Apoptosis Rate Of Hippocampal Neurons Research Articles

Dendrobium nobile Lindl. alkaloids (DNLA) inhibits d-galactose-induced hippocampal neuronal senescence through the SIRT1-FoxO1-autophagy axis

Dendrobium nobile is a well-known traditional Chinese herbal medicine, and DNLA (Dendrobium nobile Lindl. alkaloids) is the active ingredient to have anti-brain aging effects in laboratory animals. The aim of the present study is to examine the anti-aging effects of DNLA in d-galactose induced primary hippocampal neuron senescence. Primary hippocampal neuron cultures were pretreated with DNLA (35 and 350 ng/mL) for 24 h, then exposed to d-galactose (50 mM) for 48 h. Cell viability was evaluated by the CCK-8 assay. Flow cytometry analysis was performed to evaluate the apoptosis rate of hippocampal neurons. β-Galactosidase staining was used to detect the senescence of neurons. Western blot analysis was used to detect the protein expression. The results show that DNLA markedly recovered the reduction in cell viability, inhibited the d-gal-induced hippocampal neuron apoptosis, decreased the senescence-associated β-galactosidase activity, and attenuated the protein expression of senescence marker p53 and p16 in d-galactose induced senescent hippocampal neurons. Furthermore, DNLA enhanced autophagy activity by increasing LC3-II/LC3-I ratio, Beclin1, and by decreasing p62 in d-galactose induced senescent hippocampal neurons. Moreover, DNLA upregulated the expression of autophagy-regulator SIRT1 and FoxO1. These novel findings demonstrate that DNLA is effective in protecting against d-galactose induced senescent hippocampal neurons in vitro. The anti-aging mechanisms of DNLA appear to be mediated, at least in part, through activated Sirt1-FoxO1-autophagy axis.

Effects of electroacupuncture pretreatment on long-term postoperative cognitive dysfunction and neuron-inflammation in aged rats

To observe the effects of electroacupuncture pretreatment on postoperative cognitive dysfunction (POCD), neuronal apoptosis and neuron-inflammation in aged rats. Thirty-six male SD rats aged 20 months were randomly divided into sham operation group, model group and electroacupuncture (EA) group, with 12 rats in each group. The POCD rats model was prepared by internal fixation of left tibial fracture. Five days before modeling, EA stimulation (2 Hz/15 Hz, 1 mA, 30 min) was applied to "Zusanli" (ST36), "Hegu" (LI4) and "Neiguan" (PC6) on the unaffected side of rats in the EA group, once a day for consecutive 5 d. The learning and memory abilities of rats were evaluated by water maze test 31-35 days after operation. The apoptosis of hippocampal neurons was observed by Tunel/NeuN double staining. The expressions of high mobility group protein B1 (HMGB1) and phosphorylated (p)-nuclear factor (NF)-κB in microglia cells in hippocampal dentate gyrus were detected by immunofluorescence staining. The expression levels of interleukin (IL)-6 and IL-1β in the hippocampus were detected by Western blot. Compared with the sham operation group, the escape latency was prolonged (P<0.05); the frequency of crossing the original platform, ratio of the swimming distance and the time in the target quadrant of the Morris water maze were significantly decreased (P<0.05); the apoptosis rate of hippocampal neurons was significantly increased (P<0.05); the expressions of HMGB1 and p-NF-κB in microglia cells in the dentate gyrus and the expression levels of IL-6 and IL-1β in hippocampus were increased (P<0.05) in the model group. Compared with the model group, the results of the above indexes were all opposite (P<0.05) in the EA group. EA preconditioning can regulate hippocampal inflammatory response, alleviate neuronal apoptosis rate and long-term cognitive dysfunction in aged rats with POCD, the mechanisms may be related to the inhibition of microglia HMGB1/NF-κB pathway in hippocampal dentate gyrus.

Magnesium-L-threonate exhibited a neuroprotective effect against oxidative stress damage in HT22 cells and Alzheimer’s disease mouse model

BACKGROUNDOxidative stress results in the production of excess reactive oxygen species (ROS) and triggers hippocampal neuronal damage as well as occupies a key role in the pathological mechanisms of neurodegenerative disorders such as Alzheimer’s disease (AD). A recent study confirmed that magnesium had an inhibitory effect against oxidative stress-related malondialdehyde in vitro. However, whether Magnesium-L-threonate (MgT) is capable of suppressing oxidative stress damage in amyloid β (Aβ)25-35-treated HT22 cells and the AD mouse model still remains to be investigated.AIMTo explore the neuroprotective effect of MgT against oxidative stress injury in vitro and in vivo, and investigate the mechanism.METHODSAβ25-35-induced HT22 cells were preconditioned with MgT for 12 h. APPswe/PS1dE9 (APP/PS1) mice were orally administered with MgT daily for 3 mo. After MgT treatment, the viability of Aβ25-35-treated HT22 cells was determined via conducting cell counting kit-8 test and the cognition of APP/PS1 mice was measured through the Morris Water Maze. Flow cytometry experiments were applied to assess the ROS levels of HT22 cells and measure the apoptosis rate of HT22 cells or hippocampal neurons. Expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), hypoxia-inducible factor (HIF)-1α, NADPH oxidase (NOX) 4, Aβ1-42 and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway proteins was quantified by Western blot.RESULTS In vitro data confirmed that Aβ25–35-induced HT22 cells had a significantly lower cell viability, higher ROS level and higher apoptosis rates compared with those of control cells (all P < 0.001). MgT prevented the Aβ25-35-triggered oxidative stress damage by elevating viability and decreasing ROS formation and apoptosis of HT22 cells (all P < 0.001). APP/PS1 mice exhibited worse cognitive performance and higher apoptosis rate of hippocampal neurons than wild-type (WT) mice (all P < 0.01). Meanwhile, significant higher expression of Aβ1-42 and NOX4 proteins was detected in APP/PS1 mice than those of WT mice (both P < 0.01). MgT also ameliorated the cognitive deficit, suppressed the apoptosis of hippocampal neuron and downregulated the expression of Aβ1-42 and NOX4 proteins in APP/PS1 mouse (all P < 0.05). Moreover, MgT intervention significantly downregulated HIF-1α and Bax, upregulated Bcl-2 and activated the PI3K/Akt pathway both in vitro and in vivo (all P < 0.05).CONCLUSIONMgT exhibits neuroprotective effects against oxidative stress and hippocampal neuronal apoptosis in Aβ25-35-treated HT22 cells and APP/PS1 mice.

Protective effects of dexmedetomidine on cerebral ischemia/reperfusion injury via the microRNA-214/ROCK1/NF-\u03baB axis

BackgroundCerebral ischemia/reperfusion injury (CIRI) is a complication of surgical procedure associated with high mortality. The protective effect of dexmedetomidine (DEX) on CIRI has been explored in previous works, yet the underlying molecular mechanism remains unclear. Our study explored the protective effect of DEX and its regulatory mechanism on CIRI.MethodsA CIRI rat model was established using middle cerebral artery occlusion (MCAO). Neurological deficit scores for rats received MCAO modeling or DEX treatment were measured. Cerebral infarction area of rats was detected by TTC staining, while damage of neurons in hippocampal regions of rats was determined by hematoxylin-eosin (HE) staining. Apoptosis rate of neurons in hippocampal regions was examined by TUNEL staining. The dual-luciferase assay was performed to detect the binding of microRNA-214 (miR-214) to Rho-associated kinase 1 (ROCK1).ResultsDEX treatment significantly reduced infarction area of MCAO rats and elevated miR-214 expression. Injection of miR-214 inhibitor attenuated the effect of DEX in MCAO rats by increasing the area of cerebral infarction in rats and apoptosis rate of hippocampal neurons. ROCK1 was targeted and negatively regulated by miR-214. The overexpression of ROCK1 led to activation of NF-κB to aggravate CIRI.ConclusionTherapeutic effects of DEX on CIRI was elicited by overexpressing miR-214 and impairing ROCK1 expression and NF-κB activation. Our finding might provide novel insights into the molecular mechanism of DEX in rats with CIRI.

Effect of hydrogen on mitochondrial biosynthesis in hippocampus of mice with sepsis-associated encephalopathy

Objective To evaluate the effect of hydrogen on mitochondrial biosynthesis in the hippocampus of mice with sepsis-associated encephalopathy (SAE). Method Two hundred and twenty-four healthy clean-grade male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups (n=56 each) using a random number table method: sham operation group (group SH), sham operation plus hydrogen group (group SH+ H2), group SAE, and SAE plus hydrogen group (group SAE+ H2). The model of SAE was established by cecal ligation and puncture in anesthetized mice.Group SH+ H2 and group SAE+ H2 inhaled 2% hydrogen for 1 h starting from 1 and 6 h after operation, respectively.Twenty mice were selectde to record the postoperative 7-day survival rate.The remaining animals were sacrificed at 24 h after operation, and brain tissues were taken for examination of the pathological changes in hippocampal CA1 region (with a light microscope) and for determination of neuronal apoptosis (by TUNEL), mitochondrial membrane potential (MMP) (by fluorescence spectrophotometry) and ATP content (by a bioluminescence assay). The apoptosis rate was calculated.The expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) in hippocampus was determined by Western blot at 6, 12 and 24 h after operation. Results Compared with group SH, the postoperative 7-day survival rate was significantly decreased, the apoptosis rate of hippocampal neurons was increased, the contents of MMP and ATP were decreased, and the expression of PGC-1α was up-regulated in SAE and SAE+ H2groups (P 0.05). Compared with group SAE, the postoperative 7-day survival rate was significantly increased, and the apoptosis rate of hippocampal neurons was decreased, the contents of MMP and ATP were increased, and the expression of PGC-1α was up-regulated (P<0.05), and the pathological changes of hippocampal tissues were significantly attenuated in group SAE+ H2. Conclusion The mechanism by which hydrogen mitigates SAE may be related to promoting mitochondrial biosynthesis in mice. Key words: Hydrogen; Sepsis-associated encephalopathy; Mitochondria; Biosynthesis

Effects of laparotomy on cognitive function in rats with traumatic brain injury and the realationship with calcium overload

Objective To evaluate the effect of laparotomy on cognitive function in rats with traumatic brain injury and the relationship with calcium overload. Methods One hundred and fifty healthy male Wistar rats, aged 2~3 months, weighing 190~220 g, were assigned into 5 group (n=30 each) using a random number table: blank group(group B), surgery group of blank rats(group BS), surgery group of sham rats(group SS), traumatic brain injury rats group(group T), surgery group of traumatic brain injury rats(group TS). TBI model was made in rats of group T and TS by Feeney method.Rats in group SS were only treated with cranial bone window without crainocerebral impact.Both group B and BS were normal rats. Then the rats in group BS, SS and TS group underwent laparotomy under sevoflurane anesthesia (the operation time was about 3 hours) and the rats in group B and T inhaled pure oxygen for 3 hours after 20 days. One day before surgery, 3 and 7 d after surgery, 10 rats in each group were randomly selected for Morris water maze test. The hippocampus tissue of 10 rats were taken after the water maze test. The apoptosis rate and calcium concentration of hippocampal neurons were measured by flow cytometry, and the expression level of cleaved caspase-3 in hippocampal tissues was determined by Western blot. Results One day before surgery, compared with group B(the escape latency(9.8±0.8)s, the number of crossing platform (5.8±0.8), the apoptosis rate of hippocampal neurons (2.5±0.9)%, calcium ion concentration (2.3±0.2), the expression of cleaved caspase-3 (0.22±0.07) ), the escape latency of group T and group TS were prolonged (group T: (25.5±0.7)s, P<0.05; group TS: (25.1±1.1)s, P<0.05), the number of crossing platform decreased (group T: (2.7±0.8), P<0.05; group TS: (2.8±0.6), P<0.05), the apoptosis rate of hippocampal neurons increased (group T: (5.3±0.6)%, P<0.05; group TS: (5.2±1.0)%, P<0.05), calcium ion concentration increased (group T: (3.7±0.4), P<0.05; group TS: (3.6±0.5), P<0.05) and the expression of cleaved caspase-3 increased (group T: (0.45±0.07), P<0.05; group TS: (0.44±0.05), P<0.05), the differences were statistically significant. Compared with the group SS, the escape latency (3 d after surgery: group SS: (23.8±1.3)s, group TS: (56.4±2.5)s, P<0.05; 7 d after surgery: group SS: (16.6±1.8)s, group TS: (38.1±2.1)s, P<0.05)of the rats in group TS were prolonged, the number of crossing platform decreased (3 d after surgery: group SS: (2.9±0.6) , group TS: (1.1±1.1) , P<0.05; 7 d after surgery: group SS: (4.2±1.2) , group TS: (1.7±1.3), P<0.05), the apoptosis rate of hippocampal neurons (3 d after surgery: group SS: (4.8±0.6)%, group TS: (14.4±0.6)%, P<0.05; 7 d after surgery: group SS: (3.8±1.1)%, group TS: (9.6±1.3)%, P<0.05), calcium ion concentration (3 d after surgery: group SS: (3.1±0.3), group TS: (6.4±0.5), P<0.05; 7 d after surgery: group SS: (2.6±0.3), group TS: (4.8±0.4), P<0.05) , the expression of cleaved caspase-3 (3 d after surgery: group SS: (0.42±0.03), group TS: (0.88±0.08), P<0.05; 7 d after surgery: group SS: (0.33±0.05), group TS: (0.63±0.06), P<0.05) in the hippocampus increased after surgery (P<0.05). Compared with the T group, the escape latency (3 d after surgery: group T: (18.6±2.0)s, group TS: (56.4±2.5)s, P<0.05; 7 d after surgery: group T: (13.8±2.6)s, group TS: (38.1±2.1)s, P<0.05) of the rats in group TS were prolonged, the number of crossing platform (3 d after surgery: group T: (3.4±0.5), group TS: (1.1±1.1), P<0.05; 7 d after surgery: group T: (4.3±1.2), group TS: (1.7±1.3), P<0.05) decreased, the apoptosis rate of hippocampal neurons (3 d after surgery: group T: (4.4±0.7)%, group TS: (14.4±0.6)%, P<0.05; 7 d after surgery: (3.3±1.3)%, group TS: (9.6±1.3)%, P<0.05), calcium ion concentration (3 d after surgery: group T: (3.4±0.4), group TS: (6.4±0.5), P<0.05; 7 d after surgery: group T: (3.0±0.3), group TS: (4.8±0.4), P<0.05), the expression of cleaved caspase-3 (3 d after surgery: group T: (0.40±0.04), group TS: (0.88±0.08), P<0.05; 7 d after surgery: (0.35±0.02), group TS: (0.63±0.06), P<0.05) in the hippocampus increased after surgery(P<0.05). Conclusion Laparotomy can aggravate the cognitive impairment of rats with traumatic brain injury and cause postoperative cognitive impairment, which may be related to the increased degree of calcium overload and the increased rate of hippocampal neuron apoptosis. Key words: Laparotomy; Traumatic brain injury; Postoperative complications; Cognitive impairment

The neuroprotective mechanism of 2-arachidonoylglycerol 2-AG against non-caspase-dependent apoptosis in mice hippocampal neurons following MCAO.

ObjectiveIn this study, the neuroprotective mechanism of 2-arachidonoylglycerol 2-AG against non-caspase-dependent apoptosis in mice hippocampal neurons following MCAO was investigated.MethodOne hundred and fifty healthy clean male C57BL/6 mice were randomly divided into 3 groups: sham group, model group and 2-AG treatment group, 50 mice in each group. A modified Zea Longa method was used to establish a model of middle cerebral artery occlusion (MCAO) in mice. The apoptosis rate and mitochondrial membrane potential of hippocampal nerve cells were measured by flow cytometry. The mRNA expressions of AIF, Endo G and BNIP3 in hippocampal tissues were determined by qPCR. Western blot was used to determine the protein expressions of AIF, Endo G and BNIP3 in the mitochondria of hippocampal tissue.ResultsThe apoptosis rate of hippocampal neurons in the group treated with 2-AG was significantly lower than that of the model (P<0.01), which indicated that 2-AG could inhibit the apoptosis of hippocampal neurons induced by MCAO. However, the mitochondrial membrane potential of hippocampal neurons in the group treated with 2-AG was significantly higher than that of the model (P<0.01), indicating that 2-AG could improve the mitochondrial membrane potential of hippocampal neurons in MCAO mice. Real-time quantitative PCR (qPCR) showed that 2-AG could inhibit the gene expressions of AIF, Endo G and BNIP3 in hippocampal tissues. Western blot results showed that 2-AG could inhibit the secretions of AIF, Endo G and BNIP3 into cytoplasm in mitochondria.ConclusionEndocannabinoids 2-AG had a protective effect on neurons injury, and the mechanism was possibly associated with the protection of the brain nerve cells in the hippocampus and the integrity of the mitochondrial function. Endocannabinoids 2-AG may inhibit the non-caspase-dependent apoptosis pathway, so as to exert its nerve protective effect.

Effect of sevoflurane anesthesia on cognitive impairment in rats with traumatic brain injury

Objective To evaluate the effect of sevoflurane anesthesia on cognitive impairment in rats with traumatic brain injury. Methods One hundred and and twenty healthy male Wistar rats, aged 2-3 months, weighing 190-220 g, were assigned into 4 groups (n=30 each) using a random number table method: control group (group C), traumatic brain injury group (group T), sevoflurane anesthesia group (group S), and traumatic brain injury plus sevoflurane anesthesia group (group T+ S). A 40 g hammer was freely dropped onto the left parietal bone window from a height of 20 cm to establish the traumatic brain injury model in T and T+ S groups.Twelve days later, S and T+ S groups inhaled 3% sevoflurane for 3 h, and C and T groups inhaled pure oxygen for 3 h. On 1 day before anesthesia and 3 and 7 days after anesthesia, 10 rats in each group were randomly selected for performing Morris water maze test.Rats were sacrificed after the end of Morris water maze test, and the hippocampal tissues were obtained for determination of the apoptosis rate of hippocampal neurons, cytoplasmic calcium concentration [Ca2+ ]i (by flow cytometry), expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) (by immunohistochemistry), and expression of caspase-3 and caspase-12 (by Western blot). Results Compared with group C, the escape latency was significantly prolonged, the number of crossing platform was decreased, the apoptosis rate of hippocampal neurons and [Ca2+ ]i were increased, and the expression of caspase-3, caspase-12, GRP78 and CHOP in hippocampal tissues was up-regulated in S, T and T+ S groups (P<0.05). Compared with T and S groups, the escape latency was significantly prolonged, the number of crossing platform was decreased, the apoptosis rate of hippocampal neurons and [Ca2+ ]i were increased, and the expression of caspase-3, caspase-12, GRP78 and CHOP in hippocampal tissues was up-regulated in group T+ S (P<0.05). Conclusion Sevoflurane anesthesia can accentuate cognitive impairment in rats with traumatic brain injury, and the mechanism may be related to aggravating the degree of endoplasmic reticulum stress-induced calcium overload and increasing the apoptosis rate of hippocampal neurons. Key words: Anesthetics, inhalation; Brain injuries, traumatic; Cognition disorders

Effects of leptin on brain injury and long-term cognitive function in rats undergoing orthotopic liver transplantation

Objective To evaluate the effects of leptin on brain injury and long-term cognitive function in rats undergoing orthotopic liver transplantation. Methods Ninety clean-grade male Sprague-Dawley rats, aged 3 months, weighing 200-250 g, were divided into 3 groups by a random number table method: sham operation group(S group), liver ischemia-reperfusion(I/R) group(I/R group) and leptin group(L group), with 18 rats in each group.Orthotopic liver transplantation was performed to establish the model of liver I/R injury in I/R and L groups.Leptin 1 mg/kg was intraperitoneally injected at the onset of ischemia in L group, and the equal volume of normal saline was given instead of leptin in S and I/R groups.Twelve rats in each group were sacrificed at 3 days after operation, and brains were removed for examination of the pathological changes in hippocampal CA1 region(with a light microscope) and for determination of apoptosis in hippocampal neurons(by TUNEL assay) and expression of aquaporin 4(AQP4) and protein kinase C(PKC) in the hippocampus(by Western blot). The apoptosis rate was calculated.The remaining 6 rats in each group underwent a Morris water maze test at 30 days after surgery to evaluate long-term cognitive function. Results Compared with S group, the apoptosis rate of hippocampal neurons was significantly increased, the expression of AQP4 and PKC was up-regulated, the escape latency was prolonged, and the time of staying at the platform quadrant was shortened in I/R and L groups(P<0.05). Compared with I/R group, the apoptosis rate of hippocampal neurons was significantly decreased, the expression of AQP4 and PKC was down-regulated, the escape latency was shortened, and the time of staying at the platform quadrant was prolonged in L group(P<0.05). Conclusion Leptin can reduce the brain damage in rats undergoing orthotopic liver transplantation, the mechanism may be related to down-regulating the expression of PKC and AQP4, and leptin can also improve long-term cognitive function after orthotopic liver transplantation in rats. Key words: Leptin; Liver transplantation; Reperfusion injury; Cognition disorders

Electroacupuncture Reduced Apoptosis of Hippocampal Neurons in Mice with Cerebral Infarction by Regulating the Notch3 Signaling Pathway.

This study is designed to explore the effect of electric acupuncture on the apoptosis of hippocampal neurons through the Notch3 signaling pathway. A middle cerebral artery occlusion (MCAO) mouse model was generated by thread embolization. A Y-maze was used to test cognitive function (learning and memory). The apoptosis factor caspase-3 was identified through an enzyme-linked immunosorbent assay (ELISA). TTC staining was used to evaluate the cerebral infarction area. Apoptosis of hippocampal neurons was identified by TUNEL staining and flow cytometry. The expression levels of genes and proteins related to the Notch3 signaling pathway were assessed by qRT-PCR and Western blot. It was determined that 7days of electroacupuncture treatment improved recovery from cerebral infarction. Electroacupuncture therapy reduced the cerebral infarction area in mice and decreased the apoptosis of hippocampal neurons. The apoptosis rate of hippocampal neurons and the area of cerebral infarction were significantly lower in the electroacupuncture group than in the MCAO group (P < 0.01), while the apoptosis rate of hippocampal neurons and the area of cerebral infarction were significantly higher in the inhibitor group than in the electroacupuncture group (P < 0.01). The expression levels of genes related to the Notch3 signaling pathway were significantly increased in the electric acupuncture treatment group compared with the MCAO group (P < 0.01). The results showed that electroacupuncture inhibited the apoptosis of hippocampal neurons through the Notch3 signaling pathway, while the expression levels of genes related to the Notch3 signaling pathway were significantly higher in the inhibitor group than in the electroacupuncture group (P < 0.01). Electroacupuncture inhibited the apoptosis of hippocampal neurons in mice with cerebral infarction by stimulating the Notch3 signaling pathway and triggering corresponding protein expression.

Effect of acupuncture on cholinergic anti-inflammatory pathway in hippocampus of aged rats during global cerebral ischemia-reperfusion

Objective To investigate the effect of acupuncture on cholinergic anti-inflammatory pathway in the hippocampus of aged rats during global cerebral ischemia-reperfusion (I/R). Methods Ninety-six clean-grade healthy male Sprague-Dawley rats, aged 17-22 months, weighing 440-580 g, were divided into 3 groups (n=32 each) using a random number table method: sham operation group (group S), global cerebral I/R group (group I/R) and acupuncture group (group AP). Global cerebral I/R was induced by 4-vessel occlusion method described by Pulsinelli in group I/R and group AP.Baihui and Fengchi were stimulated for 14 consecutive days before ischemia in group AP.Four rats were sacrificed at 1, 3, 5 and 7 days of reperfusion, and brains were removed for determination of neuronal apoptosis by TUNEL.Four rats were sacrificed at 1, 3, 5 and 7 days of reperfusion, and brains were removed for determination of the expression of α7 nicotinic acetylcholine receptor (α7nAChR), choline acetyltransferase (ChAT), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the hippocampal CA1 region by Western blot.The apoptosis rate was calculated. Results Compared with group S, the apoptosis rate of hippocampal neurons was significantly increased, and the expression of α7nAChR, ChAT, TNF-α and IL-1β was up-regulated at each time point of reperfusion in I/R and AP groups (P<0.05). Compared with group I/R, the apoptosis rate of hippocampal neurons was significantly decreased, the expression of α7nAChR and ChAT was up-regulated, and the expression of TNF-α and IL-1β was down-regulated at each time point of reperfusion in group AP (P<0.05). Conclusion The mechanism by which acupuncture mitigates global cerebral I/R injury may be related to activating cholinergic anti-inflammatory pathway in the hippocampus of aged rats. Key words: Acupuncture; Reperfusion injury; Brain; Aged; Cholinergic anti-inflammatory pathway

Effect of icariin on apoptosis in hippocampal neurons cultured in high glucose

OBJECTIVETo investigate effect of icariin on apoptosis in hippocampal neurons cultured in high glucose, and the possible mechanism behind the action. METHODSHippocampus was obtained from newborn 24 h Sprague-Dawley rats and then primarily cultured. Then hippocampal neurons were divided into normal control group, high glucose group, icariin group, icariin + protein kinase B (Akt) inhibitor group and Akt agonist group. After each group was cultured in different conditioned medium for 72 h, we detected the apoptosis of neurons with flow cytometry, and the expression of Akt, p-Akt, B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) with western blot. RESULTSCompared with the normal control group, the apoptosis rate of hippocampal neurons increased significantly (P < 0.01), and p-Akt/Akt and Bcl-2/Bax decreased significantly (P < 0.01) in high glucose group. Compared with the high glucose group, the apoptosis rate of hippocampal neurons decreased significantly (P < 0.01), and p-Akt/Akt and Bcl-2/Bax increased significantly (P < 0.05) in icariin group and Akt agonist group. Compared with the icariin + Akt inhibitor group, the apoptosis rate of hippocampal neurons decreased significantly (P < 0.01), and p-Akt/Akt and Bcl-2/Bax increased significantly (P < 0.01) in icariin group. CONCLUSIONIcariin could reduce the apoptosis of neurons cultured in high glucose, which may be achieved by increasing the phosphorylation of Akt protein in Akt signal pathway, then increasing the expression of Bcl-2 and inhibiting the expression of Bax.

Effects of Huangpu Tongqiao capsule on apoptosis of Alzheimer's disease cell model

The loss of hippocampal neurons is one of the main pathological features of Alzheimer's disease (AD), which is related to the apoptosis of hippocampal neurons. Huangpu Tongqiao capsule is used for the treatment of AD, but the underlying mechanism is still unclear. This study is to investigate the mechanism of neuroprotective effect of Huangpu Tongqiao capsule in the treatment of AD, through observing the effect of Huangpu Tongqiao capsule containing serum on cell injury of primary cultured hippocampal neurons induced by Aβ₂₅₋₃₅ via inhibiting the cell apoptosis. Primary cultured hippocampal neurons were cultured and identified by MAP-2 immunofluorescence staining, and cell growth state was observed by inverted microscope. The Huangpu Tongqiao capsule containing serum was prepared using the method of serum pharmacology. MTT assays were used to measure the optimum concentration range of Huangpu Tongqiao capsule containing serum, and optimum Aβ concentration for establishing the AD model. After primary cultured hippocampal neurons AD cell model was induced by Aβ₂₅₋₃₅, cell survival rate was detected by MTT, cell apoptosis rate was assayed by flow cytometry, and protein expressions of Bax, Cyt C and caspase-3 were determined by Western blot analysis. The results showed that the primary cultured hippocampal neurons were cultured successfully, and cells grew mature at seventh days; Compared with normal group, the survival rate of hippocampal neurons in AD cell model group was decreased, the apoptosis rate of hippocampal neurons was increased, and the protein expressions of Bax, Cyt C and caspase-3 were increased (P<0.05, P<0.01); Compared with AD cell model group, the survival rate of hippocampal neurons in Huangpu Tongqiao capsule containing serum group was increased, the apoptosis rate of hippocampal neurons was decreased, and the protein expressions of Bax, Cyt C and caspase-3 were decreased (P<0.05, P<0.01). These findings suggest that Huangpu Tongqiao capsule containing serum has a neuroprotective effect on cell injury of the primary cultured hippocampal neurons induced by Aβ₂₅₋₃₅, and its effect on the treatment of AD is associated with the inhibition the apoptosis of hippocampal neurons.

Protective effects of notoginsenoside R1 on cerebral ischemia-reperfusion injury in rats.

The objective of this study was to investigate the protective effect of notoginsenoside R1 (NGR1) on cerebral ischemia-reperfusion injury (CIRI) in rats, and its molecular mechanism, to provide new insights into the diagnosis and treatment of CIRI. Sixty Sprague-Dawley rats were randomly divided into four groups including the sham-operation group (Sham), cerebral ischemia-reperfusion model group (CIR), NGR1 treatment group (NGR1), and nimodipine positive control group (NDC) with 15 rats each. Bilateral common carotid arteries occlusion was used to establish the rat CIRI model. The area of cerebral infarction at the end of reperfusion was calculated by triphenyl tetrazolium chloride staining. Apoptosis of hippocampal neurons in each group was detected by Annexin V/propidium iodide double staining. Hippocampal expression of brain-derived neurotrophic factor (BDNF) mRNA, and Bcl-2 and Bax protein at the end of reperfusion were measured by RT-qPCR and western blot analysis, respectively. Data were analyzed by SPSS software analysis to ensure statistical significance. At the end of reperfusion, the area of cerebral infarction in the NGR1 and NDC groups was significantly smaller than that of the CIR group. Apoptosis analysis showed that compared with the CIR group, the apoptosis rate of hippocampal neurons was significantly decreased in the NGR1 and NDC groups. RT-qPCR and western blot analysis showed that at the end of reperfusion, higher levels of BDNF mRNA and the anti-apoptotic factor, Bcl-2, and lower levels of the pro-apoptotic factor, Bax, in the hippocampus were found in the NGR1 and NDC groups compared with the CIR group. The protective effect of NGR1 on CIRI was significantly stronger than that of nimodipine. In conclusion, NGR1 can reduce the area of cerebral infarction, reduce apoptosis of hippocampal neurons, and protect rats from CIRI. Those effects were achieved by activating the expression of BDNF and Bcl-2, and by inhibiting the expression of Bax.

Neuroprotective effects of the Chinese Yi-Qi-Bu-Shen recipe extract on injury of rat hippocampal neurons induced by hypoxia/reoxygenation

ObjectiveTo explore the protective effects of the Chinese Yi-Qi-Bu-Shen recipe (YB) against neuronal injury induced by hypoxia–reoxygenation, which has shown beneficial effect in improving the brain function of type 2 diabetics likely through its antihyperglycemic, antioxidant activity, and investigate its mechanisms. MethodsThe bilateral hippocampus was collected from newborn rats to establish single cell suspension. On the 10th day, the primarily cultured hippocampal neurons were randomly divided into five groups: the normal group (NG), the hypoxia/reoxygenation group (HG), and groups protected with small, medium and large dosages of YB (SG, MG and LG, respectively). The YB-protected groups were treated with different concentrations of YB containing serum before reoxygenation. The metabolic rate of MTT, the malondialdehyde (MDA) content, and the activity of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) were measured with assay kits. The apoptosis rate of hippocampal neurons were tested using flow cytometry analysis. RT-PCR was used to evaluate the mRNA expressions of bcl-2 and bax genes. ResultsThe SOD activity, the cell survival rate, the bcl-2/bax ratio, and the bcl-2mRNA expression in the HG group were significantly lower (all P<0.01), but the levels of MDA and LDH, the apoptosis rate, and the bax mRNA expression were higher (all P<0.01) than those in the NG group. The SOD activity, the cell survival, the bcl-2mRNA expression, and the bcl-2/bax ratio were significantly higher in all of the YB-protected groups (all P<0.01), but the level of MDA and LDH, the apoptosis rate, and the bax mRNA expression were lower (P<0.01, P<0.05) than those in the HG group in a dose dependent manner. ConclusionThe YB extract has a protective effect on hippocampal neurons against injury induced by hypoxia/reoxygenation, through its antioxidant activity and the regulation of apoptosis.

Protective Effects of Rg2on Hypoxia-induced Neuronal Damage in Hippocampal Neurons

We investigated the neuroprotective effects of Rg(2) in anoxic cultured hippocampal neurons of newborn rats. The cells were divided into a control group, nimodipine group (5 μmol/L), Rg(2) (0.025 mmol/L), and Rg(2) (0.05 mmol/L) group. The apoptosis rate of hippocampal neurons was measured by flow cytometry with staining of PI. The intracellular calcium ion [Ca(2+)]i was observed with fluorospectrophotometer determined by fluorescent probe Fluo-2/AM. The contents of MDA and NO and the activities of SOD in the supernatants of cells were determined by biochemical methods. The results demonstrated that Rg(2) reduced the hypoxia-induced apoptosis, decreased the calcium overload in neurons, increased the activities of SOD, and decreased the contents of MDA and NO in the supernatants of cells. Our study suggests that Rg(2) has a neuroprotective effect against hypoxia-induced neuronal damage in hippocampal neurons mediated by anti-apoptosis, blocking calcium over-influx into neuronal cells, eliminating the free radicals, and increasing the activities of anti-oxidative enzymes to inhibit the oxidative damages caused by anoxic.

Protective effects of bone marrow mesenchymal stem cells with stable expression of Bcl-2 on ischemia- hypoxia of hippocampal neurons

The protective effect of bone marrow mesenchymal stem cells (MSCs) with stable expression of Bcl-2 on the in vitro ischemia-hypoxia of hippocampal neurons was investigated in the present study. Mouse Bcl-2 cDNA was amplified by RT-PCR followed by Lentivirus-mediated gene transfection into MSCs. Puromycin resistance was detected to screen MSCs undergoing successful transfection, and the mRNA expression of Bcl-2 was determined by RT-PCR. Serum and oxygen deprivation was performed in normal MSCs and those undergoing transfection aiming to mimic ischemia injury in vitro, and flow cytometry was used to detect the apoptosis rate of these cells. Neurogenic induction of Bcl-2 expressing MSCs was carried out followed by detection of neuron markers. The rat hippocampal neurons were isolated from neonate rats. The Bcl-2 expressing MSCs and hippocampal neurons were cocultured, and the protective effect of Bcl-2 expressing MSCs on the ischemia-hypoxia of hippocampal neurons was observed. Following transfection, MSCs had stable and efficient expression of Bcl-2 after screening. When compared with normal MSCs, the number of apoptotic Bcl-2 expressing cells was significantly decreased and that of survival cells markedly increased after serum and oxygen deprivation. In addition, the Bcl-2 expressing MSCs had the potent differentiation into neurons. In the coculture system, Bcl-2 expressing MSCs could remarkably reduce the apoptosis rate of hippocampal neurons after ischemia-hypoxia (P < 0.01). The MSCs undergoing Bcl-2 transfection had stable Bcl-2 expression, which can protect them from serum and oxygen deprivation induced apoptosis and protect hippocampal neuron against ischemia-hypoxia induced apoptosis in vitro. Furthermore, the Bcl-2 transfecion does not affect the differentiation of MSCs into neurons.