血清NEP、PTX3及H-FABP在慢性心力衰竭的表达水平及与疾病的相关性分析

摘要 目的：探讨血清脑啡肽酶（NEP）、正五聚蛋白3（PTX3）及心型脂肪酸结合蛋白（H-FABP）在慢性心力衰竭（CHF）的表达水平及其与疾病严重程度的相关性。方法：选入我院2019年10月~2021年10月收治的110例CHF患者作为观察组,并根据美国纽约心脏病学会（NYHA）标准进行心功能分级,另取同期健康志愿者50例作为对照组。比较两组之间、观察组不同心功能分级之间的血清NEP、PTX3及H-FABP的表达水平,并分析三者与CHF严重程度的相关性。出院后随访90 d,记录主要心脏不良事件（MACE）情况。结果：观察组血清NEP和LVEF水平明显低于对照组（P＜0.05）,而PTX3、H-FABP及LVEDD显著高于对照组（P＜0.05）;且随着NYHA分级升高,NEP和LVEF逐渐降低（P＜0.05）,PTX3、H-FABP及LVEDD逐渐提高（P＜0.05）,各组间差异显著（P＜0.05）;相关性分析结果显示：PTX3、H-FABP与NYHA分级和LVEDD存在明显正相关（P<0.05）,与LVEF呈明显负相关（P<0.05）,NEP与NYHA分级、LVEF和LVEDD均无明显相关性（P＞0.05）。MACE患者血清NEP水平显著低于非MACE患者,血清PTX3和H-FABP水平显著高于非MACE患者,差异有显著意义（P＜0.05）。结论：血清NEP、PTX3和H-FABP均是CHF诊断的重要生物学标志物,尤其是PTX3和H-FABP与病情严重程度和预后存在密切关系,联合检测对CHF早期诊断、病情和预后评估具有重要意义。     

MiR-29b-3p aggravates cardiac hypoxia/reoxygenation injury via targeting PTX3

Our current research aimed to decipher the role and underlying mechanism with regard to miR-29b-3p involving in myocardial ischemia/reperfusion (I/R) injury. In the present study, cardiomyocyte H9c2 cell was used, and hypoxia/reoxygenation (H/R) model was established to mimic the myocardial I/R injury. The expressions of miR-29b-3p and pentraxin 3 (PTX3) were quantified deploying qRT-PCR and Western blot, respectively. The levels of LDH, TNF-α, IL-1β and IL-6 were detected to evaluate cardiomyocyte apoptosis and inflammatory response. Cardiomyocyte viability and apoptosis were examined employing CCK-8 assay and flow cytometry, respectively. Verification of the targeting relationship between miR-29b-3p and PTX3 was conducted using a dual-luciferase reporter gene assay. It was found that miR-29b-3p expression in H9c2 cells was up-regulated by H/R, and a remarkable down-regulation of PTX3 expression was demonstrated. MiR-29b-3p significantly promoted of release of inflammatory cytokines of H9c2 cells, and it also constrained the proliferation and promoted the apoptosis of H9c2 cells. Additionally, PTX3 was inhibited by miR-29b-3p at both mRNA and protein levels, and it was identified as a direct target of miR-29b-3p. PTX3 overexpression could reduce the inflammatory response, increase the viability of H9c2 cells, and inhibit apoptosis. Additionally, PTX3 counteracted the function of miR-29b-3p during the injury of H9c2 cells induced by H/R. In summary, miR-29b-3p was capable of aggravating the H/R injury of H9c2 cells by repressing the expression of PTX3.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.     

OPA1 cleavage depends on decreased mitochondrial ATP level and bivalent metals

OPA1, an intra-mitochondrial dynamin GTPase, is a key actor of outer and inner mitochondrial membrane dynamic. OPA1 amino-terminal cleavage by PARL and m-AAA proteases was recently proposed to participate to the mitochondrial network dynamic in a DeltaPsi(m)-dependent way, and to apoptosis. Here, by an in vitro approach combining the use of purified mitochondrial fractions and mitochondrial targeting drugs, we intended to identify the central stimulus responsible for OPA1 cleavage. We confirm that apoptosis induction and PTPore opening, as well as DeltaPsi(m) dissipation induce OPA1 cleavage. Nevertheless, our experiments evidenced that decreased mitochondrial ATP levels, either generated by apoptosis induction, DeltaPsi(m) dissipation or inhibition of ATP synthase, is the common and crucial stimulus that controls OPA1 processing. In addition, we report that ectopic iron addition activates OPA1 cleavage, whereas zinc inhibits this process. These results suggest that the ATP-dependent OPA1 processing plays a central role in correlating the energetic metabolism to mitochondrial dynamic and might be involved in the pathophysiology of diseases associated to excess of iron or depletion of zinc and ATP.     

Overexpression of the lncRNA FER1L4 inhibits paclitaxel tolerance of ovarian cancer cells via the regulation of the MAPK signaling pathway

To determine how the lncRNA FER1L4 in ovarian cancer cells influences paclitaxel (PTX) resistance, we examined the expression level of FER1L4 in human ovarian epithelial cell lines IOSE80 and HOSEpiC and human ovarian cancer cell lines OVCAR-3, Caov-3, and SKOV3 through RNA isolation and quantitative polymerase chain reaction (qRT-PCR). SKOV3 cell lines were treated with PTX. The cell survival rate and apoptosis rate of SKOV3 and SKOV3-PR at different PTX dose levels were evaluated. Next, qRT-PCR was performed to detect the expression of FER1L4 in SKOV3 and SKOV3-PR cell lines. SKOV3-PR cell lines were transfected with pcDNA3.1 as the control group (SKOV3-PR/pcDNA3.1) or pcDNA3.1-FER1L4 to upregulate the expression level of FER1L4 (SKOV3-PR/pcDNA3.1-FER1L4). The level of cell survival, apoptosis, and colony formation were compared between the two groups using MTT, flow cytometry analysis, and colony formation assay. To reveal the molecular mechanism, we measured the relative protein phosphorylation level of ERK and MAPK in SKOV3, SKOV3-PR, SKOV3-PR/pcDNA3.1, and SKOV3-PR/pcDNA3.1-FER1L4 groups using an enzyme-linked immunosorbent assay. The effects of SB203580 (a p38 MAPK inhibitor) on PTX were also investigated to reveal the function of the MAPK pathway on the PTX tolerance of SKOV3. In comparison with normal ovarian epithelial cells, FER1L4 was downregulated. The FER1L4 level was decreased in human ovarian cancer cells with drug resistance than in common ovarian cancer cells. The upregulation of FER1L4 could promote the PTX sensitivity of ovarian cancer cells. The increased level of FER1L4 could suppress the PTX resistance of ovarian cancer cells through the inhibition of the MAPK signaling pathway.     

Metabolic activation of A549 human airway epithelial cells by organic dust: a study based on microphysiometry

A Cytosensor microphysiometer, which measures extracellular acidification rate (ECAR), was used to study the early metabolic activation by organic dust from a swine confinement building in a human airway epithelial cell line, A549. The dust is known to cause an intense airway inflammatory reaction following inhalation in vivo and cytokine release in vitro. Dimethyl amiloride (DMA) was used to study sodium/proton exchanger (NHE) activity in cells growing at different cell densities. Exposing cells at low density to dust induced an initial release of acid not involving NHE, followed by a sustained DMA-sensitive NHE activation. In cells near high density, NHE was not activated during exposure resulting in a modest increase in ECAR. Exposing cells at high density resulted in a bi-phasic ECAR pattern; an initial increase in proton release followed by an inhibition of ECAR below baseline. Pretreatment with pertussis toxin (PTX), an inhibitor of receptor/G(i alpha)-coupled signal transductions did not affect ECAR in low and medium density cells, but abolished the inhibition of ECAR in high-density cells. The dust did not prevent forskolin-induced cAMP accumulation and PTX did not affect cAMP in near-confluent cells suggesting the PTX-effect to be cAMP-independent. The ECAR response to organic dust was similar to that of lipopolysaccharide (LPS) except for high-density cells where PTX did not influence the LPS-induced decrease in ECAR below baseline. In summary, the organic dust induces PTX-sensitive (cAMP independent) signalling in near-confluent A549 epithelial cells and, depending on cell density opposing effects on NHE activity during exposure.     

Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in β₂-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G_i/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G_i/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser¹¹⁷⁷, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr¹⁴, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G_i/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser¹¹⁷⁷ leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr¹⁴, through a G_i/o-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.     

Long-term dark rearing induces permanent reorganization in retinal circuitry

Recent data challenged the assumption that light has little effect on retina development. Here, we report evidence that dark rearing permanently changes the synaptic input to GCs. A reduced spontaneous postsynaptic currents (SPSCs) frequency was found in retinal GCs from rats born and raised in the dark for three months. Glutamate antagonists (CNQX and AP-5) reversibly reduced SPSCs frequency in control and dark-reared (DR) retinae. The GABA antagonist picrotoxin (PTX) reduced SPSCs frequency in control retinas, but increased SPSCs frequency in DR, mainly by presynaptic action on excitatory currents. In DR animals exposed to normal cyclic light for 3 months, SPSCs frequency remained lower then in control rats and increased following PTX, suggesting that long-term dark rearing induces permanent modifications of the retinal circuitry. Our results strongly support the idea that light stimulation plays a role in establishing normal synaptic input to GCs.     

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo.In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.     

Functional lysophosphatidic acid receptors expressed in Oryzias latipes

Lysophosphatidic acid (LPA) signaling is known to play biological and pathophysiological roles in many types of animals. Medaka (Oryzias latipes) is an experimental fish that can be easily maintained, propagated, and analyzed, and whose genome has been completely sequenced. However, there is limited information available regarding medaka LPA receptors. Here, using information from the medaka genome database, we examine the genomic structures, expression, and functions of six LPA receptor genes, Lpar1–Lpar6. Our analyses reveal that the genomic structures of Lpar1 and Lpar4 are different from those deduced from the database. Functional analyses using a heterologous expression system demonstrate that all medaka LPA receptors except for LPA_5b respond to LPA treatment with cytoskeletal changes. These findings provide useful information on the structure and function of medaka LPA receptor genes, and identify medaka as a useful experimental model for exploration of the biological significance of LPA signaling.