Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1ß (IL-1ß) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Exploring the hub genes and mechanisms of Daphne altaica treating esophageal squamous cell carcinoma based on network pharmacology and bioinformatics analysis.

PURPOSE: Esophageal squamous cell carcinoma (ESCC), is a frequent digestive tract malignant carcinoma with a high fatality rate. Daphne altaica (D. altaica), a medicinal plant that is frequently employed in Kazakh traditional medicine, and which has traditionally been used to cure cancer and respiratory conditions, but research on the mechanism is lacking. Therefore, we examined and verified the hub genes and mechanism of D. altaica treating ESCC. METHODS: Active compounds and targets of D. altaica were screened by databases such as TCMSP, and ESCC targets were screened by databases such as GeneCards and constructed the compound-target network and PPI network. Meantime, data sets between tissues and adjacent non-cancerous tissues from GEO database (GSE100942, GPL570) were analyzed to obtain DEGs using the limma package in R. Hub genes were validated using data from the Kaplan-Meier plotter database, TIMER2.0 and GEPIA2 databases. Finally, AutoDock software was used to predict the binding sites through molecular docking. RESULTS: In total, 830 compound targets were obtained from TCMSP and other databases. In addition, 17,710 disease targets were acquired based on GeneCards and other databases. In addition, we constructed the compound-target network and PPI network. Then, 127 DEGs were observed (82 up-regulated and 45 down-regulated genes). Hub genes were screened including TOP2A, NUF2, CDKN2A, BCHE, and NEK2, and had been validated with the help of several publicly available databases. Finally, molecular docking results showed more stable binding between five hub genes and active compounds. CONCLUSIONS: In the present study, five hub genes were screened and validated, and potential mechanisms of action were predicted, which could provide a theoretical understanding of the treatment of ESCC with D. altaica.

Oridonin relieves depressive-like behaviors by inhibiting neuroinflammation and autophagy impairment in rats subjected to chronic unpredictable mild stress.

Major depressive disorder (MDD) is a severe life-threatening disorder with increasing prevalence. However, the mechanistic interplay between depression, neuroinflammation, and autophagy is yet to be demonstrated. This study investigated the effect of Oridonin on CUMS-induced depression, neuroinflammation, and autophagy impairment. Male 4-week-old Sprague-Dawley rats were subjected to chronic unpredictable mild stress (CUMS), some of which were injected with Oridonin, fluoxetine (FLX), or their combination at different durations of CUMS. CUMS significantly increased the levels of cytokines (IL-1ß, IL-18, and caspase-1), reduced autophagy-related protein levels (Beclin-1, p62, Atg5, and LC3B), and caused microglia cells activation. Oridonin prevented and reversed the depressive-like behavior. Furthermore, it has a stronger and longer-lasting antidepressant effect than FLX. And the antidepressant effect of Oridonin in combination with fluoxetine was greater than that of high-dose fluoxetine alone. In addition, Oridonin significantly normalized autophagy-related protein levels, and reduced levels of cytokines by blocking the interaction between NLRP3 and NEK7. Similarly, Oridonin abolished levels of cytokines and reversed autophagy impairment in LPS-activated BV2 cells. All these results supported our hypothesis that Oridonin possesses potent anti-depressive action, which might be mediated via inhibition of neuroinflammation and autophagy impairment by blocking the interaction between NLRP3 and NEK7.

Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interaction.

The activation of the nucleotide oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is related to the pathogenesis of a wide range of inflammatory diseases, but drugs targeting the NLRP3 inflammasome are still scarce. In the present study, we demonstrated that Licochalcone B (LicoB), a main component of the traditional medicinal herb licorice, is a specific inhibitor of the NLRP3 inflammasome. LicoB inhibits the activation of the NLRP3 inflammasome in macrophages but has no effect on the activation of AIM2 or NLRC4 inflammasome. Mechanistically, LicoB directly binds to NEK7 and inhibits the interaction between NLRP3 and NEK7, thus suppressing NLRP3 inflammasome activation. Furthermore, LicoB exhibits protective effects in mouse models of NLRP3 inflammasome-mediated diseases, including lipopolysaccharide (LPS)-induced septic shock, MSU-induced peritonitis and non-alcoholic steatohepatitis (NASH). Our findings indicate that LicoB is a specific NLRP3 inhibitor and a promising candidate for treating NLRP3 inflammasome-related diseases.

Betulin, a Newly Characterized Compound in Acacia auriculiformis Bark, Is a Multi-Target Protein Kinase Inhibitor.

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.

Berberine Directly Targets the NEK7 Protein to Block the NEK7-NLRP3 Interaction and Exert Anti-inflammatory Activity.

Berberine (BBR), a traditional Chinese medicine, has therapeutic effects on a variety of inflammation-related diseases, but its direct proteomic targets remain unknown. Using activity-based protein profiling, we first demonstrated that BBR directly targets the NEK7 protein via the hydrogen bond between the 2,3-methylenedioxy and 121-arginine (R121) residues. The fact that R121 is located precisely within the key domain involved in the NEK7-NLRP3 interaction allows BBR to specifically block the NEK7-NLRP3 interaction and successively inhibit IL-1ß release, independent of the NF-κB and TLR4 signaling pathways. Moreover, BBR displays in vivo anti-inflammatory efficacy in a NEK7-dependent manner. Therefore, we consider NEK7 to be a key target of BBR in the treatment of NLRP3-related inflammatory diseases, and the development of novel NEK7-NLRP3 interaction inhibitors might be easily achieved using NEK7 as a target.

Inhibiting the NLRP3 Inflammasome.

Inflammasomes are protein complexes which are important in several inflammatory diseases. Inflammasomes form part of the innate immune system that triggers the activation of inflammatory cytokines interleukin (IL)-1ß and IL-18. The inflammasome most studied in sterile inflammation and non-communicable disease is the NLRP3 inflammasome. Upon activation by diverse pathogen or disease associated signals, NLRP3 nucleates the oligomerization of an adaptor protein ASC forming a platform (the inflammasome) for the recruitment and activation of the protease caspase-1. Active caspase-1 catalyzes the processing and release of IL-1ß and IL-18, and via cleavage of the pore forming protein gasdermin D can drive pyroptotic cell death. This review focuses on the structural basis and mechanism for NLRP3 inflammasome signaling in the context of drug design, providing chemical structures, activities, and clinical potential of direct inflammasome inhibitors. A cryo-EM structure of NLRP3 bound to NEK7 protein provides structural insight and aids in the discovery of novel NLRP3 inhibitors utilizing ligand-based or structure-based approaches.

[Molecular regulative mechanisms of NLRP3 inflammasome activation in diabetic nephropathy and interventional effects of Chinese herbal medicine].

The progression of renal damage in diabetic nephropathy(DN)is closely related to Nod-like receptor protein3(NLRP3)inflammasome activation. The characteristics of NLRP3 inflammasome activation include the changed expression and combination levels of NLRP3, apoptosis-associated speck-like protein(ASC)and pro-caspase-1, the increased expression levels of caspase-1, interleukin(IL)-1ß and IL-18 and the excessive release levels of the relative inflammatory mediators. Its molecular regulative mechanisms involve the activation of multiple signaling pathways including reactive oxygen species(ROS)/thioredoxin-interacting protein(TXNIP)pathway, nuclear factor(NF)-κB pathway, nuclear factor erythroid-related factor 2(Nrf2)pathway, long non-coding RNA(lncRNA)pathway and mitogen-activated protein kinases(MAPKs)pathway. In addition, more importantly, never in mitosis aspergillus-related kinase 7(Nek7), as a kinase regulator, could target-combine with NLRP3 at upstream to activate NLRP3 inflammasome. Some extracts of Chinese herbal medicines(CHMs)such as quercetin, curcumin, cepharanthine, piperine and salidroside, as well as Chinese herbal compound prescriptions such as Wumei Pills both could treat NLRP3 inflammasome to ameliorate inflammatory renal damage in DN. Therefore, accurately clarifying the targets of anti-inflammatory CHMs and Chinese herbal compound prescriptions delaying DN progression by targeting the molecular regulative mechanisms of NLRP3 inflammasome activation will be one of the development directions in the future.

Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity.

Oridonin (Ori) is the major active ingredient of the traditional Chinese medicinal herb Rabdosia rubescens and has anti-inflammatory activity, but the target of Ori remains unknown. NLRP3 is a central component of NLRP3 inflammasome and has been involved in a wide variety of chronic inflammation-driven human diseases. Here, we show that Ori is a specific and covalent inhibitor for NLRP3 inflammasome. Ori forms a covalent bond with the cysteine 279 of NLRP3 in NACHT domain to block the interaction between NLRP3 and NEK7, thereby inhibiting NLRP3 inflammasome assembly and activation. Importantly, Ori has both preventive or therapeutic effects on mouse models of peritonitis, gouty arthritis and type 2 diabetes, via inhibition of NLRP3 activation. Our results thus identify NLRP3 as the direct target of Ori for mediating Ori's anti-inflammatory activity. Ori could serve as a lead for developing new therapeutics against NLRP3-driven diseases.

Phosphorylation of the mitotic regulator protein Hec1 by Nek2 kinase is essential for faithful chromosome segregation.

Hec1 (highly expressed in cancer) plays essential roles in chromosome segregation by interacting through its coiled-coil domains with several proteins that modulate the G(2)/M phase. Hec1 localizes to kinetochores, and its inactivation either by genetic deletion or antibody neutralization leads to severe and lethal chromosomal segregation errors, indicating that Hec1 plays a critical role in chromosome segregation. The mechanisms by which Hec1 is regulated, however, are not known. Here we show that human Hec1 is a serine phosphoprotein and that it binds specifically to the mitotic regulatory kinase Nek2 during G(2)/M. Nek2 phosphorylates Hec1 on serine residue 165, both in vitro and in vivo. Yeast cells are viable without scNek2/Kin3, a close structural homolog of Nek2 that binds to both human and yeast Hec1. When the same yeasts carry an scNek2/Kin3 (D55G) or Nek2 (E38G) mutation to mimic a similar temperature-sensitive nima mutation in Aspergillus, their growth is arrested at the nonpermissive temperature, because the scNek2/Kin3 (D55G) mutant binds to Hec1 but fails to phosphorylate it. Whereas wild-type human Hec1 rescues lethality resulting from deletion of Hec1 in Saccharomyces cerevesiae, a human Hec1 mutant or yeast Hec1 mutant changing Ser(165) to Ala or yeast Hec1 mutant changing Ser(201) to Ala does not. Mutations changing the same Ser residues to Glu, to mimic the negative charge created by phosphorylation, partially rescue lethality but result in a high incidence of errors in chromosomal segregation. These results suggest that cell cycle-regulated serine phosphorylation of Hec1 by Nek2 is essential for faithful chromosome segregation.

Expression of a constitutively active Ca2+/calmodulin-dependent kinase in Aspergillus nidulans spores prevents germination and entry into the cell cycle.

The unique gene for Ca2+/calmodulin-dependent protein kinase (CaMK) has been shown to be essential in Aspergillus nidulans. Disruption of the gene prevents entry of spores into the nuclear division cycle. Here we show that expression of a constitutively active form of CaMK also prevents spores from entering the first S phase in response to a germinating stimulus. Expression of the constitutively active kinase induces premature activation of NIMEcyclin B/NIMXcdc2 in G0/G1. As NIMXcdc2 is present in spores, the elevation of maturation promotion factor activity may be secondary to the early production of NIMEcyclin B or post-translation modification of maturation promotion factor. The expression of the constitutively active CaMK also results in the appearance of NIMA kinase activity within 1 h of the germinating signal. These results support the contention that the activities of maturation promotion factor and NIMA are coincidentally regulated in A. nidulans and suggest that the unscheduled appearance of one or both of these activities may be sufficient to prevent A. nidulans spores from entering into DNA synthesis.