| Background:Cognitive impairment in schizophrenia has been discussed for about a century. In 1970s, Gallhofer first proposed that there was another symptomin addition to positive symptoms and negative symptomsof schizophrenia, the third class of core symptoms components, namely cognitive impairment.Cognitive function is to reflect the objective reality of the human brain characterized by feeling, perception, memory and thought processes, andthe ability to properly handle complex tasks (receive information, coding, storage, and extraction).It is generallybelieved that cognitive dysfunction in schizophrenia involves with temporal lobe, frontal and subcortical areas and their closely linked areas. The incidence of cognitive impairment in schizophrenia is at least 40%-60%, especially in attention, memory and executive function. The degree of cognitive impairment in schizophreniais closely related to social and occupational function, which is thekey factor in schizophrenia return to society. The main clinical manifestations of cognitive impairment in schizophrenia were:attention disorder, memory impairment, and executive dysfunction.Most patients with schizophrenia have certain memory impairment, this disorder is extensive, but not selective. It relates to each of the major components of the memory system, including working memory, short-term memory, and long-term memory. Schizophrenia, memory disorders is stable in the development of the disease,and it reflects flaws in hippocampal function.Brain-derived neurotrophic factor (BDNF) is widely distributed in the brain, which plays a key role in neuronal growth, development, and differentiation. BDNF binding to its high affinity receptor TRKB promotes hippocampal LTP, plays a pivotal role in the formation and maintenance of learning and memory functionthrough activity-dependent synaptic plasticity. More and more evidence suggests that the incidence of schizophrenia may be associated with microscopic changes in neural development. As one of the core symptoms of schizophrenia, cognitive dysfunction is also closely related to changes in neural development and plasticity, in view of the important role of BDNF in these areas, study of correlation between BDNF and cognitive function in schizophrenia is significant important. Moreover, the molecular mechanism of learning and memory, the neural activity causes an increase in the flow of calcium ions, and thus the activation of intracellular signal transduction. Both MAPK/ERK, CaMIV and PKA could actived CREB, but BDNF primarily through activation of the TrkB receptor and ERK/CREB pathway to complete the promotion of the role of memory, studies have shown that BDNF/ERK/CREB is important for hippocampal formation and maintenance of spatial memory.WSKY (warm-supplementing kidney yang) is an herbal prescription that has been used for treatment in psychiatric conditions in Traditional Chinese Medicine (TCM). According to TCM, schizophrenia is a syndrome caused by a yin-yang imbalance characterized by spleen and stomach weakness and inadequate kidney yin. Herbal prescriptions such as WSKY to warm-supplementing kidney yang or strong kidney yin is believed to improve deficits associated with schizophrenia. Recently, we reported that WSKY capsule added on risperidone significantly enhanced cognitive function, social function, and improved the quality of life compare with placebo added to risperidone. In the current study, we focus on the action mechanism of WSKY on cognition enhancement.The 1st part WSKY affect rat model of long-term memory impairment in schizophrenia and BDNF expression in the hippocampusPurpose:1. Using HPLC in preliminary analysis and identification of the active ingredients in WSKY.2. Using subchronic MK-801 treatment to establish a rat schizophrenia model ofglutamate hypofunction.3. To observe the effect of oral administration of WSKY onspatial learning and memory fuction of rat model of schizophrenia.4. To observe the effect of WSKY onhippocampal BDNF expression in rat model of schizophrenia.Method:Production process and quality control program of WSKY were in accordance with the preliminary experiments, and use of HPLC to analyze of the and identify WSKY main active ingredientspreliminary; 60 juvenile male SD rats (weighting 100+5g) were randomly divided into six groups:Schizophrenia model group MK-801 (0.05mg/kg) by intraperitoneal injection of 14 days (1 times/day), the use of open-field test, forced swim test and Morris water maze test the reliability and validity of schizophrenia model; control group received normal saline; treatment groups were administrated withWSKY orally 2h before intraperitoneal injection MK-801, divided into:WSKY25+MK-801 group, WSKY100 + MK-801 group, WSKY250 + MK-801 group and WSKY500+ MK-801 group (mg/kg, W/W), oral administered WSKY for 14 days. Utilization of Morris water maze test to check WSKY on learning and memory function, recorded indicators include escape time and swimming speed in training, the number of crossing and time in the target quadrant in probe trail; In the end of behavioral test,rats were sacrificed for immunohistochemistry was used to detect the impact of WSKY on BDNF expression in the rat hippocampus.Results:1. HPLC analysis and identification:Through process improvement and quality control of WSKY production, stability of WSKY was comply with the requirements of the latter part of the experiment. Main ingredients include:icariin, lobetyolin, astragalus saponin, cinnamic aldehyde, hesperidin, ukraine head alkali.2. Open field test:30 minutes after the last MK-801 injected intraperitoneally, total distance in the open field test of rats in model group and control group were found withno significant difference (P> 0.05).3. Forced swimming test:Test was took in 30 minutes after the last MK-801 injected intraperitoneally, compared to the control group,rats inmodel group show a significantly longer immobility time (P<0.05).4. Morris water maze test:Repeated-measure ANOVA revealed a significant day effect on escape latency (F4,167=600.837; p<0.01), indicating that, across groups, rats improved their spatial learning effectively with increased training. MK-801-injected rats showed significant spatial reference memory function impairment during training days compared with control group (F1,23=502.759, P<0.001), while MK-801 treated rats performance still significantly improved by consecutive training (F4,115=37.14, P<0.01). We also found that a significant treatment effect on escape latency, WSKY treatment of schizophrenia animals at dose of 25mg/Kg,100mg/Kg,250mg/Kg showed significant SLM performance improvement compared with MK-801 treated group(WSKY 25mg/Kg group, F1,27=10.248, P<0.05; WSKY 100mg/Kg group F1,27=206.159, P<0.01; WSKY 250mg/Kg group F 1,27=273.908, P<0.01, respectively compared with MK-801 group), which indicated that WSKY ameliorate the SLM impairment induce by MK-801 injection. Interestingly, WSKY at dose of 500mg/Kg showed no significant difference in training compared with MK-801 group (F1,27=0.622, P>0.05) (Fig.B.3). However, there was no significant difference of swimming speed in each group.Furthermore, we investigated the effects of WSKY on spatial memory formation. Probe trials were conducted to assess the spatial memory at 24 h after last training. As shown in Fig.B.5, the WSKY treated group at dose of 100mg/Kg and 250 mg/Kg spent more time than the MK-801 group in the target quadrant of the pool in which the hidden platform was previously located (WSKY 100 mg/Kg:29.1 ± 4.9 vs. MK-801:21.5 ± 2.5, p=0.039, p< 0.01; WSKY 250mg/Kg:32.31±3.65 vs. MK-801:21.5 ± 2.5, p=0.002, p< 0.01). In addition, the 250mg/Kg WSKY treatment showed significantly increased numbers of platform crossings when compared with MK-801 treated group (WSKY 250mg/Kg:2.33±0.58 vs. MK-801:0.67 ± 0.58, p< 0.01). Thisindicated that WSKY improved the spatial learning and memory of rats treated by MK-801.5. Immunohistochemistry:Compared with the control group, model group hippocampal CA1 region (p<0.01) and DG (p<0.01) BDNF expression was significantly decreased; BDNF expression in WSKY250+MK-801 group DG area showed a significant increase (p<0.01), while there is an upward trend in the CA1 region, but there was no significant difference (p>0.05).Conclusion:1. Stability of WSKY was fullfilled with the requirements of the latter part of the experiment. Main ingredients include:icariin, lobetyolin, astragalus saponin, cinnamic aldehyde, hesperidin, ukraine head alkali.2. Subchronic intraperitoneal injection of low doses of MK-801 (0.05mg/Kg) for 14 days show a significant damage on spatial learning and memory functions (SLM)in rats, and extended the immobility time in FST, but there were no impact on Spontaneous activityand locomotor activity. Indicating the cognitive impairment and the validity of this schizophrenia model.3. After the treatment with different doses WSKY, motor function in rats was not affected, and some dose improved cognitive function. The result showed no dose-dependent trend but inverted "U"-shaped overall trend, further analysis was considered to take to study complex pharmaceutical machenism of WSKY.4. Improvement of cognitive function by WSKY maybe connected with increased expression of BDNF in hippocampal DG area.The 2nd part WSKY influence on hippocampal neurons BDNF and its signal transduction pathwayPurpose:1. Hippocampal neuronswere treated with WSKY containing serum and blank serum, to investigate the effect of WSKY on hippocampal neurons BDNF/ERK/CREB signaling pathway.2. To observe the effect WSKY of normal (without MWM training) rat hippocampus BDNF/ERK/CREB signal transduction pathway.Method:In vitro parts:(1)Hippocampus from embryonic day 16-18 (E16-18) rat pups were used forprimary hippocampal neurons culture, Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. (2) Juvenile(PND28) rats administered with WSKY (250mg/Kg) orally,2 times/day, 1h after 5thadiministration, Blood was collected from the abdominalaorta afteranesthesia, standing for 2h, then centrifugation, inactivated in 56 ? for 30min, filter-sterilized and stored at-80 ? spare. (3) Experimental groups:5% blank serum,10% blank serum,15% blank serum,5% WSKYcontainingserum,10% WSKYcontainingserum,15% WSKYcontainingserum, and control group (serum-free culture). (4) WesternBlotting assay:proBDNF, mature BDNF, ERK1/2, pERK1/2, CREB, pCREB protein expression were analysed with Wetern blotting, PKA inhibitor H-89 and MEK inhibitor U-0126 was used to treat cells, then activity and expression of ERK and CREB was detected with WB.In vivo parts:(1) 18 SD (without MWM training) rats were randomly divided into WSKY treatment group (WSKY100mg/kg,250mg/kg) and control group (saline) administered orally 2 weeks, 1/days,2h after the last administration, rat hippocampus were collected immediately, WesternBlotting was used for detecting changes in hippocampal BDNF/ERK/CREB signal transduction pathway.Results:In vitro parts:(1) Compared to the control group, pro-BDNF (p<0.01) mature-BDNF (p<0.01) expression were increased significantly in each serum (including blank serum and medicated serum) treated groups; Compare with each concentrations of blank serum treated groups, there were no significant difference in pro-BDNF expression in WSKY containing serum groups (P>0.05); While compared blank serum group, WSKY containing serum play a promoting role in expression mature-BDNF (p<0.05). (2) Compared with the blank serum groups, WSKY containing serum show no different effect on expression of ERK (P>0.05), and CREB (P>0.05), but WSKY containing serum treatmentshows a significant correlation with increased phosphorylated ERK1/2 (p<0.01) and phosphorylated CREB (p<0.01). Ant this effect can be blockd by MEK inhibitor U-0126 (p<0.01), but not by the PKA inhibitor H-89 (p>0.05).In vivo results: In line with results in vitro, hippocampal mature BDNF in WSKY100 (p<0.01) and WSKY250 group (P<0.01) were significantly increased, andWSKY maybe promot the phosphorylation of ERK (p<0.01) and CREB (p<0.01).Conclusion:In vitro experiments showed that WSKY had no significantly promoting inhippocampal pro-BDNF expression, but it can significantly promote pro--BDNF cleaved to mature-BDNF, and in vivo experiments WSKY significantly enhance protein expression mature-BDNF, indicating regulation WSKY on maturation of BDNF. In addition, effect of WSKY on BDNF/ERK/CREB in rat without MWM training indicating WSKY positive effectin cognitive functionmay be associated with activation of BDNF/ERK/CREB signal transduction pathway. |
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