Taurochenodeoxycholic acid (TCDCA) is one of the main effective components of bile acid, playing critical roles in apoptosis and immune responses through the TGR5 receptor. In this study, we reveal the interaction between TCDCA and TGR5 receptor in TGR5-knockdown H1299 cells and the regulation of inflammation via the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding (CREB) signal pathway in NR8383 macrophages. In TGR5-knockdown H1299 cells, TCDCA significantly activated cAMP level via TGR5 receptor, indicating TCDCA can bind to TGR5; in NR8383 macrophages TCDCA increased cAMP content compared to treatment with the adenylate cyclase (AC) inhibitor SQ22536. Moreover, activated cAMP can significantly enhance gene expression and protein levels of its downstream proteins PKA and CREB compared with groups of inhibitors. Additionally, TCDCA decreased tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-12 through nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activity. PKA and CREB are primary regulators of anti-inflammatory and immune response. Our results thus demonstrate TCDCA plays an essential anti-inflammatory role via the signaling pathway of cAMP-PKA-CREB induced by TGR5 receptor.
Animals ; Cell Line ; Cyclic AMP/metabolism* ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Cytokines/metabolism* ; Humans ; Inflammation ; Macrophages ; Rats ; Receptors, G-Protein-Coupled/metabolism* ; Signal Transduction/drug effects* ; Taurochenodeoxycholic Acid/pharmacology*

OBJECTIVE: To observe the direct intervention effects of electroacupuncture (EA) and non-steroid anti-inflammatory drugs (NSAIDs) on pain memory, and to explore their effects on cAMP/PKA/cAMP pathway in anterior cingulate gyrus (ACC). METHODS: Fifty clean healthy male SD rats were randomly divided into a control group, a model group, an indomethacin group, an EA group and a sham EA group, 10 rats in each group. Except the control group, the pain memory model was established in the remaining four groups by twice injection of carrageenan at foot; 0.1 mL of 2%λ-carrageenan was subcutaneously injected at the left foot of rats; 14 days later, when the pain threshold of rats of each group returned to the basic level, the second injection was performed with the same procedure. The rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36) for 30 min; the rats in the indomethacin group was treated with indomethacin intragastric administration with the dose of 3 mg/kg; the rats in the sham EA group was treated with EA without electricity at the point 0.3 mm forward "Zusanli" (ST 36) with the depth of 2 mm for 30 min; the rats in the control group was not given any invention. All the above interventions were performed 5 h, 1 d, 2 d and 3 d after the second injection of 2% λ-carrageenan. The left-side paw withdrawal thresholds (PWT) were observed before the first injection, 4 h, 3 d, 5 d after the first injection, before the second injection and 4 h, 1 d, 2 d, 3 d after the second injection. Three days after the second injection, the number of positive cells of cAMP, p-PKA, p-CREB and the number of positive cells of protein co-expression in the right ACC brain area were detected by immunofluorescence, and the relative protein expression of p-PKA and p-CREB were detected by Western blot. RESULTS: Compared with the control group, the PWTs in the model group decreased significantly 4 h, 3 d and 5 d after the first injection and 1 d, 2 d and 3 d after the second injection (<0.05); compared with the control group, the positive expression of cAMP, p-PKA and p-CREB in the right ACC brain area in the model group increased significantly (<0.05), and the number of positive cells of the co-expression of cAMP/p-PKA and p-PKA/p-CREB also increased significantly (<0.05). Compared with the model group, indomethacin group and sham EA group, the PWTs in the EA group were increased significantly 1 d, 2 d and 3 d after the second injection (<0.05); compared with the model group, indomethacin group and sham EA group, the positive expression of p-PKA and p-CREB in the right ACC brain area in the EA group decreased significantly (<0.05), and the number of positive cells of co-expression of cAMP/p-PKA and p-PKA/p-CREB was decreased significantly (<0.05). Compared with the model group and sham EA group, the positive expression of cAMP in the right ACC brain area was decreased in the EA group (<0.05). CONCLUSION EA have a direct intervention effect on pain memory, which have significant advantage over NSAIDs in the treatment of chronic pain. The advantage effect of EA on pain memory may be related to the inhibition of cAMP/PKA/CREB pathway in ACC area.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; therapeutic use ; Cyclic AMP ; metabolism ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Electroacupuncture ; Gyrus Cinguli ; metabolism ; Male ; Pain Threshold ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

OBJECTIVETo search for an appropriate animal model that is more closely related to human to study cAMP-response element binding protein target gene Staufen and to identify its location.

METHODSThe phylogenetic tree was constructed with Staufen protein (STAUFEN) sequences of different species, and the most suitable animal model was selected by analyzing relativity among them. The Staufen fragments were amplified with reverse transcription-PCR and inserted into a vector and then the sub-clone was transformed into bacteria, selected, amplified, extracted and sequenced. Staufen probes were in vitro transcribed and hybridized in situ on the cryosections of the mouse brain. The cryosections were stained and observed.

RESULTSThe clustering patterns of the phylogenetic tree indicated that the mouse and human Staufen1 had 99.7% protein sequences similarity. The mRNA of Staufen was located in CA1, CA2, CA3 and DG hippocampus regions shown by in situ hybridization.

CONCLUSIONThe mouse is a preferable animal model for research on Staufen transcription in hippocampus.

Animals ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hippocampus ; metabolism ; In Situ Hybridization ; Mice ; RNA, Messenger ; genetics ; RNA-Binding Proteins ; genetics ; metabolism

OBJECTIVE: To explore the role of phosphorylation of cAMP response element binding protein (CREB) in the incision-induced pain hypersensitivity. METHODS: A longitudinal incision was made in one plantar hind paw of isoflurane-anesthetized rats. Spinal cords were removed at various postoperative time after behavior test. Phosphorylation of CREB was determined by immunohistochemistry and double-labeling immunofluorescence. Morphine and gabapentin were intraperitoneally injected before the behavior test and were used to determine the interaction between phosphorylation of CREB and morphine and gabapentin. RESULTS: After the hind-paw incision, phosphorylation of CREB was enhanced in the ipsilateral lumbar spinal cord (P<0.05). The enhancement of p-CREB was mainly in the neurons in the dorsal horn of the spinal cord. All these were shown by double-labeling technique and p-CREB was mainly in the neurons. Intraperitoneal injection of morphine prevented the increased phosphorylation of CREB in the spinal cord and inhibited the mechanical allodynia induced by the incision (P>0.05). Gabapentin didn't inhibit the phosphorylation of CREB (P<0.05) but partly inhibited the mechanical allodynia. CONCLUSION Incision induces the phosphorylation of CREB in the spinal cord, and the increase of p-CREB is mainly in the neurons. Phosphorylation of CREB in the spinal cord contributes to the pain hypersensitivity induced by surgical incision.
Animals ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Female ; Foot ; surgery ; Neurons ; metabolism ; Pain Threshold ; Pain, Postoperative ; metabolism ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism

Trichomonas vaginalis is a flagellated lumen-dwelling extracellular protozoan parasite that causes human trichomoniasis via sexual intercourse. Human neutrophils play a crucial role in acute tissue inflammatory responses in T. vaginalis infection. In this study, we investigated the signaling mechanism of neutrophil responses when stimulated with T. vaginalis-derived secretory products (TvSP), which were collected from 1x10(7) live trichomonads. Incubation of human neutrophils isolated from peripheral blood with TvSP induced up-regulation of IL-8 protein secretion. In addition, stimulation with TvSP induced phosphorylation of NF-kappaB and CREB in neutrophils. Moreover, TvSP-induced IL-8 production was also significantly inhibited by pretreatment of neutrophils with ikappaB inhibitor or CREB inhibitor. These results suggest that transcription factors NF-kappaB and CREB are involved in IL-8 production in human neutrophils induced by stimulation with T. vaginalis infection.
Cyclic AMP Response Element-Binding Protein/*metabolism ; Human Experimentation ; Humans ; Interleukin-8/*metabolism ; Male ; NF-kappa B/*metabolism ; Neutrophils/*immunology ; Phosphorylation ; Trichomonas vaginalis/*immunology

OBJECTIVETo investigate the effects of enriched environment and impoverished environment on the learning and memory ability of manganese-exposed mice and the mechanism.

METHODSForty female Kunming mice were randomly and equally divided into 4 group: control group (CG), standard environment and manganese exposure group (SEG), enriched environment and manganese exposure group (EEG), and impoverished environment and manganese exposure group (IEG). The mouse model of manganese poisoning was established by intraperitoneal injection of manganese chloride. The learning and memory ability was tested by Morris water maze. The expression of cAMP response element-binding protein (CREB) in area CA1 of the hippocampus was measured by immunohistochemistry.

RESULTSIn place navigation test, the SEG had a significantly longer escape latency than the CG (P < 0.05), and the EEG had a significantly shorter escape latency than the SEG (P < 0.05); there was no significant difference in escape latency between IEG and SEG (P > 0.05). In spatial probe test, the EEG had a significantly greater number of platform crossings than the SEG (P < 0.05), and the IEG had a significantly smaller number of platform crossings than the SEG (P < 0.05). The expression of CREB in area CA1 of the hippocampus was significantly lower in IEG and SEG than in CG (P < 0.05), and it was significantly higher in EEG than in SEG (P < 0.05).

CONCLUSIONIn the enriched environment, the learning and memory ability of manganese-exposed mice can be improved, which may be due to the increased expression of CREB in the hippocampus.

Animals ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Disease Models, Animal ; Environment ; Female ; Hippocampus ; drug effects ; metabolism ; Learning ; drug effects ; Manganese Poisoning ; metabolism ; Memory ; drug effects ; Mice

OBJECTIVE: To observe the effect of chronic unpredicted sequence of mild stress on the expression of cAMP-dependent protein kinase A(PKA) and phosphorylated cAMP-responsive element binding protein (P-CREB) in hippocampus of rats and the antagonism of antidepressors (fluoxetine). METHODS: Thirty-six male Sprague Dawley rats were randomly and equally allocated to 3 groups: A normal control group, a model group, and a fluoxetine group. All rats except the control group were singly housed and exposed to an unpredicted sequence of mild stressors. The different distribution and expression of PKA and P-CREB in the hippocampus of rats in different groups were investigated with immunohistochemistry and Westernblot technique. RESULTS: The positive PKA and P-CREB cells in the hippocampus of normal controls were the pyramidal cells and the granule cells. The PKA and P-CREB protein expression levels in the hippocampus of model rats were significantly lower than those of the normal controls (P<0.05). The PKA and P-CREB protein expression levels in the hippocampus of the fluoxetine group were significantly higher than those of the model group (P<0.05). CONCLUSION Chronic unpredicted mild stress can affect the PKA and P-CREB expression in hippocampus of rats and fluoxetine has antagonism against it.
Animals ; Antidepressive Agents, Second-Generation ; antagonists & inhibitors ; Cyclic AMP Response Element-Binding Protein ; biosynthesis ; genetics ; Cyclic AMP-Dependent Protein Kinases ; biosynthesis ; genetics ; Depression ; etiology ; metabolism ; Fluoxetine ; antagonists & inhibitors ; Hippocampus ; metabolism ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological ; metabolism

With the deepening research on pathogenesis of depression, the focus has diverted from the mechanism of regulating monoamines to the basic pathophysiology of depression and the long-term mechanism of antidepressant treatments. cAMP response element binding protein (CREB) in the brain, especially in the hippocampi, as a converging agent of many intracellular signaling transduction pathways is getting increasing attention. To better understand the basic pathophysiology of depression and the long-term mechanism of antidepressant treatments, it is significant to make clear the correlation between hippocampal CREB and antidepressant treatments. This review mainly refers to the formation of CREB and its distribution in hippocampi, the upstream signaling transduction pathways of hippocampal CREB and antidepressant treatments, and the possible antidepressant mechanisms by regulating hippocampal CREB.
Animals ; Antidepressive Agents ; therapeutic use ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Depression ; drug therapy ; physiopathology ; Hippocampus ; drug effects ; metabolism ; Humans ; Signal Transduction

OBJECTIVETo investigate the effect of acute blood loss on postoperative cognitive function of aged spontaneously hypertensive rats (SHRs).

METHODSForty aged male SHRs were randomized into sham hemorrhage group (group A, n=13), 20% hemorrhage group (group B, n=13) and 40% hemorrhage group (group C, n=14). The rats were subjected to acute loss from the femoral artery and subsequent fluid replacement with lactated Ringer's Solution (3 folds of the blood loss volume). All the rats underwent Morris water maze test to assess the visuospatial memory and learning ability, and were then decapitated to observe the pathological changes of the hippocampus.

RESULTSThe latency of reference memory in group C was significantly prolonged after the operation (P=0.002), but the working memory and learning capacity showed no significant differences between the 3 groups. Immunochemistry did not reveal significant differences in p-CREB expressions in the hippocampal CA1 region among the groups, but volume reduction of some neurons was noted in the CA1 region in group C.

CONCLUSIONVarying degrees of acute hemorrhage can result in different effect on postoperative cognition in aged SHR. Acute hemodilutional anemia to 40% of baseline can cause reference memory impairment with cell volume reduction of the neurons in the hippocampal CA1 region but does not affect the working memory and learning capacity or p-CREB expression.

Animals ; CA1 Region, Hippocampal ; cytology ; metabolism ; Cognition ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hemorrhage ; physiopathology ; Male ; Maze Learning ; Memory ; Neurons ; pathology ; Rats ; Rats, Inbred SHR

OBJECTIVETo investigate the effects of cAMP-response element binding protein-1 (CREB-1) on transforming growth factor-b3 (TGF b3) mRNA expression and promoter activity in hepatic stellate cells (HSCs).

METHODSFreshly isolated HSCs from rats were divided into six groups: CREB-1 expression plasmid transfected group (C), siRNA-CREB-1 plasmid transfected group (S), negative control group (N), forskolin treated group (F), H-89 treated group (H), and blank group (B). Rats in each group were further sub-divided according to whether (+) or not (-) they were exposed to exogenous TGF b3. TGF b3 mRNA expression was measured by real time quantitative PCR. HSCs of the C, S, N, F, H and B groups were transfected with the TGF b3 promoter luciferase reporter plasmid (PGL3-TGF b3-P; W group), the TGF b3 promoter luciferase reporter plasmid with CRE mutation (PGL3-basic-TGF b3P-mCRE; M group) and the renilla luciferase control plasmid (pRL-SV40; control group). TGF b3 promoter activity was assessed by luciferase reporter assays.

RESULTSCompared to N(-), the TGF b3 mRNA expression was reduced to 0.69+/-0.15 in S(-) (P less than 0.05) and increased to 4.68+/-2.76 in C(-) (P more than 0.05). Compared to B(-), the TGF b3 mRNA expression was reduced to 0.57+/-0.08 in H(-) (P less than 0.05). The differences between N(+) and N(-), S(+) and S(-), B(+) and B(-), and H(+) and H(-) were all significant (P less than 0.05). The values of TGF b3 promoter activity in S(W), N(W), and C(W) were 0.062+/-0.013, 0.122+/-0.011, and 0.165+/-0.016 (P less than 0.05), but the changes of TGF b3 promoter activity in S(M), N(M), and C(M) were not significant (P more than 0.05). The values of TGF b3 promoter activity in H(W), B(W), and F(W) were 0.154+/-0.010, 0.188+/-0.016, and 0.276+/-0.031 (P less than 0.05), but the changes of TGF b3 promoter activity in H(M), B(M), and F(M) were not significant (P more than 0.05).

CONCLUSIONIncreased levels of CREB-1 mRNA or p-CREB-1 up-regulate the TGF b3 mRNA expression and promoter activity in rat HSCs. The CRE site in the TGF b3 promoter is critical for this effect, and the gene's activity becomes significantly decreased when the site is missing. Exogenous TGF b3 enhances expression of endogenous TGF b3 in rat HSCs.

Animals ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hepatic Stellate Cells ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Rats ; Transforming Growth Factor beta3 ; genetics