Effect of Jieyuwan on depression-like behaviors and expression of BDNF in hippocampus and prefrontal cortex of WKY rats

AIM:To investigate the mechanism of depression and its development, and to study the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex of Wistar-Kyoto (WKY) rats treated with Jieyuwan. METHODS:Adult male WKY rats were used as an animal model of endogenous depression. Wistar rats of the same strain were selected as control group. WKY rats were randomly divided into model group, citalopram group and Jieyuwan group. After intragastric administration for 21 d, the changes of depression-like behaviors were observed by sucrose preference test and forced swimming test. Immunofluorescence and Western blot were used to detect the expression of BDNF in the hippocampus and prefrontal cortex. RESULTS:WKY rats showed significant depression-like behaviors, and the expression of BDNF was significantly decreased in the hippocampus and prefrontal cortex (P<0.01). The reduction of neuronal axons in hippocampus was also observed. After drug treatment, the depression-like behaviors of WKY rats were significantly attenuated, and the expression of BDNF and the number of axons were increased (P<0.01). CONCLUSION:Jieyuwan effectively attenuates the depression-like behaviors of WKY rats, and BDNF is a key factor in its antidepressant effect. Our findings further confirm the involvement of BDNF in the development of depression.     

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 BDNF expression in cerebral cortex and hippocampus on ability of learning and memory in APP/PS1 transgenic mice

AIM: To investigate the expression changes of brain-derived neurotrophic factor (BDNF) in the cerebral cortex and hippocampus and their effects on the ability of learning and memory in the wild-type (WT) mice and APP/PS1 transgenic mice. METHODS: WT mice and APP/PS1 transgenic mice were selected as study subjects. Aβ plaques, apoptosis rate and BDNF expression in the cerebral cortex and hippocampus of WT mice and APP/PS1 transgenic mice were detected by the methods of Congo red staining, TUNEL, immunofluorescence and Western blot. The abilities of learning and memory were determined by Morris water maze test. RESULTS: The Aβ plaques appeared in the cerebral cortex and hippocampus of APP/PS1 transgenic mice, and the number of Aβ plaques in 12-month-old mice was larger than that in 6-month-old mice (P<0.05). The number of apoptotic neurons in the cerebral cortex and hippocampus of 12-month-old APP/PS1 transgenic mice was larger than that of WT mice (P<0.01). The expression level of BDNF in the cerebral cortex and hippocampus of WT mice was higher than that of APP/PS1 transgenic mice (P<0.01). The Morris water maze test showed that the escape latency in APP/PS1 transgenic mice was longer than that in WT mice, and the times across the platform quadrant in 60 s was less than that in WT mice (P<0.01). The swim-tracking path of APP/PS1 transgenic mice was disordered and irregular. CONCLUSION: The expression of BDNF in the cerebral cortex and hippocampus of APP/PS1 transgenic mice was lower than that of WT mice, accompanied by increased neuronal apoptosis and decreased spatial learning and memory ability. The decrease in learning and memory ability may be related to decreased BDNF expression in the cerebral cortex and hippocampus of APP/PS1 transgenic mice, leading to increased neuronal apoptosis, which may be one of the pathological mechanisms of Alzheimer disease.     

Expression of EAAT3 in prefrontal cortex and hippocampus in CPP reinstatement rats induced by morphine

AIM: To investigate the role of excitatory amino acid transporter 3(EAAT3) in prefrontal cortex and hippocampus in morphine relapse by detecting the changes of EAAT3 expression in prefrontal cortex and hippocampus in conditioned place preference (CPP) reinstatement rat model induced by morphine.METHODS: Forty adult male SD rats, weighing 200-250 g, were randomly divided into 5 groups with 8 rats each: control group, CPP establishment group (Es), CPP extinction group (Ex), reinstatement 2 h group (Re2) and reinstatement 4 h group (Re4).Intraperitoneal (ip) injection of morphine was applied at a constant dose (10 mg/kg) for 10 days to the established CPP model.Normal saline instead of morphine was used to induce CPP extinction for 10 days.CPP was reinstated following a single priming injection of morphine (2.5 mg/kg).After the CPP behavior test, the rats were sacrificed, and the prefrontal cortex and hippocampus were collected for detecting the levels of EAAT3 by Western blotting.RESULTS: The accumulated time the rats spent in the drug-paired chamber was significantly longer in Es group, Re2 group and Re4 group than that in control group (P<0.05).Compared with control group, the expression of EAAT3 in prefrontal cortex significantly decreased both in Es group and Re4 group (P<0.05).No significant change of EAAT3 in hippocampus among groups was observed (P>0.05), while EAAT3 in hippocampal CA1 area significantly increased in Es group and Ex group as compared with control group (P<0.05).CONCLUSION: The expression of EAAT3 in prefrontal cortex decreases both in CPP establishment and reinstatement models, indicating that down-regulation of EAAT3 in prefrontal cortex may partly participate in the formation of opium relapse.     

Change of depression-like behavior in chronic alcoholism and withdrawal model,and co-mechanism of depression and chronic alcoholism in mice

AIM: To investigate the behavior of depression in chronic alcoholism and withdrawal model of mice, and to explore the co-mechanism of alcoholism and depression. METHODS: A novel model of chronic alcoholism was constructed in this study. The animals were divided into normal control group, and alcohol 7 d, 14 d, 21 d and 28 d groups. The mice were given alcohol preference test on the 6th, 13th, 20th and 27th days. After the test, alcohol were withdrawn for 1 d, then the next day the mice were given behavior test of depression. After the test, the mice were sacrificed. The contents of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) were detected by HPLC. The expression of cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) was detected by Western blot. RESULTS: The mice showed an obvious drinking phenomenon, and the immobility time of forced swimming test and tail suspension test was significantly increased, with increasing drinking days and withdrawal times. 5-HT level in 7 d group mice only increased in frontal cortex (P < 0.05). However, compared with control group, 5-HT levels in hippocampus and cortex were decreased on the 21th and 28th days (P < 0.01). NE levels in 21 d and 28 d groups were decreased in hippocampus and frontal cortex (P < 0.05), and no significant change was observed in 7 d and 14 d groups. The protein levels of p-CREB and BDNF were significantly decreased in hippocampus and frontal cortex of 12 d and 28 d groups (P < 0.05), and no significant change was observed in 7 d group and 14 d group. CONCLUSION: The co-mechanism of alcoholism, withdrawal and depression is related to 5-HT. 5-HT-cAMP-CREB-BDNF signaling pathway may be a common mechanism for alcoholism and depression.     

Phosphodiesterase 4 inhibitor rolipram prevents chronic alcoholism and withdrawal-induced depression-like behaviors in mice

AIM: To investigate the effect of phosphodiesterase 4 (PDE4) inhibitor rolipram on the levels of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), cAMP response element-binding protein (CREB), phosphorylated CREB (p-CREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus and the prefrontal cortex (PFC) of alcoholism model mice.METHODS: The mice (n=60) were randomly divided into control group, control+rolipram group, alcoholism model group, and alcohol+rolipram (0.1, 0.5 and 1 mg/kg) groups. The mice were given alcohol preference test on days 6, 13, 20 and 27. After the test, the mice received withdrawal of alcohol for 1 d. On day 28, the mice were given behavior test of depression, and after the test, the mice were sacrificed. The cAMP levels in the hippocampus and PFC were detected by ELISA, and the protein levels of PKA, CREB, p-CREB and BDNF were detected by Western blot.RESULTS: The mice showed an obvious drinking phenomenon (P<0.01), and the immobility time of forced swimming test and tail suspension test was significantly increased (P<0.01), with increasing drinking days and withdrawal times. However, chronic treatment with rolipram for 28 d reversed this phenomenon. Moreover, the cAMP levels in the hippocampus and PFC were significantly decreased after 28 d alcohol treatment (P<0.01), and pretreatment with rolipram (1 mg/kg) obviously reversed this decrease (P<0.01). Parallel to these changes of cAMP, the protein levels of PKA, p-CREB and BDNF were also decreased in the hippocampus and PFC (P<0.01), and 28 d rolipram administration inhibited the decreased cAMP, PKA, p-CREB and BDNF levels in the hippocampus. Moreover, 28 d rolipram administration also reversed decreased cAMP, PKA and p-CREB in the PFC.CONCLUSION: Rolipram treatment protects against alcohol-induced depression-like behaviors, and also reduces alcohol drinking. These effects may be related to PDE4-cAMP-PKA-CREB-BDNF pathway.     

Effect of naoluo xintong on expression of Fas,Fas L in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats

AIM: To investigate the effets of naoluo xintong on the expression of Fas, FasL protein in hippocampus CA1 area and Fas mRNA in the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats. METHODS: The local cerebral ischemia /reperfusion model was established by intraluminal thread occlusion of the middle cerebral arteries (MCAO), the middle cerebral arteries of rats were occluded for 2 hours and reperfused for 1, 3 and 7 days. The animals were divided into pseudo surgery group(sham group), model group, Yiqi group, Huoxue group and naoluo xintong group. Using the techniques of immuno-histochemical staining and in situ hybridization, the expression of Fas and FasL was observed in hippocampus CA1 area, the expression of Fas mRNA was also observed in the cortex of frontal and parietal lobe. RESULTS: A value of Fas and FasL protein expression or A value and positive unit of Fas mRNA expression in control group were higher than those in sham in hippocampus CA1 area, the cortex of frontal or parietal lobe after local cerebral ischemia/reperfusion in MCAO rats (P<0.01). A value and/or positive unit of their expression in naoluo xintong group were lower than those in control group (P<0.05 or P<0.01). A value and/or positive unit of their expression in Yiqi and Huoxue groups were higher than those in naoluo xintong group for 3 and/or 7 days (P<0.05 or P<0.01). CONCLUSION: naoluo xintong could resist neuron apoptosis, alleviate pathologic injury after local cerebral ischemia/reperfusion in MCAO rats by inhibiting the expression of Fas, FasL protein and Fas mRNA.     

Preliminary study on pyroptosis involved in cerebral ischemia-reperfusion injury

AIM:To investigate the changes of pyroptosis in hippocampus and cortex at different time points after cerebral ischemia-reperfusion, and to explore its mechanism from NLRP3-mediated classical pyroptosis pathway, and to analyze the role of pyroptosis in different parts of cerebral injury. METHODS:SD rats were randomly divided into sham operation group (sham group) and model group (MCAO/R group). The rats in model group was further divided into cerebral ischemia-reperfusion 6 h group (MCAO/R 6 h group), 12 h group (MCAO/R 12h group)and 24 h group (MCAO/R 24 h group). The rat model was established on rats by middle cerebral artery occlusion and reperfusion (MCAO/R) induced by modified right-side thread method. Neurologic function score, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and morphological observation were used to evaluate the degree of nervous cell injury. TUNEL and caspase-1 immunofluorescence double staining were used to detect pyroptosis. The protein expression of NLRP3, cleaved caspase-1, pro-caspase-1 and interleukin-1β (IL-1β) was determined by Western blot. RESULTS:Neurological damage occurred at different times after cerebral ischemia-reperfusion. TTC staining showed that the volume of cerebral infarction gradually increased with the prolongation of reperfusion time (P<0.05). The hippocampal CA1 area and cortical area showed typical morphological features such as loose tissue structure, interstitial edema, disordered arrangement of nerve cells, deepening of nucleus staining, nuclear fragmentation and decreased cell number. Immunofluorescence double staining showed that there was a phenomenon of pyroptosis at different time after cerebral ischemia-reperfusion. The pyroptosis of hippocampal CA1 and cortical area was most obvious at 12 h and 24 h after reperfusion (P<0.05). Western blot analysis showed that the expression of NLRP3, cleaved caspase-1, pro-caspase-1 and IL-1β in NLRP3-mediated classic pyroptosis pathway was regulated in different degrees after cerebral ischemia-reperfusion. The protein expression of NLRP3 in hippocampus was significantly increased at 12 h and 24 h after reperfusion (P<0.05), and the protein expression of NLRP3 in cortex was significantly increased at 6 h after reperfusion (P<0.05). The protein expression of pro-caspase-1 in hippocampus was significantly increased at each time points of reperfusion (P<0.05), and the protein expression of pro-caspase-1 in the cortex was significantly increased at 24 h after reperfusion (P<0.05). The protein expression of cleaved caspase-1 in the hippocampus was significantly increased at 12 h after reperfusion (P<0.05), and increased in the cortex at 24 h after reperfusion (P<0.05). The protein expression of IL-1β in the hippocampus was significantly increased at 24 h after reperfusion (P<0.05), and increased in the cortex at 6 h after reperfusion (P<0.05). CONCLUSION:Pyroptosis is involved in neuronal injury after cerebral ischemia-reperfusion. The classic pyroptosis pathway plays an important regulatory role in hippocampus and cortex, especially in hippocampus, suggesting that hippocampus is the main part of secondary nerve impairment induced by pyroptosis and inflammation after cerebral ischemia-reperfusion.     

Protective effect of BNDF on vascular endothelial cells with H2O2-induced oxidative injury

AIM: To study the protective effect of brain-derived neurotrophic factor (BDNF) on vascular endothelial cells with H₂O₂-induced oxidative injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and the oxidation injury model of HUVECs was established by treatment with H₂O₂. The oxidatively injured HUVECs were cultured with different concentrations (1, 10 and 100 μg/L) of BDNF. At the same time, the control group (no injury), PBS treatment after H₂O₂ injury group and TrkB inhibitor group (with 100 μg/L BDNF and 1: 1 000 TrkB inhibitor) were also set up. The viability of the HUVECs was detected by MTT assay. The levels of LDH, MDA, SOD and GSH were measured. The releases of NO, ET-1 and ICAM-1 were analyzed by ELISA. The changes of ROS production and cell apoptosis were evaluated by flow cytometry. The protein levels of TrkB, p-TrkB, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with uninjured control group, in H₂O₂ oxidative injury plus PBS treatment group, the viability of the cells was decreased significantly, the LDH and MDA levels were increased significantly and the activities of SOD and GSH were decreased significantly. The NO secretion was decreased, and the ET-1 and ICAM-1 concentrations were increased significantly. The ROS content and apoptotic rate were increased significantly. The protein levels of cleaved caspase-3 and Bax were increased but Bcl-2 protein expression was decreased significantly. Compared with PBS treatment group, in H₂O₂-injured HUVECs treated with different concentrations of BDNF, the cell viability was gradually increased, the LDH and MDA levels were decreased and the activities of SOD and GSH were increased gradually. The secretion of NO was increased but ET-1 and ICAM-1 were decreased gradually. The ROS content and apoptotic rate were decreased significantly. The TrkB and p-TrkB levels were significantly increased significantly, the protein expression of cleaved-caspase 3 and Bax was decreased gradually and the Bcl-2 protein expression increased gradually. The role of BDNF was inhibited by TrkB inhibitor. CONCLUSION: BDNF protects HUVECs from oxidative injury by binding with TrkB to activate the BDNF-TrkB signaling pathways.     

Effects of dexmedetomidine on behaviors and expression of BDNF and mTOR in hippocampus of depressive rats

AIM: To investigate the effects of dexmedetomidine (DEX) on the behaviors and the expression of brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) in the hippocampus of depressive rats. METHODS: Sprague-Dawley (SD) rats were randomly divided into 5 groups: sham operation group, model group, and DEX (2.5, 5 and 10 μg/kg) groups. The rats were randomly selected in each group (n=12). The rat depression model was established by chronic unpredictable mild stress and ovariectomy. The rats in DEX groups received daily DEX treatment via intraperitoneal injection for 21 d. The forced swimming immobility time (FSIT) and open-field test were used to evaluate the antidepressant effect of DEX. Escape latency and times of crossing the flat were evaluated by Morris water maze. The histological changes of hippocampal neurons were determined by Nissl staining. The mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were detected by RT-qPCR. The protein expression of IL-1β, IL-6, TNF-α and BDNF, and the phosphorylation levels of protein kinase A (PKA), cAMP response element-binding protein (CREB), tropomyosin-related kinase B (TrkB), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and mTOR in hippocampus were evaluated by Western blot. RESULTS: Compared with model group, the FSIT was significantly reduced and the spontaneous activity was markedly increased in DEX groups. The damage of the hippocampal neurons was obviously attenuated, the escape latency was obviously decreased, and times of crossing the flat were markedly increased (P<0.05 or P<0.01). The levels of IL-1β, IL-6 and TNF-α were obviously decreased, and the protein levels of p-PKA, p-CREB, BDNF, p-TrkB and p-PI3K, p-Akt, p-mTOR in hippocampal tissues were obviously increased (P<0.05 or P<0.01). CONCLUSION: Dexmedetomidine improves the behaviors and the spatial learning and memory ability of depressive model rats, which may be related to its anti-inflammatory effects, as well as up-regulating the protein levels of BDNF and p-TrkB, and activating PI3K/Akt/mTOR signaling pathway in the hippocampus.     

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.     

Effects of Xiaoyaosan on imiquimod induced psoriatic lesions and depression neurotransmitters in mouse model

AIM: To observe the effect of Xiaoyaosan decoction on the psoriatic lesions and depression neurotransmitters induced by imiquimod in mice. METHODS: BALB/c male mice were randomly divided into control group, model group, methotrexate group and Xiaoyaosan high, medium and low dose groups, 6 mice in each group. Imiquimod (IMQ, 5%) was used on the back of the animals to induce psoriasis-like lesions in the mice. The psoriasis area and seve-rity index (PASI) were evaluated for daily scoring. The sugar water preference experiment was conducted to explore the behavioral differences in the mice. The morphological changes and epidermal thickness of the lesions were observed under light microscopy. Immunohistochemical method was used to detect the expression of CD3 on T lymphocyte surface. The expression of Ki67 in the skin lesions was detected by immunofluorescence. The contents of monoamine neurotransmitters such as adrenaline (AD), gamma-aminobutylic acid (GABA), glutamate (Glu), dopamine (DA) and their metabolites in the hippocampus and hypothalamus of mice were detected by high performance liquid chromatography-mass spectrometry (HPLC-MS). RESULTS: Compared with model group, the back skin lesions of Xiaoyaosan each dose group and methotrexate group were significantly improved, and the PASI score and epidermal thickness were both lower than those in model group (P<0.05). The expression levels of Ki67 and CD3⁺ T cells in Xiaoyaosan group and methotrexate group were lower than those in model group (P<0.05). Compared with model group, the body mass change range of Xiaoyaosan high-dose group and blank control group was significantly smaller than that in model group (P<0.05). The sugar water preference rate in blank control group was significantly higher than that in model group (P<0.01). Compared with model group, the sugar water preference rate in methotrexate and Xiaoyaosan groups showed a certain increase trend, but no statistical diffe-rence was observed. Compared model group, the levels of 3, 4-Dihydroxypheny-lacetic acid (DOPAC), AD, GLU and GABA levels in the mouse hippocampus in blank control group were decreased significantly (P<0.01), while the levels of DA and homovanillic acid (HVA) had no significant difference (P>0.05). No significant difference of DA, DOPAC, HVA and GLU levels in the mouse hypothalamus was observed between blank control group and model group (P>0.05), while the content of AD and GABA in the mouse hypothalamus in blank control group was lower than that in model group. The AD content of the hypothalamus in high-dose Xiaoyaosan group was significantly higher than that in model group (P<0.01), and the HVA content of the hypothalamus in low-dose Xiaoyaosan group was significantly higher than that in model group (P<0.01). PASI score was negatively correlated with the content of DOPAC, AD, GLU and GABA in the hippocampus and the content of AD, GLU and GABA in the hypothalamus, those were, the more severe the back skin lesion was, the lower the expression of depression-related neurotransmitters were, indicating the aggravation of depression in the mice. CONCLUSION: Xiaoyaosan improves the skin lesions induced by imiquimod in the mice with psoriasis, improves the behavior of depression in the mice with psoriasis, and up-regulates the expression of depression-related monoamine neurotransmitters. The expression of depression-related neurotransmitters is negatively correlated with the skin lesions induced by imiqumod in the mice with psoriasis. The degree of depression is increased with the aggravation of psoriatic lesions.     

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.     

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.     

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 curcumin on learning-memory ability and expression of HMGB1 and JNK in rat model of Alzheimer disease

AIM: To evaluate the effect of curcumin on impaired learning-memory ability and the expression of high mobility group box protein 1 (HMGB1) and c-Jun N-terminal kinase (JNK) in a rat model of Alzheimer disease (AD). METHODS: Male Sprague-Dawley rats, weighing 250~270 g, were randomly divided into 4 groups (n=9): blank control group (group A), model group (group B), curcumin treatment group (group C, curcumin injected intraperitoneally at 100 mg·kg-1·d-1 for 6 consecutive days) and solvent control group (group D). The rats of AD model were induced by injection of ibotenic acid into the nucleus basalis of Meynert (NBM) bilaterally. All rats were trained in Morris maze to assess the ability of learning and memory. The expression of HMGB1 and JNK in the hippocampus was detected by the methods of immunohistochemistry and Western blotting. RESULTS: Compared with group A, the average escape latency (AEL) in groups B and D were obviously longer (P<0.05), while AEL in group C in the 5th and 6th days were significantly shorter (P<0.05). The releases of HMGB1 in the CA1 and CA3 areas in groups B and D from the nucleus were abundant. Compared with groups B and D, HMGB1 in hippocampal CA1 and CA3 areas in group C secreted out of the nucleus decreased obviously (P<0.05). No significant difference of the release of HMGB1 between group A and group C was observed (P>0.05). No significant difference in the expression of HMGB1 in the hippocampus among the 4 groups was found (P>0.05). However, compared with groups B and D, the expression of JNK in group C was decreased obviously (P<0.05). CONCLUSION: Curcumin significantly improves the learning and memory ability of AD rats. The probable mechanisms may be related to inhibiting the release of HMGB1 from the nucleus of hippocampal neurons and decreasing the expression of JNK in the hippocampus.     

Phosphatidylinositol 3-kinase regulates hypoxia-inducible factor 1α in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension

AIM:To investigate relationship among phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in lung of rats with hypoxia-inducible pulmonary hypertension. METHODS:Forty male adult Wistar rats were randomly divided into five groups (eight rats in each group):control group (C group) and groups with hypoxia for 3, 7, 14 and 21 days (H3, H7, H14 and H21 group). Mean pulmonary arterial pressure (mPAP), right ventric hypertrophy index (RVHI) and vessel morphometry were measured. The levels of HIF-1α mRNA expression in lung tissue was measured by in siteu hybridization (ISH). The protein expression of HIF-1α，VEGF and phosphorylated protein kinase β (P-AKT) were observed by immunohistochemistry or Western blotting. RESULTS:mPAP increased significantly 7 days after hypoxia ［(23.53±1.78) mmHg］, peaked 14 days after hypoxia, then remained on the high level. Pulmonary artery remodeling index (extern diameter 100 μm) and RVIH became evident 14 days after hypoxia. Expression of P-AKT protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima and tunica media in all hypoxia rats. HIF-1α mRNA staining was poorly positive in control，hypoxia for 3 days and hypoxia for 7 days, but began to increase significantly 14 days after hypoxia (0.305±0.104, P<0.05), then remained stable. Expression of HIF-1α protein in control group was poorly positive, but was up-regulated in pulmonary arterial tunica intima in all hypoxic rats. In pulmonary arterial tunica media, the levels of HIF-1α protein was markedly up-regulated after 3 days (0.029±0.019, P<0.05 ), reached its peak 7 days after hypoxia (0.232±0.008, P<0.05), then tended to decline 14 days and 21 days after hypoxia. Expression of VEGF protein began to increase 7 days after hypoxia (0.188±0.018, P<0.05), reached its peak 14 days after hypoxia (0.238±0.017, P<0.05), then remained on the high level in pulmonary arterial tunica intima. Linear correlation analysis showed that P-AKT, HIF-1α mRNA, VEGF and mPAP were correlated with vessel the morphometry and RVHI (P<0.01). P-AKT was positively correlated with HIF-1α and VEGF (tunica intima). CONCLUSION:P-AKT, HIF-1α and VEGF are all involved in the pathogenesis of hypoxia-induced pulmonary hypertension in rats.     

Effects of hypoxia-inducible factor-1 signal pathway on proliferation in hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the effect of hypoxia on the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC); and to evaluate the role of hypoxia-inducible factor-1α(HIF-1α), iNOS, P-ERK1/2 protein expression in hypoxic pulmonary hypertension (HPH) pathogenesis.METHODS: Cultured rat PASMC were divided into normoxic group; hypoxic group; hypoxia+ADM(adrenomedulin) group, hypoxia+L-NAME(iNOS inhibitor) group; hypoxia+PD98059 group. Proliferation was investigated by MTT and PCNA. Apoptosis was examined by flow-cytometry. Westen blotting was used to measure protein expression of HIF-1α, P-ERK1/2 and iNOS. RESULTS: (1) A value of 24 h-hypoxia was significantly higher than that in the normoxic group (P<0.01). In the hypoxia+PD98059 group, ADM was significantly lower than that in hypoxia group, whereas A value of the hypoxia+L-NAME was significantly higher than that in hypoxic group and normoxic group (P<0.01). (2) PCNA was positive in PASMC after 24 h hypoxia (P<0.01). PD98059, ADM inhibited the expression of PCNA significantly (P<0.01), whereas L-NAME increased the expression of PCNA significantly (P<0.01). (3) Apoptosis index was not significantly difference among the different groups (P>0.05). (4) HIF-1α, iNOS and P-ERK1/2 expression was poorly positive in normoxic group, positive after hypoxia for 4h (P<0.01), reaching its peak at 8 h hypoxia (P<0.01), HIF-1α, P-ERK1/2 expression declined after 24 h hypoxia. L-NAME promoted the expression of HIF-1α, PD98059 inhibited the expression of HIF-1α, iNOS and P-ERK1/2 partly. ADM inhibited the expression of HIF-1α partly, promoted the expression of iNOS. CONCLUSION: (1) Hypoxia stimulates the proliferation of PASMC, and has no obvious effects on the apoptosis of PASMC. (2) HIF-1 plays an importent role in the proliferation of hypoxic PASMC.     

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.