group (0.91±0.65). Compared with sham operation group, the expression of fractalkine in ischemia/reperfusion group was decreased. The expression of fractalkine in bFGF group was mainly higher than that in ischemia/reperfusion group (P<0.05).CONCLUSION: Up-regulation of fractalkine may be one of the molecular mechanisms of bFGF to protect neurons against ischemia/reperfusion injury.     

Stability of cerebral focal ischemia/reperfusion model induced by monofilament in Kunming mice

AIM: To study the stability of mouse cerebral ischemia/reperfusion model induced by the method of monofilament. METHODS: Sixty male Kunming mice were divided into 3 groups according to the body weight: group A (18-21 g), group B (22-28 g) and group C (30-35 g). Ischemia/reperfusion (I/R) model was made by middle cerebral artery occlusion (MCAO) with nylon monofilament. To evaluate the mouse MCAO model, the method of PRM2 laser Doppler was used to detect the cerebral blood flow, the neurological deficit scores were determined by Longa standard and infarction volume was detected with TTC staining. The content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were also measured by ELISA. RESULTS: The successful rates of model establishment in both group A and group B were higher than that in group C (P＜0.05), especially the highest in group B . The mortality in group A was significantly higher than that in group B and group C (P＜0.05). The behavior scores and cerebral infarct volume in group A and group B were significantly higher than those in group C (P＜0.05). Obvious brain injury and neurological deficits were also observed in group A and group B with the higher content of MDA and the lower activity of SOD in the cerebral cortex of the injury side. CONCLUSION: There are three important factors to ensure the success and stability of MCAO mouse model induced by monofilament, i.e. the diameter of monofilament matching the body weight of the mice, the suitable length of monofilament within the blood vessel, as well as the maintaining of proper room temperature during experiment. The MDA content and SOD activity are also effective indexes for evaluating the cerebral I/R injury.     

Nerve growth factor attenuates cochlear injury induced by brain ischemia and reperfusion in rats

AIM: To investigate the effect of nerve growth factor (NGF) on auditory and cochlear damage induced by brain ischemia and reperfusion. METHODS: The rats with brain ischemia and reperfusion were divided into two groups at random. In the experimental group, the rats were injected intramuscularly with NGF . In control group, the animals were injected with normal saline instead of NGF. Then the hearing loss and cochlear structural changes of rats in both groups were compared. RESULTS: It was found that the hearing loss of rats in NGF group were less significantly than that of control group ( P< 0.01) after 60 min and 24 h reperfusion following 30 min ischemia. Scanning and transmission microscopy showed that the damage of the outer hair cells and the spiral neurons of rats in NGF group was much slighter than that of control group. CONCLUSION: NGF prevents auditory and cochlear injury induced by brain ischemia and reperfusion.     

Protective effect of spermine on rats with acute cerebral ischemia/reperfusion injury

AIM: To study the effects and the possible mechanisms of exogenous spermine on the rats with acute transient focal cerebral ischemia/reperfusion (I/R) injury.METHODS: The rat model of focal cerebral ischemia/reperfusion was established by middle cerebral artery occlusion (2 h) and reperfusion (2 h). Healthy adult SD rats were divided into 5 groups;sham group,I/R group and spermine(4,20 and 40 mmol/L)groups.The degree of cerebral injury was evaluated by neurological deficit score, infracted volume, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The morphological changes of the brain were observed by HE staining and electron microscopy. RESULTS: Compared with I/R group, the neurological deficit score, infracted volume and the content of MDA were decreased, the SOD activity was increased and the ultrastructural changes were improved in spermine-treated groups. CONCLUSION: Exogenous spermine has a protective effect against acute focal cerebral ischemia/reperfusion injury. The mechanisms may be related to scavenging free radical by spermine.     

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.     

Effects of Astragalus polysaccharide on expression of HSP70, PKB and P53 in rat cerebral cortex with cerebral ischemia and reperfusion

AIM:To explore the molecular effects of Astragalus polysaccharide(AP) on improving nervous functions and preventing neuronal apoptosis in rat cerebral cortex with cerebral ischemia and reperfusion. METHODS:One hundred and twenty male Wister rats were randomly divided into sham operation group(SOG), model groups(MG-1 d, 3 d and 7 d), low-dose AP treatment groups(L-APTG-1 d, 3 d and 7 d), and high-dose AP treatment groups(H-APTG-1 d, 3 d and 7 d). The right middle cerebral artery of the rats in MG and AGTG was intercepted by operation to induce ischemic brain injury. The rats in L-APTG and H-APTG were treated with AP at the doses of 5 mg/kg and 15 mg/kg by intraperitoneal injection, respectively. On the 1st day, 3rd day and 7th day after operation, those animals were sacrificed to collect the brain specimens for the study after cerebral blood flow reperfusion and determination of neurological deficit scores. The structural changes of the neurons were observed under electron microscope. Apoptosis was analyzed by flow cytometry. The protein levels of heat-shock protein 70(HSP70), protein kinase B(PKB) and P53 in cerebral corical neurons were determined by immunohistochemical staining and Western blotting. RESULTS:The neurological deficit scores and the apoptotic rate of cerebral cortical neurons in H-APTG were significantly lower than those in MG and L-APTG(P<0.05). The structures of the neurons in H-APTG, such as ribosome endoplasmic reticulum, nucleolus, Golgi complex, mitochondria, etc, were better than those in MG and L-APTG. On the 1st day, 3rd day and 7th day, the protein levels of HSP70 and PKB in cerebral cortical neurons in H-APTG were significantly higher than those in L-APTG, which were significantly higher than those in MG(P<0.05). However, the P53 protein level in H-APTG was significantly lower than that in L-APTG, which was significantly lower than that in MG(P<0.05). CONCLUSION:AP improves nervous functions and inhibits neuronal apoptosis during ischemia and reperfusion. The molecular mechanisms are associated with variations of protein expression in cerebral cortical neurons, such as promotion of HSP70 and PKB and inhibition of P53.     

Effect of tetramethylpyrazine on paw MMP-13 protein expression and serum VEGF,IL-17 and IL-23 levels in rats with collagen-induced arthritis

AIM：To investigate the mechanism of tetramethylpyrazine (TMP) in treating collagen-induced arthritis (CIA) in rats. METHODS：The rat model of type II collagen-induced arthritis was established and the rats were randomly divided into normal control group, CIA group and TMP treatment group. The protein expression of matrix metalloproteinase 13 (MMP-13) in paw tissues was detected by immunohistochemical staining and Western blotting. The histopathological changes of the skin in the rat paws were observed with HE staining. The levels of vascular endothelial growth factor (VEGF), interleukin 17 (IL-17) and interleukin 23 (IL-23) in the serum of CIA rats were detected by ELISA. RESULTS：Compared with CIA group, TMP at a dose of 100 mg/kg significantly reduced the weight loss in CIA rats and decreased the protein expression of MMP-13 by 31.82%. TMP also attenuated the pathological changes of the paw subcutaneous tissues. TMP obviously decreased the levels of VEGF by 33.33%, IL-17 by 27.40% and IL-23 by 33.33% in the serum. CONCLUSION：TMP significantly inhibits the development of CIA by decreasing the protein expression of MMP-13, inhibiting the inflammatory cell infiltration and vascular proliferation, and reducing the production of VEGF, IL-17 and IL-23 in CIA 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.     

Effect of electroacupuncture on astrocyte morphology of ischemia cortex penumbra zone in cerebral infarct rats

AIM: Effect of electron acupuncture on astrocyte morphology of cerebral ischemic penumbra zone in rats was observed.METHODS: The brain infarct model in male Sprague-Dawley rats was made by thread embolism cerebral middle artery.Immunochemistry method was used to observe the change of astrocyte morphology in cerebral ischemic penumbrazone.RESULTS: There was no statistically difference in astrocyte morphology between cerebral infarct group and electroacupuncture treatment group.CONCLUSION: Acupoint stimulation of 5 and 10 Hz electro-acupuncture with a current intensity of 2 mA may not affect on astrocytic morphology.     

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

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

Effects of NBP on expression of VEGF and HIF-1α in HUVECs under the condition of oxygen-glucose deprivation

AIM: To investigate the mechanism that dl-3-n-butylphthalide (NBP) protects cells from the induced by oxygen-glucose deprivation (OGD).METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Endothelial injury was assessed by measuring the changes of chromatin morphology and MTT method. The protein levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were determined by immunofluorescence and quantitatively analyzed with the software IPP. Western blotting was applied to verify the results.RESULTS: NBP at the concentrations of 0.01 to 100 μmol/L dose-dependently protected against OGD-induced cell damage. Compared with OGD group, NBP enhanced OGD-induced the expression of VEGF and HIF-1α, and the difference was statistically significant. The expression of VEGF and HIF-1α reached to the peak at the time points of 6 h and 8 h after OGD, respectively.CONCLUSION: Under the condition of OGD, NBP enhances the expression of HIF-1α in HUVECs, subsequently promotes the expression of downstream VEGF, and eventually elevates the survival of the cells.