Lung injury after ischemia-reperfusion of small intestine in rats involves apoptosis of type II alveolar epithelial cells mediated by TNF-α and activation of Bid pathway

Although ischemia-reperfusion (I/R) of small intestine is known to induce lung cell apoptosis, there is little information on intracellular and extracellular molecular mechanisms. Here, we investigated the mechanisms of apoptosis including the expression of Fas, Fas ligand (FasL), Bid, Bax, Bcl-2, cytochrome c, and activated caspase-3 in the rat lung at various time-points (0–24 h) of reperfusion after 1-h ischemia of small intestine. As assessed by TUNEL, the number of apoptotic epithelial cells, which were subsequently identified as type II alveolar epithelial cells by electron microscopy and immunohistochemical double-staining, increased at 3 h of reperfusion in the lung. However, intravenous injections of anti-TNF-α antibody decreased the number of TUNEL-positive cells, indicating involvement of tumor necrosis factor-α (TNF-α) in the induction of lung cell apoptosis. Western blotting and/or immunohistochemistry revealed a marked up-regulation of Fas, FasL, Bid, Bax, cytochrome c and activated caspase-3 and down-regulation of Bcl-2 in lung epithelial and stromal cells at 3 h of reperfusion. Our results indicate that I/R of small intestine results in apoptosis of rat alveolar type II cells through a series of events including systemic TNF-α, activation of two apoptotic signaling pathways and mitochondrial translocation of Bid.     

Phosphorylation by protein kinase Cα regulates RalB small GTPase protein activation, subcellular localization, and effector utilization

Ras-like (Ral) small GTPases are regulated downstream of Ras and the noncanonical Ral guanine nucleotide exchange factor (RalGEF) effector pathway. Despite RalA and RalB sharing 82% sequence identity and utilization of shared effector proteins, their roles in normal and neoplastic cell growth have been shown to be highly distinct. Here, we determined that RalB function is regulated by protein kinase Cα (PKCα) phosphorylation. We found that RalB phosphorylation on Ser-198 in the C-terminal membrane targeting sequence resulted in enhanced RalB endomembrane accumulation and decreased RalB association with its effector, the exocyst component Sec5. Additionally, RalB phosphorylation regulated vesicular trafficking and membrane fusion by regulating v- and t-SNARE interactions. RalB phosphorylation regulated vesicular traffic of α5-integrin to the cell surface and cell attachment to fibronectin. In summary, our data suggest that phosphorylation by PKCα is critical for RalB-mediated vesicle trafficking and exocytosis.     

Pim protein kinase-3 is regulated by TNF-α and promotes endothelial cell sprouting

Tumor necrosis factor-α (TNF-α) plays an important role in pathological angiogenesis associated with inflammatory response. Pim-3 kinase belonging to serine/threonine protein kinases is a potent suppressor of myc-induced apoptosis. We have recently demonstrated that Pim-3 plays an essential role in endothelial cell (EC) spreading and migration. In this study, we showed that TNF-α transiently increased Pim-3 mRNA expression, and this was mediated through Tumor necrosis factor-α receptor-1 (TNFR1) pathway in ECs. TNF-α could promote stabilization of Pim- 3 mRNA in ECs. Small-interfering RNA (siRNA)-mediated gene knockdown of Pim-3 significantly impaired TNF-α-induced formation of EC membrane protrusions in vitro. Furthermore, Pim-3 silencing inhibited EC sprouting in subcutaneous Matrigel in vivo. eNOS mRNA abundance was lower in Pim-3 siRNA transfected ECs compared with the control ECs. These observations suggest that Pim-3 plays a role in TNF-α-induced angiogenesis.     

Pro-inflammatory effect of fibrinogen and FDP on vascular smooth muscle cells by IL-6, TNF-α and iNOS

AimsAtherosclerosis is a chronic inflammatory response of the arterial wall to multiple endothelial injuries. As one of the inflammatory markers, fibrinogen has been implicated in pathogenesis of atherosclerosis. But, it is not completely understood whether atherogenesis of fibrinogen is related to its pro-inflammatory effect on vascular smooth muscle cells (VSMCs). The purpose of the present study was to observe effects of fibrinogen and fibrin degradation products (FDP) on interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS) generation in rat VSMCs.Main methodsRat VSMCs were cultured, and fibrinogen and FDP were used as stimulants for IL-6, TNF-α, and iNOS. IL-6 and TNF-α level in the supernatant were measured by ELISA, mRNA expression of IL-6, TNF-α, and iNOS were assayed with RT-PCR, and protein expression of iNOS was detected with western blot and immunocytochemistry.Key findingsFibrinogen and FDP both significantly stimulated mRNA and protein expressions of IL-6, TNF-α and iNOS in VSMCs in time- and concentration-dependent ways. The pro-inflammatory potency of FDP is higher than fibrinogen, which seems to mean that smaller fragments of the protein have greater pro-inflammatory activity. Fibrinogen and FDP promote more protein expressions of IL-6 and TNF-α compared to iNOS, suggesting that fibrinogen and FDP produce a pro-inflammatory effect on VSMCs mainly by IL-6 and TNF-α.SignificanceThese findings are helpful to better understand pro-inflammatory effect of fibrinogen on VSMCs involved in atherogenesis, and imply a therapeutic strategy targeting hyperfibrinogenemia in atherosclerosis.     

Bcl-3, induced by Tax and HTLV-1, inhibits NF-κB activation and promotes autophagy

The human T cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes an aggressive leukemia known as adult T cell leukemia (ATL). The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway, which is perceived as the primary cause of ATL. Bcl-3, a member of the NF-κB inhibitor (IκB) family, is highly expressed in many HTLV-1-infected T cell lines and ATL cells. However, the role of Bcl-3 in Tax-induced NF-κB activation has not been fully elucidated. Here, we show that Tax induces Bcl-3 expression, which in turn negatively regulates the Tax-induced NF-κB activation. Interestingly, both Bcl-3 up-regulation and NF-κB inhibition promote the autophagy process in HTLV-1-infected cells. Consistent with this, over-expression of Bcl-3 also results in enhancement of rapamycin-, pifithrin-α- or starvation-induced autophagy in control cells. Together, these data demonstrate that Bcl-3 acts as a negative regulator of NF-κB activation and promotes autophagy in HTLV-1-infected cells.     

Polydatin suppresses the development of lung inflammation and fibrosis by inhibiting activation of the NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 inflammasome and the nuclear factor-κB pathway after Mycoplasma pneumoniae infection

Mycoplasma pneumoniae (MP) can infect both the upper and lower respiratory tracts. Polydatin (PD), a traditional Chinese medicine, is known to have anti-inflammation and antifibrosis properties. However, the protective effects of PD against MP pneumonia (MPP) remain unclear. So, the aim of this study was to describe the therapeutic effects and underlying mechanisms of PD against MPP. BALB/c mice were assigned to three groups: a normal control group, MP infection group, or PD-treated MP infection group. BEAS-2B cells transfected with or without NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 (NLRP3) were used to confirm the protective mechanisms of PD. Immunohistochemical analysis, Western blot analysis, enzyme-linked immunosorbent assay, and flow cytometry were used in this study. The results showed that PD treatment suppressed MP-induced lung injury in mice by suppressing the expression of inflammatory factors and inhibiting the development of pulmonary fibrosis. Meanwhile, PD treatment inhibited activation of the NLRP3 inflammasome and nuclear factor κB (NF-κB) pathway. Overexpression of NLRP3 reversed the protective effect of PD against MP-induced injury of BEAS-2B cells. Taken together, these results indicate that PD treatment suppressed the inflammatory response and the development of pulmonary fibrosis by inhibiting the NLRP3 inflammasome and NF-κB pathway after MP infection.     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

Dexmedetomidine improves acute stress-induced liver injury in rats by regulating MKP-1, inhibiting NF-κB pathway and cell apoptosis

Acute stress is a frequent and unpredictable disease for many animals. Stress is widely considered to affect liver function. However, the underlying mechanism by which dexmedetomidine (DEX) attenuates acute stress-induced liver injury in rats remains unclear. In this study, we used forced swimming for 15 min and acute 3-hr restraint stress model. Behavioral tests and changes in norepinephrine levels confirmed the successful establishment of the acute stress model. Acute stress-induced liver injury, evidenced by hematoxylin and eosin-stained pathological sections and increased serum aminotransferase and aspartate aminotransferase levels, was reduced in DEX-treated livers. Reactive oxygen species and oxidative stress levels were dramatically decreased with DEX treatment compared with acute stress-induced liver injury. DEX significantly reduced acute stress-induced liver inflammation and apoptosis, as assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and inflammation and apoptosis-related protein levels. DEX treatment also effectively inhibited acute stress-induced c-Jun N-terminal kinase (JNK), P38, and BAD signaling pathway activation, and significantly induced MKP-1 activation. Thus, DEX has a protective effect on acute-stress-induced liver injury by reducing inflammation and apoptosis, which suggests a potential clinical application for DEX in stress syndrome.     

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.     

Dexmedetomidine alleviates lipopolysaccharide-induced acute kidney injury by inhibiting the NLRP3 inflammasome activation via regulating the TLR4/NOX4/NF-κB pathway

Dexmedetomidine (DEX) prevents kidney damage caused by sepsis, but the mechanism of this effect remains unclear. In this study, the protective molecular mechanism of DEX in lipopolysaccharide (LPS)-induced acute kidney injury was investigated and its potential pharmacological targets from the perspective of inhibiting oxidative stress damage and the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation. Intraperitoneal injection of DEX (30 μg/kg) significantly improved LPS (10 mg/kg) induced renal pathological damage and renal dysfunction. DEX also ameliorated oxidative stress damage by reducing the contents of reactive oxygen species, malondialdehyde and hydrogen peroxide, and increasing the level of glutathione, as well as the activity of superoxide dismutase and catalase. In addition, DEX prevented nuclear factor-kappa B (NF-κB) activation and I-kappa B (IκB) phosphorylation, as well as the expressions of NLRP3 inflammasome-associated protein and downstream IL-18 and IL-1β. The messengerRNA (mRNA) and protein expressions of toll-like receptor 4 (TLR4), NADPH oxidase-4 (NOX4), NF-κB, and NLRP3 were also significantly reduced by DEX. Their expressions were further evaluated by immunohistochemistry, yielding results were consistent with the results of mRNA and protein detection. Interestingly, the protective effects of DEX were reversed by atipamezole-an alpha 2 adrenal receptor (α₂AR) inhibitor, whereas idazoxan-an imidazoline receptor (IR) inhibitor failed to reverse this change. In conclusion, DEX attenuated LPS-induced AKI by inhibiting oxidative stress damage and NLRP3 inflammasome activation via regulating the TLR4/NOX4/NF-κB pathway, mainly acting on the α₂AR rather than IR.     

Overexpression of tissue factor pathway inhibitor in CHO-K1 cells results in increased activation of NF-κB and apoptosis mediated by a caspase-3 independent pathway

There is now circumstantial evidence that tissue factor pathway inhibitor (TFPI) is not only a major anticoagulant, but also has proapoptotic properties. The current study was designed to address the role of TFPI on signalling pathways and apoptosis. The non-TFPI expressing cell line CHO-K1 was stably transfected with pcDNA3.1/V5-His-TOPO-TFPI and control cells were established by transfecting the CHO-K1 cells with pcDNA3.1/V5-His-TOPO. Sodium butyrate (NaBut) has been shown to induce the expression of recombinant proteins. Here we have used NaBut to increase the expression of TFPI as assessed by qRT-PCR and ELISA. Compared to the control cells, TFPI induced apoptosis in a concentration dependent manner as measured by a cell death detection assay. Independent of caspase-3 activation an increased cleavage of PARP was detected in the TFPI expressing cells. This was accompanied by downregulation of Bcl-XL, elevated levels of Bax, and increased translocation of the apoptosis initiating factor. Increased DNA binding activity of NF-κB was revealed by electrophoretic mobility shift assay when the TFPI level was elevated by NaBut together with an increased translocation of the NF-κB subunit p65. The results indicate that TFPI affected the apoptotic activity through a process independent of caspase-3, and was also able to increase the activation of the NF- κB pathway.     

miR-29b ameliorates atrial fibrosis in rats with atrial fibrillation by targeting TGFβRΙ and inhibiting the activation of Smad-2/3 pathway

Objective

Atrial fibrillation (AF) is a major cause of stroke with lifetime risks. microRNAs (miRNAs) are associated with AF attenuation, yet the mechanism remains unknown. This study investigated the functional mechanism of miR-29b in atrial fibrosis in AF.

Methods

The AF rat model was established by a 7-day intravenous injection of Ach-CaCl₂ mixture. AF rats were injected with adeno-associated virus (AAv)-miR-29b and TGFβRΙ overexpression plasmid. AF duration was recorded by electrocardiogram. Atrial fibrosis was observed by Masson staining. Expressions of COL1A1, COL3A1, TGFβRΙ, TGFβΙ, miR-29b and Smad-2/3 pathway-related proteins in atrial tissues were detected by RT-qPCR and Western blot. Binding sites of miR-29b and TGFβRΙ were predicted and their target relationship was verified by dual-luciferase reporter assay.

Results

miR-29b was poorly expressed and expressions of COL1A1, COL3A1, TGFβRΙ, and TGFβ1 were increased in atrial tissues of AF rats. miR-29b overexpression alleviated atrial fibrosis, reduced expressions of COL1A1, COL3A1, and TGFβ1, and shortened AF duration in AF rats. TGFβRΙ was highly expressed in atrial tissues of AF rats. miR-29b targeted TGFβRΙ. TGFβRΙ overexpression overcame the improving effect of miR-29b overexpression on AF. miR-29b overexpression decreased ratios of p-Smad-2/3 and Smad-2/3 and inhibited the Smad-2/3 pathway.

Conclusion

miR-29b might mitigate atrial fibrosis in AF rats by targeting TGFβRΙ and inhibiting the Smad-2/3 pathway.

     

The antimicrobial peptide, tilapia hepcidin 2-3, and PMA differentially regulate the protein kinase C isoforms, TNF-α and COX-2, in mouse RAW264.7 macrophages

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, were previously studied. Herein, we report the differential modulation of protein kinase C (PKC)-associated proteins by TH2-3, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), in RAW264.7 macrophages. Treatment with TH2-3 at 40 or 80 μg/ml did not affect the cell morphology, but TH2-3 at 120 μg/ml produced morphological changes similar to those after treatment with PMA in RAW264.7 cells. The coexistence of the PKC inhibitor, Ro-31-8220, prevented morphological changes induced by either PMA or 120 μg/ml TH2-3 in RAW264.7 cells. Since PMA is known to induce expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, activation of the TNF-α promoter in response to TH2-3 and PMA treatments in lipopolysaccharide (LPS)-stimulated cells was compared. In LPS-stimulated RAW264.7 cells, TNF-α promoter activity was significantly suppressed by TH2-3, but not by PMA. In addition, PMA activated prostaglandin synthase-associated cyclooxygenase (COX)-2 proteins on the cell surface, while the presence of TH2-3 inhibited its expression. Western blotting demonstrated that the expressions of PKC-μ, phosphorylated (p)-PKCμ at serine (S)-744, and p-PKCδ were activated by PMA, but were suppressed by TH2-3. In addition, p-PKC at S-916 was activated by TH2-3 and inhibited by PMA. In conclusion, the differential regulation of PKC isoforms by PMA and TH2-3 may influence morphological changes and regulation of TNF-α in RAW264.7 cells.     

The açaí flavonoid velutin is a potent anti-inflammatory agent: blockade of LPS-mediated TNF-α and IL-6 production through inhibiting NF-κB activation and MAPK pathway

Recent studies have shown that some flavonoids are modulators of proinflammatory cytokine production. In this study, velutin, a unique flavone isolated from the pulp of açaí fruit (Euterpe oleracea Mart.), was examined for its effects in reducing lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in RAW 264.7 peripheral macrophages and mice peritoneal macrophages. Three other structurally similar and well-studied flavones, luteolin, apigenin and chrysoeriol, were included as controls and for comparative purposes. Velutin exhibited the greatest potency among all flavones in reducing TNF-α and IL-6 production. Velutin also showed the strongest inhibitory effect in nuclear factor (NF)-κB activation (as assessed by secreted alkaline phosphatase reporter assay) and exhibited the greatest effects in blocking the degradation of inhibitor of NF-κB as well as in inhibiting mitogen-activated protein kinase p38 and JNK phosphorylation; all of these are important signaling pathways involved in production of TNF-α and IL-6. The present study led to the discovery of a strong anti-inflammatory flavone, velutin. This compound effectively inhibited the expression of proinflammatory cytokines TNF-α and IL-6 in low micromole levels by inhibiting NF-κB activation and p38 and JNK phosphorylation.