Effects of different active constituents of Gynostemma pentaphyllum on mitochondrial energy metabolism-related proteins in endothelial cells induced by ox-LDL

AIM To investigate the effects of different components of Gynostemma pentaphyllum ［gypenosides (Gps), gypenoside XLIX (GpXLIX) and ginsenoside Rb3 (GRb3)］ on mitochondrial energy metabolism-related proteins in endothelial cells induced by oxidized low-density lipoprotein (ox-LDL). METHODS EA.hy926 cells were divided into control group, model group, Gps group, GpXLIX group and GRb3 group. The cells in control group were cultured only in DMEM complete medium. The cells in model group were treated with 100 mg/L ox-LDL for 48 h. The cells in Gps group, GpXLIX group and GRb3 group were treated with 100 mg/L ox-LDL for 24 h, and then treated with Gps, GpXLIX and GRb3 at 100 mg/L for another 24 h, respectively. The ATP content in each group was detected by ELISA. The expression levels of mitochondrial energy metabolism-related proteins, cytochrome C oxidase subunit 5a （Cox5a）, NADH:ubiquinone oxidoreductase core subunit S1 （Ndufs1）, ATP synthase F1 subunit alpha (ATP5a) and cytochrome C (Cyt C）, were determined by Wes automatic Western blot quantitative analysis system and Western blot. RESULTS Compared with control group, the ATP content in model group was decreased (P<0.01). After drug intervention, the ATP content increased to different degrees in Gps group, GpXLIX group and GRb3 group (P<0.01). The results of Wes automatic Western blot quantitative analysis system were consistent with those of Western blot. These results showed that compared with control group, the protein expression of Cox5a, Ndufs1 and ATP5a in model group was decreased, and the protein expression of Cyt C was increased (P<0.01). After intervention, the protein expression of Cox5a, Ndufs1 and ATP5a was increased and the protein expression of Cyt C was decreased in Gps group, GpXLIX group and GRb3 group (P<0.05 or P<0.01). Among them, the effect of Gps on the protein expression of Cox5a, Ndufs1 and Cyt C was significantly stronger than those of the 2 monomer components, and the effect of GRb3 was found to be superior in the 2 monomer components. The effect of GpXLIX on ATP5a protein was superior to the other 2 components. CONCLUSION Gynostemma total saponins and related active ingredients protect ox-LDL-induced endothelial cells by affecting mitochondrial energy metabolism-related proteins, thereby preventing and treating atherosclerosis.     

Effect of Ganoderma lucidum spores on cytochrome C,mitochondrial Ca2+, HSP70 and BDNF in brain of epilepsy rats

AIM: To investigate the effects of Ganoderma lucidum spores on superoxide dismutase(SOD),malondialdehyde(MDA),total antioxidative capacity(T-AOC), cytochrome C, heat-shock protein 70 (HSP70), mitochondrial Ca²⁺ and brain-derived neurotrophic factor(BDNF) in the brain tissues of epilepsy rats.METHODS: The rat chronic epilepsy model was by intraperitoneal injection of pentetrazole(PTZ) at a subconvulsant dose (32 mg/kg).Flame atomic absorption method was used to detect the content of mitochondrial Ca²⁺,and spectrophotometer colorimetry was used to measure SOD activity,MDA content,T-AOC and cytochrome C levels in rat brain tissues. HSP70 and BDNF were determined by immunohistochemical method.RESULTS: The contents of mitochondrial Ca²⁺ and cytochrome C were higher, and the content of intracytoplasmic cytochrome C in the rat brain tissues was obviously lower in Ganoderma lucidum spores group than that in epileptic model group. Compared to epileptic model group, the activity of SOD and T-AOC in cytoplasm of the rat brain tissues decreased while MDA increased, and the numbers of BDNF-positive cells in cerebral cortex and hippocampus were significantly increased in Ganoderma lucidum spores group. The positive neuron population of HSP70 in hippocampus, basal nucleus and cortex was significantly higher in Ganoderma lucidum spores group than that in epilepsy model group.CONCLUSION: Ganoderma lucidum spores attenuate the impairment of neuronal mitochondria induced by seizure, and accelerate the expression of BDNF, resulting in restoring the energy metabolism in mitochondrion, thus alleviating the impairment and apoptosis of the brain tissues.     

Altered expression of G protein-coupled inwardly rectifier potassium channels (GIRK) subunit 1 and 2 in hippocampus of chronic temporal epileptic rats induced by kainic acid

AIM: G protein-coupled inwardly rectifier potassium (GIRK) channel are distributed widely in mammalian brain. In CNS, GIRK 1/2 seems to be the predominant heterotetramers which play a pivot role in the regulation of the excitability of neurons and may contribute to the resting potential by leading to a hyperpolarization of membrane potential and reduction of the action potential frequency. In the context, the Weaver mouse is the first neurological abnormality directly linked to a genetic point mutation in the GIRK2 protein which includes spontaneous seizure. GIRK2 knock out mice showed normal development but more susceptible than normal mice to seizure induced by GABA antagonist. Here, we report that the mRNA and protein expression of GIRK subunit 2 is altered in kainic acid(KA)-induced epileptic rat hippocampus. METHODS: Rats were injected with kainate 14 mg/kg intraperitoneally to establish an acute and chronic temporal lobe epilepsy model. At chronic spontaneous seizure stage, by using of in situ hybridization, immunocytochemistry and Western blotting, the GIRK 1,2 mRNA and protein were analyzed quantitatively in the dentate gyreus, CA1, CA3 regions of hippocampus. RESULTS: GIRK1,2 mRNA and proteins were expressed abundantly in all regions of hippocampus. KA induced seizures and caused a significant increase in GIRK2 mRNA abundance and immunoreacitivity; only GIRK1 mRNA was increased significantly, but no difference was found by Western blotting protocol. CONCLUSION: GIRK1,2 mRNA and protein expression in the hippocampus of epileptic rat brain is up-regulated, which may be an adaptive response to over-excitability of neuron networks and prevent the over-excitability spread in hippocampus (DG-CA3-CA1).     

Molecular mechanism of BMSC intracerebral transplantation in improving learning and memory abilities of AD mice

AIM: To investigate the effect of bone marrow mesenchymal stem cell (BMSC) transplantation on learning and memory abilities and pathological changes of Alzheimer disease (AD) mice and the molecular mechanisms. METHODS: C57/BL6 wild-type (WT) and transgenic (Tg) mice were randomly divided into 4 groups:WT/PBS group, WT/BMSCs group, Tg/PBS group and Tg/BMSCs group. The mice were administered with PBS or BMSCs via intracerebroventricular injection. Spatial learning and memory abilities of the mice were evaluated by Morris water maze test on the 3rd day after surgery. Real-time PCR was applied to detect the mRNA expression of CX3C chemokine ligand 1 (CX3CL1), CX3C chemokine receptor 1 (CX3CR1), IL-1β, TNF-α, Nurr1, YM1, insulin-degrading enzyme (IDE) and matrix metalloproteinase 9 (MMP9). The protein levels of CX3CL1 and Aβ42 were measured by ELISA. Western blot was used to detect the protein expression of postsynaptic density protein 95 (PSD95) and synaptophysin (SYP). RESULTS: The transplanted BMSCs were observed near the hippocampus of APP/PS1 mice on the 10th postoperative day. The escape latency of the mice in Tg/PBS group was significantly longer than that in the WT/PBS mice (P<0.05). Compared with Tg/PBS group, the escape latency of Tg/BMSCs group was significantly shorter (P<0.05), and the mRNA and protein levels of CX3CL1 in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.01). The results of immunohistofluorescence staining showed that BMSC transplantation promoted the activation of microglia in the brain of WT and Tg mice. The mRNA expression of YM1 was up-regulated in WT/BMSCs group and Tg/BMSCs group (P<0.05). Compared with WT/PBS mice, the mRNA expression of TNF-α in the cortex and hippocampus of Tg/PBS group was significantly increased (P<0.05), and the mRNA expression of Nurr1 in the cortex was significantly decreased (P<0.01). Meanwhile, the mRNA expression of TNF-α in the cortex of Tg/BMSCs mice was decreased (P<0.01) and the mRNA expression of CX3CR1 and Nurr1 was up-regulated compared with Tg/PBS group (P<0.05). The results of Western blot showed that the protein levels of PSD95, p85, p110 and p-Akt in Tg/BMSCs group were significantly higher than those in Tg/PBS group (P<0.05). Finally, BMSC transplantation reduced the protein level of Aβ42 in APP/PS1 mice (P<0.05), and increased the mRNA expression of IDE and MMP9 in the hippocampus (P<0.05). CONCLUSION: BMSC transplantation modulates neuroinflammatory responses and promotes neuroprotective factor and synaptic protein expression, thus improving the learning and memory abilities in the APP/PS1 mice, which may be achieved by up-regulating the expression of CX3CL1.     

Neuroprotective effect of wortmannin on epileptic rats by inhibiting autophagy

AIM: To investigate the role of autophagy in hippocampus injury induced by seizures and to observe the neuroprotective effects of autophagy inhibitor wortmannin(WM) on epileptic rats.METHODS: The Wistar rats were randomly divided into control group, model groups at 2 h, 8 h, 16 h, 24 h or 72 h after seizure induction by pilocarpine, and WM pretreatment group. The methods of HE and Nissl staining were used to evaluate the hippocampus injury. The expression of microtubule-associated protein 1 light chain 3(LC3) was detected by Western blotting. The ratio of LC3II to LC3I was calculated and used to represent the activity of autophagy. RESULTS: The significant increase in the ratio of LC3II to LC3I began at 2 h, peaked at 24 h, and maintained at high level at least to 72 h after seizure induction. Obvious neural injury and neuron depletion were observed in hippocampus CA1 area at 24 h after seizure induction. The number of surviving neurons at 24 h was sharply decreased in rats with seizures(75.50±5.92) as compared to the controls(110.67±18.56, P＜0.01). WM significantly decreased the neuron depletion induced by seizures(100.88±18.73, P＜0.05). Moreover, WM significantly decreased the ratio of LC3II to LC3I in rats with seizures at 24 h(P＜0.05). CONCLUSION: Autophagy was activated in hippocampus injury induced by seizures. WM reduces the transformation of LC3II to LC3I to inhibit the autophagy activated by seizures. WM has neuroprotective effect on epileptic rats by increasing the surviving neurons in hippocampus CA1 area.     

Comparative proteomics of myocardial mitochondrial proteins in aging rats

AIM:To identify the proteins related to aging in the mitochondria of the heart. METHODS:Mitochondria were isolated from the hearts of adult (10-week) and old (12-month) rats (n=3). Total proteins extracted from the mitochondria were applied to the two-dimensional gel electrophoresis. The differentially expressed protein spots evaluated by a software were subjected to in-gel digestion, and analyzed by the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). ATP content in the heart was also measured. RESULTS:Eighty-four differentially expressed protein spots were observed, and fifteen of them were analyzed by MALDI-TOF- MS. Four proteins including creatine kinase, peroxiredoxin 2, Tu translation elongation factor and isocitrate dehydrogenase were up-regulated and 11 proteins including succinate dehydrogenase, malate dehydrogenase, aconitate hydratase, NADH dehydrogenase, ATP synthase H+ transporting, ATP synthase beta chain, heat shock protein 60, glucose-regulated protein 78, prohibitin, Aldehyde dehydrogenase 2 and voltage-dependent anion channel exhibited down-regulation in the aging group compared to the adult one. ATP content in the heart of old rats was significantly reduced as compared to that in adult rats (n=5, P<0.01). CONCLUSION:Significant changes in the mitochondrial protein expression in the aging heart were identified by the 2-DE electrophoretogram with high resolution and reproducibility. The functions of these identified proteins need to be further investigated.     

Poly adenosine diphosphate-ribose polymerase regulates the expression of nuclear factor-κB and related inflammatory factors in rat hippocampus after epilepsy

AIM: To investigate the time course of nuclear factor-κB (NF-κB) and the effects of 3-aminobenzamide (3-AB) on the expressions of NF-κB, interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) in hippocampus after seizures. METHODS: Epilepsy were induced by kainic acid through cerebral ventricular injection. Western blotting was used to detect NF-κB p65 expression in nucleus at various experiment groups. Moreover, mRNA and protein expressions of IL-1β and COX-2 in different experiment groups were determined by RT-PCR and Western blotting analysis. RESULTS: NF-κB p65 immunoreactivity began to increase in the nuclear fraction at 2 h (P<0.05), kept rising at 12 h (P<0.05) and returned to control level at 24 h after epilepsy seizures. Furthermore, 3-AB sharply decreased the accumulation of NF-κB p65 in nucleus (P<0.05). In addition, 3-AB significantly decreased the mRNA and protein expressions of IL-1β and COX-2 which obviously increased in hippocampus at 6 h after epilepsy seizures (P<0.05). CONCLUSION: Seizures triggers NF-κB nucleus translocation and promotes the expressions of IL-1β and COX-2 in hippocampus. In addition, poly (adenosine diphosphate-ribose) polymerase inhibition by 3-AB suppresses NF-κB associated inflammatory pathway in epileptic rat hippocampus.     

Gene and protein alterations in the hippocampus of rats with temporal lobe epilepsy

AIM:To examine the expression profiles of both genes and proteins in hippocampus of rats with temporal lobe epilepsy (TLE) for revealing the molecular mechanisms of TLE and looking for the candidate targets and new therapeutic approaches in clinical practice.METHODS:Rat temporal lobe epilepsy was induced by administration of lithium chloride and pilocarpine (LiCl-PILO).The expression spectra of genes and proteins were constructed through the techniques of cDNA microarray,two-dimensional (2D) electrophoresis and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Subsequently,the differentially expressed genes and proteins were identified and analyzed.RESULTS:There were 192 genes of differential expression observed in hippocampal tissues of LiCl-PILO-induced temporal lobe epilepsy,and 159 genes have been registered in Genbank database,in which 84 genes were up-regulated while 75 genes were down-regulated.78 protein spots of differential display were screened out,in which 31 proteins were detected to be down-regulated and 47 were up-regulated.Finally,5 proteins were identified.CONCLUSION:These genes and proteins found in our study may play pivotal roles in the pathogenic mechanisms of epilepsy and may promise new therapeutic targets for refractory epilepsy in the future.     

Role of NF-κB in pentetrazole-induced repeated seizure in developing rats

AIM:To investigate the role of NF-κB in pentetrazole-induced repeated seizure in developing rats with the inhibitor of NF-κB pyrrolidine dithiocarbamate (PDTC). METHODS:10-day-old Wistar rats (n=72) were prepared for epilepsy model and divided into three groups at random: the PTZ group, the PDTC+PTZ group and the control group. The behavioral changes, the cells morphology and neurons counts in hippocampus, the expression of NF-κB, BrdU (5-bromo, 2-deoxyuridine) immunoreactive cells in hippocampus and the mossy fiber sprouting were observed.RESULTS:(1) The NF-κB expressed in PTZ group was significantly higher than that in PDTC+PTZ group and control group (P<0.01). (2) The dentate gyrus granule cell count in PTZ group was significantly higher than that in control group (P<0.05). In PDTC+PTZ group cell counts in CA1, CA3 and hilar region were significantly lower than those in PTZ group (P<0.05). (3) The BrdU-immunoreactive cells counts in dentate gyrus in PTZ group and PDTC+PTZ group were significantly higher than those in control group (P<0.01), but in PDTC+PTZ group BrdU-immunoreactive cell count was significantly lower than that in PTZ group (P<0.01). Correlate analyzes between NF-κB expression and BrdU-immunoreactive cell counts/granule cell counts showed positive correlation (P<0.01). (4) The mossy fiber sprouting in both PTZ and PDTC+PTZ group was observed. However, the degrees of sprouting showed no significant differences between two groups. CONCLUSION:NF-κB plays a crucial role in epilepsy of developing rats. It encourages neurogenesis and protects neurons in hippocampus, but has no significant effect on mossy fiber sprouting.     

Poly(adenosine diphosphate ribose) glycohydrolase regulates apoptosis-inducing factor and inflammatory factors in rat hippocampus after seizures

AIM: To investigate the effects and molecular mechanisms of poly(adenosine diphosphate ribose) glycohydrolase (PARG) on rat hippocampus neurons after seizures and to study the effects of gallotannin on the expression of apoptosis-inducing factor (AIF), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in rat hippocampus after seizures. METHODS: Seizures were induced by kainic acid (KA). The damage of hippocampus neurons was evaluated by Nissl staining. The protein expression levels of poly(adenosine diphosphate ribose) (PAR), AIF, IL-1β and TNF-α were detected by Western blotting analysis. RESULTS: The number of damaged hippocampal pyramidal neurons in gallotannin-treated group was significantly lower than that in KA-treated group (P<0.05). The expression of PAR was increased in gallotannin-treated group compared with KA-treated group(P<0.05).AIF in mitochondrial fraction increased and its accumulation in nucleus fraction decreased in gallotannin-treated group compared with KA-treated group (P<0.05). In addition, gallotannin significantly decreased the protein expression of IL-1β and TNF-α, which were obviously increased in hippocampus after seizures (P<0.05). CONCLUSION: PARG inhibition by gallotannin has the neuroprotective effect on the damage of hippocampal neurons induced by seizures. In addition, gallotannin suppresses the translocation of AIF from mitochondria to nucleus and the expression of IL-1β and TNF-α in rat hippocampus after seizures.     

Artificial Calculus Bovis inhibits neuron loss in hippocampus and hilus and protects the GAD positive cells in hippocampus of epileptic rats

AIM:To probe into the anti-epilepsy action of artificial Calculus Bovis, by observing its effect on the behavioral of the experimental epileptic rats, neuron loss in the hippocampus and hilus, and GAD positive cell alteration in the hippocampus. METHODS:SD rats were divided into three groups:group A (artificial Calculus Bovis treatment group);group B (acute epilepsy group) and group C (control group). A model of acute epilepsy rats was established by PTZ. The rat’s behavioral alteration was observed by the Racine’ scale. The neurons in the hippocampus and hilus were calculated by Nissl staining. The GAD positive cells were observed by immunohistochemical staining. RESULTS:The latency of the first seizure in group A was longer than that in group B, while the seizure times in group A was less than that in group B. Besides, in group A, both the neuron loss amount in the hippocampus and hilus and the GAD positive cell loss amount in the hippocampus were less than those in group B. CONCLUSION:The artificial Calculus Bovis prolonged the latency of the first seizure time, decreased the frequency of seizure, and prevented the neuron loss and protected the GAD positive cells.     

Astragalus injection inhibits expression of Apaf-1 in rat hippocampus after cerebral ischemia and reperfusion

AIM: To investigate the effect of Astragalus injection on the expression of apoptotic protease-activating factor 1 (Apaf-1) in the hippocampus of global cerebral ische-mia-reperfusion rats. METHODS: Male SD rats were randomly divided into 4 groups with 30 each: sham operation group, cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group, and cerebral ischemia-reperfusion+vehicle group. The global cerebral ischemia-reperfusion model of the rats was established by 4-vessel occlusion. The rats in cerebral ischemia-reperfusion group, cerebral ischemia-reperfusion+Astragalus injection group and cerebral ischemia-reperfusion+vehicle group were further divided into 7 subsets, according to the reperfusion time of 0 h, 0.5 h, 2 h, 6 h, 24 h, 72 h and 120 h. After reperfusion, the brains were removed at the corresponding time points. The protein expression of Apaf-1 in hippocampal neurons was detected by immunohistochemistry and Western blotting. The mRNA expression of Apaf-1 was observed by RT-PCR. RESULTS: Compared with sham operation group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h increased obviously in cerebral ischemia-reperfusion group (P<0.05). Compared with cerebral ischemia-reperfusion group, the expression of Apaf-1 at mRNA and protein levels at all time points except 0 h and 120 h decreased obviously in cerebral ischemia-reperfusion+Astragalus injection group (P<0.05). However, those in cerebral ischemia-reperfusion+vehicle group had no obvious change (P>0.05). CONCLUSION: Astragalus injection inhibits the expression of Apaf-1 at mRNA and protein levels in hippocampus of global cerebral ischemia-reperfusion rats, thus inhibiting the apoptosis of hippocampal neurons.     

Effect of Ganoderma lucidum spores on IL-1β and c-Fos in the brain tissue of epilepsy rats

AIM: To observe the influence of Ganoderma lucidum spores on the cytokine IL-1β and c-Fos in the brain tissue of epilepsy rats. METHODS: The IL-1β level was examined by radioimmunoassay and the c-Fos expression was measured by mmunohistochemical assay. RESULTS: Comparing the Ganoderma lucidum spores group with the epilepsy model group: c-Fos positive cell count in brain cortex and hippocampus in treatment group was obviously reduced. IL-1β level in brain tissue was also reduced obviously. CONCLUSION: Ganoderma lucidum spores effectively reduces cytokine IL-1β in brain tissue of epilepsy rats, improves the immunity dysfunction and plays a role in anti-epilepsy through suppressing c-Fos expression in epilepsy rats brain tissue and blocking of LRG.     

Effect of Buyanghuanwu decoction on the expression of ERK2 and CaMKⅡβ in hippocampus tissue and on ability of learning and memory in vascular dementia rats

AIM: To investigate the effect of Buyanghuanwu decoction, a Chinese medicine, on the ability of learning and memory in the rats with vascular dementia (VD) and on the protein expression of extracellular signal-regulated kinase 2(ERK2) and calcium/calmodulin-dependent protein kinase Ⅱβ(CaMKⅡβ) in hippocampus CA1 area.METHODS: The rats were divided into 4 groups: sham group, VD group, VD+Buyanghuanwu decoction group and VD+nimodipine group. The VD rat model was prepared by Pulsinelli's four-vessel occlusion. At 7th day, 14th day or 28th day after operation, the behaviors of the rats were tested by Morris water maze. The morphological changes of the neurons in hippocampus CA1 area were observed by HE staining 30 d after operation. Western blotting was used to observe the protein expression of ERK2 and CaMKⅡβ in the brain tissues of hippocampal CA1 area of the VD rats. RESULTS: Compared with sham group, the pathological changes such as irregular arrangement, coagulation necrosis and obvious deletion in the neurons of hippocampus CA1 area in VD group appeared significantly. The obstacle of learning and memory ability was observed and the protein expression of ERK2 and CaMKⅡβ in hippocampal CA1 area was significantly decreased (P<0.05). Compared with VD group, the neurons in hippocampal CA1 area of VD+Buyanghuanwu decoction group and VD+nimodipine group were in eumorphism, lined up in order, and the structure was close to that in sham group. The ability of learning and memory also significantly improved (P<0.05). The protein expression of ERK2 and CaMKⅡβ in hippocampal CA1 area significantly increased (P<0.05). CONCLUSION: Buyanghuanwu decoction promotes the protein expression of ERK2 and CaMKⅡβ in hippocampus CA1 area to protect the neurons from injury, builds up the synapses and promotes the ability of learning and memory in VD rats.     

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.     

Neuroprotective effect of novel Rho kinase inhibitor FSD-C10 on a mouse model of Alzheimer disease

AIM: To explore the neuroprotective effect of novel Rho kinase inhibitor FSD-C10 on Alzheimer disease (AD) model of APP/PS1 double transgenic (Tg) mice. METHODS: Male APP/PS1 Tg mice (n=20) at 8 months of age were randomly divided into 2 groups:model group and FSD-C10 treatment group. The mice were treated with normal saline or FSD-C10 (25 mg·kg^-1·d^-1) by intraperitoneal injection, once daily for 2 months. Age-and sex-matched wild-type (WT) mice without treatment were used as the control. The Morris water maze (MWM) test and SMART 3.0 behavioral record system were applied to examine and analyze the spatial cognitive function of the mice. The protein levels and distribution of Aβ, p-tau and synapse-associated proteins such as synaptophysin, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and postsynaptic density protein 95 (PSD-95) were determined by immunofluorescence staining. The protein levels of phosphorylated amyloid precursor protein at Thr668[p-APP(Thr668)], beta-site APP-cleaving enzyme 1 (BACE1) and synapse-associated proteins in the brain were analyzed by Western blot. RESULTS: Compared with model group, FSD-C10 treatment significantly improved the cognitive function of the APP/PS1 Tg mice, accompanied by reduced Aβ deposition and p-tau level, increased protein level of p-APP (Thr668) in the central nervous system, decreased expression of BACE1, and increased expression of synapse-associated proteins in the brain of the mice (P<0.05). CONCLUSION: FSD-C10 has neuroprotective potential in the APP/PS1 Tg mice. The mechanism may be related to enhancing the non-amyloidogenic APP cleavage pathway, reducing the production of Aβ oligomers, the deposition of senile plaques and the amount of tau protein, up-regulating synapse-associated proteins, and increasing synaptic plasticity.     

Role of gap junction in pentylenetetrazol-induced epilepsy in rats

AIM: To study the role of gap junction(GJ) in the pathogenesis of epilepsy. METHODS: The expression of connexin(Cx) 32 and Cx43 was evaluated in penlylenetetrazol(PTZ)-induced epilepsy rats at different time points with immunohistochemistry and real-time RT-PCR. The uncoupler of gap junction,carbenoxolone (CBX), and anti-epilepsy drug,carbamazepine (CBZ), were introduced to investigate the role of gap junction in epilepsy. RESULTS: The expression of Cx32 in the cortex and hippocampus of rats increased 2 h after PTZ administration, and was more obvious 8 h later, so was the expression of Cx43. CBX not only notably inhibited the expression of Cx32 and Cx43, but also suppressed the seizures. CBZ had no obvious effect on Cx32/43 expression. Real-time RT-PCR examination showed that the level of Cx32 mRNA went up rapidly 2 h after seizures, and began to decrease 10 h later. The level of Cx43 mRNA in PTZ group was higher than that in control group from 2 h to 5 h. CBX inhibited both the seizures and the increase in the mRNA expression of Cx32/43, while CBZ only suppressed the seizures and did not affect the expression of Cx32/43. CONCLUSION: GJ between neurons is reinforced after epileptic activities and takes part in the pathophysiological mechanism of synchronization and epilepsy. CBZ has no effect on the expression of Cx32 and Cx43, indicating that the anti-epileptic mechanism of CBZ is independent of GJ.     

Proteomic study of myocardial mitochondria with ischemia/reperfusion injury and pinacidil postconditioning in isolated rat hearts

AIM: To investigate the protective effect of pinacidil postconditioning on rat myocardium suffering ischemia/reperfusion injury by mitochondrial proteomics. METHODS: Langendorff apparatus was used to establish the model of myocardial ischemia/reperfusion injury. Sprague-Dawley rats were randomly divided into 2 groups: pinacidil postconditioning group (Pina group) and ischemia/reperfusion injury group (I/R group). After 20 min of perfusion with K-H solution, the perfusion was suspended for 40-min (global ischemia) follow by 60 min of reperfusion in I/R group. In Pina group at the end of 40 min global ischemia, the isolated hearts were perfused with K-H solution containing pinacidil (50 μmol/L) for 2 min followed 58-min perfusion with regular K-H solution. Total proteins extracted from the mitochondria were applied to the two-dimensional gel electrophoresis (2-DE). The differentially expressed protein spots over 2 times were evaluated by a software. Then they were subjected to in-gel digestion, and analyzed by spectrometry. RESULTS: The expression levels of NDUFA10, NDUFS2 and NDUFV2 were elevated but those of IDHA and ECH1 were decreased in Pina group compared with I/R group. Interestingly, 2 spots in the 2-DE map were identified as ATPase subunit δ. The expression levels of one spot was elevated, while the other was decreased. CONCLUSION: Pinacidil postconditioning may decrease the degree of increased expression levels of NDUFA10, NDUFS2 and NDUFV2, promote the expression of IDHA and ECH1, and induce the phosphorylation of ATPase subunit δ, which may be related to the protective mechanism of pinacidil postconditioning.     

Primary screening of stress protective proteins in heat-preconditioned fibroblast NIH-3T3

AIM：To establish stress adaptation model of mouse fibroblast cell line NIH-3T3 to analyze the effect of stress and adaptation on protein synthesis,and to screen for stress adaptation related proteins.METHODS：A stress-adapted cell model was established by thermal preconditioning (42 ℃,20 min).Total cytolytes were separated by 2-DE,analyzed by PDQUEST software,and the selected differential expression spots were detected by MOLDI-TOF.The effect of stress and adaptation on protein synthesis was studied.The stress adaptation related spots were identified by PMF.RESULTS：Comparative proteomics method by 2-DE was used to find different distributions of total proteins of heat stress group and thermal preconditioning group.Expression-increased protein spots were found almost limited in low molecular weight range in directly stress group,whereas expression-increased protein spots in thermal preconditioning group have more extensive molecular weight distribution.PMF results showed that tubulinβ,vimentin,eIF-4AI,Eno1 protein might be related to stress adaptation.CONCLUSION：2-DE analysis suggested,cell might favor to synthesize low small molecular weight protein to deal with hostile stress.Cellular protein storage might be increased by preconditioning,and may play a protection role during successive stress.The increased expression of tubulinβ，vimentin,eIF-4AI,Eno1 protein suggests that cytoskeletons,protein synthesis pathway and glycometabolism pathway may play an important role in stress adaption.     

Dexmedetomidine reduced isoflurane-induced neuroapoptosis by inhibiting activation of p38 and JNK proteins in hippocampus of neonatal rats

AIM：To investigate the effect of dexmedetomidine (Dex) on neuronal apoptosis induced by isoflurane (Iso) and its relationship with the expression of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) proteins in the hippocampus of neonatal rats. METHODS：Forty-eight neonatal SD rats at postnatal day 7 were randomly divided into control group (Con), Dex group, Iso group and Iso combined with Dex (Iso+Dex) group. Rats in Iso and Iso+Dex groups were exposed to 0.75% Iso for 6 h, while rats in Con and Dex groups were exposed to air for 6 h. Rats were intraperitoneally injected with 25 μg·kg-1 Dex (Dex and Iso+Dex groups) or 150 μL saline (Con and Iso groups) 20 min before exposure and 2 and 4 h after exposure. After the termination of anesthesia, the neuronal apoptosis in hippocampal CA1 region was detected by TUNEL staining, and the protein expression of cleaved caspase-3, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK) and JNK in hippocampal tissues was detected by Western blotting. RESULTS：The number of TUNEL positive cells in hippocampal CA1 region of the rats in Iso group was increased by 447.57% (P<0.01) compared with Con group, while Dex significantly inhibited the increased TUNEL positive cells in Iso group by 75.18% (P<0.01). The expression of cleaved caspase-3 protein in Iso group was increased by 126.29% (P<0.01) compared with Con group, while Dex reversed the increased cleaved caspase-3 protein expression (P<0.01). Iso significantly increased the phosphorylation of p38 and JNK proteins (P<0.01), while Dex reversed the increased p-p38 and p-JNK proteins (P<0.01). CONCLUSION：Dex attenuates Iso-induced neuroapoptosis in the hippocampus of neonatal rats through inhibiting the phosphorylation of p38 and JNK proteins.