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Objective To investigate the analgesic effect and mechanism of Buyang Huanwu Decoction on diabetic peripheral neuropathy(DPN)rats.Methods Sixty rats were divided into normal group,model group,low-,medium-and high-dose groups of Chinese medicine,and high-dose + H-89[protein kinase A(PKA)inhibitor]group,with 10 rats in each group.Except for the normal group,rats in all other groups were fed with high-fat and high-sugar chow combined with intraperitoneal injection of streptozotocin(STZ)method to construct DPN model.At the end of drug administration,the foot thermal pain threshold of rats was detected,the motor nerve conduction velocity(MNCV)and sensory nerve conduction velocity(SNCV)of rats was measured,the intraepidermal nerve fiber(IENF)in the epidermis was observed by immunohistochemistry,and serum fasting insulin(FINS),total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),insulin resistance index(HOMA-IR),and the interleukin(IL)-1β,IL-6,tumor necrosis factor α(TNF-α),vascular endothelial growth factor(VEGF),angiopoietin 1(Ang-1),CD34 levels,cyclic adenosine monophosphate(cAMP)concentration in the sciatic nerve tissues were detected by enzyme-linked immunosorbent assay(ELISA),and Western Blot assay to detect the PKA and the carbohydrate responsive element binding(CREB)in the sciatic nerve tissues.Results Compared with the normal group,foot thermal pain threshold,TC,TG,LDL-C,HOMA-IR,IL-1β,IL-6 and TNF-α levels were significantly increased in the model group(P＜0.05),HDL-C,FINS,VEGF,Ang-1,CD34,IENF,MNCV and SNCV values,cAMP concentration levels,PKA and CREB phosphorylation levels were significantly reduced(P＜0.05).Compared with the model group,the above indexes were significantly improved in the low-,medium-and high-dose groups of Chinese medicine(P＜0.05)in a dose-dependent manner.Compared with the Chinese medicine high-dose + H-89 group,all the indexes were reversed in the Chinese medicine high-dose group.Conclusion Buyang Huanwu Decoction can improve insulin resistance and lipid metabolism,reduce limb pain,improve local microcirculation disorder,and protect nerve function in DPN rats,which reflects the therapeutic characteristics of"activating blood circulation and relieving pain".The pain-relieving effect of Buyang Huanwu Decoction may be related to the improvement of local microcirculation,inhibition of inflammatory factor release and regulation of cAMP/PKA/CREB signaling pathway protein expression.
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Objective:To explore the therapeutic effect and mechanism of Chaihu Yueju decoction on model rats induced by chronic unpredictable mild stress （CUMS）. Method:The 60 SD rats were randomly divided into normal group， model group， Chaihu Yueju decoction low， medium and high-dose groups（0.3， 0.6， 1.2 g·kg-1） and fluoxetine hydrochloride group（0.2 mg·kg-1）， 10 for each group. The model rats was established by chronic unpredictable mild stress for 5 weeks， and then Chaihu Yueju decoction and fluoxetine hydrochloride were given to the corresponding treatment group by gavage for 3 weeks. In the last week of gavage， Morris water maze training and testing were conducted. After the last day of gavage， sugar water preference and other behavioral experiment were tested. The sugar water preference test was used to detect the degree of pleasure deficiency in rats before and after treatment， the open field test was used to detect the depression of rats before and after treatment， the spatial memory ability was tested by Morris water maze. Western blot was used to detect the levels of tumor necrosis factor-α（TNF-α）， 5-hydroxytryptamine receptor 1A （5-HT1A）， extracellular signal-regulated kinase （ERK） and synapse associated protein in hippocampus of each group， Gloji and Nissl staining was used to observe the changes of dendritic spines and Nissl bodies in the hippocampus CA3. Result:Compared with normal group， the weight， sugar water preference rate， the scores of horizontal and vertical movement were significantly reduced （P<0.05）， and the immobility time was significantly prolonged in model group （P<0.05）. Meanwhile， the ability of learning and memory in model group decreased （P<0.05）. Western blot results showed that the levels of TNF-α was significantly increased （P<0.05）， and the levels of 5-HT1A， p-ERK， cyclic adenosine phosphate reactive element binding protein （CREB）， p-CREB， Synapsin-1，Synaptophysin， glutamate receptor subtype-1（GluR-1）and postsynaptic membrane protein-95（PSD-95） in model group were significantly decreased （P<0.05）. Gloji and Nissl staining results showed that the density of dendritic spines and the number of Nissl body in the hippocampal CA3 of the model group was obviously attenuate （P<0.05）. The results show that the neurons were evidently damaged. Compared with model group， the weight， sugar water preference rate， the scores of horizontal and vertical movement were clearly increased （P<0.05） and the immobility time was significantly shortened in fluoxetine hydrochloride group， middle dose and high dose Chaihu Yueju decoction group （P<0.05）. Western blot results showed that the levels of synaptophysin， GluR-1 and PSD-95 were significantly increased in fluoxetine hydrochloride group， middle dose and high dose Chaihu Yueju decoction group. The level of TNF-α was significantly decreased （P<0.05）， the levels of 5-HT1A， p-ERK， CREB， p-CREB and Synapsin-1 were remarkably increased （P<0.05）， especially the high dose group of Chaihu Yueju decoction. Gloji and Nissl staining results showed that the density of dendritic spines and the number of Nissl body in the hippocampal CA3 of high dose group were similarly increased in a dose-dependent manner. Conclusion:Chaihu Yueju decoction could improves the weight， the depressive despair， autonomous activity ability and learning and memory ability of DP model rats. Its mechanism is closely related to attenuating the inflammatory reaction and enhancing the levels of 5-HT1A receptor protein， ERK and synapse related protein， then activating 5-HT/CREB and ERK/CREB signaling pathways， increasing the number and distribution of dendritic spines and repairing damaged neurons in the DP model rat's hippocampus.
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Objective @#This study aimed to investigate the effect of cAMP-responsive-element-binding protein (CREB) overexpression on the differentiation of human stem cells from the apical papilla (hSCAPs), stimulated by transforming growth factor- beta (TGF-β1). @*Methods@#Cells were isolated from human immature third molars via enzymatic digestion. Four experimental groups were set up: ①a control group, receiving normal mineralization inducer (α-MEM, 10% FBS, 10 mmol/L β-sodium glycerophosphate, 50 μg/mL vitamin C, 10 nmol/L dexamethasone); ② a TGF-β1 group, receiving normal mineralization inducer and 5 μg/mL TGF-β1; ③ a TGF-β1+LV-empty group, receiving normal mineralization inducer and the transfected empty virus vector with 5 μg/mL TGF-β1; and ④ a TGF-β1+ov-CREB group, receiving normal mineralization inducer and the transfected CREB-overexpressing viral vector, with 5 μg/mL TGF-β1. The transfected cells were cultured in odontogenic medium in the presence or absence of TGF-β1 for 2 weeks. Alizarin red staining was used to detect mineralized nodules, and the mRNA expression of the mineralization genes runt-related transcription factor 2 (RUNX2), dentin sialophosphoprotein (DSPP) and alkaline phosphatase (ALP) was measured by qPCR. @*Results @#Compared with the control group (1.12 ± 0.11), TGF-β1 inhibited the deposition of calcium minerals (0.67 ± 0.12) (P < 0.05) via hSCAPs and inhibited the mRNA expression of RUNX2 (0.60 ± 0.03), DSPP (0.43 ± 0.12) and ALP (0.69 ± 0.05) (P < 0.05). In contrast, overexpression of CREB attenuated the effect of TGF-β1 on hSCAPs, resulting in the development of a high number of mineralized nodules (1.27 ± 0.10) (P < 0.01) and increased RNA levels of RUNX2 (1.33 ± 0.07), DSPP (1.32 ± 0.11) and ALP (1.26 ± 0.03) (P<0.05) compared with those in the TGF-β1 group. @*Conclusion@#Overexpressed CREB promotes odontogenic differentiation of hSCAPs by interfering with TGF-β1.
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Objective To investigate the changes of calmodulin kinase Ⅱ (CaMK Ⅱ) and cAMP responsive element binding protein(CREB) expressing in primary cultured hippocampal neurons and its relationship with learning and memory deficit after 2000 μW/cm2 electromagnetic radiation.Methods Primary cultured hippocampal neurons in vitro were randomly divided into normal control group,sham-radiated group,and 1 h/d,2 h/d,3 h/dradiation groups.The neurons in the radiation groups were received microwave exposure of 2000 μW/cm2.The change of CaMK Ⅱ and CREB protein in hippocampal neurons of each group of rats were measured with western blot,and the expression of CaMK Ⅱ and CREB mRNA in hippocampus were determined by RT-PCR.Results Compared with control group((0.78 ± 0.07),(0.62 ± 0.12)),the expression of CaMK Ⅱ protein (1 h/d(0.59 ±0.05),2h/d(0.44 ±0.08),3h/d(0.18 ±0.04)) and its mRNA(1h/d(0.41 ±0.08),2h/d(0.34 ±0.04),3h/d(0.24 ±0.02)) was obviously decreased (P＜0.05).Compared with control group((0.69 ±0.10),(0.80 ±0.12)),the expression of CREB protein(1h/d(0.49 ±0.05),2h/d(0.4 ±0.04),3h/d(0.17 ±0.03))and its mRNA (1 h/d (0.68 ± 0.11),2h/d (0.53 ± 0.08),3h/d (0.30 ± 0.03)) was obviously decreased after radiation(P＜0.05).Conclusion Electromagnetic radiation of 2000 μW/cm2 exposure could weaken the learning and memory abilities of rats and the decreases in the expression of CaMK Ⅱ and CREB protein and their mRNA in hippocampus may be involved in the pathophysiological process of learning and memory deficit.
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Objective To investigate the effect of ketamine on cAMP response element binding protein pbosphorylation(p-CREB)in hippocampus of neonatal rats.Methods Seventy-five 7-day old SD rats of both sexes were randomly divided into 3 groups(n=25 each):control group(group C)and 2 ketamlne groups(group K1,K2)which received 7 subcutaneous injections of ketamine 10 and 20 mg/kg respectively at 90 min intervals.The animsla were decapitated at 24 h after fwst ketamine injection.The brains were immediately removed and the hippocampi were isolated for detection of neuronal apoptosis by TUNEL.Apoptosis index wag calculated.The expression of p-CREB Wag meagured by immuno-histochemistry and the expression of BDNF mRNA and Bcl-2 mRNA was detected by RT-PCR.Cognitive function Wag agsessed using Morris water maze test at 6 weeks after first ketamine injection.Results The apoptosis index Wag significantly increased while the expression of CREB,BDNF mRNA and Bcl-2 mRNA was down-regulated in group K1 and K2 as compared with group C.The apoptosis index Wag significantly higher and the expression of p-CREB and BDNF mRNA and Bcl-2 mRNA Wag significantly lower in group K2 than in group K1.The latent period of escape was significantly longer in group K2 than in group C and K1.Conclusion Ketamine 20 mg/kg administered in neonatal rats can decrease cognitive function when they grow up by increasing neuronal apoptosis induced by down-regulatlon of the expression of p-CREB,BDNF and Bcl-2.
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Objective:To study the function and regulation of CREB signaling in rat hypothalamic neurons by exogeneous corticotropin-releasing hormone(CRH). Methods:Perifused primary cultures of rat hypothalamic neurons. Intracelluar free Ca2+ concentr ation([Ca2+]i) was assayed at 32℃ by fluorescence analysis,the coverslip was mounted in a Delta Scan spectrofluorimeter(Photon Technology International);and phospho-CREB by western blot. Results:The basal lower intracellular Ca2+ level of perifused primary cultures of rat hypothalamic neurons cultured on coverslipy was markedly increased by CRH,and significantly attenuated by application of nifedipine(Nif) or CP-154526. CRH may cause the increase of phospho-CREB in the neuron of hypothalamus,while CP-154526,Nif can significantly inhibit the production of phospho-CREB. Conclusion: CRH may excite hypothalamic neurons directly suggest that phospho-and[Ca2+]i,suggesting a role of CRH type 1 receptor in the activation of CRH gene expression.
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Objective To investigate the activation of extracellular signal-regulated kinase(ERK) and cAMP-responsive element binding protein(CREB) in the anterior cingulate cortex(ACC) induced by peripheral injection of formalin.Methods 5% formalin was injected subcutaneously into the unilateral hindpaw.The rats were perfused or killed and the ACC dissected at different time points.The activation of phosphorylated ERK(pERK) and phosphorylated CREB(pCREB) in the ACC were observed by immunohistochemical and Western blotting techniques.Results Subcutaneous injection of formalin induced the activation of pCREB and pERK in the ACC.Formalin-induced the activation of pERK and pCREB in the ACC peaked at 3 min and 30 min after stimulation,respectively.The total ERK and CREB had no significant changes.Conclusion The ACC could receive peripheral noxious information and the ERK and CREB in the ACC might involve in the processing of the primary unpleasentness of pain.
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AIM To investgate the changes in the expression of CREB and p-CREB in the cortex of ethanol dependent rats. On other hand, the effects of fluoxetine on these changes were also studied. METHODS Ethanol was administered in drinking water at the concentration of 6% V/V, for 28 d. The expression of CREB and p-CREB in the rat cortex were measured by immunohistochemistry methods. RESULTS It was found that chronic ethanol treatment and its withdrawal did not modify the expression of CREB in the rat cortex. However, the levels of p-CREB in the frontal and piriform cortex of rats were significantly decreased during ethanol withdrawal, and respectively decreased 57.28%?55.84% compared to control rats at 24 h after ethanol withdrawal. When fluoxetine (ip, 10 mg?kg -1) were administried concurrently with ethanol, it can significantly dampen alcohol withdrawal symptoms and antagonize the down-regulation of the phosphorylation of CREB in the frontal and piriform cortex of rats at 24 h after ethanol withdrawal, the expression of p-CREB protein respectively increased 292 58%,128 44% compared with rats intaking ethanol. CONCLUSION CREB may be one of the substance of the signal transduction process for ethanol dependence. Fluoxetine can reverse the decreased p-CREB levels in rat cortex induced by ethanol withdrawal, which may be the part reason of fluoxetine dampening alcohol withdrawal symptoms.