This study aims to investigate the effects of baicalein(BAI) on lipopolysaccharide(LPS)-induced human microglial clone 3(HMC3) cells, with a focus on suppressing inflammatory responses and elucidating the potential mechanism underlying the therapeutic effects of BAI on ischemic stroke via modulating the cAMP-PKA-NF-κB/CREB pathway. The findings have significant implications for the application of traditional Chinese medicine in treating cerebral ischemic diseases. First, the safe dosage of BAI was screened, and then an inflammation model was established with HMC3 cells by induction with LPS for 24 h. The cells were assigned into a control group, a model group, and high-, medium-, and low-dose(5, 2.5, and 1.25 μmol·L~(-1), respectively) BAI groups. The levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in cell extracts, as well as the levels of interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α), and cyclic adenosine monophosphate(cAMP) in the cell supernatant, were measured. Western blot was performed to determine the expression of protein kinase A(PKA), phosphorylated cAMP-response element binding protein(p-CREB), and nuclear factor-kappa B p65(NF-κB p65). Hoechst 33342/PI staining was employed to assess cell apoptosis. High and low doses of BAI were used for treatment in the research on the mechanism. The results revealed that BAI at the concentrations of 10 μmol·L~(-1) and below had no impact on normally cultured HMC3 cells. LPS induction at 200 ng·mL~(-1) for 24 h reduced the SOD activity and increased the MDA content in HMC3 cells. However, 5, 2.5, and 1.25 μmol·L~(-1) BAI significantly increased the SOD activity and 5 μmol·L~(-1) BAI significantly decreased the MDA content. In addition, BAI ameliorated the M1 polarization of HMC3 cells induced by LPS, as indicated by cellular morphology. The results of ELISA demonstrated that BAI significantly lowered the levels of TNF-α, IL-1β, IL-6, and cAMP in the cell supernatant. Western blot revealed that BAI up-regulated the protein levels of PKA and p-CREB while down-regulating the expression of NF-κB p65. Hoechst 33342/PI staining results indicated that BAI mitigated the apoptosis of HMC3 cells. Overall, the results indicated that BAI had protective effects on the HMC3 cells induced by LPS, and could inhi-bit inflammatory response and improve cell apoptosis, which might be related to the regulation of the cAMP-PKA-NF-κB/CREB pathway.
Humans ; NF-kappa B/metabolism* ; Microglia ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Lipopolysaccharides/pharmacology* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Superoxide Dismutase/metabolism*

BACKGROUND: Anterior thalamic nuclei (ATN) deep brain stimulation (DBS) is an effective method of controlling epilepsy, especially temporal lobe epilepsy. Mossy fiber sprouting (MFS) plays an indispensable role in the pathogenesis and progression of epilepsy, but the effect of ATN-DBS on MFS in the chronic stage of epilepsy and the potential underlying mechanisms are unknown. This study aimed to investigate the effect of ATN-DBS on MFS, as well as potential signaling pathways by a kainic acid (KA)-induced epileptic model. METHODS: Twenty-four rhesus monkeys were randomly assigned to control, epilepsy (EP), EP-sham-DBS, and EP-DBS groups. KA was injected to establish the chronic epileptic model. The left ATN was implanted with a DBS lead and stimulated for 8 weeks. Enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining were used to evaluate MFS and levels of potential molecular mediators in the hippocampus. One-way analysis of variance, followed by the Tukey post hoc correction, was used to analyze the statistical significance of differences among multiple groups. RESULTS: ATN-DBS is found to significantly reduce seizure frequency in the chronic stage of epilepsy. The number of ectopic granule cells was reduced in monkeys that received ATN stimulation (P < 0.0001). Levels of 3',5'-cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in the hippocampus, together with Akt phosphorylation, were noticeably reduced in monkeys that received ATN stimulation (P = 0.0030 and P = 0.0001, respectively). ATN-DBS also significantly reduced MFS scores in the hippocampal dentate gyrus and CA3 sub-regions (all P < 0.0001). CONCLUSION ATN-DBS is shown to down-regulate the cAMP/PKA signaling pathway and Akt phosphorylation and to reduce the number of ectopic granule cells, which may be associated with the reduced MFS in chronic epilepsy. The study provides further insights into the mechanism by which ATN-DBS reduces epileptic seizures.
Adenosine Monophosphate ; Anterior Thalamic Nuclei ; Cyclic AMP-Dependent Protein Kinases ; Deep Brain Stimulation ; Epilepsy/therapy* ; Epilepsy, Temporal Lobe/therapy* ; Hippocampus ; Humans ; Mossy Fibers, Hippocampal ; Signal Transduction

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

OBJECTIVE: To investigate the effect of protein kinase A (PKA) activation on aggregation funetion of platelets in vitro. METHODS: The peripheral blood of healthy adults were collected, and the washed platelets were gained from collected peripheral blood. The washed platelets were treated with PKA activator Forskolin, then the platelet aggregation was induced by using Ristocetin, Thrombin, Collagen and ADP respectively, the platelet aggregation level was detected by the platelet aggregator. RESULTS: Compared with the controls, 5 μmol/L forskolin significantly inhibited ADP and collagen-induced platelet aggregation (P＜0.001), and showed mild inhibiting effect on Thrombin-induced platelet aggregation (P＜0.05). 2.5-10 μmol/L forskolin significantly inhibited ADP and Collagen -induced platelet aggregation (P＜0.001); but not showed significantly inhibitory effects on Ristocetin-induced platelet aggregation (P＞0.05). CONCLUSION PKA activation inhibits agonists-induced platelet aggregation.
Blood Platelets ; Cyclic AMP-Dependent Protein Kinases ; Humans ; Platelet Aggregation ; Platelet Aggregation Inhibitors ; Ristocetin ; Thrombin

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*

BACKGROUND/OBJECTIVES: Myocardial infarction (MI) is caused by extensive myocardial damage attributed to the occlusion of coronary arteries. Our previous study in a rat model of ischemia/reperfusion (I/R) demonstrated that administration of arabinoxylan (AX), comprising arabinose and xylose, protects against myocardial injury. In this study, we undertook to investigate whether psyllium seed husk (PSH), a safe dietary fiber containing a high level of AX (> 50%), also imparts protection against myocardial injury in the same rat model. MATERIALS/METHODS: Rats were fed diets supplemented with PSH (1, 10, or 100 mg/kg/d) for 3 d. The rats were then subjected to 30 min ischemia through ligation of the left anterior descending coronary artery, followed by 3 h reperfusion through release of the ligation. The hearts were harvested and cut into four slices. To assess infarct size (IS), an index representing heart damage, the slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC). To elucidate underlying mechanisms, Western blotting was performed for the slices. RESULTS: Supplementation with 10 or 100 mg/kg/d of PSH significantly reduces the IS. PSH supplementation (100 mg/kg/d) tends to reduce caspase-3 generation and increase BCL-2/BAX ratio. PSH supplementation also upregulates the expression of nuclear factor erythroid 2-related factor 2 (NRF2), and its target genes including antioxidant enzymes such as glutathione S-transferase mu 2 (GSTM2) and superoxide dismutase 2 (SOD2). PSH supplementation upregulates some sirtuins (NAD+-dependent deacetylases) including SIRT5 (a mitochondrial sirtuin) and SIRT6 and SIRT7 (nuclear sirtuins). Finally, PSH supplementation upregulates the expression of protein kinase A (PKA), and increases phosphorylated cAMP response element-binding protein (CREB) (pCREB), a target protein of PKA. CONCLUSIONS: The results from this study indicate that PSH consumption reduces myocardial I/R injury in rats by inhibiting the apoptotic cascades through modulation of gene expression of several genes located upstream of apoptosis. Therefore, we believe that PSH can be developed as a functional food that would be beneficial in the prevention of MI.
Animals ; Apoptosis ; Arabinose ; Blotting, Western ; Caspase 3 ; Coronary Vessels ; Cyclic AMP Response Element-Binding Protein ; Cyclic AMP-Dependent Protein Kinases ; Diet ; Dietary Fiber ; Functional Food ; Gene Expression ; Glutathione Transferase ; Heart ; Infarction ; Ischemia ; Ligation ; Models, Animal ; Myocardial Infarction ; Psyllium ; Rats ; Reperfusion ; Sirtuins ; Superoxide Dismutase ; Xylose

Objective To observe the effect of low-frequency pulsed electromagnetic fields(PEMFs) on bone formation in rat osteoblasts(ROBs) and explore the mechanism of action of the cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cyclic adenosine effect binding protein(CREB) signaling pathway.Methods The skulls of newborn Wistar rats were harvested,and the ROBs were obtained by multiple enzymatic digestion methods for subculture. After treatment with 50 Hz 0.6 mT PEMFs for 3,6,and 9 days,the alkaline phosphatase(ALP) concentration in ROBs was detected;after 0,15,30,60,90,and 120 min,the expression of bone formation-related factor(RUNX2),the protein expression of osteogenesis-associated transcription factor(OSX),the cAMP concentration,as well as the protein expressions of p-PKA,p-CREB,and CREB were detected. The p-CREB nuclear translocation was observed. After interference with IFT88 by RNA interference,the expressions of RUNX2,OSX,p-PKA,and p-CREB protein in ROBs were detected.Results After treatment with PEMFs for 3,6,and 9 days,the ALP activity values in ROBs were 24.356±4.911,37.688±2.151,and 39.922±5.486,respectively,which were significantly higher than 18.531±2.401(P=0.0121),33.675±4.366(P=0.0324),and 36.574±1.339(P=0.0134) in the control groups. RUNX2 and OSX activities in ROBs were significantly higher than untreated group after PEMFs treatment for 30(P=0.0042 and P=0.0058),60(P=0.0097 and P=0.0079),and 90 min(P=0.0083 and P=0.0098). After PEMFs treatment for 30(P=0.0012) and 60 min(P=0.0035),the cAMP concentrations in ROBs were significantly higher than that in untreated group. After PEMFs treatment for 15(P=0.0018),30(P=0.0087),90(P=0.0250),and 120 min(P=0.0350),the p-PKA levels in ROBs were significantly higher than that in the untreated group. After PEMFs treatment for 15(P=0.0075),30(P=0.0017),60(P=0.0074),and 90 min(P=0.0096),the level of p-CREB in the ROBs was significantly higher than in the untreated group. After PEMFs treatment of ROBs for 15 min,CREB phosphorylated and accumulated in the nuclei. PKA and p-PKA were co-localized with primary cilia and stained,and it was found that p-PKA was localized on the primary cilia. After the primary cilia was removed by RNA interference,the protein expression levels of p-PKA(F=78.602,P=0.0270),p-CREB(F=76.082,P=0.0089),RUNX2(F=41.064,P=0.0230) and OSX(F=57.524,P=0.0310) were significantly lower than those of the non-interfered group.Conclusion PEMFs promote bone formation in ROBs by activating the primary cilia-associated cAMP/PKA/CREB signaling pathway.
Animals ; Cyclic AMP-Dependent Protein Kinases ; Electromagnetic Fields ; Osteoblasts ; Osteogenesis ; Rats ; Rats, Wistar

PURPOSE: Goserelin is a drug used for chemical castration. In a rat model, we investigated whether surgical and chemical castration affected memory ability through the protein kinase A (PKA)/cyclic adenosine monophosphate response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) and c-Raf/mitogen-activated protein kinases-extracellular signal–regulated kinases (MEK)/extracellular signal–regulated kinases (ERK) pathways in the hippocampus. METHODS: Orchiectomy was performed for surgical castration and goserelin acetate was subcutaneously transplanted into the anterior abdominal wall for chemical castration. Immunohistochemistry was done to quantify neurogenesis. To assess the involvement of the PKA/CREB/BDNF and c-Raf/MEK/ERK pathways in the memory process, western blots were used. RESULTS: The orchiectomy group and the goserelin group showed less neurogenesis and impaired short-term and spatial memory. Phosphorylation of PKA/CREB/BDNF and phosphorylation of c-Raf/MEK/ERK decreased in the orchiectomy and goserelin groups. CONCLUSIONS: Short-term memory and spatial memory were affected by surgical and chemical castration via the PKA/CREB/BDNF and c-Raf/MEK/ERK signaling pathways.
Abdominal Wall ; Adenosine Monophosphate ; Blotting, Western ; Castration ; Cyclic AMP-Dependent Protein Kinases ; Down-Regulation ; Goserelin ; Hippocampus ; Immunohistochemistry ; Memory ; Memory, Short-Term ; Models, Animal ; Neurogenesis ; Orchiectomy ; Phosphorylation ; Phosphotransferases ; Spatial Memory

Vitamin D deficiency is a common health issue around the world. We therefore evaluated the associations of semen quality with both serum and seminal plasma vitamin D levels and studied the mechanisms underlying these by incubating spermatozoa with 1,25(OH)₂D In vitro. Two hundred and twenty-two men were included in our study. Vitamin D was detected using an electrochemiluminescence method. Spermatozoa used for In vitro experiments were isolated by density gradient centrifugation. Positive relationships of serum 25(OH)D with semen volume and seminal plasma fructose were identified. Seminal plasma 25(OH)D level showed no relationship with serum 25(OH)D level, while it was inversely associated with sperm concentration and positively correlated with semen volume and sperm kinetic values. In vitro, sperm kinetic parameters increased after incubation with 1,25(OH)₂D, especially upon incubation for 30 min with it at a concentration of 0.1 nmol l^-1. Under these incubation conditions, the upward migration of spermatozoa increased remarkably with increasing adenosine triphosphate (ATP) concentration. The concentration of cyclic adenosine monophosphate (cAMP) and the activity of protein kinase A (PKA) were both elevated, and the PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89) reversed the increase of ATP production. The concentrations of cytoplasmic calcium ions and nicotinamide adenine dinucleotide (NADH) were both enhanced, while mitochondrial calcium uniporter (MCU) inhibitor, Ruthenium 360 (Ru360) did not reverse the increase of ATP production. Therefore, seminal plasma vitamin D may be involved in regulating sperm motility, and 1,25(OH)₂D may enhance sperm motility by promoting the synthesis of ATP both through the cAMP/PKA pathway and the increase in intracellular calcium ions.
Adenosine Triphosphate/metabolism* ; Adult ; Calcium/metabolism* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Humans ; Male ; Semen/metabolism* ; Semen Analysis ; Signal Transduction/physiology* ; Sperm Motility/physiology* ; Spermatozoa/metabolism* ; Vitamin D/pharmacology* ; Vitamin D Deficiency/blood* ; Wit and Humor as Topic ; Young Adult

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l^-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l^-1), whereas higher concentrations (>5 μmol l^-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca²⁺-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.
8-Bromo Cyclic Adenosine Monophosphate/pharmacology* ; Acrosome/metabolism* ; Acrosome Reaction/drug effects* ; Calcimycin/pharmacology* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors* ; Exocytosis/drug effects* ; Guanine Nucleotide Exchange Factors/metabolism* ; Humans ; Male ; Protein Kinase Inhibitors/pharmacology* ; Signal Transduction/drug effects* ; Spermatozoa/metabolism* ; Thapsigargin/pharmacology*