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.     

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.     

Flavonoids from stem and leaf of Scutellaria baicalonsis Georgi inhibit PHF abnormality and regulatory mechanism of protein phosphatase in rats' brain induced by okadaic acid

AIM: To investigate the effect of flavonoids from stem and leaf of Scutellaria baicalonsis Georgi (SSF) on paired helical filament (PHF) abnormality and the regulatory mechanism of protein phosphatase (PP) in rats' brain induced by okadaic acid (OA). METHODS: Male Sprague-Dawley (SD) rats were microinjected with OA (200 ng/kg) by the lateral ventricle to establish a memory impairment model. Morris water maze was used to screen the memory impairment model. The successful model rats were continuous intragastric infusion (ig) SSF for 36 days. The relative protein expression of PHF, PP1, PP2A-Cα, PP2A-Cβ, PP2CA and PP2CB in the rat cerebral cortex and hippocampus were detected by Western blot. GinKgo biloba leaf flavonoids (GLF) were used as positive control drug. RESULTS: Compared with the sham-operated rats, the relative protein expression of PHF in the cerebral cortex and hippocampus and PP1 in cortex of model rats were significantly increased (P<0.01), and the protein expression of PP2A-Cα, PP2A-Cβ in the cerebral cortex and hippocampus and PP2CB in the hippocampus were decreased (P<0.05), while the relative protein expression of PP2CA and PP2CB in the cortex were significantly increased (P<0.01). SSF reversed the abnormality in the protein expression of PHF, PP2A-Cα and PP2A-Cβ in rat cortex and hippocampus and PP1 in rat cortex induced by OA (P<0.01), which had no significant effect on the relative protein expression of PP2CA and PP2CB. GLF also showed similar results to SSF. CONCLUSION: SSF significantly reduces the abnormal formation of PHF in rats' brain induced by OA, which may be related to the regulation of PP1, PP2A-Cα and PP2A-Cβ expression, but not with PP2CA and PP2CB expression.     

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.     

The effect of buyanghuanwu decoction on expression of AMPA receptor GluR1 subunit in mRNA and protein levels in rat hippocampus with vascular dementia

AIM: To observe the effect of buyanghuanwu decoction, a Chinese medicine, on the expression of AMPA receptor GluR1 subunit in mRNA and protein levels in rat hippocampus with vascular dementia (VD). METHODS: One hundred and forty-four rats were randomly divided into 4 groups: sham-operation group, VD model group, nimodipine group and buyanghuanwu decoction treatment group. The rat model of VD was built up by the method of 4 vessel occlusion. The VD rats were intragastrically treated with buyanghuanwu decoction suspension (pharmacognostic 50 g·kg-1·d-1) and nimodipine suspension (20 mg·kg-1·d-1) for 30 d. The learning and memory abilities were evaluated by Morris water maze testing. The change of GluR1 protein in hippocampal neurons in each group of rats was measured with immunohistochemistry and Western blotting techniques. The expression of GluR1 mRNA in hippocampus was determined by real-time fluorescence quantitative PCR. RESULTS: Compared to sham-operation group, the average escaping latency period (s) of Water maze tests in VD rats prolonged significantly and cross-platform time (numbers/min) shortened distinctly (P<0.05). The VD rats treated with buyanghuanwu decoction significantly improved the above-mentioned learning and memory performances (P<0.05); no significant difference of above-mentioned learning and memory performances among the rats in sham-operation group, nimodipine group and buyanghuanwu decoction treatment group was observed (P>0.05). Compared to the rats in sham-operation group, the mRNA and protein levels of GluR1 were apparently decreased in VD rats (P<0.05). The mRNA and protein levels of GluR1 in the neurons of hippocampus in buyanghuanwu decoction treated VD rats were higher than those in the model animals (P<0.05), and no difference was discovered in the rats among sham operation group, buyanghuanwu decoction treatment group and nimodipine group (P>0.05). CONCLUSION: Buyanghuanwu decoction improves the learning and memory abilities in VD rats. The therapeutic mechanism is associated with lessening the neuron injury on CA1 field in hippocampus and restoring the mRNA and protein expression of GluR1.     

The SDF-1/CXCR4 axis repairs hypoxia-ischemia brain damage via-regulating oriented differentiation of mesenchymal stem cells

AIM:To explore the effect of stromal cell-derived factor-1(SDF-1)/CXC chemokine receptor 4(CXCR4)axis on the neural differentiation of rat mesenchymal stem cells(rMSCs) and recovery from hypoxia-ischemia brain damage(HIBD). METHODS:The rMSCs were isolated from the rat bone marrow, and expanded in vitro. The mRNA and protein levels of CXCR4 in rMSCs treated with SDF-1α(10 μg/L) and hypoxia for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h were detected by RT-PCR, Western blotting and flow cytometry. The mRNA and protein levels of SDF-1α in the hippocampus of the rats with hypoxia-ischemia for 1 d, 3 d, 5 d, 7 d, 14 d and 21 d were also detected by the same methods. The protein levels of neuron-specific enolase(NSE) and glial fibrillary acidic protein(GFAP), as well as the positive rate of neural-induced rMSCs pretreated with AMD3100(a CXCR4 antagonist) at dose of 5 mg/L were determined by Western blotting and immunocytochemistry. RESULTS:Compared with the normal controls, both mRNA and protein levels of CXCR4 increased in rMSCs exposed to hypoxia for 6 h and 12 h, and the results were also confirmed by flow cytometry. As expected, the mRNA level of SDF-1α in the hippocampus of HIBD rats was higher than that in normal control rats(P<0.01). Moreover, the mRNA expression of CXCR4 was extremely up-regulated in rMSCs treated with SDF-1α at concentration of 10 μg/L, and the results were also confirmed by Western blotting and flow cytometry analysis(P<0.01). The protein levels and positive cell numbers of NSE and GFAP were extremely decreased in rMSCs pretreated with AMD3100 at concentration of 5 mg/L. CONCLUSION:Compared with normal rats, SDF-1α level in the hippocampus of the rats with hypoxia-ischemia is increased. Hypoxia and micro-dose of SDF-1α induce the expression of CXCR4 in rMSCs, while CXCR4 antagonist reduces the neural differentiation of rMSCs, suggesting that SDF-1/CXCR4 axis may be deeply involved in the neural differentiation of rMSCs during the process of repairing HIBD.     

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.     

Synthetic kainate acid induces seizures in rats

AIM: To observe the effect of synthetical kainic acid (SKA) on inducing epileptic seizure in rats. METHODS: Forty Wistar rats were divided into normal control group, 5 mg/kg, 10 mg/kg and 12 mg/kg SKA-treated groups and kainic acid (KA) positive control group. The SKA was injected intraperitoneally. The changes of the epileptic behaviors were observed for 8 h successively and the electroencephalography was recorded for 3.5 h. RESULTS: SKA induced epileptic seizures at the dose of 5-12 mg/kg by intraperitoneal injection. No apparently difference between SKA and KA on the changes of epileptic behaviors or electroencephalography was observed. However, the epileptic seizure induced by SKA showed obvious stages, regular pattern and action steady. In addition, SKA caused lower fatality than KA did. CONCLUSION: SKA induces epileptic seizure in rats and the dose of 10 mg/kg by intraperitoneal injection is the optimal.     

Astrocyte proliferation in the rat hippocampus after middle cerebral artery occlusion

AIM: To study rat astrocyte proliferation in ipsilateral hippocampus following focal cerebral ischemia. METHODS: Ischemia was induced by temporary middle cerebral artery occlusion (MCAO). In hippocampus of rats at 3, 7 and 30 days after MCAO, the numbers and anatomic distribution of glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry. The protein expression of GFAP and proliferating cell nuclear antigen (PCNA) in the ipsilateral hippocampus were analyzed by Western blot analysis. RESULTS: Astrocytes appeared hypertrophic, with increased process thickness and numbers at 7 days after MCAO, and the highest density of astrocytes were seen at 30 days in the CA1, CA2 regions of the ipsilateral hippocampus. Western blot analysis revealed that GFAP levels were normal at 3 days, but increased by 7 days and remained elevation at 30 days. Western blot analysis of PCNA protein also revealed identified upregulation PCNA at 3 days after MCAO and the expression peaked at 7 days. CONCLUSION: This study demonstrates that focal cerebral ischemia in the rat results in a rapid response, a process often referred to as reactive astrogliosis or glial scarring, from resident astrocytes of the ipsilateral hippocampus to the side of ischemia.     

Effect of ascorbic acid on chaperone-mediated autophagy in hippocampus of rats with seizures

AIM: To investigate the role of chaperone-mediated autophagy (CMA) and oxidative stress in hippocampal injury induced by seizures and the neuroprotective effect of antioxidant ascorbic acid (AA) on epileptic rats. METHODS: The rats were randomly divided into control group, group with 24 h seizures induced by pilocarpine, group with 24 h seizures and AA pretreatment, and group of AA treatment alone. Both mRNA and protein levels of lysosome-associated membrane protein type 2a (LAMP2a) were tested by RT-PCR and Western blotting to evaluate the activity of CMA. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were also measured by chemical methods. RESULTS: The significant increases in mRNA and protein levels of LAMP2a were found in hippocampus of the rats with 24 h seizures. The level of MDA highly increased and the level of SOD obviously decreased in the rats with seizures. AA pretreatment significantly decreased the levels of LAMP2a and MDA induced by seizures, but increased the activity of SOD. CONCLUSION: Both CMA and oxidative stress are activated in hippocampal injury induced by seizures. AA inhibits CMA and reduces oxidative stress activated by seizures by reducing LAMP2a and MDA, and has a potential antiepileptic function.     

Expression of DR1 and DR2 mRNA and protein in rat pathological cardiac hypertrophy

AIM:To observe the expression profile of DR1, DR2 mRNA and protein in rat heart during pathological cardiac hypertrophy. METHODS:Hypertrophy model was established using renal artery coarctation (AC). Hypertrophy coefficient, left ventricular pressure and collagen were determined 35 days after the operation. RT-PCR, in situ hybridization, immunofluorescence, Western blotting, and integrating image analysis system were applied to examine DR1, DR2 mRNA and protein expressions and location in rat heart. RESULTS:mRNA and protein of DR1 and DR2 were detected in normal rat heart. DR2 mRNA in vascular smooth muscle cells was higher than that in ventricular muscle and the atrium muscle cells. Compared with the normal group, DR1, DR2 mRNA and protein were remarkably decreased in AC group than those in control group. CONCLUSION:Dopamine receptor D1, D2 mRNA and protein expresse in the normal rat heart and their expression decreases in cardiac hypertrophy.     

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

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

Expression of phosphorylated cell cycle related proteins in developing and aging rat hippocampus

AIM: To observe the expression of phosphorylated cell cycle related proteins cyclin-dependent kinase (CDK) and the retinoblastoma protein (Rb) in the developing and aging rat hippocampus. METHODS: The immunofluorescent staining for anti-phospho- CDK2, anti-phospho- CDC2, anti-phospho-Rb and anti-NeuN antibodies was used to determine their expressions and distributions in Wistar rat hippocampus at different ages. The expressions of phosphorylated cell cycle related proteins were also assessed by Western blotting analysis. RESULTS: (1) The number of neurons of CA1 area in hippocampus decreased during the period from 1 day to 15 months. (2) The positive expressions of phospho- CDK2 and phospho-Rb increased with aging. (3) No apparent change in the expression of phospho- CDC2 in the hippocampus of all the five groups was observed. CONCLUSION: The expressions of phospho-CDK2 and phospho-Rb suggest that differentiated neuron can re-enter the cell cycle and more neurons enter the cell cycle in the aged rat brain. Re-entering the cell cycle of differential neurons probably results in apoptosis.     

Effects of ginsenoside Rb1 on proteome from hippocampus of rats with epilepsy induced by Coriaria lactone

AIM: To explore the mechanism of development of Coriaria lactone (CL)-induced epilepsy and the neuroprotective effects of ginsenoside Rb1 (GRb1) on the process of epilepsy by identifying the proteins related to CL and GRb1 in rat hippocampus with two-dimensional electrophoresis (2-DE) technology.METHODS: The rat model of epilepsy was induced by CL. The rats in control group, CL epileptic group and GRb1 +CL epileptic group were decapitated and the hippocampus were collected. Two-dimensional electrophoresis was applied to separate the proteins from each group. Analysis of 2-DE maps was used to determine differential expression of proteins by ImageMaster 2D Platinum 5.0, and some differentially expressed protein spots were picked up for further identification by matrix-assisted laser sorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS).RESULTS: Six proteins were successfully identified. Among these, the expression of brain creatine kinase, endophilin-A1 and UPF0628 protein C10orf96 homolog was lower in GRb1+CL epileptic group than those in CL epileptic group. The expression of cytochrome P-450, phosducin-like protein and bridging integrator 3 protein was lower in GRb1+CL epileptic group than those in control group.CONCLUSION: The protein expression profiles are significantly different among control group, CL epileptic group and GRb1+CL epileptic group. The identified proteins may be related to the neuroprotective effects of GRb1. Among these, brain creatine kinase, endophilin-A1 and UPF0628 protein C10orf96 homolog may be related to CL-induced seizures.     

Role of hypoxia stimulated expression of connective tissue growth factor in renal interstitial fibrosis

AIM: To study the expression of connective tissue growth factor (CTGF) induced by hypoxia, and the role and mechanism of hypoxia on promoting renal interstitial fibrosis. METHODS: Renal interstitial fibrosis was induced by unilateral ureteral obstruction (UUO) in rat animal model for 9 days as in vivo studies; marker of hypoxia-HIF-1α mRNA and protein, the expression of CTGF in the obstructed kidneys were assessed by RT-PCR, immunohistochemistry and Western blotting respectively. In vitro, normal rat kidney interstitial fibroblast cells (NRK-49F) were exposed to hypoxia (1%O2) for up to 6 hours, hypoxia was confirmed by detecting the expression of HIF-1α protein in cells, cellular level of CTGF mRNA and protein were assessed by RT-PCR and Western blotting respectively. RESULTS: Neither HIF-1α mRNA nor HIF-1α protein was expressed in the kidney from sham-operated group of rats. High level of HIF-1α mRNA were occurred, and strongly HIF-1α positive immunostaining were seen in the tubular and interstitial cells in kidney from UUO rats. Expression and location of CTGF protein were paralleled and relevant with the expression of HIF-1α protein in kidney of UUO rats. In cultured NRK-49F cell line, subjected to hypoxia even for 6 hours stimulated the expression of CTGF mRNA and protein. CONCLUSION: Our results indicated that hypoxia could stimulate the expression of CTGF mRNA and protein in kidney from UUO rats, which may in turn contribute to renal interstitial fibrosis.     

Effects of baicalin on CA46 cell proliferation inhibition and apoptosis induction

AIM: To investigate the effects of baicalin on proliferation inhibition and apoptosis induction in human Burkitt lymphoma cell line CA46 and to explore its underlying mechanisms. METHODS: CA46 cells were exposed to baicalin at different dosages and its proliferation inhibition was detected by MTT assay. The ability of baicalin to induce CA46 cell apoptosis was examined by Annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation. The mRNA expressions of c-myc and bcl-2 were detected by RT-PCR, and the protein expressions of c-Myc, Bcl-2, caspase-3 precursor (procaspase-3) and poly ADP-ribose polymerase (PARP) were detected by Western blotting. RESULTS: Baicalin remarkably inhibited the CA46 cell proliferation, with an IC50 value of 10 μmol/L. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, and its earlier and later stages were detected by annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation, respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cells decreased in a time-dependent manner. Western blotting showed that the protein expressions of c-Myc, Bcl-2, procaspase-3 and PARP (116 kD) in baicalin treated CA46 cells were down-regulated in a time-dependent manner, while the expression of PARP(85 kD) was up-regulated. CONCLUSION: Baicalin efficiently induces proliferation inhibition and apoptosis in CA46 cells, which may be related with the down-regulation of c-Myc and Bcl-2 expressions, as well as the up-regulation of caspase-3 activity.     

Effects of extract of Ginkgo biloba on cognitive function and apoptosis of hippocampus neuronal cells in type 1 diabetic rats

AIM: To investigate the protective mechanism of extract of Ginkgo biloba (EGB) on apoptosis of hippocampus neuronal cells in type 1 diabetic encephalopathic rats. METHODS: Thirty-six male Sprague-Dauley rats were divided into 3 groups: control group, diabetic group and EGB-treated group. Streptozotocin was injected intraperitoneally to the animals in later two groups to induce diabetes. The rats in EGB-treated group were injected intraperitoneally with EGB, and the same volume of normal saline was injected to the rats in other groups. At the end of the 12th week, the spatial learning and memory abilities of rats in each group were examined by Morris water maze test. Blood glucose and serum insulin concentration were measured. The neuron densities in hippocampus were measured by Image-Pro Plus 6.0 software. The expressions of Bax, Bcl-2, caspase-3 were assayed by Western blotting and immunohistochemistry. RESULTS: Compared to control group, the level of blood glucose (P<0.01), the protein expression of Bax (P<0.01) and caspase-3 (P<0.01) in hippocampus neuronal cells, and the ratio of Bax/Bcl-2 (P<0.01) and the escape latency (P<0.01) in diabetic group, were significantly increased, while the serum insulin concentration (P<0.01), the neuronal density (P<0.05) in CA1,CA2 hippocampal regions and the platform searching score (P<0.01) were significantly deceased. After treated with EGB, the serum insulin concentration (P<0.05), the neuronal density (P<0.05) in CA1,CA2 hippocampal regions and the platform searching score (P<0.01) were significantly increased, while the level of blood glucose (P<0.01), the protein expression of Bax (P<0.05), caspase-3 (P<0.05) in hippocampus neuronal cells, the ratio of Bax/Bcl-2 (P<0.01) and the escape latency (P<0.05) were significantly deceased than those in diabetic group. The protein expression of Bcl-2 in hippocampus neuronal cells did not alter in any experimental rats. CONCLUSION: EGB improves the spatial learning and memory capacity in diabetic rats by decreasing the expression of Bax, Bax/Bcl-2 ratio and down-regulating caspase-3 to reduce neurocyte apoptosis and increase the neuron density in CA1, CA2 hippocampal regions, suggesting that effective regulation of neuron apoptosis associated genes may be one of the mechanisms for EGB to treat diabetic encephalopathy.     

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.     

Expression of dopamine receptor in rat anoxia-reoxygenation injured cardiac tissue

AIM: To investigate the expression of dopamine receptor (DR) in rat cardiac tissue and the relationship with the anoxia-reoxygenation injury (ARI).METHODS: The rat cardiac ARI model in vitro was rebuilt on Langendorf device.RT-PCR and Western blotting were used to detect the mRNA and protein expression of DR1and DR2.The activity of LDH in coronary effluent volume was assayed with ultraviolet spectrophotometer.The ultrastructure of cardiac tissue was determined by transmission electron microscope.RESULTS: The mRNA and protein expressions of DR1and DR2 in rat cardiac tissue increased at anoxia 40 min (P<0.05)，markedly increased at reperfusion 1 h (P<0.01),reached the highest level at 2 h,but lightly decreased at 3 h and 4 h.The activity of LDH increased and the myocardial ultrastructure injured seriously after reperfusion.CONCLUSION: The expressions of DR1 and DR2 in rat cardiac tissue increased firstly then decreased during anoxia-reoxygenation processes.The two kinds of receptors may take part in the cardiac anoxia-reoxygenation injury.     

Effects of TGF-β1 and signal protein Smad3 on rat cardiac myocyte hypertrophy

AIM: To investigate the effects of TGF-β1 and signal protein Smad3 on rat cardiac myocyte hypertrophy. METHODS: The total protein was analyzed by flow cytometry and the ANF mRNA expression was measured by RT-PCR to judge the hypertrophy of cultured neonatal cardiac myocytes. Smad3 mRNA expression in cardiac myocytes was measured by RT-PCR, and the protein expression of Smad3 was analyzed by Western blotting. RESULTS: TGF-β1 significantly increased the total protein in cardiac myocytes and promoted ANF mRNA expression, compared with control group. In cultured neonatal myocytes, AS-ODN of Smad3 inhibited myocyte hypertrophy induced by TGF-β1. Smad3 mRNA and protein expression increased at 15 min after incubated with TGF-β1, reached the peak at 1 h, and declined at 4 h. CONCLUSION: TGF-β1 and signal protein Smad3 may participate in the progress of rat cardiac myocyte hypertrophy.