Ginsenoside Re promotes human sperm capacitation through nitric oxide-dependent pathway

The regulation of sperm capacitation is important for successful fertilization. Ginsenosides, the biologically effective components of ginseng, have been found to enhance intracellular nitric oxide (NO) production and the latter has recently been indicated to play a significant role in modulation of sperm functions. We investigated the effect of Ginsenoside Re on human sperm capacitation in vitro and the mechanism by which the Ginsenosides play their roles. Spermatozoa were separated by Percoll and incubated with 0, 1, 10, or 100 microM of Ginsenoside Re. The percentages of spontaneous and lysophosphatidylcholine (LPC)-induced acrosome reaction (AR), as a measure of sperm capacitation, were assayed with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA). The intracellular cGMP level was measured by [(3)H] cGMP radioimmunoassay system. The results showed that the percentages of both spontaneous and LPC-induced AR and intracellular cGMP level were significantly enhanced by Ginsenoside Re with a concentration-dependent manner. Sodium nitroprusside (SNP, 100 nM), a NO donor, mimicked the effects of Ginsenoside Re. And pretreatment with a NOS inhibitor N(omega)-nitro-l-arginine methyl ester (L-NAME, 100 microM) or a NO scavenger N-acetyl-l-cysteine (LNAC, 1 mM) completely blocked the effects of Ginsenoside Re. Furthermore, the AR-inducing effect of Ginsenoside Re was significantly reduced in the presence of the soluble guanylate cyclase inhibitor LY83583 or cGMP-dependent protein kinase (PCK) inhibitor KT5823, whereas addition of the cGMP analogue 8-Br-cGMP significantly increased the AR of human spermatozoa. Data suggested that Ginsenoside Re is beneficial to sperm capacitation and AR, and that the effect is accomplished through NO/cGMP/PKG pathway.     

大豆异黄酮对大鼠乳腺癌细胞内cAMP/PKA信号途径的影响

本实验研究了大豆异黄酮对SHZ-88大鼠乳腺癌细胞内cAMP／PKA信号途径的影响。实验设3组：空白对照组、50μg／ml大豆黄酮及15μg／ml染料木素组。采用放射免疫测定法（RIA）检测了胞内cAMP的浓度、腺苷酸环化酶（adenylate cyclase,AC）和磷酸二酯酶（phosphodiesterase,PDE）的活性,用（γ-^32P）ATP掺入法测定cAMP依赖性PKA的活性,半定量RT-PCR法分析cAMP反应元件结合蛋白（cAMP response element binding protein,CREB）mRNA表达的变化。结果表明：在处理后5min,大豆黄酮组和染料木素组细胞的cAMP浓度分别比对照组升高了9．5％和11．0％（P〈0．05）：10min时,分别比对照组升高31．0％和40.3％（P〈0．01）。3组细胞的AC活性在处理时间内没有明显变化。但在处理后5min,大豆黄酮组和染料木素组细胞的PDE活性分别降至对照组的71．8％和71．6％（P〈0．05）。处理后20min,大豆黄酮组和染料木素组细胞PKA活性分别上升到对照组的125．8％和122．3％（P〈0．05）;到40min时仍维持在高水平。大豆黄酮组和染料木素组细胞CREB mRNA的表达量在处理后3h分别比对照组增加31．6％和51．1％（P〈0．05）;6h后开始下降。这些结果提示,大豆异黄酮能够激活大鼠乳腺癌细胞内cAMP／PKA信号途径;而且是通过抑制磷酸二酯酶的活性,导致胞内cAMP浓度升高而实现的。     

双歧杆菌调节VIP/cAMP/PKA和mTOR通路改善溃疡性结肠炎小鼠的实验研究

摘要 目的：探讨双歧杆菌MIMBb75通过调节血管活性肠肽（VIP）/环磷酸腺苷（cAMP）/蛋白激酶A（PKA）和哺乳动物雷帕霉素靶蛋白（mTOR）通路对溃疡性结肠炎（UC）小鼠的影响。方法：BALB/c小鼠随机分为正常对照（NC）组、结肠炎模型（UC）组、Mesalazine组和MIMBb75低、高剂量组、MIMBb75高剂量+VIP antagonist组、MIMBb75高剂量+MHY1485组（每组10只）,除NC组外均采用5%葡聚糖硫酸钠（DSS）诱导UC模型。治疗结束后,观察小鼠的一般情况及UC疾病活动指数（DAI）,检测小鼠肠道组织病理损伤、结肠组织中髓过氧化物酶（MPO）活性、肠道菌群多样性（Chao指数、Shannon指数和Simpson指数）及结肠组织VIP、cAMP、PKA、水通道蛋白3（AQP3）、mTOR、核糖体蛋白S6激酶（S6K1）的mRNA和蛋白水平。结果：与UC组相比,MIMBb75低、高剂量组和Mesalazine组小鼠的体重升高、DAI评分降低,组织病理损伤得到改善,结肠长度增加,MPO活性降低,Chao指数、Shannon指数和Simpson指数升高;VIP、cAMP、PKA、AQP3的mRNA水平和VIP、cAMP、AQP3蛋白的表达及PKA的磷酸化水平升高,mTOR和S6K1 mRNA及其蛋白的磷酸化水平降低（P<0.05）。与MIMBb75高剂量组相比,MIMBb75高剂量+VIP antagonist组VIP、cAMP、PKA、AQP3的mRNA水平和VIP、cAMP、AQP3蛋白的表达及PKA的磷酸化水平降低（P<0.05）;MIMBb75高剂量+MHY1485组mTOR和S6K1 mRNA及其蛋白的磷酸化水平升高（P<0.05）。VIP antagonist和MHY1485均能逆转MIMBb75对UC小鼠的保护作用,使其结肠损伤加重,MPO活性增高（P<0.05）。结论：双歧杆菌可改善UC小鼠的结肠损伤,增加肠道菌群的多样性,这可能与激活VIP/cAMP/PKA通路、抑制mTOR通路有关。     

cAMP/PKA signalling reinforces the LATS–YAP pathway to fully suppress YAP in response to actin cytoskeletal changes

Actin cytoskeletal damage induces inactivation of the oncoprotein YAP (Yes‐associated protein). It is known that the serine/threonine kinase LATS (large tumour suppressor) inactivates YAP by phosphorylating its Ser127 and Ser381 residues. However, the events downstream of actin cytoskeletal changes that are involved in the regulation of the LATS–YAP pathway and the mechanism by which LATS differentially phosphorylates YAP on Ser127 and Ser381 in vivo have remained elusive. Here, we show that cyclic AMP (cAMP)‐dependent protein kinase (PKA) phosphorylates LATS and thereby enhances its activity sufficiently to phosphorylate YAP on Ser381. We also found that PKA activity is involved in all contexts previously reported to trigger the LATS–YAP pathway, including actin cytoskeletal damage, G‐protein‐coupled receptor activation, and engagement of the Hippo pathway. Inhibition of PKA and overexpression of YAP cooperate to transform normal cells and amplify neural progenitor pools in developing chick embryos. We also implicate neurofibromin 2 as an AKAP (A‐kinase‐anchoring protein) scaffold protein that facilitates the function of the cAMP/PKA–LATS–YAP pathway. Our study thus incorporates PKA as novel component of the Hippo pathway.     

Bmp2 regulates Serpinb6b expression via cAMP/PKA/Wnt4 pathway during uterine decidualization

Serpinb6b is a novel member of Serpinb family and found in germ and somatic cells of mouse gonads, but its physiological function in uterine decidualization remains unclear. The present study revealed that abundant Serpinb6b was noted in decidual cells, and advanced the proliferation and differentiation of stromal cells, indicating a creative role of Serpinb6b in uterine decidualization. Further analysis found that Serpinb6b modulated the expression of Mmp2 and Mmp9. Meanwhile, Serpinb6b was identified as a target of Bmp2 regulation in stromal differentiation. Treatment with rBmp2 resulted in an accumulation of intracellular cAMP level whose function in this differentiation program was mediated by Serpinb6b. Addition of PKA inhibitor H89 impeded the Bmp2 induction of Serpinb6b, whereas 8‐Br‐cAMP rescued the defect of Serpinb6b expression elicited by Bmp2 knock‐down. Attenuation of Serpinb6b greatly reduced the induction of constitutive Wnt4 activation on stromal cell differentiation. By contrast, overexpression of Serpinb6b prevented this inhibition of differentiation process by Wnt4 siRNA. Moreover, blockage of Wnt4 abrogated the up‐regulation of cAMP on Serpinb6b. Collectively, Serpinb6b mediates uterine decidualization via Mmp2/9 in response to Bmp2/cAMP/PKA/Wnt4 pathway.     

Cholesterol enhances neuron susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1

Cholesterol is a major component of membrane lipid rafts. It is more abundant in the brain than in other tissues and plays a critical role in maintaining brain function. We report here that a significant enhancement in apoptosis in rat cerebellar granule neurons (CGNs) was observed upon incubation with 5mM K(+) /serum free (LK-S) medium. Cholesterol enrichment further potentiated CGN apoptosis incubated under LK-S medium. On the contrary, cholesterol depletion using methyl-beta-cyclodextrin protected the CGNs from apoptosis induced by LK-S treatment. Cholesterol enrichment, however, did not induce apoptosis in CGNs that have been incubated with 25mM K(+) /serum medium. Mechanistically, increased I(K) currents and DNA fragmentation were found in CGNs incubated in LK-S, which was further potentiated in the presence of cholesterol. Cholesterol-treated CGNs also exhibited increased cAMP levels and up-regulation of Kv2.1 expression. Increased levels of activated form of PKA and phospho-CREB further supported activation of the cAMP/PKA pathway upon treatment of CGNs with cholesterol-containing LK-S medium. Conversely, inhibition of PKA or small G protein Gs abolished the increase in I(K) current and the potentiation of Kv2.1 expression, leading to reduced susceptibility of CGNs to LK-S and cholesterol-induced apoptosis. Our results demonstrate that the elevation of membrane cholesterol enhances CGN susceptibility to apoptotic stimuli via cAMP/PKA/CREB-dependent up-regulation of Kv2.1. Our data provide new evidence for the role of cholesterol in eliciting neuronal cell death.     

Alteration of the human sperm surface during in vitro capacitation as assessed by lectin-induced agglutination

Human sperm incubated in vitro in BWW medium containing 35 mg/ml human serum albumin acquire the capacity to penetrate the human zona pellucida and to fuse with the zona-free hamster oocyte. We have studied changes in lectin-induced agglutination of human sperm during incubation in this medium to detect alterations in the sperm surface which may be correlated with the acquisition of these functions. Sperm incubated for 1, 6, or 24 hr were combined with two-fold dilutions of lectins for 30 min at 37°C, in 5% CO₂, balance air. When pooled data from five donors were analyzed, the average sperm agglutination titer of wheat germ agglutinin (WGA), phytohemagglutinin-E (PHA), Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), and Pisum sativum agglutinin (PSA) was found to increase significantly (P ≤ 0.06) with incubation in vitro, although there was considerable variation between ejaculates. Ulex europaeus and Dolichos biflorus agglutinins did not agglutinate human sperm (≤250 μg/ml). Results of this screening demonstrate the alteration of sperm surface components during in vitro incubation and suggest that WGA, PHA, LCA, and PSA may prove useful in efforts to correlate changes in the sperm surface with the ability of the sperm to fertilize the egg.     

Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells

Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARγ and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 μM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1 μM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.     

FPP modulates mammalian sperm function via TCP-11 and the adenylyl cyclase/cAMP pathway

Fertilization promoting peptide (FPP; pGlu-Glu-ProNH₂), which is found in seminal plasma, promotes capacitation but inhibits spontaneous acrosome loss in mammalian spermatozoa in vitro. Adenosine, known to modulate the adenylyl cyclase (AC)/cAMP pathway, elicits these same responses whereas FPP + adenosine produces an enhanced response, leading to the hypothesis that FPP and adenosine modulate the same signal transduction pathway but act via different receptors. TCP-11, the product of a t-complex gene, is the putative receptor for FPP: Fab fragments of anti-TCP-11 antibodies have the same effect as FPP on mouse spermatozoa and Gln-FPP, a competitive inhibitor of FPP, also competitively inhibits responses to the Fab fragments. In the present study, specific binding of ³H-FPP to sperm membranes was significantly inhibited by 200 nM Gln-FPP and anti-TCP-11 Fab fragments (1/25 dilution), thus confirming that FPP, Gln-FPP, and Fab fragments compete for the same binding site. In addition, spermatozoa treated with A23187 to induce the acrosome reaction bound significantly less ³H-FPP than untreated cells, suggesting that a large proportion of the FPP binding sites are associated with the acrosomal cap region; TCP-11 is located in this region. In other experiments, 100 nM FPP significantly stimulated cAMP production in mouse sperm membranes, permeabilized cells and intact cells. Furthermore, Gln-FPP inhibited production of cAMP in response to FPP but not to adenosine (10 μM) or its analogue NECA (100 nM), supporting the involvement of two different receptors. Finally, anti-TCP-11 Fab fragments (1/25 dilution) significantly stimulated cAMP production, whereas low Fab (1/200; nonstimulatory when used alone) plus adenosine (10 μM) significantly enhanced the stimulation of capacitation by adenosine. These results support the hypotheses that TCP-11 is the receptor for FPP and that FPP↔TCP-11 interactions modulate AC/cAMP. Mol. Reprod. Dev. 51:468–476, 1998. © 1998 Wiley-Liss, Inc.     

Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

The present study was designed to investigate the role of endogenous sulfur dioxide (SO₂) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO₂ inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO₂ synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO₂ suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO₂ did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO₂ dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO₂ simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO₂ could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO₂-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO₂ exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling.     

Iberiotoxin and clofilium regulate hyperactivation,acrosome reaction,and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.     

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

针刺对缺氧缺血性脑损伤大鼠脑神经功能及5-HTR1A/cAMP/PKA信号通路的影响机制研究

摘要 目的：探讨与研究针刺对缺氧缺血性脑损伤大鼠脑神经功能及5-HTR1A/cAMP/PKA信号通路的影响机制。方法：研究时间为2022年6月到2022年12月,SPF级健康雄性SD大鼠36只平分为空白组、模型组、针刺组,每组各12只大鼠。空白组不进行造模,针刺组在造模完成1周后进行针刺治疗,空白组、模型组不进行治疗。结果：所有大鼠都顺利完成实验,无死亡大鼠出现。针刺组、模型组治疗后2周与4周的神经功能评分都显著高于空白组（P<0.05）,针刺组的神经功能评分与模型组相比显著降低(P<0.05)。针刺组、模型组治疗后2周与4周的脑缺氧缺血组织体积都高于空白组(P<0.05),针刺组的脑缺氧缺血组织体积与模型组相比显著降低（P<0.05）。针刺组、模型组治疗后2周与4周的血清超氧化物歧化酶活力低于空白组（P<0.05）,血清丙二醛含量高于空白组（P<0.05）,针刺组与模型组对比也有显著差异（P<0.05）。针刺组、模型组治疗后2周与4周的大脑组织5-HTR1A蛋白、cAMP蛋白、PKA蛋白相对表达水平明显低于空白组（P<0.05）,针刺与模型组相比显著提高（P<0.05）。结论：针刺在缺氧缺血性脑损伤大鼠的应用能激活5-HTR1A/cAMP/PKA信号通路,能提高超氧化物歧化酶活力,降低血清丙二醛含量,能改善大鼠的脑神经功能,降低脑缺氧缺血组织体积。     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

Cre transgene results in global attenuation of the cAMP/PKA pathway

Use of the cre transgene in in vivo mouse models to delete a specific ‘floxed'' allele is a well-accepted method for studying the effects of spatially or temporarily regulated genes. During the course of our investigation into the effect of cyclic adenosine 3′,5′-monophosphate-dependent protein kinase A (PKA) expression on cell death, we found that cre expression either in cultured cell lines or in transgenic mice results in global changes in PKA target phosphorylation. This consequently alters gene expression profile and changes in cytokine secretion such as IL-6. These effects are dependent on its recombinase activity and can be attributed to the upregulation of specific inhibitors of PKA (PKI). These results may explain the cytotoxicity often associated with cre expression in many transgenic animals and may also explain many of the phenotypes observed in the context of Cre-mediated gene deletion. Our results may therefore influence the interpretation of data generated using the conventional cre transgenic system.     

参仙升脉口服液联合曲美他嗪对缓慢性心律失常患者外周血单个核细胞中cAMP/PKA信号通路相关蛋白水平的影响

摘要 目的：观察缓慢性心律失常（BA）经参仙升脉口服液联合曲美他嗪治疗4周后的临床疗效及对外周血单个核细胞中环磷酸腺苷( cAMP) /蛋白激酶A（PKA）信号通路相关蛋白水平的影响。方法：将2017年8月~2020年8月期间我院收治的126例BA患者随机分为两组,对照组和观察组,各63例。对照组接受曲美他嗪治疗,观察组接受参仙升脉口服液联合曲美他嗪治疗,观察并对比两组24 h动态心电图指标（最慢心率、静息状态下心率、24 h平均心率）、心功能指标[左室射血分数(LVEF)、心输出量(CO)、心脏指数(CI)]、疗效、外周血单个核细胞中cAMP/PKA信号通路相关蛋白（cAMP蛋白、PKA蛋白）水平及不良反应。结果：观察组的临床总有效率90.48%（57/63）高于对照组的71.43%（45/63）,差异有统计学意义（P<0.05）。治疗4周后,与对照组比较,观察组静息状态下心率、24 h平均心率、最慢心率均较高（P<0.05）。治疗4周后,观察组LVEF、CO、CI高于对照组（P<0.05）。治疗4周后,观察组外周血单个核细胞中cAMP蛋白、PKA蛋白水平高于对照组（P<0.05）。两组不良反应发生率组间对比无差异（P>0.05）。结论：参仙升脉口服液联合曲美他嗪治疗BA患者,可改善治疗效果,促进24h动态心电图、心功能指标改善,其主要作用机制可能与调节cAMP/PKA信号通路相关蛋白表达有关。     

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 μM IBMX without LIF, 10 μM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways. This work was partly funded by the Millennium Research Fund National University of Ireland Galway.