Gigantol ameliorates CCl4-induced liver injury via preventing activation of JNK/cPLA2/12-LOX inflammatory pathway

Arachidonic acid (AA) signaling pathway is an important constituent of inflammatory processes. In our previous study, it was found that dihydro-stilbene gigantol relieved hepatic inflammation in mice with CCl4-induced acute liver injury. This study aimed to investigate the involvement of arachidonate metabolic cascade in this process. Our results showed CCl4 activated AA metabolism with the evidence of cPLA2 phosphorylation, which was dependent on the MAPK/JNK activation. Pretreatment with JNK inhibitor SU3327 or gigantol abolished the cPLA2 activation, along with the attenuation of liver damage. Besides, gigantol markedly decreased immune cells activation. Metabolomic analysis revealed that gigantol universally reversed the upregulation of major AA metabolites in injured mouse livers induced by CCl4, especially 12-hydroxyeicosatetraenoic acid (12-HETE). Gigantol also decreased the mRNA and protein expression of platelet-, and leukocyte-type 12-lipoxxygenase (LOX) in the liver. Furthermore, pan-LOX inhibitor nordihydroguaiaretic acid (NDGA) and specific 12-LOX inhibitors baicalein and ML351 attenuated the liver injury to the same extent as gigantol. Overall, our study elucidated a comprehensive profile of AA metabolites during hepatic inflammation caused by CCl4, highlighting the role of 12-LOX-12-HETE pathway in this process. And gigantol alleviated liver inflammation partly through inhibiting the JNK/cPLA2/12-LOX pathway.

Interactions between Cytosolic Phospholipase A2 Activation and Mitochondrial Reactive Oxygen Species Production in the Development of Ventilator-Induced Diaphragm Dysfunction

Cytosolic phospholipase A2 (cPLA2) has been reported to be critical for infection-induced mitochondrial reactive oxygen species (ROS) production and diaphragm dysfunction (DD). In the present study, we aim to investigate whether cPLA2 was involved in ventilator-induced diaphragm dysfunction (VIDD). Our results showed that mechanical ventilation (MV) induced cPLA2 activation in the diaphragm with excessive mitochondrial ROS generation and muscle weakness. Specific inhibition of cPLA2 with CDIBA resulted in decreased mitochondrial ROS levels and improved diaphragm forces. In addition, mitochondria-targeted antioxidant MitoTEMPO attenuated ventilator-induced mitochondrial oxidative stress and downregulated cPLA2 activation in vivo. Both CDIBA and MitoTEMPO were able to attenuate protein degradation, muscle atrophy, and weakness following prolonged MV. Furthermore, laser Doppler imaging showed that MV decreased diaphragm tissue perfusion and induced subsequent hypoxia. An in vitro study also demonstrated a positive association between cPLA2 activation and mitochondrial ROS generation in C2C12 cells cultured under hypoxic condition. Collectively, our study showed that cPLA2 activation positively interacts with mitochondrial ROS generation in the development of VIDD, and ventilator-induced diaphragm hypoxia serves as a possible contributor to this positive feedback loop.

LIM kinase-1 selectively promotes glycoprotein Ib-IX–mediated TXA2 synthesis, platelet activation, and thrombosis

Key Points Role for LIMK1 in GPIb-IX–dependent cPLA2 activation, TXA2 synthesis, and platelet activation independent of its role in actin polymerization. LIMK1 is important in arterial thrombosis in vivo but appears to be dispensable for hemostasis, suggesting a new antithrombotic target.

Both Kdr and Flt1 play a vital role in hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization

Key Points Both VEGFA and VEGFB and their receptors, Kdr and Flt1, are involved in retinal neovascularization.

Agonist-Induced Thromboxane A2 Generation Requires Signaling Through Apoptosis Signal-Regulating Kinase 1 (ASK1): Genetic Ablation of Ask1 Severely Affects In Vivo Thrombosis

Abstract 187 Oxidative stress is a major contributing factor for cardiovascular diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family, which responds to a diverse array of cellular stresses including oxidative stress. Although its downstream signaling effectors, the JNK and p38 MAP kinases, have been shown to be present and activated in platelets, the presence of ASK1 is not known. Here we show that ASK1 is highly expressed in human platelets. During activation of platelets by a variety of physiological agonists, ASK1 is rapidly activated by phosphorylation of threonine (T845) in its activation loop. In order to determine the physiological significance of activation of ASK1 we used Ask1 knockout mouse. Genetic ablation of Ask1 significantly delayed tail-bleeding time (P=0.2×10−9). While all WT mice stopped bleeding with an average bleeding time of 100 s, the Ask1 null mice had an average bleeding time of 576 s, with 5 out of 9 mice that did not stop bleeding for up to 10 min. A carotid artery injury induced by 10% FeCl3 showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine as determined by increased survival and a lack of exclusion of Evans blue dye from the lungs. To determine the cause of such a severe defect in thrombosis, we evaluated ex vivo platelet functions. We found that low doses of agonist-induced platelet aggregation were significantly reduced in Ask1 null mice compared to WT mice. Defective aggregation was found to be due in part to impaired activation of fibrinogen receptor as indicated by reduced fibrinogen binding. When the ability of Ask1 platelets to release platelet granular contents was analyzed, both a- and d-granule secretion were significantly inhibited as assessed by P-selectin exposure and 14C-serotonin release, respectively. Interestingly, aspirin treatment had no effect on the amount of 14C-serotonin release from Ask1 null platelets, but reduced the amount in WT platelets, bringing it to the same level as that of Ask1 null platelets, suggesting that agonist-induced thromboxane generation was impaired in the Ask1 null platelets. This was further confirmed by the complete lack of thrombin-induced TxA2 production in Ask1 null platelets as compared to WT. Since cPLA2 is a key enzyme in the pathway of TxA2 generation, we analyzed the effect of the absence of Ask1 on cPLA2 activation. We found a complete absence of cPLA2 activation-induced by thrombin in Ask1 null platelets while a robust activation of cPLA2 occurred in WT. Additionally, agonist-induced activation of the p38 MAP kinase, known to be involved in cPLA2 activation, was absent in Ask1 null platelets. Furthermore, the TxA2 mimetic U46619-induced platelet aggregation was also greatly reduced in Ask1 null platelets, suggesting that signaling through the TxA2 receptor is also impaired in Ask1 null platelets. These in vitro and in vivo results strongly suggest that ASK1 plays a major role in the regulation of hemostasis and thrombosis by regulating a number of signaling events that are critical for proper platelet function. Disclosures: No relevant conflicts of interest to declare.