Neutrophil extracellular traps (NETs) are net-like chromatin fibers that can trap and kill microorganisms. Although several anti-inflammatory effects of intravenous anesthetics have been reported, it has not been investigated whether intravenous anesthetics influence NET formation. To compare the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on phorbol myristate acetate (PMA)-induced NET formation and analyze the associated signaling pathways. PMA-stimulated NETs formed in the absence or presence of intravenous anesthetics were stained with SYTOX Green and then quantified. Inhibitors were applied to investigate the related mechanism, which was confirmed by western blotting, and ROS were detected. The neutrophils incubated with propofol showed the lowest degree of NET formation compared with those incubated with the other intravenous anesthetics. Propofol significantly reduced the level of myeloperoxidase (MPO)-derived HOCl but not that of superoxide. Aminopyrine, an MPO inhibitor, markedly decreased the number of PMA-induced NETs, indicating the involvement of HOCl in the inhibitory effect of propofol on NET formation. According to western blotting results, the level of p-ERK was reduced by propofol during PMA-induced NET formation. The ERK inhibitor PD98059 decreased NET formation but did not inhibit PMA-induced HOCl generation, and aminopyrine did not reduce ERK phosphorylation. Through this study, we define a new anti-inflammatory effect of intravenous anesthetics. Of the four intravenous anesthetics tested, propofol was the most potent inhibitor of NET formation. Moreover, propofol resulted in a decrease in PMA-induced NET formation by two independent mechanisms: inhibition of HOCl and p-ERK.

The development of endocrine therapy resistance in the luminal A subtype of breast cancer is related to the appearance of protective autophagy. The bioactive component from the root of licorice, 18β-glycyrrhetinic acid (18β-GA), has many antitumor properties. Whether 18β-GA can modulate autophagy to inhibit proliferation of the luminal A subtype is still unclear. The proportion of apoptosis caused by 18β-GA in MCF-7 and T-47D cells was determined using flow cytometry. The autophagy marker, LC3-II conversion, was investigated using Western blotting, and a Premo^TM Tandem Autophagy Sensor Kit. We found that the concentration (150-μM) of 18β-GA caused caspase-dependent apoptosis and LC3-II accumulation or blocked autophagic flux. Moreover, 18β-GA-mediated apoptosis was improved using rapamycin but reversed by 3-methyladenine (3-MA) addition. The phosphorylation level of Jun-amino-terminal kinase (JNK) was increased significantly in the 18β-GA treatment and combined incubation using rapamycin. A JNK inhibitor (SP600125) significantly inhibited 18β-GA-mediated apoptosis, LC3-II accumulation and rescued the numbers of MCF-7 and T-47D colony formation. Especially, 18β-GA can inhibit xenograft tumor growth in BALB/c nude mice. These data indicate the combination of 18β-GA with rapamycin or 3-MA can sensitize or decrease MCF-7 and T-47D cells to 18β-GA-induced apoptosis, respectively. 18β-GA modulated autophagy is cytotoxic to luminal A subtype breast cancer cells through apoptosis promotion and JNK activation.

Autophagy is a potential target for the treatment of triple negative breast cancer (TNBC). Because of a lack of targeted therapies for TNBC, it is vital to find optimal agents that avoid chemoresistance and metastasis. Flavopereirine has anti-proliferation ability in cancer cells, but whether it regulates autophagy in breast cancer cells remains unclear. A Premo™ Tandem Autophagy Sensor Kit was used to image the stage at which flavopereirine affects autophagy by confocal microscopy. A plasmid that constitutively expresses p-AKT and siRNA targeting p38 mitogen-activated protein kinase (MAPK) was used to confirm the related signaling pathways by Western blot. We found that flavopereirine induced microtubule-associated protein 1 light chain 3 (LC3)-II accumulation in a dose- and time-dependent manner in MDA-MB-231 cells. Confocal florescent images showed that flavopereirine blocked autophagosome fusion with lysosomes. Western blotting showed that flavopereirine directly suppressed p-AKT levels and mammalian target of rapamycin (mTOR) translation. Recovery of AKT phosphorylation decreased the level of p-p38 MAPK and LC3-II, but not mTOR. Moreover, flavopereirine-induced LC3-II accumulation was partially reduced in MDA-MB-231 cells that were transfected with p38 MAPK siRNA. Overall, flavopereirine blocked autophagy via LC3-II accumulation in autophagosomes, which was mediated by the AKT/p38 MAPK signaling pathway.

Anesthetics have immunomodulatory effects, but the use of different assay systems has contributed to inconsistent results in the literature. IL-1β and reactive oxygen species (ROS) secreted by phagocytes are important factors that protect against Staphylococcus aureus infection. In this study, the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on IL-1β secretion, ROS, and bacterial survival in S. aureus-infected RAW264.7 cells were evaluated. S. aureus-infected RAW264.7 cells with or without intravenous anesthetic treatment were established as the experimental model. Cell supernatants were subjected to ELISAs to measure secreted IL-1β. Cell pellets were subjected to qPCR and western blot analyses to analyze IL-1β mRNA and protein levels. Luminol chemiluminescence assays were used to detect ROS, and bacterial survival was determined by counting the colony forming units at the beginning and end of the infection. Compared with the levels after treatment with the other intravenous anesthetics, secreted IL-1β levels were lowest in the supernatant of S. aureus-infected RAW264.7 cell cultures after propofol treatment, but propofol did not decrease IL-1β mRNA or protein expression. However, thiamylal sodium and midazolam decreased IL-1β mRNA and protein expression in a dose-dependent manner. Additionally, propofol substantially decreased S. aureus-stimulated ROS and phagocytosis. Bacterial survival was strongly increased by propofol treatment. Of the four intravenous anesthetics, propofol was the most potent inhibitor of IL-1β secretion and ROS level in S. aureus-infected RAW264.7 cells; moreover, propofol resulted in an increase in bacterial survival by inhibiting ROS and phagocytosis.

Urinary tract infections (UTIs) are prevalent bacterial infection, with uropathogenic Escherichia coli (UPEC) as the primary causative agent. The outer membrane of UPEC contains a lipopolysaccharide (LPS), which plays crucial roles in the host's immune response to infection. Neutrophils use neutrophil extracellular traps (NETs) are mechanism by which neutrophils defend against bacterial infections. However, the exact mechanism by which a bacterial LPS induces NET formation is not well understood. Therefore, the objective of this study is to identify the possible mechanism of LPS-mediated NETs and dissect the LPS domains of UPEC that predominantly modulate NET formation and NET-mediated killing. To investigate the mechanism of bacterial LPS-induced NET formation, we constructed UPEC CFT073 mutants that had rfaD, rfaL and the wzzE deleted with individual LPS biosynthetic genes including the inner core synthase, O-antigen ligase and O-antigen polymerase, respectively. Subsequently, we evaluated the NET/reactive oxygen species (ROS)/IL-1β induction abilities and assessed the activation of toll-like receptor 4 (TLR4)/JNK signaling by CFT073 and its mutants. The results showed that the O-antigen of CFT073 LPS is essential for inducing NET formation through TLR4/JNK/NOX pathways. Inhibition of either pathway significantly decreased the production of ROS, induction of NETs, and secretion of IL-1β. Our results demonstrate that CFT073 LPS is essential for inducing ROS-dependent NETs and IL-1β secretion from neutrophils. This study also provides evidence for the crucial roles of O-antigen in the immune response to UPEC infection, as well as its potential as a therapeutic target for the treatment of UTIs.