Erythropoietin attenuates cerebral ischemia-induced cognitive dysfunction through stimulation of hippocampal CREB phosphorylation in C57BL/6 mice

AIM: To investigate the neuroprotective effect of erythropoietin (EPO) on cognitive dysfunction and neuronal injury in hippocampal CA1 region induced by cerebral ischemia in mice. METHODS: Male C57BL/6 green fluorescent protein-transgenic mice were randomly divided into 3 groups: sham operation group (sham), ischemia/reperfusion group (I/R) and EPO-treated group. Transient cerebral global ischemia was induced by bilateral common carotid artery occlusion (2-VO). The step-down test was used to measure the capacity of learning and memory of the animals in each group. Nissl staining was applied to examine the neuronal number in hippocampal CA1 region. The expression of phosphorylated cAMP response element-binding protein (pCREB) was determined by Western blotting. Alterations of dendritic morphology in hippocampal CA1 region were evaluated using laser scanning confocal microscopy and Neurolucida software. RESULTS: Transient cerebral ischemia caused deficits of spatial learning and memory, and delayed neuronal death and loss of dendritic spines in hippocampal CA1 region were also obvious. The EPO treatment significantly improved the cognitive function in cerebral ischemic mice, and the protein expression of pCREB was obviously increased. At the same time, neuronal death and loss of dendritic spines were reduced in hippocampal CA1 region. CONCLUSION: Erythropoietin increases the protein expression of pCREB, and reduces neuronal death and loss of dendritic spines. These processes may be responsible for erythropoietin-mediated neuroprotective effects and the improvement of cognitive function in cerebral ischemic mice.     

Effect of intermittent hypobaric hypoxia preconditioning on expression of Ngb and Bcl-2 in hippocampal CA1 region in rats with global cerebral ischemia-reperfusion

AIM:To study the effect of intermittent hypobaric hypoxia preconditioning (IHHP) on the expression of neuroglobin (Ngb) and Bcl-2 in hippocampal CA1 region in the rats with global cerebral ischemia-reperfusion. METHODS:The Wistar rats were randomly divided into sham group, IHHP control group, global cerebral ischemia-reperfusion group (I/R group), and IHHP+I/R group. The 4-vessel occlusion rat model of Pulsinelli was performed in the rats in I/R group and IHHP+I/R group, in which the common carotid artery was occluded for 8 min before reperfusion. Thionine staining and immunohistochemical staining were used to observe the histological changes of the hippcampus and the expression of Ngb and Bcl-2 in the hippocampal CA1 region. RESULTS:A significant increase in the quantity of surviving cells in the hippocampal CA1 region was observed in IHHP+I/R group as compared with I/R group. There was a significant increase in the expression of Ngb and Bcl-2 in the hippocampal CA1 region in IHHP+I/R group as compared with I/R group. CONCLUSION: Through the up-regulation of hippocampal Ngb and Bcl-2 expression, intermittent hypobaric hypoxia preconditioning may play a role in neuroprotection by reducing hippocampal neuronal apoptosis from ischemia-reperfusion.     

Protective roles of hypobaric hypoxic pretreatment on hippocampus neurons in mice

AIM: This study was designed to investigate the protective role of hypobaric hypoxic pretreatment (HHPT) on hippocampal neurons in Babl/c inbred mice. METHODS: HHPT was produced by simulating a 7 000 m high altitude 2.5 h/d for 3 d. At 36 h after last time decompression, three subgroups consisted of both the control and pretreatment mice were subjected to the 12 000 m high altitude hypobaric hypoxia for 4 h, the severe ischemia produced by bilateral common carotid artery occlusion for 18 min, and the severe ischemia/hypoxia produced by permanently ligation of right lateral common carotid artery followed by a 8 000 m high altitude hypobaric hypoxia for 4 h, respectively. The extents of protection to hippocampal CA1 neurons by HHPT were evaluated by accounting the number of intact neurons between HHPT and control group. RESULTS: The results indicated that HHPT protected hippocampal neurons against severe hypobaric hypoxia, severe ischemia, and ischemia combined with hypobaric hypoxia. CONCLUSION: Hypobaric hypoxic pretreatment induces a delayed protection to hippocampal neurons against hypoxic and ischemic injuries.     

Changes of dendritic morphology and spine density in hippocampal CA1 pyramidal cells of chronic cerebral ischemic rats

AIM: To investigate the changes of dendritic morphology and spine density in hippocampal CA1 pyramidal cells of the chronic cerebral ischemic rats. METHODS: The model of chronic cerebral ischemia was established by permanent occlusion of the bilateral common carotid arteries (2VO) in rats. Two weeks, 4 weeks or 8 weeks later, the behavior of the rats in each group was evaluated through the Morris water maze to select the successful modeling, and the brains were collected for processing Golgi staining. The changes in dendritic branch and length, and spine density in hippocampal CA1 pyramidal cells were observed under optical microscope. RESULTS: Compared with sham-operated group, dendritic branch and length in model group was significantly reduced in 4-week group and 8-week group (P<0.01), and spine density in model group were significantly reduced in 2-week, 4-week and 8-week groups (P<0.01). With prolonged ischemia, dendritic branch and length, and spine density in model group were all significantly reduced (P<0.05). CONCLUSION: Chronic cerebral ischemia leads to traumatic changes in dendrites and spines in hippocampal CA1 pyramidal cells, which constitutes the pathophysiological basis in the progressive cognitive dysfunction.     

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

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

Neuroprotective effect of astrocyte protein phosphatase 2A up-regulation on APP/PS1 double transgenic mice

AIM: To investigate the protective effects of astrocyte protein phosphatase 2A(PP2A) up-regulation on APP/PS1 double transgenic mice.METHODS: An eGFP-wtPP2A lentivirus with glial fiber acidic protein promoter was constructed to specifically increase PP2A expression in the astrocytes. The mice were divided into wild -type mice+vector virus group(Con), APP/PS1 transgenic mice+vector virus group(APP/PS1) and APP/PS1 transgenic mice+eGFP-wtPP2A lentivirus group(PP2A) by lateral ventricular injection of the lentivirus. Four weeks after injection of the virus, the immunofluorescence of brain slices were used to detect the level of β-amyloid protein(Aβ). Golgi staining was used to detect the changes of dendritic spine density and morphology. Electron microscopy was applied to detect the thickness of postsynaptic density(PSD). The Morris water maze test was applied to examine the learning and memory abilities of the mice.RESULTS: Up-regulation of PP2A in the astrocytes attenuated Aβ level increasing in APP/PS1 group. Up-regulation of PP2A in the astrocytes significantly attenuated both decreases in the dendritic spine density and the percentage of mushroom-like dendritic spines in the hippocampal CA3 region of APP/PS1 mice. Up-regulation of PP2A in the astrocytes significantly attenuated the reduced thickness of PSD in APP/PS1 group. Up-regulation of PP2A in the astrocytes attenuated the escape latency extending in APP/PS1 group.CONCLUSION: Up-regulation of PP2A in the astrocytes reduces AD-like pathological changes, and attenuates synaptic impairment, synaptic plasticity deficits and cognitive impairment in the APP/PS1 double transgenic mice.     

Effect of NAC on oxidant stress and apoptosis in the hippocampal CA1 region of rats exposured to chronic intermittent hypoxia

AIM: To investigate the effect of N-acetylcystein (NAC) on oxidant stress, neuron apoptosis in the hippocampal CA1 region of rats exposured to chronic intermittent hypoxia (CIH). METHODS: 30 healthy male Wistar rats were randomly divided into three groups of 10 each, a CIH group, a NAC therapeutic group and a control group. The levels of MDA and SOD were detected by colorimetric method. Immunohistochemistry was used to examine the expression of p-JNK and TUNEL was used to detect the neuron apoptosis in the hippocampal CA1 region. RESULTS: The level of MDA in NAC group were lower than that in CIH group［(1.71±0.43) μmol/g protein vs （1.37±0.26) μmol/g protein, P<0.05)］. The activity of SOD in NAC group was higher than that in CIH group［(44.94±14.01) 103 NU/g protein vs(57.66±14.07) 103 NU/g protein, P<0.05)］. The expression of p-JNK protein and the apoptotic indices ［(0.39±0.16), (0.20±0.11)］ in NAC group were significantly lower than those in CIH group ［(0.53±0.10), (0.32±0.18), all P<0.05］. CONCLUSION: NAC protects hippocampal neuron from apoptosis by suppressing the oxidant stress in the hippocampal CA1 region and inhibiting the activation of JNK signaling pathway.     

4-PBA attenuates damage of primary hippocampal neurons via inhibiting endoplasmic reticulum stress

AIM:To investigate the inhibitory effect of 4-phenylbutyric acid (4-PBA), an aromatic short-chain fatty acid with the functions of endoplasmic reticulum (ER) molecule chaperon, on ER stress-induced decreases in both dendritic spine density and synaptic protein expression. METHODS:Primary hippocampal neurons were cultured from neonatal rats. The primary hippocampal neurons were transfected with enhanced green fluorescent protein plasmids at DIV (day in vitro) 5~7. At DIV 20, the neurons were divided into DMSO group, tunicamycin group and tunicamycin+4-PBA (treated with 4-PBA 1 h before tunicamycin) group. The expression levels of ER stress marker protein BiP and synaptic proteins were detected by Western blot. After immunofluorescence staining, the neurons were morphologically observed under confocal laser scanning microscope, and the density of dendritic spines was analyzed. At last, the cell viability was measured by MTT assay. RESULTS:Tunicamycin induced ER stress in the primary hippocampal neurons, characterized by significantly increased level of BiP in tunicamycin group, which was reduced in tunicamycin+4-PBA group (P<0.05). 4-PBA inhibited tunicamycin-induced decreases in both dendritic spine density and synaptic protein expression in the primary hippocampal neurons. 4-PBA attenuated tunicamycin-induced decrease in the cell viability. CONCLUSION:4-PBA not only attenuates ER stress of primary hippocampal neurons but also inhibits the decreases in both dendritic spine density and synaptic protein expression induced by tunicamycin.     

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.     

Acute cerebral ischemia activates EphB2/ephrin-B1/NMDA receptor signaling pathway to promote hippocampal neurogenesis in mice

AIM To investigate the effect of acute cerebral ischemia on hippocampal neurogenesis in mice and its possible mechanism involving EphB2/ephrin-B1/NMDA receptor signaling pathway. METHODS C57BL/6 mice (n=52) were randomly divided into sham group and acute cerebral ischemia group (model group). The model of acute cerebral ischemia in mice was established by bilateral common carotid artery occlusion. The pathological changes of the hippocampal CA1 region in mice were observed by HE staining. The learning and memory functions of the mice were assessed by Morris water maze. The BrdU positive cells and doublecortin (DCX) protein expression were observed by immunofluorescence staining for detecting hippocampal neurogenesis. The mRNA and protein expression levels of EphB2, ephrin-B1, reelin, microtubule-associated protein-2 (MAP-2) and NMDA receptor subunits NR2A and NR2B in the hippocampus were determined by RT-qPCR and Western blot. RESULTS The neuronal damage in the hippocampal CA1 region was significant (P<0.01), and the learning and memory functions were significantly decreased in the cerebral ischemia mice(P<0.01), suggesting that the cerebral ischemia model was successfully established. The BrdU positive cells and DCX protein expression were increased significantly (P<0.01), indicating that neurogenesis occurred in the hippocampus after cerebral ischemia. At the same time, the mRNA and protein expression levels of EphB2, ephrin-B1, reelin, MAP-2, NR2A and NR2B in the hippocampus were also significantly up-regulated (P<0.05). CONCLUSION Acute cerebral ischemia promotes the proliferation of hippocampal neural stem cells and endogenous neurogenesis, which may be related to the activation of EphB2/ephrin-B1/NMDA receptor signaling pathway.     

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 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.     

Effect of hypercapnia on lysyl oxidase-dependent collagen cross-linking and hypoxia-induced pulmonary hypertension in rat

AIM: To investigate the effect of hypercapnia on hypoxia-induced pulmonary hypertension and the changes of lysyl oxidase (LOX) and extracellular matrix collagen cross-links in the rat. METHODS: Sprague-Dawley rats were randomly divided into 4 groups:normoxia group, hypoxia group, hypercapnia group and hypoxia+hypercapnia group. LOX activity was detected by fluorescence spectrophotometry. LOX protein expression was detected by immunohistochemistry and Western blot. The mRNA expression of LOX in the pulmonary artery was detected by real-time PCR. RESULTS: The levels of mean pulmonary artery pressure (mPAP), RV/(LV+S) and WA/TA in hypoxia group were significantly higher than those in normoxia group (P<0.01). Moreover, the levels of mPAP and RV/(LV+S) in hypoxia+hypercapnia group were significantly lower than those in hypoxia group (P<0.01). However, no significant difference of mPAP and RV/(LV+S) between hypercapnia group and normoxia group was observed. In hypoxia group, the collagen cross-links in the lung tissue was significantly higher than that in normoxia group and hypercapnia group (P<0.01). Importantly, collagen cross-links in the lung tissue of hypoxia+hypercapnia group was significantly lower than that in hypoxia group (P<0.01). There was no significant difference in collagen cross-links between hypercapnia group and normoxia group. The expression of LOX at mRNA and protein levels and its activity in the pulmonary arteries of hypoxia group were significantly increased as compared with normoxia group (P<0.01). Furthermore, the expression of LOX at mRNA and protein levels and its activity in the pulmonary arteries in hypoxia+hypercapnia group were lower than those in hypoxia group (P<0.01). CONCLUSION: Hypoxia not only up-regulates LOX but also promotes collagen cross-linking in the rat lung, which contributes to the development of pulmonary hypertension. Hypercapnia inhibits hypoxia-induced LOX expression and collagen cross-linking, therefore impairing the progress in hypoxia-induced pulmonary hypertension.     

Relationship between Th17 cells infiltrated in lungs and pulmonary vascular reconstruction under hypobarric hypoxia

AIM: To investigate the changes of retinoid-related orphan receptor γt(RORγt) mRNA and interleukin-17(IL-17) protein in the lung tissue under hypobaric hypoxia, and the relationship between Th17 cells and hypoxic pulmonary vascular reconstruction. METHODS: Male BALB/c mice(n=50) were randomly divided into control group and 3 d, 7 d, 14 d and 28 d hypobaric hypoxia groups. The mice in hypobaric hypoxia groups were housed in a hypobaric hypoxia chamber(simulated altitude of 6 000 m) for 3 d, 7 d, 14 d or 28 d. The mice in control group were housed in normal pressure and oxygen environment. The hemodynamic data were recorded by cardiac catheterization. The hypertrophy of right ventricle was evaluated by the ratio of weight of the right ventricle to the weight of the left ventricle plus interventri-cular septum, and the right ventricular weight over body weight. The spleen was collected and the proportions of the Th17(CD4⁺IL-17⁺RORγt⁺) cells were detected by flow cytometry. The serum levels of IL-4, IL-6 and IL-17 and the change of IL-17 in the lung tissue were measured by ELISA. The mRNA expression of RORγt in the spleen and lung tissues was measured by RT-qPCR. RESULTS: Compared with control group, the mouse right ventricular systolic pressure, the hypertrophy index of right ventricle and the serum IL-17 level were significantly elevated in hypoxia groups, which was consistent with the results of flow cytometry. The mRNA expression of RORγt in the lung tissue was also significantly increased in 7 d, 14 d and 28 d hypoxia groups. The expression of IL-17 in the lung tissue was significantly increased in 14 d and 28 d hypoxia groups. CONCLUSION: Hypoxia promotes differentiation of Th0 cells to Th17 cells in the spleen. The Th17 cells infiltrated in the lung tissue under hypobarric hypoxia are involved in pulmonary vascular reconstruction.     

Effect of intraoperative dexmedetomidine intervention on perioperative neurocognitive disorders in aged mice

AIM To investigate the effect of dexmedetomidine (DEX) on perioperative neurocognitive disorders (PND) in aged mice and its mechanism. METHODS The PND animal model was established by hepatic left lateral lobectomy in C57BL/6J aged mice, and the effects of intraoperative DEX intervention on postoperative cognitive behavior in aged mice were evaluated by Morris water maze and Y maze experiments. The effects of intraoperative DEX intervention on the changes of neurons in hippocampal CA1 region of aged mice were observed by Nissl staining and TUNEL staining. The effect of intraoperative DEX intervention on long-term potentiation in hippocampal CA1 region was recorded by patch-clamp technique. The effects of intraoperative DEX intervention on hippocampal astrocyte activation in aged mice were detected by immunofluorescence and immunohistochemistry. The effects of intraoperative DEX intervention on the protein levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and cleaved caspase-3 in hippocampal CA1 region of aged mice were determined by Western blot. RESULTS Intraoperative DEX intervention attenuated postoperative cognitive dysfunction in aged mice (P<0.01). Intraoperative DEX intervention significantly inhibited surgery-induced hippocampal neuron loss (P<0.01), reversed surgery-induced decrease in field excitatory postsynaptic potential amplitude, and reduced surgery-induced increases in the protein levels of TNF-α, IL-6 and cleaved caspase-3 in hippocampal CA1 region of aged mice (P<0.01). CONCLUSION Dexmedetomidine protects hippocampal neurons and improves postoperative cognitive function in aged mice by inhibiting hippocampal astrocyte activation and reducing neuronal inflammation and neuronal apoptosis.     

Expression relevance of GATA-1 and miR-451a in erythroid differentiation of K562 cells under hypoxia

AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O₂) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells.     

Effects of hypoxia on the expression of heparanase and invasiveness of SKOV3 ovarian cancer cell line

AIM: To evaluate the effects of hypoxia on the expression of heparanase and the invasiveness of SKOV3 ovarial carcinoma cell line. METHODS: SKOV3 cells were incubated at either normoxia (37 ℃, 5％CO2, 21％O2) or hypoxia (37 ℃, 5％CO2, 1％O2) condition for 12 h, 24 h and 36 h. RT-PCR and Western blotting were used to detect mRNA and the protein expressions of heparanase under different conditions. Cell invasiveness was measured by matrigel invasion assay. RESULTS: Compared to normoxia group, the heparanase mRNA expression level in hypoxia group was increased and in 12 h hypoxia group was the highest. The heparanase protein expression in hypoxia group was also significantly increased (P<0.01) and the expression of heparanase in hypoxia group was also different (P<0.05). Compared to normoxia group, the level of cell invasion was markedly increased in 12 h, 24 h and 36 h groups (P<0.05). During 12-36 h hypoxia period, the increase in hypoxia-induced invasiveness in SKOV3 cell line showed a time-dependent manner (P<0.05). Meanwhile, there was a positive correlation between the expression of HPA and the invasiveness of SKOV3 cells (r=0.8530, P<0.05). CONCLUSION: Invasion of SKOV3 cells in hypoxia condition correlates with heparanase level. Hypoxia plays an important role in the augmentation of the heparanase expression and the invasiveness of human ovarian cancer.     

Effects of Chinese traditional medicine-selected recipe Q0409 on ability of learning and memory in SAM-P/8 mice

AIM: To investigate the effects of Chinese traditional medicine-selected recipe Q0409 on the ability of learning and memory in SAM-P/8 mice. METHODS: Total 91 mice (4-month-old SAM-P/8 mice, SAM-R/1 mice and Kunming mice) were used in the study, in which the male and female animals were labeled separately. According to the performance of Morris water maze test, the mice were divided into 5 groups randomly. The mice were fed with different drugs or distilled water for 60 d (from 4 months to 6 months). The mice were fed with the drugs from 61 d to 65 d, and 1 h later each time, the Morris water maze test was carried out. After this Morris test were finished at 65 d, the mice were killed immediately and their hippocampal tissues were isolated. Half of the hippocampal tissues were added with precooled normal saline and made into 10% (g/mL) homogenate for detecting the protein content and acetyl cholinesterase (AChE) activity. The other half was fixed with 4% paraformaldehyde and embedded with paraffin for immunohistochemical staining of amyloid β-protein (Aβ). RESULTS: Compared with model group, the results of navigation training and spatial probe training in Morris water maze test were significantly improved (P<0.05), and the activity of AChE in the hippocampal homogenate was significantly decreased (P<0.05) in Q0409 treatment group. No difference in Q0409 group was observed compared with control group and positive drug (huperzine A) group. Immunohistochemical staining showed no typical "senile plaques" in the male mice of Q0409 group, while there was shallower and smaller brown staining in the hippocampus of the female mice of Q0409 group. The positive area of Aβ deposition decreased in the CA1 area of hippocampal tissues in Q0409 group. These results were similar to those in positive drug group. CONCLUSION: Q0409 improves the ability of learning and memory in SAM-P/8 mice, which is related to the inhibition of AChE activity and the reduction of Aβ protein deposition in the hippocampus. The effects is similar to those of huperzine A.     

Effect of Jieyuwan on dendritic spines in prefrontal cortex and hippocampus in WKY depression-like rats

AIM To observe the changes of dendritic spines in prefrontal cortex and hippocampus of Wistar-Kyoto (WKY) depression-like rats, and to explore the effects of Jieyuwan (JYW) on them. METHODS The male WKY rats were selected as the experimental group, and the same strain of Wistar rats were selected as the control group. Firstly, sucrose preference test, open-field experiment and forced swimming test were used to detect the behavior changes in the rats as their baseline. Then, all WKY rats were randomly divided into model (WKY+NaCl) group, WKY+JYW group and WKY+citalopram group. All WKY rats and Wistar rats (Wistar+NaCl group) were administered intragastrically for 21 d, and the changes of behavior after administration were detected by the same behavioral methods. Golgi staining was used to observe the pathological characteristics of dendritic spines in the prefrontal cortex and hippocampus, and Western blot was used to detect the protein expression level of postsynaptic density protein-95 (PSD-95) in the prefrontal cortex and hippocampus. RESULTS Before administration, WKY rats clearly showed depression-like behavior, the density of dendritic spines in the prefrontal cortex and hippocampus decreased significantly (P<0.01), and the protein expression level of PSD-95 was significantly reduced (P<0.01). After treatment with the drugs, the depression-like behavior of WKY rats was significantly attenuated, the density of dendritic spines in the prefrontal cortex and hippocampus increased (P<0.01), and the protein expression level of PSD-95 also increased (P<0.01). CONCLUSION Jieyuwan significantly attenuates the depression-like behavior of WKY rats, and affects the structural changes of dendritic spines and the expression of PSD-95 protein, which further proves that dendritic spines may be one of the importantearly structural changes in depression.     

Effect of DEC1 gene silencing on viability,invasion and migration of human breast cancer MDA-MB-231 cells under hypoxia

AIM: To investigate the effect of differentiated embryonic chondrocyte gene 1 (DEC1) expression silencing on viability, invasion and migration of human breast cancer MDA-MB-231 cells and its possible mechanism under hypoxia. METHODS: The expression of DEC1 was detected by RT-qPCR and Western blot in breast cancer MDA-MB-231 cells under normoxia and hypoxia. MDA-MB-231 cells were transfected with the siRNA targeting DEC1 and the protein levels of DEC1, Smad3 and phosphorylated Smad3 (p-Smad3) were examined under hypoxia. Subsequently, the changes in the viability, invasion and migration abilities of MDA-MB-231 cells were analyzed by CCK-8 assay, Transwell experiment and Scratch test, respectively. RESULTS: The expression of DEC1 in MDA-MB-231 cells under hypoxia was higher than that in the MDA-MB-231 cells under normoxia condition at both mRNA and protein levels (P<0.05). The viability, invasion and migration abilities of MDA-MB-231 cells in siRNA-DEC1 group were decreased significantly as compared with control group (P<0.01). Besides, the protein level of p-Smad3 in the MDA-MB-231 cells in siRNA-DEC1 group was lower than that in negative control group under hypoxia condition (P<0.05). CONCLUSION: Down-regulated DEC1 expression significantly decreases the viability, invasion and migration abilities of breast cancer MDA-MB-231 cells by blocking the TGF-β/Smad3 signaling pathway under hypoxia condition.