anisomycin and Necrosis

Acute liver failure (ALF) is a life-threatening disease characterized by abrupt and extensive hepatic necrosis and apoptosis, resulting in high mortality. The approved drug, N-acetylcysteine (NAC), is only effective for acetaminophen (APAP)-associated ALF at the early stage. Thus, we investigate whether fluorofenidone (AKF-PD), a novel antifibrosis pyridone agent, protects against ALF in mice and explore its underlying mechanisms.. ALF mouse models were established using APAP or lipopolysaccharide/D-galactosamine (LPS/D-Gal). Anisomycin and SP600125 were used as JNK activator and inhibitor, respectively, and NAC served as a positive control. Mouse hepatic cell line AML12 and primary mouse hepatocytes were used for in vitro studies.. AKF-PD pretreatment alleviated APAP-induced ALF with decreased necrosis, apoptosis, reactive oxygen species (ROS) markers, and mitochondrial permeability transition in liver. Additionally, AKF-PD alleviated mitochondrial ROS stimulated by APAP in AML12 cells. RNA-sequencing in the liver and subsequent gene set enrichment analysis showed that AKF-PD significantly impacted MAPK and IL-17 pathway. In vitro and in vivo studies demonstrated that AKF-PD inhibited APAP-induced phosphorylation of MKK4/JNK, while SP600125 only inhibited JNK phosphorylation. The protective effect of AKF-PD was abolished by anisomycin. Similarly, AKF-PD pretreatment abolished hepatotoxicity caused by LPS/D-Gal, decreased ROS levels, and diminished inflammation. Furthermore, unlike NAC, AKF-PD, inhibited the phosphorylation of MKK4 and JNK upon pretreatment, and improved survival in cases of LPS/D-Gal-induced mortality with delayed dosing.. In summary, AKF-PD can protect against ALF caused by APAP or LPS/D-Gal, in part, via regulating MKK4/JNK pathway. AKF-PD might be a novel candidate drug for ALF.

Topics: Acetaminophen; Animals; Anisomycin; Hepatocytes; Lipopolysaccharides; Liver; Liver Failure, Acute; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Necrosis; Pyridones; Reactive Oxygen Species

Necroptosis is an essential pathophysiological process in cerebral ischemia-related diseases. Therefore, targeting necroptosis may prevent cell death and provide a much-needed therapy. Ansiomycin is an inhibitor of protein synthesis which can also activate c-Jun N-terminal kinases. The present study demonstrated that anisomycin attenuated necroptosis by upregulating CHIP (carboxyl terminus of Hsc70-interacting protein) leading to the reduced levels of receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3) proteins in two in vitro models of cerebral ischemia. Further exploration in this research revealed that losing neither the co-chaperone nor the ubiquitin E3 ligase function of CHIP could abolish its ability to reduce necroptosis. Collectively, this study identifies a novel means of preventing necroptosis in two in vitro models of cerebral ischemia injury through activating the expression of CHIP, and it may provide a potential target for the further study of the disease.

Topics: Animals; Anisomycin; Anti-Bacterial Agents; Apoptosis; Cell Hypoxia; Female; Gene Expression Regulation, Enzymologic; Glucose; Mice; Necrosis; Neuroblastoma; Oxygen; Rats; Rats, Sprague-Dawley; Ubiquitin; Ubiquitin-Protein Ligases

Apoptosis occurring during ischaemia /reperfusion contributes independently to tissue damage, and involves activation of the stress-kinase, p38 MAPK during reperfusion. Ischaemic preconditioning (IPC) protects against ischaemia/reperfusion mediated necrosis and apoptosis. The role of p38 MAPK in the protective effect of preconditioning against apoptosis is unknown. Pharmacologic preconditioning with isoproterenol (beta-PC) also protects against necrosis, but it is not known whether it protects against apoptosis.. The aim of the study was to investigate whether the protective effect of IPC against apoptosis is related to activation of p38 MAPK and whether beta-PC also protects against apoptosis.. Isolated perfused rat hearts were used to study the effect of ischaemia and reperfusion on apoptosis and infarct size. Ischaemic preconditioning was elicited by 3 x 5 min global ischaemia, and beta-PC by 5 min isoproterenol 10(-7) M. For infarct size hearts were subjected to regional ischaemia for 35 min followed by 120 min reperfusion. Infarct size was determined by the tetrazolium staining technique, and expressed as percentage of area at risk. For markers of apoptosis hearts were subjected to global ischaemia of 25 min plus 30 min reperfusion. Apoptosis was determined by Western blot using antibodies against caspase-3 and PARP. p38 MAPK activation was inhibited by SB203580 (1 microM) administration 10 min prior to commencing ischaemia, and bracketing the IPC and beta-PC preconditioning protocols. p38 MAPK was activated by administration of anisomycin (5 microM) 10 min prior to index ischaemia in one protocol, and 10 min during reperfusion in non-preconditioned as well as IPC and beta-PC hearts. Results were analysed using ANOVA and a Newman-Keuls post-hoc test.. In the apoptosis model using global ischaemia, IPC and beta-PC both resulted in a significant decrease in p38 MAPK activation at the end of reperfusion when compared to non-preconditioned hearts. This was accompanied by a significant decrease in apoptosis as measured with both caspase-3 activation and PARP cleavage. Inhibiting p38 MAPK by administration of SB203580 10 min prior to ischaemia resulted in a significant reduction in both markers of apoptosis. Bracketing the triggering phase of either IPC or beta-PC with SB203580 resulted in attenuated p38 MAPK activation during reperfusion and did not abolish the protective effect of IPC or beta-PC against apoptosis. Activating p38 MAPK with anisomycin prior to ischaemia resulted in a reduction of markers of apoptosis, whereas activation of p38 MAPK with anisomycin during reperfusion did not exacerbate apoptosis in any groups, exept for an increase in PARP cleavage in IPC hearts. In the model of regional ischaemia, IPC and beta-PC reduced infarct size significantly, and to the same extent as inhibition of p38 MAPK by administration of SB203580 10 min prior to ischaemia. Bracketing the triggering phase of either IPC or beta-PC did not abolish the reduction in infarct size. Activating p38 MAPK during reperfusion was accompanied by an increase in infarct size only in IPC hearts, but not in beta-PC hearts.. These results indicate that (1) Both IPC and beta-PC elicit protection against apoptosis and necrosis, (2) activation of p38 MAPK is not a trigger of preconditioning against apoptosis and necrosis and (3) activation of p38 MAPK during reperfusion increases necrosis only if ischaemia is used to precondition hearts, but not with pharmacologic preconditioning with isoproterenol.

Topics: Adrenergic beta-Agonists; Animals; Anisomycin; Apoptosis; Enzyme Activators; Imidazoles; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Isoproterenol; Models, Animal; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Necrosis; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Pyridines; Rats