Effects of EGb761 on synaptophysin expression in hippocampus of vascular dementia rats

AIM: To investigate the effects of Ginkgo biloba extract 761 (Egb761) on synaptophysin (SYN) expression in hippocampus of vascular dementia (VD) rats.METHODS: VD rat models, established by repeatedly cerebral ischemia/reperfusion, were randomly divided into two groups: model group and EGb761 treated group (both n=30), and another 30 condition-matched rats were selected as the sham-operated controls. Spatial learning and memory abilities of rats were assessed by Morris water maze (MWM) task, and SYN expression in hippocampal formation of rats in different groups was detected by immunohistochemical technique and image analysis.RESULTS: The MWM escape latency (EL) in model group was highly longer than that in the sham-operated group (P<0.01), while the EL of EGb761-treated group was significantly shorter than that in model group, but still longer than that in the sham-operated group at 1 m, 2 m and 4 m after VD modeling operation (P<0.05 or P<0.01). Immunohistochemical analysis showed that the SYN immunoreactive expression in hippocampal formation in model group greatly decreased and mean optical density of SYN expression highly increased compared with both sham-operated group and EGb761-treated group at three time points (P<0.01).CONCLUSION: EGb761 can increase the expression of SYN in hippocampus, which may be one of important mechanisms of EGb761 in improving learning and memory dysfunction of VD rats.     

Effect of concentrated decoction of Chinese herbal compound Buyanghuanwutang on cAMP-PKA-CREB signaling pathway in hippocampus of rats with vascular dementia

AIM: To evaluate the role of concentrated decoction of Chinese herbal compound Buyanghuanwutang (BYHWT) in cyclic adenosine monophosphate(cAMP)-protein kinase A(PKA)-cAMP response element-binding protein(CREB) signaling pathway in hippocampus of rats with vascular dementia (VD). METHODS: The rats were randomly divided into sham operation group (sham-operated rats treated with normal saline), VD model group (VD rats treated with normal saline), BYHWT treatment group (VD rats treated with BYHWT) and nimodipment treating group (VD rats treated with nimodipine). The rat model of VD was build by the method of four-vessel occlusion. The rats in all 4 groups were administered with the corresponding reagents for successive 30 days. The content of cAMP was measured by radioimmunoassay. The expression of PKA catalytic subunit (PKAc) was observed by Western blotting. The changes of DNA-binding activity of CREB in rat hippocampus were detected by electrophoretic mobility shift assay. RESULTS: The content of cAMP, the expression of PKAc and the DNA-bingding activity of CREB in the hippocampus of VD rats were lower than those in the hippocampus of sham-operated rats (P<0.01). The above indexes in both nimodipine treatment group and BYHWT treatment group were definitely higher than those in VD model group (P<0.01). CONCLUSION: BYHWT increases the content of cAMP, the expression of PKAc and the DNA-binding activity of CREB in VD rat hippocampus, thus strengthening the cAMP-PKA-CREB signaling pathway.     

Changes of MARCKS mRNA expression in rat hippocampus with acute multi-cerebral infarction

AIM: To observe the dynamic alteration of myristoylated alanine-rich C kinase substrate （MARCKS） mRNA expression in rat hippocampus with acute multi-cerebral infarction, and discuss the relationship between the alteration of hippocampus MARCKS gene and ischemia damage. METHODS: The acute multi-cerebral infarction model was established by method of Kaneko. Neurological function deficits were evaluated in the behavior test. The consequences of cerebral ischemic damage were examined by histopathological analyses. The MARCKS mRNA expression was measured by semi-quantitative PCR. RESULTS: The rats in acute multi-cerebral infarction group showed different level changes of neurological function deficits. The hippocampus damage of histopathology became significant 24h after ischemia. At the same time, the MARCKS mRNA expression was upregulated at the area of rats hippocampus during ischemia, and its overexpression started 1h after ischemia, and reached maximum7d after ischemia. CONCLUSION: MARCKS mRNA of rat hippocampus overexpresses during acute cerebral ischemia. This MARCKS mRNA overexpression is related with hippocampus ischemia damage.     

Histopathological observation of cerebral cortex and hippocampus in the mouse with synthetic vascular dementia

AIM:To observe pathomorphological changes in cerebral cortex and hippocampus in the mouse with synthetic vascular dementia.METHODS:The synthetic vascular dementia model was produced in the mouse. Animals were killed 7 d, 15 d, and 30 d after the operation, brain tissues were removed and embedded in paraffin. Section of 8μm thickness were stained with hematoxylin-eosin(HE)and Nissl methods, and observed with light microscope.RESULTS:The cerebral cortex in the mouse became thinner on the seventh day, karyopyknosis in partial nervous cells was formed, the number of local neurons was reduced, sieve structure was observed, and glial cells pro liferated, with the similar results 15 d and 30 d afteroperation.Model mouseπs hippocampal cells in CA₁ area were reduced and almost disappeared 30 d after operation.At the same time, glial cells were abundantly proliferated, tu bercles were formed.Cells in CA₂, CA₃ area were also reduced and hippocampal sclerosis occurred.CONCLUSION:Delayed necrosis of hippocampal pyramidal cells may be the pathological basis of ischemia cerebral vascular dementia.     

Effect of YIGU capsule on IGF-I mRNA and protein expression in rat osteoblasts in vitro

AIM: The effects of YIGU capsule on proliferation and IGF-I mRNA protein expressions in osteoblasts were studied. METHODS: (1) Forty 12-month old Sprague-Dawley female rats were divided randomly into four groups (YIGU capsule high dose group, medium dose group and low dose group; saline group), the drug-containing serum and control serum were prepared. (2) The new-born Sprague-Dawley rat osteoblasts were cultured with different YIGU capsule drug-containing serum at different concentrations and different exposure time. MTT method was used to observe proliferation of osteoblasts. (3) RT-PCR method was used to measure the relative IGF-I mRNA levels and ELISA method was used to measure IGF-I secretion at different exposure time. (4) ELISA method was used to measure IGF-I secretion at different exposure time. RESULTS: (1) Proliferation of osteoblasts was more than the control groups after 48, 72 and 96 h, respectively (P<0.01); (2) The relative IGF-I mRNA levels and IGF-I protein expression were higher than those in control group after 48, 72 and 96 h, respectively (P<0.01 or P<0.05). CONCLUSIONS: It was suggested that YIGU capsule drug-containing serum promoted proliferation, IGF-I mRNA and protein expression. These results may be parts of the mechanisms of YIGU capsule to prevent and treat osteoporosis.     

Tubulin expression of basal cells in patients with Alzheimer's disease and vascular dementia

AIM: To investigate quantitatively the tubulin expression of basal cells in patients with Alzheimer's disease (AD) and vascular dementia (VD) and evaluate its significance in diagnosing Alzheimer's disease. METHODS: α-tubulin of basal cells was examined using LSAB immunohistochemical technique, and analyzed qualitatively and quantitatively by image analyzer. RESULTS: The integrated absorbance (IA, 261.43±25.21) and the average absorbance (AA, 1.89±0.14) in the AD group were significantly higher than those (IA, 120.55±19.71 and AA, 0.85±0.14, respectively) in the VD group (P<0.01). CONCLUSION: These data suggest that the α-tubulin of basal cells could be served as a extraneuronal biological marker in diagnosing Alzheimer's disease early.     

Effect of benefiting-bone Capsule on IL-6 mRNA and protein expression in rat osteoblasts in vitro

AIM: The effects of benefiting-bone Capsule (BBC) containing serum on IL-6 mRNA and protein expression in osteoblasts were studied.METHODS: (1) The neonate Sprague-dawley rat osteoblasts were cultured and divided into three groups: group Ⅰ (containing deactivating serum with BBC), group Ⅱ (containing deactivating serum without BBC) and group Ⅲ (DMEM medium group)； (2) RT-PCR was used to measure the relative IL-6 mRNA levels； (3) The radioimmunoassay method was used to examine IL-6 protein in the supernatant of the cultured osteoblasts. RESULTS: (1) The relative IL-6 mRNA levels was lower in group I than the control (P<0.05); (2) The IL-6 protein expression in osteoblasts was also lower in group I than the control (P<0.05).CONCLUSIONS: These results suggest BBC drug-containing serum can down-regulate the expression of IL-6 mRNA and protein in osteoblasts, which may be one of the mechanisms of BBC preventing and treating osteoporosis.     

Differential regulating effect on estrogen receptor α and β expression in female rat hippocampus and cortex regions between different types of estrogen regents

AIM: To study the regulatory effect two different estrogen reagents on expressions of estrogen receptor α and β in female rat hippocampus and cortex regions. METHODS: 12 cycles after ovariectomy, female rats were orally injected with premarin or progynova for 3 cycles before sacrificed. Semiquantitative RT-PCR was used to detect the ERs mRNA expression and SP immunohistochemistry was performed to measure the ERs protein distribution and expression. RESULTS: In premarin group, ER α mRNA levels in both hippocampus and cortex tissues decreased significantly compared with control. ER α protein level in hippocampus was lower than that in the control. However, ER α protein level in cortex had no statistical difference. ER β mRNA in the two regions and ER β protein in cortex had no statistical differences compared with control, while ER β protein level in hippocampus was higher than that in the control. In progynova group, both mRNA and protein levels of ER β increased significantly in the two regions compared with the control, and ER α mRNA level also increased in hippocampus, but ER α mRNA level in cortex and ER α protein levels in the above two regions showed no statistical differences. CONCLUSION: There were differential regulatory effects on ER α and ER β expression in female rat cognitive regions between the two different types of estrogen reagents, which may be one of the mechanisms of varied effects in different estrogen replacement therapy reagents.     

Dynamic expressions of synapsin I and phosphorylated synapsin I in hippocampus of vascular dementia rats

AIM: To observe the dynamic changes of synapsin I expression and its phosphorylation in hippocampus in vascular dementia (VD) rats. METHODS: Eighty SD rats were randomly divided into sham-operated group (n=40) and VD model group (n=40), and the latter were established by repeatedly clipping the common carotid arteries with an intraperitoneal injection of sodium nitroprusside solution in anesthetized condition. The synaptic ultrastructural changes in hippocampal CA1 region and the expression levels of synapsinⅠ and phosphorylated synapsinⅠin hippocampus were observed by TEM and immunohistochemical staining method respectively in both sham-operated group and VD model group at 15 d, 1 month, 2 months and 4 months time points. RESULTS: No obviously pathological changes to CA1 area synapse were found in SO group. In model group rats, synaptic circa membrane ambiguity and fusion, synaptic circa membrane structure decreased the postsynaptic density, reduced synaptic vesicles and vesicle cluster. Above pathological changes became gradually severe along with the time prolongation after model-making operation. Compared with sham-operated group, the expression of synapsin I significantly reduced in CA1 region (P<0.01). However, no significant change in molecular layer of DG region (P>0.05) in model group was observed. The number of p-synapsin I positive neurons in DG and hippocampal CA1 region was less in model group than that in sham-operated group (P<0.05, P<0.01). The average absorbance values of p-synapsin I positive neurons in DG and hippocampal CA1 region in model group were decreased at 15 d and 1 month time points (P<0.01), but increased in CA1 region (P<0.01) and unchanged in DG at 2 months and 4 months time points (P>0.05). CONCLUSION: The damaged synaptic structure and depressed expression of synapsin I and its phosphorylation in presynaptic parts of hippocampus induced by repeatedly cerebral ischemia/reperfusion may contribute to the synaptic transmission disorders, especially the presynaptic disorder which may be one of the important pathogenesis of the initiation and development in vascular dementia rats.     

Effect of homocysteine on expression of interleukin-8 mRNA and protein in THP-1 macrophages

AIM: To investigate the effect of homocysteine (Hcy) on expression of interleukin-8 (IL-8) mRNA and protein in THP-1-derived macrophages (THP-1 macrophages). METHODS: Cultured THP-1 monocytes were induced to macrophages by 0.1 μmol/L PMA treatment for 72 hours, then the differentiated THP-1 macrophages were incubated with homocysteine (0.01 mmol/L-0.20 mmol/L) for 24 hours, or with 0.10 mmol/L Hcy for various time up to 48 hours. IL-8 protein in THP-1 supernatants was measured by ELISA, and IL-8 mRNA expression was detected by semiquantitive RT-PCR. RESULTS: Compared with control, Hcy significantly increased the expression of IL-8 protein in a concentration-dependent manner. 0.05 mmol/L, 0.10 mmol/L and 0.20 mmol/L Hcy increased IL-8 production by 1.28 fold, 1.32 fold and 1.55 fold, respectively (P＜0.01). IL-8 production were elevated significantly 3 h after treatment with 0.10 mmol/L Hcy. In addition, Hcy also increased IL-8 mRNA expression in a concentration-and time-dependent manner. CONCLUSION: Hcy may contribute to atherogenesis by inducing IL-8 expression and secretion in THP-1 macrophages.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial 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.     

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.     

Effect of safflower injection on expression of COX-2 mRNA and protein in chronic hypoxic hypercapnic rat pulmonary arterioles

AIM:To study the effect of safflower injection on expression of COX-2 mRNA in chronic hypoxic hypercapnic rat pulmonary arterioles.METHODS: Sprague-Dawley rats were randomly divided into normal control group, hypoxic hypercapnic group (B), hypoxic hypercapnia+ safflower injection group (C). The concentration of TXB2 and 6-Keto-PGF1α in plasma and in lung were detected by the technique of radioimmunoassay. COX-2 mRNA was observed in arterioles from rats by the technique of in situ hybridization. RESULTS: ① Mean pulmonary arterial pressure(mPAP), weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) were much higher in B group than those in control group. No significant difference of mean carotid arterial pressure(mCAP) was observed in three groups. ② The concentration of TXB2 and the ratio of TXB2/6-keto-PGF1α were significantly higher in B group than those in control group. ③ Light microscopy showed that vessel wall area/total area, the density of medial smooth muscle cells and the thickness of medial smooth cell layer were significantly higher in B group than those in control group. Electron microscopy showed proliferation of medial smooth muscle cells and collagenous fibers in pulmonary arterioles in B group. Safflower injection reversed the changes mentioned above. ④ Expression of COX-2 mRNA in pulmonary arterioles was much higher in C group than those in B group. Differences of COX-1 mRNA in pulmonary arterioles were not significant between these two groups.CONCLUSION:Safflower injection increases the expression of COX-2 mRNA in chronic hypoxic hypercapnic rat pulmonary arterioles, indicating an important mechanism that safflower injection inhibits the formation of hypoxic hypercapnia pulmonary hypertension and pulmonary vessel remodeling.     

The synergistic effect of endothelin-1 and basic fibroblast growth factor on rat vascular smooth muscle cell proliferation

AIM: The synergistic effect of basic fibroblast growth factor (bFGF) and endothelin-1 (ET-1) on rat aortic vascular smooth muscle cells (VSMC) proliferation was observed. The possible mechanism of the synergism was also investigated. METHODS: BrdU incorporation and cell counting method were adopted to value the pro-proliferative effect of VSMC. Western blotting was used to observe the variation of bFGF and FGFR-1 isoforms expression. RESULTS: bFGF and ET-1 could promote VSMC proliferation separately, and synergistically in combination. The synergism was dose- and time-dependent. ET-1 increased all the three bFGF isoforms and FGFR-1 protein level in dose- and time-dependent manner. In addition, after exhaustion of intracellular PKC, the upregulation effects of ET-1 on bFGF and FGFR-1 expressions in VSMC both inclined. CONCLUSION: bFGF and ET-1 had synergistic effect on VSMC proliferation. ET-1 may increase the responsiveness of VSMC to bFGF through modulation of bFGF isoforms together with FGFR-1, which was PKC-dependent.     

Protective effect of allitridi on hippocampus of rats with cerebral ischemia-reperfusion and P53 expression

AIM: To observe the protective effect of allitridi on hippocampal neuron of rats with cerebral ischemia-reperfusion (I/R) injury and to investigate its effects on P53 expression in hippocampus.METHODS: The global cerebral ischemia-reperfusion models were established by 4-vessel occlusion. Allitridi at doses of 10, 20 or 30 mg/kg was injected through rat’s tail vein, half dose at 30 min before brain ischemia and another half dose at 10 min after reperfusion were injected, respectively. The hippocampus of rat was removed 24 h after reperfusion. Toluidine blue staining was applied to estimate morphologic changes. Flow cytometry was used to evaluate neuronal apoptosis rate of hippocampus. Immunohistochemistry was used to observe the expression of P53 protein.RESULTS: Compared with sham group, survival neuronal density in I/R group was significantly depressed. The rate of neuronal apoptosis and the expression of P53 protein were significantly increased. Allitridi significantly increased the number of survival neurons in hippocampus compared to I/R group. Meanwhile, allitridi remarkably inhibited the rate of neuronal apoptosis and the expression of P53 protein.CONCLUSION: Allitridi has protective role against brain ischemia reperfusion injury. The mechanism may be involved in blocking P53 protein expression in hippocampus of rats with ischemia-reperfusion.     

Effects of baicalin on glial fibrillary acidic protein and nuclear factor-κB expression in juvenile rat hippocampus after status convulsion

AIM: To study the effects of baicalin (BC) on glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) expression and neuronal apoptosis in juvenile rat hippocampus after status convulsion (SC). METHODS: One hundred and ninety five juvenile male Sprague-Dawley rats were randomly divided into 3 groups: normal saline pretreatment group (NS group), SC group and SC with BC pretreatment group (BC group). Each of these 3 groups would be subdivided into 5 subgroups sacrificed at 4 h, 12 h, 24 h, 48 h and 72 h after SC. The rat SC model was prepared by lithium-pilocarpine chemical method. The protein expression of GFAP and NF-κB was detected by the method of immunohistochemistry. The mRNA expression of GFAP was detected by RT-PCR. The neuronal apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: Compared with NS group, the GFAP positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of GFAP was significantly reduced in BC group (P<0.05). Compared with NS group, the NF-κB positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of NF-κB was significantly reduced in BC group. RT-PCR showed that the expression trend of GFAP mRNA was similar to that of the protein. Compared with NS group, the TUNEL positive cells in the hippocampus CA1 area in SC group increased significantly 12 h after SC (P<0.01), and reached a peak at 48 h. After the intervention with BC, the TUNEL positive cells decreased significantly between 12~48 h after SC (P<0.05 or P<0.01), but the number of TUNEL positive cells remained significantly greater than that in NS group (P<0.05). CONCLUSION: The expression of GFAP and NF-κB in the hippocampus increased after SC in rats. Baicalin decreases the expression of GFAP and NF-κB in hippocampus of rats with pilocarpine-induced seizures, and reduces the number of neuronal apoptosis, suggesting that baicalin may protect against the brain damage caused by status convulsion.     

Effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells

AIM: To assess the effect of estrogen on the gene expression of caveolin-1 in rat vascular smooth muscle cells (VSMCs). METHODS: Wistar rats were ovariectomized and subjected to subcutaneous implantation of placebo pellets (OVX+V group) or estradiol pellets (OVX+E group). 2 weeks after implantation, the expression of caveolin-1 gene in endothelium-denuded aortic tissue was examined by RT-PCR. Furthermore, Northern blotting was used to analyze the mRNA expression of caveolin-1 in cultured rat VSMCs. RESULTS: RT-PCR showed that expression of caveolin-1 gene was significantly higher in OVX+E group than that in OVX+V group. Northern blot analysis showed that the mRNA expression of caveolin-1 was higher in VSMCs pretreated with 17β-estradiol (17β-E₂) than that in VSMCs without 17β-E₂ pretreatment. CONCLUSION: Estrogen up-regulates the gene expression of caveolin-1 in the vascular wall, partially indicating the cardiovascular effect of estrogen.