Hypoxic preconditioning alleviates oxygen-glucose deprivation in PC12 cells:the involvement of autophagy

AIM: To examined the effects of hypoxic preconditioning(HPC) on oxygen-glucose deprivation(OGD)-induced PC12 cells, and to investigate its possible mechanisms of autophagy.METHODS: Cultured PC12 cells were randomly divided into control group, HPC group, 3-methyladenine(3-MA) group, HPC+OGD group, 3-MA+HPC+OGD group and OGD group. CCK-8 assay was used to detect the cell viability. The caspase-3 activity was also tested. TUNEL staining and flow cytometry were used to detect the cell apoptosis. The protein levels of apoptosis-related protein caspase-3 and autophagy-marked protein LC3-2 and beclin-1 were determined by Western blot.RESULTS: Compared with control group, the viability of PC12 cells was significantly reduced, and the activity of caspase-3 was significantly increased in OGD group. Compared with 3-MA+ HPC+OGD group and OGD group, the viability of PC12 cells was significantly increased, and the activity of caspase-3 was significantly reduced in HPC+OGD group(P<0.05). The PC12 cell injury was apparent after OGD with a great increase in the apoptotic rate(P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in HPC+OGD group(P<0.05). Compared with control group, the protein level of cleaved caspase-3 was significantly increased in OGD group(P<0.05). Compared with OGD group, the protein level of cleaved caspase-3 was significantly decreased, and the levels of LC3-2 and beclin-1 were significantly increased in HPC+OGD group(P<0.05).CONCLUSION: OGD decreases cell survival and induces apoptosis.Activation of cell autophagy may be the mechanism by which hypoxic preconditioning protects the PC12 cells from OGD induced injury.     

Influence of Bcl-2 inhibitor on Astragalus injection-reduced expression of caspase-3 in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation

AIM: To observe the influence of Bcl-2 inhibitor on the expression of caspase-3 reduced by Astra-galus injection in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation (OGD/R). METHODS: The primary rat hippocampal neurons cultured in vitro for 8 d were chosen and randomly divided into 6 groups: normal control group, model group (OGD/R group), Astragalus injection group, Astragalus injection solvent (sterile deionized water)group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. The cells in all groups were tested 24 h after they were treated with reoxygenation after deprived of oxygen and glucose for 30 min except normal control group. The cell type and rate of positive cells were observed by immunohistochemistry. The protein levels of Bcl-2 and cleaved caspase-3 in the hippocampal neurons were measured by Western blotting. The mRNA expression of caspase-3 was detected by RT-PCR. RESULTS: Compared with normal control group, the caspase-3 positive rate of the cells, the protein levels of Bcl-2 and cleaved caspase-3, and the mRNA expression of caspase-3 in model group enhanced significantly (P < 0.05). Compared with model group, the expression of Bcl-2 in Astragalus injection group obviously enhanced, while the caspase-3 positive rate of the cells, the protein level of cleaved caspase-3 and the mRNA expression of caspase-3 in the Astragalus injection group decreased significantly (P < 0.05). No significant difference in injection solvent group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group was observed (P > 0.05). The expression of Bcl-2 was decreased sharply in Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. CONCLUSION: Bcl-2 inhibitor antagonizes the inhibitory effect of Astragalus injection on caspase-3 expression in rat hippocamal neurons with OGD/R, which may be one of the possible target for the inhibitory action of Astragalus injection on the apoptosis of rat hippocampal neurons induced by OGD/R.     

Role of oxytocin in neonatal rat hippocampal neurons after hypoxic-ischemic injury

AIM: To investigate whether oxytocin has neuroprotective effects on hippocampal CA1 pyramidal neurons from neonatal rats exposed to hypoxic-ischemic brain injury and the underlying mechanisms. METHODS: An in vitro model of hypoxic-ischemic injury was used by exposing the brain slices to oxygen-glucose deprivation (OGD) solution. Acute dissociated brain slices (6~8 slices per rat) from 8 Sprague-Dawely rats of 7~10 d old were used. The slices were randomly divided into 4 groups:control group, OGD 20 min group, OGD 40 min group and OGD+oxytocin group. The effect of oxytocin on neuronal death was evaluated by TO-PRO-3 staining. Fresh brain slices from other 20 neonatal rats were divided into OGD group, OGD+oxytocin group, OGD+dVOT (oxytocin receptor antagonist)+oxytocin group, and OGD+bicucuclline (GABA_A receptor antagonist)+oxytocin group. The onset of anoxic depolarization in the hippocampal neurons treated with different drugs was recorded by whole-cell patch-clamp techniques. RESULTS: The results of TO-PRO-3 staining showed that neuronal deaths in hippocampal CA1 area were increased over the prolonged OGD time. Oxytocin significantly reduced the hypoxic-ischemic deaths. Oxytocin dramatically prolonged the onset time of anoxic depolarization after the application of OGD solution. Both dVOT and bicuculline blocked this effect. CONCLUSION: Oxytocin plays a neuroprotective role in neonatal rat hippocampal CA1 pyramidal neurons by enhancing the inhibitory synaptic transmission via oxytocin receptors. Therefore, oxytocin is useful as a candidate for neuroprotective treatment after neonatal hypoxic-ischemic brain injury.     

Protective effect of β-asarone on primary rat hippcampal neurons against hypoxia/hypoglycemia and reperfusion injury

AIM：To investigate the protective effect of β-asarone against hypoxia/hypoglycemia and reperfusion injury in primary rat hippocampal neurons. METHODS：Cell viability, the activity of caspase-3, the protein expression of p-JNK and Bcl-2, and the mRNA expression of Bcl-2 and caspase-3 were determined by MTT assay, spectrophoto-metry, Western blotting and real-time PCR. RESULTS：Compared with normal control group, the cell viability decreased and the activity of caspase-3 increased obviously, the expression of p-JNK protein and caspase-3 mRNA increased obviously, and the expression of Bcl-2 protein decreased obviously in model group (P<0.05). Compared with model group, different doses of β-asarone inhibited the changes of the above indexes (P<0.05). CONCLUSION：β-asarone inhibits JNK-mediated chondrosome signaling pathway, thereby attenuating the process of hippocampal neuron apoptosis after hypoxia/hypoglycemia and reperfusion.     

Effects of Homer1a over-expression on apoptosis and AMPK protein expression in mechanically injured neurons

AIM:To investigate the effects of Homer1a over-expression on the apoptosis and AMP-activated protein kinase (AMPK) protein expression in mechanically injured neurons. METHODS:The rat cortical neurons were isolated and cultured in vitro, and then ramdomly divided into control group, model group, empty vector group, and Exp-Homer1a group. Neuron models with mechanical injury were constructed and infected with the Homer1a over-expression vector. The mRNA expression of Homer1a was detected by qPCR. The cell viability in each group was detected by MTT assay. The activity of lactate dehydrogenase (LDH) in the supernatant of each group was measured by LDH test kit. The apoptosis level was analyzed by flow cytometry. The protein levels of Hormer1a, cleaved caspase-3, Bax, Bcl-2, p-AMPKα and AMPKα were determined by Western blot. RESULTS:Compared with control group, the viability of mechanically injured neurons was significantly decreased, the LDH activity in the supernatant and neuronal apoptotic rate were significantly increased (P<0.05), and Homer1a expression at mRNA and protein levels was significantly increased (P<0.05). Compared with model group, the LDH activity in the supernatant and neuronal apoptotic rate in Exp-Homer1a group were significantly decreased, the protein levels of cleaved caspase-3 and Bax were significantly decreased (P<0.05), and the protein levels of Bcl-2 and p-AMPKα were significantly increased (P<0.05). CONCLUSION:Over-expression of Homer1a may increase the viability of mechanically injured neurons and inhibit their apoptosis by promoting the activation of AMPKα phosphorylation.     

Rapamycin reduces SH-SY5Y cell damage induced by oxygen-glucose deprivation

AIM: To observe the effect of rapamycin (Rapa) on human neuroblastoma SH-SY5Y cell injury induced by oxygen-glucose deprivation (OGD), and to explore the role of autophagy in this process. METHODS: The SH-SY5Y cells were randomly divided into 4 groups:normal control group:the cells were cultured without OGD treatment; Rapa group:the cells were pretreated with Rapa for 1 h; OGD group:the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O₂, 94% N₂ and 5% CO₂ for 12 h; Rapa+OGD group:the cultured cells were treated with Rapa for 1 h, and then were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by determining the leakage of lactate dehydrogenase (LDH). The enzyme activity of caspase-3 was detected. TUNEL staining were used to detect the variation of cell apoptosis. The protein levels of apoptosis-related proteins Bax and Bcl-2, autophagy-related protein beclin-1 and autophagy marker protein LC3B were determined by Western blot. RESULTS: Compared with OGD group, the viability of the SH-SY5Y cells was significantly increased, and the activity of caspase-3 was significantly reduced in Rapa+OGD group (P<0.05). The SH-SY5Y cell injury was apparent after OGD with a great increase in the apoptotic rate (P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in Rapa+OGD group (P<0.05). Compared with control group, the protein level of Bcl-2 was significantly decreased (P<0.05) and the protein level of Bax was significantly increased in OGD group. Compared with OGD group, the levels of Bcl-2, beclin-1 and LC3B-Ⅱ were significantly increased and the protein level of Bax was significantly increased in Rapa+OGD group (P<0.05). CONCLUSION: Rapamycin has a protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the promotion of autophagy.     

Ganoderma lucidum extract protects rat cerebral cortical neurons from hypoxia/reoxygenation injury in vitro

AIM: To investigate the neuroprotective effect of Ganoderma lucidum extract (GLE) in an in vitro model of primary cultured neurons with oxygen and glucose deprivation (OGD). METHODS: Neuronal injury was induced by oxygen and glucose deprivation/reoxygenation (OGD/R). The neuronal injury and viability were determined by LDH leakage and XTT assay at 0 h,3 h,6 h,12 h,24 h,48 h and 72 h after OGD/R. Neuronal apoptosis was detected by flow cytometry (FCM). The expression of apoptosis-related proteins was analyzed by Western blotting.RESULTS: The viability of the neurons increased with exposure to GLE (0.1 mg/L,1 mg/L and 10 mg/L)after OGD/R. The LDH releases were also significantly reduced. GLE significantly inhibited OGD/R-induced apoptosis of cultured rat cortical neurons in a concentration-dependent and time-dependent manner(P<0.05). GLE at concentrations of 0.1 mg/L,1 mg/L and 10 mg/L inhibited the expression of caspase-3 and caspase-8 proenzyme. Additionally,GLE at concentration of 10 mg/L suppressed the expression of caspase-9 proenzyme.CONCLUSION: Our findings provide the evidence that the GLE has neuroprotective effect on cerebral ischemia. The mechanisms are related to the inhibition of caspase-3,-8 and-9 activations. GLE may be a novel and effective reagent for treating ischemic stroke.     

Expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage in neonatal A2AR knockout mice

AIM： To study the expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage (HIBD) in the neonatal adenosine A2A receptor knockout (A2AR-/-) mice. METHODS：Base on the modified Rice method, the model of HIBD was established. The total 64 C57/BL6 neonatal mice (7 days old) of A2AR-/-(KO) and corresponding wild type (A2AR+/+, WT) were randomized into sham-operated group and model group. The mice in model group were divided into 3 subgroups： 1 d after HIBD, 3 d after HIBD and 7 d after HIBD (n=8 for each group). The cortex and hippocampal CA1 region were used as the study areas. The neuronal apoptosis was detected using TUNEL assay combined with Nissl staining. The expression of p-p38 MAPK and activated caspase-3 was determined by the method of immunohistochemistry. The KO mice and WT mice were also taken from sham-operated group (SKO and SWT, n=10) and model group (MKO and MWT, n=30) 1 d after HIBD to assess the early neurological behavior. RESULTS：The apoptotic neurons, activated caspase-3 and p-p38 MAPK increased after HIBD and peaked at 1 d after HIBD in the cortex and the hippocampal CA1 region. The apoptotic neurons and the expression of activated caspase-3 in KO mice were significantly higher than those in WT mice at the same time point after HIBD. The expression level of p-p38 MAPK in KO mice were significantly higher than that in WT mice at 1 d and 3 d after HIBD. The expression of activated caspase-3 was positively correlated with the expression of p-p38 MAPK in neonatal mice after HIBD (in the cortex：r=0.957, P<0.01; in the hippocampal CA1 region： r=0.939, P<0.01). CONCLUSION：p-p38 MAPK might be involved in the aggravated neuron apoptosis and brain damage induced by A2AR knockout after neonatal HIBO.     

Effect of Astragalus injection on expression of calmodulin after hypoxia/hypoglycemia and reoxygenation in rat hippocampal neurons

AIM: To investigate the effect of Astragalus injection on the expression of calmodulin(CaM) after hypoxia/ hypoglycemia and reoxygenation in rat hippocampal neurons.METHODS: The hippocampal neurons were cultured for 8 days and divided into 4 groups: normal control group (normal control), hypoxia/hypoglycemia and reoxygenation group (model), Astragalus injection solution group (solution control) and Astragalus injection group ( Astragalus ).The cells in all groups were treated with reoxygenation and normal medium after deprived of oxygen and glucose for 30 min except normal control group.The method of immunohistochemistry was used to measure the number of caspase-3 positive neurons.The expression of CaM at mRNA and protein levels was measured at time points of 0 h, 0.5 h, 2 h, 6 h, 24 h, 48 h, 72 h and 120 h after hypoxia/hypoglycemia and reoxygenation by RT-PCR and Western blotting, respectively.RESULTS: No difference of the parameters at all time points between model group and solution control group was found.Compared with normal control group, the numbers and the percentages of caspase-3 positive cells at all time points obviously increased in model group except at 0 h and 0.5 h (P<0.05).Compared with model group, the numbers and the percentages of caspase-3 positive cells were decreased in Astragalus injection group except at 0 h and 0.5 h (P<0.05).Compared with normal control group, the protein expression of CaM in rat hippocampal neurons at all time points obviously increased in model group (P<0.05).However, the protein expression of CaM in rat hippocampal neurons at all time points obviously decreased in Astragalus injection group as compared with model group (P<0.05).Compared with normal control group, the mRNA expression of CaM in rat hippocampal neurons at all time points obviously decreased in model group (P<0.05).The mRNA expression of CaM in rat hippocampal neurons at all time points obviously increased in Astragalus injection group as compared with model group (P<0.05).CONCLUSION: Astragalus injection inhibits the protein expression of CaM, the calcium overload and the expression of caspase-3 after hypoxia/hypoglycemia and reoxygenation, thus inhibiting hippocampal neuronal apoptosis.     

Effect of autophagic inhibitor 3-methyladenine on apoptosis of hippo-campal neurons induced by Aβ(25-35)

AIM: To investigate the influence of autophagy on the apoptosis of hippocampal neurons in the rat model of Alzheimer disease.METHODS: Sprague-Dawley rats were divided into model group, autophagic inhibitior 3-methyladenine (3-MA) pretreatment group and control group.In model group, the rats were anesthetized and placed in a stereotaxic apparatus.Hippocampus CA1 area microinjection was performed and Aβ(25-35) was applied to establish the model of AD.3-MA in 0.9% saline was administered by the same way prior to Aβ(25-35) infusion.The learning and memory ability of the rats was observed by Morris water maze.The ultrastructure of the hippocampal neurons, the formation of autophagic vesicles, beclin-1 expression and cell apoptosis were detected after behavioral experiment.RESULTS: Compared with model group, the learning and memory ability of the rats in 3-MA group significantly impaired (P<0.05) and the apoptotic rate of the hippocampal neurons significantly increased (P<0.05).Moreover, the expression of beclin-1 was declined.In model group, hippocampal neurons showed double membrane wrapped in the autophagic vacuoles, and the neuronal damages were significantly milder than that in 3-MA group.CONCLUSION: Decrease in the levels of neuronal autophagy increases the neuronal apoptosis, indicating that increasing neuronal autophagy may have therapeutic potential for AD.     

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

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

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.     

Inhibitory effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease rats via SIRT1/NF-κB signaling pathway

AIM: To investigate the effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease (AD) rats via SIRT1/NF-κB signaling pathway and its mechanism. METHODS: AD rat model was established by intragastric administration of AlCl₃ and intraperitoneal injection of D-galactose. After treated with butylphthalide at 25 mg/kg (low dose), 50 mg/kg (medium dose) and 100 mg/kg (high dose), the effects of butylphthalide on the morphology of hippocampal neurons, apoptosis rate, and the protein levels of Bcl-2, Bax, cleaved caspase-3 and the SIRT1/NF-κB signaling pathway associated proteins were determined by HE staining, flow cytometry and Western blot, respectively. After treated with SIRT1 agonist SRT1720 and inhibitor sirtinol, the role of SIRT1/NF-κB signaling pathway in hippocampal neuronal apoptosis was observed. On the basis of giving 50 mg/kg butylphthalide, sirtinol was administered, and the effect of butylphthalide on neuronal apoptosis regulated by SIRT1/NF-κB signaling pathway was evaluated. RESULTS: The morphology of hippocampal neurons in the AD rats were improved, the apoptosis rate of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited, and the protein levels of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted by butylphthalide significantly (P<0.05). The protein expression of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted, and the apoptosis of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited by SRT1720 remarkably (P<0.05), whereas the effect of sirtinol was contrary to that of SRT1720. After sirtinol treatment, the inhibitory effect of butylphthalide on apoptosis of hippocampal neurons, the protein levels of Bax and cleaved caspase-3, and the promotion of Bcl-2 protein expression in hippocampal neurons were markedly weakened (P<0.05). CONCLUSION: Butylphthalide inhibits the apoptosis of hippocampal neurons in the AD rats by down-regulating the protein expression of Bax and cleaved caspase-3, and up-regulating the protein expression of Bcl-2 through activating SIRT1/NF-κB signaling pathway.     

Ginsenoside Rg1 promotes growth and extracellular matrix synthesis in degenerative human lumbar nucleus pulposus cells by inhibiting Wnt/β-catenin pathway

AIM: To explore the role of ginsenoside Rg1 in the growth of degenerative human lumbar nucleus pulposus cells (HNPCs). METHODS: Cultured HNPCs were subjected to oxygen-glucose deprivation (OGD) to mimic the micro-environment of degenerative HNPCs. The morphological changes of the cells in control group and OGD group were observed under optical microscope. The cells were treated with ginsenoside Rg1 at concentrations of 25, 50 and 100 μmol/L. The expression of collagen Ⅱ and aggrecan at mRNA and protein levels was determined by real-time PCR and Western blot analysis. The cell viability was measured by CCK-8 assay. The mRNA level of Ki67 was detected by real-time PCR. The apoptosis was analyzed by flow cytometry. The activity of caspase-3 was measured by a caspase-3 kit. The expression of Wnt/β-catenin pathway-related proteins was determined by Western blot. Furthermore, the expression of Wnt/β-catenin pathway-related proteins, the cell viability and apoptosis, and the expression of extracellular matrix synthesis proteins were assessed after the cells were co-treated with LiCl and 100 μmol/L ginsenoside Rg1. RESULTS: Normal HNPCs attached on the cell culture plate faster, and were almost round with rich cytoplasm. However, the cell adherence was slower, and the cells were long fusiform with decreased cytoplasm after OGD treatment, indicating that the model of degenerative HNPCs was successfully established. Compared with normal HNPCs, the expression of collagen Ⅱ and aggrecan at mRNA and protein levels was decreased in OGD group (P<0.05), which was then increased after the cells were treated with ginsenoside Rg1 at 25, 50 and 100 μmol/L (P<0.05). Compared with normal HNPCs, the cell viability and Ki67 expression were decreased in OGD group (P<0.05), which were increased after treatment with ginsenoside Rg1 (P<0.05). Meanwhile, the apoptotic rate and caspase-3 activity were significantly increased in OGD-treated cells (P<0.05), which were decreased after treatment with ginsenoside Rg1 (P<0.05). In addition, the activation of Wnt/β-catenin pathway was also inhibited by ginsenoside Rg1 treatment at dose of 100 μmol/L (P<0.05). LiCl, a Wnt/β-catenin pathway agonist, obviously decreased the protective effects of ginenoside Rg1 on OGD-induced cells (P<0.05), indicating that the Wnt/β-catenin pathway was involved in the protective effects of ginenoside Rg1 on degenerative HNPCs. CONCLUSION: Ginsenoside Rg1 promotes growth and extracellular matrix synthesis of degenerative HNPCs through inhibiting Wnt/β-catenin pathway. This study will provide a new idea for prevention and treatment of degenerative HNPCs.     

Effects of electroacupuncture pretreatment on survival,brain injury and cognitive function in rats after limb ischemia/reperfusion

AIM：To investigate the effects of electroacupuncture (EA) pretreatment on survival, brain injury and cognitive function in rats after limb ischemia/reperfusion (LI/R). METHODS：One hundred and thirty-two healthy male SD rats weighing 255~300 g were randomly divided into 3 groups (n=44 each)：sham operation group (sham), LI/R group and LI/R plus EA pretreatment (IL/R+EA) group. The LI/R model was made by a method that the bilateral femoral arteries were occluded for 3 h with atraumatic microclips followed by 48 h of reperfusion. In sham group, sham operation was performed. The EA pretreatment was conducted twice a day for 14 d prior to the LI/R event. EA pretreatment included the following acupoints：Baihui (GV20), Zusanli (ST36) and Xuehai (SP10). The survival rate within 7 d following LI/R was calculated. The changes of cognitive function were detected 48 h after reperfusion using Morris water maze test. The cerebral water content was determined by detecting the wet and dry weight. Microglial cells were evaluated following immunolabeling of Iba1 (a marker of microglia). The protein level of cleaved caspase-3 in the hippocampus was measured by Western blotting. The neuronal apoptosis was detected using TUNEL method. Meanwhile, the changes of pathological structure in hippocampus were observed under light microscope. The activity of MPO and SOD, and the content of ROS and MDA were also investigated. RESULTS：Compared with sham group, the Iba1 positive cells, the protein level of cleaved caspase-3, the apoptotic index, and the levels of ROS/MDA and MPO activity in LI/R and LI/R+EA groups increased significantly. The normal hippocampal neurons reduced, and SOD activity decreased significantly in hippocampus. The survival rates of the rats within 7 d decreased, the latency and swimming distance increased, and the number of crossing the platform reduced. Compared with LI/R group, the above indexes in LI/R+EA group were markedly improved. CONCLUSION：EA stimulation improves the survival rate and cognitive dysfunction, and reduces brain damage in LI/R rats by preventing microglial activation and attenuating oxidative stress.     

Glutamate-induced apoptosis is related with the damage of mitochondria that results in cytochrome C release into cytosol in cultured hippocampal neurons

AIM: To set up a glutamate-induced cell damage model in cultured hippocampal neurons, and to determine whether glutamate-induced neuronal apoptosis changes and whether this process is mediated by mitochondrial signal transduction pathways involving the release of cytochrome C. METHODS: Hippocampal neurons, isolated and cultured from new born Wistar rats, were exposed to various concentrations of glutamate. Extent of cell death was assessed by measuring the release of lactate dehydrogenase (LDH) in the culture media. Based on these data, an appropriate concentration of glutamate was selected, and all subsequent experiments were carried out under the concentration. Kinetics of glutamate-induced both apoptotic and necrotic cell death after exposure to glutamate for various times(3-24 h) were determined by flow cytometry and LDH release. The caspase-3 protein levels and cytochrome C release from mitochondria into cytosol in hippocampal neurons were determined by Western blotting. RESULTS: Glutamate treatment induced hippocampal neurons death in dose-dependent and time-dependent manners. A significant increase in LDH release (18.4%) was induced in the cells treated with 50 μmol/L glutamate, compared to control untreated cells(P<0.05). A significant increases in LDH release and apoptosis were observed at 6 h after glutamate treatment (P<0.05). The significant increase in the level of caspase-3 protein occurred at 3 h after glutamate exposure, which preceded neuronal cell apoptosis, and reached maximum levels at 6 h(62.4%). Treatment with glutamate induced a rapid release of cytochrome C into cytosol. Cytosolic cytochrome C showed a significant increase (P<0.05) as early as 30 min after glutamate treatment, which preceded the increase in caspase-3 level, and after 3 h, the level of cytochrome C was higher in the cytosol compared to the mitochondria. Concomitant with these changes in cytosol, the mitochondrial levels of cytochrome C decreased significantly. CONCLUSION: Exposure to 50 μmol/L glutamate induces apoptosis in cultured hippocampal neurons. The glutamate-induced apoptosis may be via the damage of mitochondria that results in cytochrome C release into cytosol, which activates caspase cascade and induces apoptotic cell death.     

Shikonin inhibits neuronal apoptosis induced by OGD through targeting PI3K/Akt signaling pathway

AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation (OGD)-induced injury model.METHODS: The neurons were pretreated with shikonin at different concentrations (0.02, 0.2, 2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase (LDH) release assay and fluorescein diacetate/propidium iodide (FDA/PI) double staining were used to detect neuronal viability and apoptosis, and then the optimal concentration of shikonin was determined. LY294002 (PI3K/Akt signaling pathway inhibitor, 1 μmol/L) was added before the addition of shikonin, and the protein level of p-Akt (Ser473) in the neurons was determined by Wes-tern blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis.RESULTS: A certain concentration (0.2~20 μmol/L) of shikonin increased the viability of impaired neurons (P<0.05) and the protein level of p-Akt (Ser473) in the neurons (P<0.05). The effect of shikonin on neuronal p-Akt (Ser473) levels and the cell death were blocked by LY294002 (P<0.05).CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.     

Effect of senegenin on H2O2-induced damage in hippocampal neurons of SD rats

AIM: To observe the effect of senegenin (Sen) on hippocampal neuron injuries induced by H₂O₂.METHODS: Hippocampal neurons were isolated from neonatal SD rats. The primarily cultured neurons were divided into control group, H₂O₂ group, Sen group and Sen+H₂O₂ group. The cell viability, the content of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) in the neurons were detected after treated with Sen. The morphological changes of nucleus of the neurons were observed by Hoechst 33258 staining. The mRNA expression of bcl-2 and bax was quantified by real-time PCR. The protein levels of Bcl-2 and bax were measured by Western blotting. The activity of caspase-3 was also assayed.RESULTS: Compared with H₂O₂ group, the levels of antioxidative enzyme were increased in Sen+H₂O₂ group (P<0.05). In addition, mRNA expression of bcl-2 increased and that of bax decreased (P<0.05) in Sen+H₂O₂ group. Moreover, Sen increased the protein level of Bcl-2, and reduced the protein level of Bax and the activity of caspase-3 in the neurons exposed to H₂O₂ (P<0.05).CONCLUSION: The protective effect of Sen on hippocampal neurons with H₂O₂ -induced injury may be involved in the mechanisms of     

Lycopene protects primary mouse cerebrocortical neurons against t-BHP-induced damage in vitro

AIM: To investigate the protective effect of lycopene on primary mouse cerebrocortical neurons exposed to tert-butyl hydroperoxide (t-BHP) and its mechanisms of in vitro.METHODS: Primary cerebrocortical neurons of newborn C57 mice were extracted and divided into normal group, t-BHP group, lycopene+t-BHP group and lycopene group. The neuronal damage was induced by t-BHP exposure for 24 h, and the cell viability was examined by MTT assay. ROS content was measured by flow cytometry, and the protein levels of Bax, Bcl-2, caspase-3, cleaved caspase-3 and cytochrome C were examined by Western blot.RESULTS: The primary mouse cortical neurons expressed MAP-2 protein. Lycopene at concentration of 4 μmol/L reversed the decrease in cell viability. Flow cytometry revealed that lycopene treatment attenuated ROS content under the condition of t-BHP exposure. In addition, the protein level of Bcl-2 was increased, and the expression of Bax, cleaved caspase-3 and cytochrome-C was suppressed in lycopene+t-BHP group.CONCLUSION: The protective effect of lycopene on cortical neurons with t-BHP-induced injury may be involved in the mechanism of neuronal antioxidative response by down-regulating caspase-3 and Bax/Bcl-2 through the mitochondrial apoptotic pathway.