Liu Y

NF-κB signaling is essential for resistance to heat stress-induced early stage apoptosis in human umbilical vein endothelial cells

Cell apoptosis induced by heat stress is regulated by a complex signaling network. We previously reported that a p53-dependent pathway is involved. Here, we present evidence that NF-κB signaling plays a crucial role in preventing heat stress-induced early apoptosis. Human umbilical vein endothelial cells (HUVECs) were examined and increased phosphorylation of p65 and IκBα were detected, without IκBα degradation. When NF-κB signaling was inhibited by BAY11-7082, or a small interference RNA (siRNA) targeting p65, a significant increase in cell apoptosis and caspase-3 activity was observed, as well as reduced expression and translocation of HSP27 into the nucleus, an accumulation of reactive oxygen species, and prolonged phosphorylation of mitogen-activated protein kinases (MAPKs). In addition, an association between HSP27 and p65 was identified which may enhance NF-κB activation. When HSP27 was overexpressed, pretreatment of HUVECs with the antioxidant, apocynin, or N-acetyl cysteine, suppressed apoptosis. Similarly, inhibition of JNK and p38 with SP600125 and SB203580, respectively, also suppressed apoptosis, whereas siRNA-mediated HSP27 knockdown and treatment with the ERK 1/2 inhibitor PD98059 did otherwise. In conclusion, these findings suggest a novel role for an NF-κB signaling pathway involving HSP27, ROS, and MAPKs that confers a protective effect against heat stress-induced cell apoptosis.

Heat Stress-Induced Disruption of Endothelial Barrier Function Is via PAR1 Signaling and Suppressed by Xuebijing Injection

Increased vascular permeability leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is central to the pathogenesis of heatstroke. Protease-activated receptor 1 (PAR1), the receptor for thrombin, plays a key role in disruption of endothelial barrier function in response to extracellular stimuli. However, the role of PAR1 in heat stress-induced endothelial hyper-permeability is unknown. In this study, we measured PAR1 protein expression in heat-stressed human umbilical venous endothelial cells (HUVECs), investigated the influences of PAR1 on endothelial permeability, F-actin rearrangement, and moesin phosphorylation by inhibiting PAR1 with its siRNA, neutralizing antibody (anti-PAR1), specific inhibitor(RWJ56110), and Xuebijing injection (XBJ), a traditional Chinese medicine used for sepsis treatment, and evaluated the role of PAR1 in heatstroke-related ALI/ARDS in mice by suppressing PAR1 with RWJ56110, anti-PAR1and XBJ. We found that heat stress induced PAR1 protein expression 2h after heat stress in endothelial cells, caused the release of endothelial matrix metalloprotease 1, an activator of PAR1, after 60 or 120 min of heat stimulation, as well as promoted endothelial hyper-permeability and F-actin rearrangement, which were inhibited by suppressing PAR1 with RWJ56110, anti-PAR1 and siRNA. PAR1 mediated moesin phosphorylation, which caused F-actin rearrangement and disruption of endothelial barrier function. To corroborate findings from in vitro experiments, we found that RWJ56110 and the anti-PAR1 significantly decreased lung edema, pulmonary microvascular permeability, protein exudation, and leukocytes infiltrations in heatstroke mice. Additionally, XBJ was found to suppress PAR1-moesin signal pathway and confer protective effects on maintaining endothelial barrier function both in vitro and in vivo heat-stressed model, similar to those observed above with the inhibition of PAR1. These results suggest that PAR1 is a potential therapeutic target in heatstroke.

Xuebijing injection reduces organ injuries and improves survival by attenuating inflammatory responses and endothelial injury in heatstroke mice

BackgroundThe pathogenesis of heatstroke is a multi-factorial process involved with an interplay among subsequent inflammation, endothelial injury and coagulation disturbances, which makes pharmacological therapy of heatstroke a challenging problem. Xuebijing injection (XBJ), a traditional Chinese medicine used to sepsis, has been reported to suppress inflammatory responses and restore coagulation disturbances. However, little is known about the role of XBJ in heatstroke.MethodsMice were treated with indicated dose of XBJ before and/or after the induction of heatstroke. Serum inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and endothelial markers, von Willebrand Factor (vWF) and E-selectin, were measured by ELISA. Liver, kidney and heart profiles including alanine aminotransferase, aspartic aminotransferase, creatinine, blood urea nitrogen, and lactate dehydrogenase, were evaluated by UniCel DxC 800 Synchron Clinical Systems, and troponin was measured by ELISA. Coagulation profiles, including thrombin time, prothrombin time, activated partial thromboplastin time, international normalized ratio, and fibrinogen were examined by STA Compact® Hemostasis System. Jejunum injury was evaluated with H&E staining. Changes in mitochondrial structure in cardiac tissue were assesed by electron microscopy.ResultsPretreatment with XBJ decreased serum pro-inflammatory cytokines including TNF-α and IL-6, as well as endothelial injury markers, vWF and E-selectin, in a dose-dependent manner in heatstroke mice. Similar protective effects were observed when XBJ was administered after, or both before and after heat insult. These protective effects lasted for over 12xa0h in mice receiving XBJ before and after heat insult. XBJ also improved survival rates in heatstroke mice, ameliorated liver, heart, and kidney injuries, including mitochondrial damage to the heart, and reduced coagulation disturbances.ConclusionsXBJ prevents organ injuries and improves survival in heatstroke mice by attenuating inflammatory responses and endothelial injury. XBJ may be a potentially useful in the prevention and treatment of heatstroke.

PAR1‑mediated c‑Jun activation promotes heat stress‑induced early stage apoptosis of human umbilical vein endothelial cells

Our previous study indicated that when human umbilical vein endothelial cells (HUVECs), which are involved in endothelial barrier function, are heat stressed, levels of protease-activated receptor 1 (PAR1) are increased significantly. In the present study, it was demonstrated that PAR1 serves a vital role in heat stress-induced HUVEC apoptosis. When the PAR1 inhibitor, SCH79797 (SCH), or a small interfering RNA (siRNA) targeting PAR1 were used to inhibit PAR1 signaling, a marked decrease in cell apoptosis, caspase-3 activity and the expression of the pro-apoptotic protein B-cell lymphoma 2 (Bcl-2) associated X (Bax), as well as increased expression of the anti-apoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), were observed. In addition, heat stress-induced apoptosis, caspase-3 activity and the expression of Bax were significantly increased following administration of the PAR1 agonist, TFLLR-NH2 or adenovirus overexpression of PAR1. This was accompanied by decreased protein expression levels of Mcl-1. Furthermore, it was identified that the DNA binding activity of the nuclear factor (NF)-κB p65 subunit increased and c-Jun activation was reduced as a result of inhibition of PAR1 signaling by SCH or siRNA-mediated PAR1 knockdown in heat stress-induced HUVECs. Additionally, our previous study reported that NF-κB p65 activation may have an anti-apoptosis effect on heat stressed HUVECs, whereas in the present study c-Jun activation had a pro-apoptosis effect on heat stressed HUVECs. Taken together, these results indicated that PAR1 signaling-mediated c-Jun activation promotes early apoptosis of HUVEC cells induced by heat stress.

Oxidative stress regulates mitogen‑activated protein kinases and c‑Jun activation involved in heat stress and lipopolysaccharide‑induced intestinal epithelial cell apoptosis

Heat stress and gut-derived endotoxinemia are common causes of multiple organ dysfunction syndrome in heat stroke patients. Evidence has demonstrated that cell apoptosis in the small intestine serves an important role in the pathogenesis of heatstroke, which leads to increased intestinal permeability to endotoxin or lipopolysaccharides (LPS) from the gut entering the circulation. However, little is known about the potential underlying mechanisms mediating heat stress combined with LPS-induced intestinal epithelial cell apoptosis. In the present study, LPS combined with heat stress induced production of reactive oxygen species (ROS), mitochondrial membrane potential disruption and cell apoptosis, which eventually led to increased intestinal permeability and reduced epithelial resistance in the IEC-6 cell line. Inductions in ROS, mitochondrial membrane potential disruption and cell apoptosis were detected by using an ROS assay kit, 5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazo carbocyanine iodide dye kit and annexin V-fluorescein isothiocyanate apoptosis kit, respectively. The effect of ROS on mitogen activated protein kinases (MAPKs) and c-Jun activation was investigated using the antioxidant drug, butylated hydroxyanisole (BHA) by western blotting. The results of the present study demonstrated that ROS is essential to activate p38, extracellular signal-regulated kinase (ERK) and c-Jun, but not c-Jun N-terminal kinase (JNK), in LPS combined with heat stress treated cells. Furthermore, ROS, and activation of p38, JNK and c-Jun, were revealed to serve pro-apoptosis roles which aggravated damage to epithelial barrier integrity, as assessed by flow cytometry using Annexin V-fluorescein isothiocyanate staining and pretreatment of cells with specific inhibitors of ROS, JNK, p38 and c-Jun (BHA, SP600125, SB203580 and c-Jun peptide, respectively). Transepithelial electrical resistance and horseradish peroxidase permeability were detected in cells treated with LPS combined with heat stress, which revealed that ERK serves an anti-apoptosis role, as determined by pretreatment of cells with PD98059, a specific inhibitor of ERK. In conclusion, these findings suggested a novel role of the ROS signaling pathway which involved activation of MAPKs and c-Jun, following LPS combined with heat stress-induced IEC-6 cell apoptosis and impairment of the epithelial barrier. These results may facilitate understanding of pathological conditions involving ROS, such as heat stroke.

Sirt1 Protects Endothelial Cells against LPS-Induced Barrier Dysfunction

Sepsis is a threatening health problem and characterized by microvascular dysfunction. In this study, we verified that LPS caused the downregulation of Sirt1 and the hyperpermeability of endothelial cells. Inhibition of Sirt1 with ex527 or Sirt1 siRNA displayed a higher permeability, while activation of Sirt1 with SRT1720 reversed the LPS-induced hyperpermeability, formation of fiber stress, and disruption of VE-cadherin distribution. In pulmonary microvascular vein endothelial cells isolated from wild-type mice, Sirt1 was attenuated upon LPS, while Sirt1 was preserved in a receptor of advanced glycation end product-knockout mice. The RAGE antibody could also diminish the downregulation and ubiquitination of Sirt1 in LPS-exposed human umbilical vein endothelial cells. An LPS-induced decrease in Sirt1 activity was attenuated by the RAGE antibody and TLR4 inhibitor. In vivo study also demonstrated the attenuating role of Sirt1 and RAGE knockout in LPS-induced increases in dextran leakage of mesenteric venules. Furthermore, activation of Sirt1 prevented LPS-induced decreases in the activity and expression of superoxide dismutase 2, as well as the increases in NADPH oxidase 4 and reactive oxygen species, while inhibition of Sirt1 aggravated the SOD2 decline. It also demonstrated that Sirt1-deacetylated p53 is required for p53 inactivation, which reversed the downregulation of β-catenin caused by LPS.

p38 MAPK‑MK2 pathway regulates the heat‑stress‑induced accumulation of reactive oxygen species that mediates apoptotic cell death in glial cells

Previous studies have demonstratedf that heat stress can induce injury of the central nervous system and lead to neuronal cell apoptosis. However, the molecular mechanisms underlying these cellular changes remain unclear. In the present study, flow cytometry was used to investigate heat-stress-induced apoptosis, and caspase-3 activation was also assessed in neurons. The role of reactive oxygen species (ROS) accumulation in the heat-stress-induced apoptosis of neurons was demonstrated using the antioxidant drug manganese (III) tetrakis (4-benzoic acid)porphyrin. The present study presents evidence that heat stress induces mitogen-activated protein kinase (MAPK) activation in rat malignant glioma F98 cells. Following the inhibition of different MAPKs with a range of specific inhibitors, SB203580 (an inhibitor of p38 MAPK), but not PD98059 (an inhibitor of extracellular signal-regulated kinases) or SP600125 (an inhibitor of c-Jun N-terminal kinases), diminished the production of ROS and apoptosis, and prevented activation of the p38-downstream kinase MAPK-activated protein kinase 2 (MK2) in neurons. Inhibiting MK2 with dominant negative adenoviral constructs or a specific inhibitor significantly decreased normal and heat-stress-induced ROS accumulation and cell apoptosis, whereas inhibition of another kinase downstream of p38 MAPK, MAPK-activated protein kinase 5, by transfection with another adenoviral construct did not exert the same effects. Taken together, these findings indicate that heat stress stimulation induces p38-MK2 pathway activation, which exerts a pro-apoptotic effect by regulating ROS accumulation in neurons.

NF‑κB regulates HSF1 and c‑Jun activation in heat stress‑induced intestinal epithelial cell apoptosis

Heat stress may induce intestinal epithelial cell apoptosis; however, the molecular mechanisms have not yet been identified. The present study used IEC‑6 rat small intestinal epithelial cells to investigate heat stress‑induced production of reactive oxygen species (ROS), which may be involved in nuclear factor (NF)‑κB activation during heat stress. IEC‑6 cells were transfected with NF‑κB p65‑specific small interfering RNA (siRNA), and observed a significant increase in cell apoptosis and caspase‑3 cleavage; however, in cells transfected with adenovirus that constitutively overexpressed p65, the opposite results were obtained. Furthermore, p65 knockdown increased the heat stress‑induced expression and activity of heat shock transcription factor 1 (HSF1); conversely, p65 overexpression slightly decreased HSF1 activity. The levels of heat stress‑induced c‑Jun phosphorylation were also examined: Knockdown of p65 resulted in a reduction of c‑Jun phosphorylation, whereas p65 overexpression resulted in increased phosphorylation. Furthermore, siRNA‑mediated knockdown of HSF1 in IEC‑6 cells significantly increased heat stress‑induced apoptosis. Cells pretreated with c‑Jun peptide, an inhibitor of c‑Jun activation, exhibited a significant reduction in apoptosis. These findings indicated that heat stress stimulation in IEC‑6 cells induced the pro‑apoptotic role of NF‑κB by regulating HSF1 and c‑Jun activation.