Regulation of Hypoxic Response Elements on the Expression of Vascular Endothelial Growth Factor Gene Transfected to Rat Skeletal Myoblasts under Hypoxic Environment

The regulation of hypoxic response elements on the expression of vascular endothelial growth factor （VEGF） gene transfected to primary cultured rat skeletal myoblasts under hypoxic environment was investigated. pEGFP-C3-9HRE-CMV-VEGF vector was constructed with molecular biology technique and transfected to primary cultured rat skeletal myoblasts by lipofectamine in vitro. Gene expression of transfected myoblasts was detected by RT-PCR, Western blot and fluorescence microscope under different oxygen concentrations and different hypoxia time. The results showed that in hypoxia group, the VEGF gene bands were seen and with the decrease of oxygen concentrations and prolongation of hypoxia time, the expression of VEGF mRNA was obviously increased. Under hypoxic environment, the expression of VEGF protein in the transfected myoblasts was significantly increased. EGFP was expressed only under hypoxic environment but not under normoxic environment. It was concluded that hypoxia promoter could be constructed with HRE and regulate the expression of VEGF gene under hypoxic and normoxic environment, which could enhance the re- liability of gene therapy.

Dual androgen-response elements mediate androgen regulation of MMP-2 expression in prostate cancer cells

Aim： To characterize the matrix metalloproteinases （MMP）-2 promoter and to identify androgen response elements （AREs） involved in androgen-induced MMP-2 expression. Methods： MMP-2 mRNA levels was determined by reverse transcription-polymerase chain reaction （RT-PCR）. MMP-2 promoter-driven luciferase assays were used to determine the fragments responsible for androgen-induced activity. Chromatin-immunoprecipitation assay and electrophoretic mobility shift assays （EMSA） were used to verify the identified AREs in the MMP-2 promoter. Results： Androgen significantly induced MMP-2 expression at the mRNA level, which was blocked by the androgen antagonist bicalutamide. Deletion of a region encompassing base pairs -1591 to -1259 （relative to the start codon） of the MMP-2 promoter led to a significant loss of androgen-induced reporter activity. Additional deletion of the 5＇-region up to -562 bp further reduced the androgen-induced MMP-2 promoter activity. Sequence analysis of these two regions revealed two putative ARE motifs. Introducing mutations in the putative ARE motifs by site-directed mutagenesis approach resulted in a dramatic loss of androgen-induced MMP-2 promoter activity, indicating that the putative ARE motifs are required for androgen-stimulated MMP-2 expression. Most importantly, the androgen receptor （AR） interacted with both motif-containing promoter regions in vivo in a chromatin immunoprecipitation assay after androgen treatment. Furthermore, the AR specifically bound to the wild-type but not mutated ARE motifs-containing probes in an in vitro EMSA assay. Conclusion： Two ARE motifs were identified to be responsible for androgen-induced MMP-2 expression in prostate cancer cells.

A peroxisome proliferator response elements regulatory system in xenopus oocytes and its application

Background Peroxisome proliferator-activated receptor-gamma （PPARγ） is a kind of ligand-activated transcription factors binding to peroxisome proliferator response element （PPRE）, a specific recognition site. It is thought to play a critical role in glucose and lipid metabolism and in inflammation control. The aim of this study was to establish a new cellular model for the quick screening of lipid-lowering drugs, which may be effective as PPAR-γ ligands on the PPRE-mediated pathway regulatory system. Methods Two plasmids were constructed： pXOE-PPARγ, in which the human PPARγ gene was in the downstream of TF Ⅲ A gene promoter, and pLXRN-PPRE-d2EGFP, in which the enhanced green fluorescent protein （EGFP） gene was subcloned into PPRE. The xenopus oocytes were injected with these two plamids, and consequently treated with prostaglandin E1, pioglitazone, and different kinds of lipid-lowering drugs. After 3 days, the oocytes were observed under a fluorescence microscope. To confirm the drug action,we injected pXOE- PPARγ plasmid into the oocytes, which then treated with prostaglandin E1 and Hawthorn flavonoids. The mass of expressed lipoprotein lipase （LPL） in the cells was determined by enzyme labeling linked immunosorbent assay （ELISA）. Results The expression of EGFP was only induced by prostagalandin E1, pioglitazone, Hawthorn flavonoids. A concentration-response relationship was seen between expressed EGFP and Hawthorn flawonids. The levels of LPL in both Hawthorn flaworvoids groups and PPARγ ligand prostagalandin E1 group injected with pXOE-PPARγ plamid increased significantly （ P 〈 0. 001 ） compared with controls, and a cocentration-response relationship was observed between LPL mass and Hawthorn flavonoids. Conclusions It is possible to establish a PPRE regulatory EGFP reporter system in xenopus oocytes to monitor the activity of PPARγ ligand. Hawthorn flavonoids can in increase the expression of gene downsteam of PPRE by effect on the PPRE pathway regulatory system.

Natural variation of GmFNSII-2 contributes to drought resistance by modulating enzyme activity in soybean

As an essential crop that provides vegetable oil and protein,soybean(Glycine max(L.)Merr.)is widely planted all over the world.However,the scarcity of water resources worldwide has seriously impacted on the quality and yield of soybean.To address this,exploring excellent genes for improving drought resistance in soybean is crucial.In this study,we identified natural variations of GmFNSII-2(flavone synthase II)significantly affect the drought resistance of soybeans.Through sequence analysis of GmFNSII-2 in 632 cultivated and 44 wild soybeans nine haplotypes were identified.The full-length allele GmFNSII-2^(C),but not the truncated allele GmFNSII-2^(A) possessing a nonsense nucleotide variation,increased enzyme activity.Further research found that GmDREB3,known to increase soybean drought resistance,bound to the promoter region of GmFNSII-2^(C).GmDREB3 positively regulated the expression of GmFNSII-2^(C),increased flavone synthase abundance and improved the drought resistance.Furthermore,a singlebase mutation in the GmFNSII-2^(C) promoter generated an additional drought response element(CCCCT),which had stronger interaction strength with GmDREB3 and increased its transcriptional activity under drought conditions.The frequency of drought-resistant soybean varieties with Hap 1(Pro:GmFNSII-2^(C))has increased,suggesting that this haplotype may be selected during soybean breeding.In summary,GmFNSII-2^(C) could be used for molecular breeding of drought-tolerant soybean.

Tonicity response element binding protein associated with neuronal cell death in the experimental diabetic retinopathy

AIM: To study the contribution of tonicity response element binding protein(Ton EBP) in retinal ganglion cell(RGC) death of diabetic retinopathy(DR).METHODS: Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin(STZ). Control mice received vehicle(phosphate-buffered saline). All mice were killed 2mo after injections, and the extent of cell death and the protein expression levels of Ton EBP and aldose reductase(AR) were examined.RESULTS: The Ton EBP and AR protein levels and the death of RGC were significantly increased in the retinas of diabetic mice compared with controls 2mo after the induction of diabetes. Terminal deoxynucleotidyl transferase(Td T)-mediated d UTP nick end labeling(TUNEL)-positive signals co-localized with Ton EBP immunoreactive RGC. These changes were increased in the diabetic retinas compared with controls.CONCLUSION: The present data show that AR and Ton EBP are upregulated in the DR and Ton EBP may contribute to apoptosis of RGC in the DR.

Icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels in the hippocampus of the senescence-accelerated mouse

At 8 weeks after intragastric administration of icariin to senescence-accelerated mice （P8 strain）, Morris water maze results showed that escape latency was shortened, and the number of platform crossings was increased. Immunohistochemical staining and western blot assay detected significantly increased levels of cyclic adenosine monophosphate response element binding protein These results suggest that icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels and improves learning and memory functions in hippocampus of the senescence-accelerated mouse.

Effects of basic fibroblast growth factor on hippocampal and parietal cortical neuronal cAMP-response element-binding protein expression in a rat model of focal cerebral ischemia/reperfusion

BACKGROUND： cAMP-response element binding protein （CREB） is a key modulator of various signaling pathways. CREB activation initiates a series of intracellular signaling pathways that promote neuronal survival. OBJECTIVE： To investigate the regulatory effects of basic fibroblast growth factor （bFGF） on cerebral neuronal CREB expression following ischemia/reperfusion injury. DESIGN, TIME AND SETTING： An immunohistochemical detection experiment was performed at the Department of Anatomy, Shenyang Medical College, between October 2006 and April 2008. MATERIALS： A total of 60 healthy, adult, Wistar rats were randomly divided into three groups： sham-operated （n =12）, ischemia/reperfusion （n = 24）, and bFGF-treated （n = 24）. Rabbit anti-rat CREB （1： 100） and biotin labeled goat anti-rabbit IgG were purchased from the Wuhan Boster Company, China. MetaMorph-evolution MP5.0-BX51 microscopy imaging system was provided by China Medical University, China. METHODS： Rat models of cerebral ischemia/reperfusion injury were developed using the suture method for right middle cerebral artery occlusion. Two-hour ischemia was followed by reperfusion. Rats from the bFGF-treated and ischemia/reperfusion groups were intraperitoneally administered endogenous bFGF （500 IU/mL, 2 000 IU/kg） or an equal amount of physiological saline. Rats from the sham-operated group underwent a similar surgical procedure, without induction of ischemia/reperfusion injury and drug administration. MAIN OUTCOME MEASURES： After 48-hour reperfusion, hippocampal and parietal cortical neuronal CREB expression was detected by immunohistochemistry, and the absorbance of hippocampal CREB-positive products was determined using MetaMorph-evolutionMP5.0-BX51 microscopy imaging system. RESULTS： The sham-operated group exhibited noticeable CREB expression in hippocampal and parietal cortical neurons. In the ischemia/reperfusion group, the CREB expression was discrete and neurons were poorly arranged. The bFGF-treated group exhibited increased CREB expression and better neuronal arrangement compared with the ischemia/reperfusion group. The mean absorbance of CREB-immunoreactive products in the hippocampus and parietal cortex was significantly higher in the ischemia/reperfusion group than in the sham-operated group （P 〈 0.05）, and significantly higher in the bFGF-treated group than in the ischemia/reperfusion group （P 〈 0.05）. CONCLUSION： bFGF significantly upregulates CREB expression in hippocampal and parietal cortical neurons following ischemia/reperfusion injury.

Finite element analysis of dynamic response and structure borne noise of gearbox

A dynamic finite element method combined with finite element mixed formula for contact problem is used to analyze the dynamic characteristics of gear system. Considering the stiffness excitation, error excitation and meshing shock excitation, the dynamic finite element model is established for the entire gear system which includes gears, shafts, bearings and gearbox housing. By the software of I-DEAS, the natural frequency, normal mode, dynamic time-domain response, frequency-domain response and one-third octave velocity grade structure borne noise of gear system are studied by the method of theoretical modal analysis and dynamic response analysis. The maximum values of vibration and structure borne noise are occurred at the mesh frequency of output grade gearing.

Investigation of Micro-mechanical Response of Asphalt Mixtures by a Three-dimensional Discrete Element Model

The micro-mechanical response of asphalt mixtures was studied using the discrete element method. The discrete element sample of stone mastic asphalt was generated first and the vehicle load was applied to the sample. A user-written program was coded with the FISH language in PFC3 D to extract the contact forces within the sample and the displacements of the particles. Then, the contact forces within the whole sample, in asphalt mastic, in coarse aggregates and between asphalt mastic and coarse aggregates were investigated. Finally, the movement of the particles in the sample was analyzed. The sample was divided into 15 areas and a figure was drawn to show how the balls move in each area according to the displacements of the balls in each area. The displacements of asphalt mastic balls and coarse aggregates were also analyzed. The experimental results explain how the asphalt mixture bears vehicle load and the potential reasons why the rutting forms from a micro-mechanical view.

Sevoflurane effects on cyclic adenosine monophosphate response element binding protein,phosphorylated cyclic adenosine monophosphate response element binding protein,and Livin expression in the cortex and hippocampus of a vascular cognitive impairment rat

BACKGROUND： Neuronal necrosis and apoptosis play important roles in the pathophysiology of cerebral ischemia and resulting cognitive impairment. However, inhibition of neuronal necrosis and apoptosis has been shown to attenuate cognitive impairment following cerebral ischemia. OBJECTIVE： To investigate the effects of sevoflurane on cyclic adenosine monophosphate response element binding protein （CREB）, phosphorylated CREB （pCREB）, and Livin expression in the cortex and hippocampus of a rat model of vascular cognitive impairment.DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed in the Chongqing Key Laboratory of Neurology between June 2007 and July 2008.MATERIALS： Sevoflurane was provided by Abbott Laboratory, UK; Morris water maze was provided by Chinese Academy of Medical Sciences, China; goat anti-rat CREB, goat anti-rat pCREB and goat anti-rat Livin antibodies were provided by Biosource International, USA. METHODS： A total of 42 female, Wistar rats were randomly assigned to the following groups： sham operation, vascular cognitive impairment, and sevoflurane treatment. The vascular cognitive impairment rat model was established by permanent bilateral occlusion of both common carotid arteries, and 1.0 MAC sevoflurane was immediately administered by inhalation for 2 hours. MAIN OUTCOME MEASURES： CREB, pCREB, and Livin expression was measured in the cortex and hippocampus by Western blot and reverse transcription-polymerase chain reaction. Behavior was evaluated with Morris water maze. RESULTS： CREB, pCREB, and Livin expression in the sevoflurane treatment group was significantly greater than the vascular cognitive impairment group （P 〈 0.01）. However, expression of CREB and pCREB was significantly less in the sevoflurane treatment and vascular cognitive impairment groups, compared with the sham operation group （P 〈 0.01）. Livin expression in the sevoflurane treatment and vascular cognitive impairment groups was significantly greater than the sham operation group （P 〈 0.01）. Learning, memory, and behavior disorders were observed in the vascular cognitive impairment group. Sevoflurane treatment significantly improved these observed disorders. CONCLUSION： Sevoflurane improved cognitive impairment due to permanent bilateral occlusion of both common carotid arteries. Improved function was associated with increased CREB, pCREB, and Livin expression in the cortex and hippocampus.

Effect of ChREBP on carbohydrate feeding regulation in mandarin fish Siniperca chuatsi

Carnivorous fish have poor tolerance to carbohydrate in feed and low utilization rate of carbohydrate.Therefore,the balance of carbohydrate and lipids in the nutrient metabolism of carnivorous fish,the ef fective conversion and utilization of carbohydrate and lipids,and the feedback regulation of feeding are the key links for the e fficient utilization of carnivorous fish feed.Carbohydrate response element binding protein(ChREBP)is a new transcription factor found in recent years in the glucose signaling pathway,and can also participate in feeding regulation.We performed in-vivo and in-vitro experiments to reveal the role of ChREBP in the glucose metabolism and feeding in mandarin fish.The mRNA expression of ChREBP and appetite regulatory factors were measured after intraperitoneal injection of glucose in mandarin fish Siniperca chuatsi and cotransfection with glucose and glucose+siRNA in the hypothalamic cells in mandarin fish.The results reveal that at hour 2 and 4 post intraperitoneal injection with 1 mg/g BW glucose,the blood glucose level of the mandarin fish increased significantly,but food intake decreased significantly,and it also displayed a significantly increased ChREBP mRNA expression levels in liver.At hour 4 post injection,hypothalamic ChREBP mRNA level was significantly increased,whereas the mRNA expression levels of appetite genes neuropeptide Y(npy)and agouti-related peptide(AgRP)were decreased significantly.When the glucose concentration was 40 mmol/L,the expression level of ChREBP mRNA in mandarin fish hypothalamic cells was significantly up-regulated,but the expression level of appetite gene npy mRNA was down-regulated.When siRNA and glucose were co-transfected into mandarin fish brain cells,the expression level of chrebp mRNA was significantly decreased,and the appetite gene npy mRNA was significantly increased.The results indicated that glucose regulated food intake through the modulation of appetite gene npy by ChREBP.

Inactivation of ERK1/2-CREB Pathway Is Implicated in MK801-induced Cognitive Impairment

Objective Schizophrenia(SZ)is associated with cognitive impairment,and it is known that the activity of cAMP response element binding protein(CREB)decreases in the brain of SZ patients.The previous study conducted by the investigators revealed that the upregulation of CREB improves the MK801-related SZ cognitive deficit.The present study further investigates the mechanism on how CREB deficiency is associated with SZ-related cognitive impairment.Methods MK-801 was used to induce SZ in rats.Western blotting and immunofluorescence were performed to investigate CREB and the CREB-related pathway implicated in MK801 rats.The long-term potentiation and behavioral tests were performed to assess the synaptic plasticity and cognitive impairment,respectively.Results The phosphorylation of CREB at Ser133 decreased in the hippocampus of SZ rats.Interestingly,among the upstream kinases of CREB,merely ERK1/2 was downregulated,while CaMKII and PKA remained unchanged in the brain of MK801-related SZ rats.The inhibition of ERK1/2 by PD98059 reduced the phosphorylation of CREB-Ser133,and induced synaptic dysfunction in primary hippocampal neurons.Conversely,the activation of CREB attenuated the ERK1/2 inhibitor-induced synaptic and cognitive impairment.Conclusion These present findings partially suggest that the deficiency of the ERK1/2-CREB pathway is involved in MK801-related SZ cognitive impairment.The activation of the ERK1/2-CREB pathway may be therapeutically useful for treating SZ cognitive deficits.

Involvement of chromatin and histone acetylation in the regulation of HIV-LTR by thyroid hormone receptor

The HIV-1 LTR controls the expression of HIV-1 viral genes and thus is critical for viral propagation and pathology. Numerous host factors have been shown to participate in the regulation of the LTR promoter. Among them is the thyroid hormone (T3) receptor (TR). TR has been shown to bind to the critical region of the promoter that contain the NFbB and Sp1 binding sites. Interestingly, earlier transient transfection studies in tissue culture cells have yielded contradicting conclusions on the role of TR in LTR regulation, likely due to the use of different cell types and/or lack of proper chromatin organization. Here, using the frog oocyte as a model system that allows replication-coupled chromatin assembly, mimicking that in somatic cells, we demonstrate that unliganded heterodimers of TR and RXR (9-cis retinoic acid receptor) repress LTR while the addition of T3 relieves the repression and further activates the promoter. More importantly, we show that chromatin and unliganded TR/RXR synergize to repress the promoter in a histone deacetylase-dependent manner.

Improvement of Copper-inducible Gene Expression System for Plant

The copper-regulated gene expression system has been developed to control spacial and temporal expression of transgene in plant. It comprises two parts: (1) ace I gene encoding copper-responsive transcription factor under the control of a constitutive or organ-specific promoter, and (2) a gene of interest under the control of a chimeric promoter consisting of the CaMV 35S (-90 to +8) promoter linked to the metal responsive element (MRE) carrying activating copper-metallothionein expression (ACE1)-binding sites. Here, the effectiveness of two different ACE1-binding cis -elements which derive from 5'-regulatory region of yeast metallothionein gene was investigated in transgenic tobacco (Nicotiana tabacum L. cv. W38). The results revealed that the MRE (-210 to -126) could increase the system inducibility by 50% - 100% compared with the previously reported MRE (-148 to -105). It is potential to use the copper-inducible system to control valuable gene traits in plant biotechnology.

Sulforaphane protects liver injury induced by intestinal ischemia reperfusion through Nrf2-ARE pathway

AIM: To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antiox-idant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R). METHODS: Rats were divided randomly into four ex-perimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfu-sion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the op-eration. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis.RESULTS: Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a signif icant increase in serum AST and ALT levels (AST: 260.13 ± 40.17 U/L vs 186.00 ± 24.21 U/L, P < 0.01; ALT: 139.63 ± 11.35 U/L vs 48.38 ± 10.73 U/L, P < 0.01), all of which were reduced by pretreatment with SFN, respectively (AST: 260.13 ± 40.17 U/L vs 216.63 ± 22.65 U/L, P < 0.05; ALT: 139.63 ± 11.35 U/L vs 97.63 ± 15.56 U/L, P < 0.01). The activity of SOD in the liver tissue decreased after intestinal I/R (P < 0.01), which was enhanced by SFN pretreatment (P < 0.05). In ad-dition, compared with the control group, SFN markedly reduced liver tissue MPO activity (P < 0.05) and elevat-ed liver tissue GSH and GSH-Px activity (P < 0.05, P < 0.05), which was in parallel with the increased level of liver Nrf2 and HO-1 expression.CONCLUSION: SFN pretreatment attenuates liver injury induced by intestinal I/R in rats, attributable to the antioxidant effect through Nrf2-ARE pathway.

Antidepressant effect of electroacupuncture regulates signal targeting in the brain and increases brain- derived neurotrophic factor levels

Electroacupuncture improves depressive behavior faster and with fewer adverse effects than antidepressant medication. However, the antidepressant mechanism of electroacupuncture remains poorly understood. Here, we established a rat model of chronic unpredicted mild stress, and then treated these rats with electroacupuncture at Yintang （EX-HN3） and Baihui （DU20） with sparse waves at 2 Hz and 0.6 mA for 30 minutes, once a day. We found increased horizontal and vertical activity, and decreased immobility time, at 2 and 4 weeks after treatment. Moreover, levels of neurotransmitters （5-hydroxytryptamine, glutamate, and y-aminobutyric acid） and protein levels of brain-derived neurotrophic factor and brain-derived neurotrophic factor-related proteins （TrkB, protein kinase A, and phosphorylation of cyclic adenosine monophosphate response element binding protein） were increased in the hippocampus. Similarly, protein kinase A and TrkB mRNA levels were increased, and calcium-calmodulin-dependent protein kinase lI levels decreased. These findings suggest that electroacupuncture increases phosphorylation of cyclic adenosine monophosphate response element binding protein and brain-derived neurotrophic factor levels by regulating multiple targets in the cyclic adenosine rnonophosphate response element binding protein signal- ing pathway, thereby promoting nerve regeneration, and exerting an antidepressive effect.

Moxibustion eases chronic inflammatory visceral pain through regulating MEK, ERK and CREB in rats

AIM To investigate the effects of herb-partitioned moxibustion(HPM) on phosphorylation of mitogen-activated extracellular signal-regulated kinase(MEK)1, extracellular signal-regulated kinase(ERK)1/2 and c AMP response element binding protein(CREB) in spinal cord of rats with chronic inflammatory visceral pain(CIVP), and to explore the central mechanism of HPM in treating CIVP.METHODS Male Sprague-Dawley rats were randomized into normal, model, HPM, sham-HPM, MEK-inhibitor and dimethyl sulfoxide(DMSO) groups. The CIVP model was established using an enema mixture of trinitrobenzene sulfonic acid and ethanol. HPM was applied at bilateral Tianshu(ST25) and Qihai(CV6) acupoints in the HPM group, while in the sham-HPM group, moxa cones and herb cakes were only placed on the same points but not ignited. The MEK-inhibitor and DMSO groups received L5-L6 intrathecal injection of U0126 and 30% DMSO, respectively. Abdominal withdrawal reflex(AWR), mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) were applied for the assessment of pain behavior. The colonic tissue was observed under an optical microscope after hematoxylin-eosin staining. Expression of phosphor(p)MEK1, p ERK1/2 and p CREB in rat spinal cord was detected using Western blotting. The levels of MEK, ERK and CREB m RNA in rat spinal cord were detected using real-time polymerase chain reaction. RESULTS Compared with the normal group, the AWR scores were increased significantly(P < 0.01) and the MWT and TWL scores were decreased significantly(P < 0.05) in the model, sham-HPM and DMSO groups. Compared with the model group, the AWR scores were decreased significantly(P < 0.01) and the MWT and TWL scores were increased significantly in the HPM and MEK-inhibitor groups(P < 0.05). Compared with the sham-HPM and DMSO groups, the AWR scores were decreased significantly(P < 0.01) and the MWT and TWL scores were increased significantly(P < 0.05) in the HPM and MEK-inhibitor groups. Compared with the normal group, the expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were increased significantly in the model, sham-HPM and DMSO groups(P < 0.01 or < 0.05). Compared with the model group, the expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups(P < 0.01 or < 0.05). Compared with the sham-HPM and DMSO groups, expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups(P < 0.01 or < 0.05). CONCLUSION HPM down-regulates protein phosphorylation of MEK1, ERK1/2 and CREB, and m RNA expression of MEK, ERK and CREB, inhibiting activation of the MEK/ERK/CREB signaling pathway in the spinal cord of CIVP rats, which is possibly a critical central mechanism of the analgesic effect of HPM.

Shuganjieyu capsule increases neurotrophic factor expression in a rat model of depression

Shuganjieyu capsule has been approved for clinical treatment by the State Food and Drug Ad-ministration of China since 2008. In the clinic, Shuganjieyu capsule is often used to treat mild to moderate depression. In the rat model of depression established in this study, Shuganjieyu capsule was administered intragastrically daily before stress. Behavioral results conifrmed that depressive symptoms lessened after treatment with high-dose （150 mg/kg） Shuganjieyu capsule. Immunohistochemistry results showed that high-dose Shuganjieyu capsule signiifcantly increased phosphorylation levels of phosphorylation cyclic adenosine monophosphate response element binding protein and brain-derived neurotrophic factor expression in the medial prefrontal cortex and hippocampal CA3 area. Overall, our results suggest that in rats, Shuganjieyu capsule effec-tively reverses depressive-like behaviors by increasing expression levels of neurotrophic factors in the brain.

Changes of CREB in rat hippocampus, prefrontal cortex and nucleus accumbens during three phases of morphine induced conditioned place preference in rats

Objective: To investigate the changes in CREB (cAMP response element binding protein) in hippocampus, PFC (prefrontal cortex) and NAc (nucleus accumbens) during three phases of morphine induced CPP (conditioned place preference) in rats, and to elucidate the role of CREB during the progress of conditioned place preference. Methods: Morphine induced CPP acquisition, extinction and drug primed reinstatement model was established, and CREB expression in each brain area was measured by Western Blot methods. Results: Eight alternating injections of morphine (10 mg/kg) induced CPP, and 8 d saline extinction training that extinguished CPP. CPP was reinstated following a priming injection of morphine (2.5 mg/kg). During the phases of CPP acquisition and reinstatement, the level of CREB expression was significantly changed in different brain areas. Conclusion: It was proved that CPP model can be used as an effective tool to investigate the mechanisms underlying drug-induced reinstatement of drug seeking after extinction, and that morphine induced CPP and drug primed reinstatement may involve acti-vation of the transcription factor CREB in several brain areas, suggesting that the CREB and its target gene regulation pathway may mediate the basic mechanism underlying opioid dependence and its drug seeking behavior.

Pachymic Acid Ameliorates Pulmonary Hypertension by Regulating Nrf2-Keap1-ARE Pathway

Objective:Pulmonary hypertension(PH)is a severe pulmonary vascular disease that eventually leads to right ventricular failure and death.The purpose of this study was to investigate the mechanism by which pachymic acid(PA)pretreatment affects PH and pulmonary vascular remodeling in rats.Methods:PH was induced via hypoxia exposure and administration of PA(5 mg/kg per day)in male Sprague-Dawley rats.Hemodynamic parameters were measured using a right ventricular floating catheter and pulmonary vascular morphometry was measured by hematoxylin-eosin(HE),a-SMA and Masson staining.MTT assays and EdU staining were used to detect cell proliferation,and apoptosis was analyzed by TUNEL staining.Western blotting and immunohistochemistry were used to detect the expression of proteins related to the Nrf2-Keapl-ARE pathway.