Journal of Neuroinflammation
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2026
·Vol. 23
·期 1
·54
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IF 11.5
Shi Xin,Dong Jing,Yang Ying,He Yi-Lin,Li Heng,Du Tong,Yang Bing,Yang Chun-Lin,Zhang Peng,Xin Tao
Background The NLRP3 inflammasome is a supramolecular complex that mediates pyroptosis and the production of inflammatory cytokines. Aberrant activation of the NLRP3 inflammasome has been implicated in the pathogenesis of various diseases, including cancers, neurodegenerative disorders, metabolic diseases, and autoimmune conditions. Methylglyoxal (MGO), a highly reactive carbonyl compound derived from glycolysis, exerts unclear effects on NLRP3 inflammasome activation. This study aimed to clarify the impact of MGO on NLRP3 inflammasome activity and its underlying mechanism. Methods NLRP3 inflammasome activation was assessed in vitro in macrophages primed with LPS and stimulated with NLRP3 activators (e.g., nigericin, ATP, or imiquimod), and in vivo using LPS-induced peritonitis and experimental autoimmune encephalomyelitis (EAE) mouse models. Techniques including immunoblotting, immunofluorescent staining, flow cytometry, ELISA, and immunoprecipitation were employed to investigate the effects of MGO on NLRP3 inflammasome activation and its mechanism of action. Results MGO significantly suppressed NLRP3 inflammasome activation in macrophages, reducing pyroptosis and mature IL-1β release. It ameliorated inflammatory cell infiltration in the mouse models of LPS-induced peritonitis and EAE. Mechanistically, MGO did not interfere with the potassium efflux or α-tubulin acetylation triggered by NLRP3 inflammasome activators, but rather MGO covalently bonded to NLRP3, interrupted the interaction between NLRP3 and NEK7, which interfered with the assembly of NLRP3 inflammasome and subsequently suppressed pyroptosis and production of mature, bioactive inflammatory cytokines. Conclusions MGO suppresses NLRP3 inflammasome activation and associated inflammation by covalently binding NLRP3 and disrupting its interaction with NEK7. This reveals a novel mechanism for MGO in innate immunity and identifies NLRP3 as a direct MGO target, suggesting potential therapeutic strategies for NLRP3-driven inflammatory diseases.
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PROTEIN AND PEPTIDE LETTERS
·
2021
·Vol. 28
·期 12
·1391-1397
·
IF 1.89
Zhang Xing-Jie,Wang Ting-Ting,Pu Yu-Kun,Zeng Lin,Zhang Rui-Han,Li Xiao-Li,Ji Xu,Xiao Wei-Lie
Background: NIMA (never in mitosis, gene A) serine/threonine kinase 7 (NEK7) is a regulator of mitosis spindle in mammals and is considered as a drug target of inflammasome related inflammatory diseases. However, most commercially available or reported recombinant NEK7 proteins are
either inactive or have low purity. These shortcomings limit the pharmacological studies and development of NEK7 inhibitors. Objective: To elucidate what causes the NEK7 low purity in E. coli, and optimize a protocol to improve the protein purity. Methods: A comparative
study of expression full length NEK7 with an N-terminal His-tag or a Cterminal His-tag was performed. His-affinity resin, ion exchange and gel filtration chromatography were used to purify NEK7. The protein was identified by mass spectrometry. The activity and folding of NEK7 were evaluated
by chemiluminescent assay and thermal shift assay. Results: Our results demonstrated that N-terminal tagged protein was toxic to E. coli, resulting in incomplete translated products. The C-terminal tagged NEK7-His6 had a much higher purity than that of an N-terminal
tag. The Ni2+ resin one-step purification led to a purity of 91.7%, meeting the criteria of most kinase assays. With two-step and three-step procedures, the protein purities were 94.7% and ~100%, respectively. The NEK7 purified in this work maintained its kinase activity
and correct conformation, and the compound-protein interaction ability. Conclusion: Our optimized protocol could produce good purity of His tagged NEK7 in E. coli, and the kinase activity and biophysical characteristics of which are preserved.
研究领域:
分子生物学
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蛋白质工程
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药理学
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炎症
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FREE RADICAL BIOLOGY AND MEDICINE
·
2026
·
IF 8
Hengchang Yao,Lichao Yang,Yawei Zhang,Qiang Wu,Kailing Xie,Hao Liu,Baojia Yao,Zhixian Jiang,Qi Sun,Zhuoxian Zeng,Lianwen Yuan
Inflammatory Bowel Disease (IBD) is a chronic inflammatory disorder where pyroptosis of intestinal epithelial cells, driven by the NEK7/NLRP3 axis, is a key pathological feature. This study aimed to identify a novel inhibitor of NIMA-related kinase 7 (NEK7) and develop a targeted delivery system to enhance its therapeutic efficacy for IBD. Through a virtual screening of FDA-approved drugs, nebivolol was identified as a potent NEK7 inhibitor. Subsequent biochemical and cellular assays confirmed that nebivolol directly binds to the LYS64 site of NEK7, inhibiting its kinase activity and disrupting its interaction with NLRP3. This inhibitory action was shown to protect intestinal epithelial cells from pyroptosis in vitro and to significantly ameliorate disease symptoms, histological damage, and molecular markers of pyroptosis in a chronic DSS mouse model of colitis, with a high safety profile. To enhance therapeutic efficacy and specificity, a quantitatively characterized targeted delivery system of lactoferrin-modified liposomes loaded with nebivolol (LF-Lipo@N) was developed. This system demonstrated superior uptake by intestinal cells via lactoferrin and lactoferrin receptors (LFRs)interaction and a more potent anti-pyroptotic effect in vitro . Crucially, the quantitative biodistribution analysis showed that the targeted LF-Lipo@N system significantly accumulated in the colon in vivo , leading to a significantly enhanced therapeutic effect compared to free nebivolol while eliminating systemic side effects on heart rate. In conclusion, the targeted delivery of nebivolol via lactoferrin-modified liposomes represents a promising and effective strategy for treating IBD by specifically inhibiting NEK7-mediated intestinal pyroptosis.
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JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY
·
2026
·
IF 3.5
Xiaoran Sun,Yifan Chen,Kunyi Liu,Yi Si,Mingda Xuan,Rui Zhang,Jiao Jiao,Ting Liu,Shuangshuang Han,Jia Wang,Weifang Yu
Objective Gastric cancer remains a major global health burden. This study aimed to investigate the biological function, clinical significance, and underlying molecular mechanism of the RNA-binding protein YTHDF3 in gastric cancer (GC) progression. Methods YTHDF3 expression was analyzed using public databases (TCGA, GEO), clinical GC specimens (IHC, RT-qPCR), and GC cell lines. Functional roles were assessed through in vitro proliferation, migration, invasion, and apoptosis assays following YTHDF3 knockdown or overexpression, and via an in vivo subcutaneous tumor model. Mechanistic insights were gained through RNA sequencing, RIP-qPCR, dual-luciferase reporter assay, co-immunofluorescence, rescue experiments, and western blotting to identify downstream targets and signaling pathways. Results YTHDF3 was significantly upregulated in GC tissues, correlating with lymph node metastasis and advanced TNM stage. In vitro, YTHDF3 knockdown inhibited GC cell proliferation, migration, and invasion while promoting apoptosis, whereas its overexpression had the opposite effects. In vivo, YTHDF3 knockdown suppressed tumor growth. Mechanistically, YTHDF3 directly bound to and stabilized NEK7 mRNA. NEK7 was highly expressed in GC and crucial for YTHDF3-mediated oncogenic effects. Furthermore, YTHDF3 promoted GC progression by activating the Wnt/β-catenin signaling pathway via NEK7, which was validated using a pathway inhibitor (IWR-1-endo). Conclusion This study identifies the YTHDF3/NEK7 axis as a key regulator of GC progression via activation of the Wnt/β-catenin pathway. YTHDF3 represents a potential prognostic biomarker and a promising therapeutic target for GC. Graphical YTHDF3 binds to and stabilizes NEK7 mRNA in an m6A-dependent manner, thereby activating the Wnt/β-catenin signaling pathway and promoting gastric cancer progression. This study identifies the YTHDF3/NEK7 axis as a potential prognostic biomarker and therapeutic target in gastric cancer.
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JOURNAL OF ETHNOPHARMACOLOGY
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2026
·
IF 6.8
Qianqian Du,Zhimei Chen,Xian Ma,Yayu Zhang,Yongqiang Fan
Ethnopharmacological relevance Notoginsenoside R1 (NGR1), a primary bioactive constituent of P. notoginseng , has potent anti-inflammatory and immunomodulatory functions, but its role and mechanism in UC remain incompletely understood and warrant further exploration. The global prevalence of ulcerative colitis (UC) is rising steadily, threatening human health. Despite advances in modern therapies, UC treatments remain limited. Aim of the study This work investigated whether NGR1 alleviates DSS-induced colitis by inhibiting the PGAM5-NEK7 interaction and NLRP3 inflammasome activation in macrophages, providing new mechanistic insights and therapeutic targets for UC. Materials and methods UC model was established via 3% DSS administration to assess the protective efficacy of NGR1. Disease activity index (DAI), body weight and colon length were determined to assess disease severity and intestinal injury. HE staining, ELISA and immunofluorescence assays were employed to assess pathological changes in the colon and liver. Macrophages were depleted via tail vein injection of clodronate liposomes. RT-qPCR, ELISA, immunofluorescence and WB were performed to quantify macrophage markers, pro-inflammatory genes and NLRP3 inflammasome-related proteins. In vitro , LPS/ATP-stimulated bone marrow-derived macrophages (BMDMs) demonstrated that NGR1 exerts a potent inhibitory effect on NLRP3 inflammasome activation. After cell treatment, CCK-8, ELISA, RT-qPCR, and WB assays were performed to detect cell viability, inflammatory cytokine levels, and the expression of related genes and proteins. Meanwhile, molecular docking was utilized to verify the interactions among PGAM5, NEK7 and NLRP3. Co-IP in 293T cells overexpressing PGAM5, NEK7 and NLRP3 further verified their interplay. Results Compared with the Model group, NGR1 intervention markedly reduced the DAI scores, ameliorated body weight loss and colon shortening, alleviated colonic and hepatic pathological damage, upregulated the expression of intestinal barrier tight junction proteins (ZO-1, Occludin and Claudin-1), and decreased LPS levels and the release of TNF-α, IL-1β and IL-6 in serum and tissues. Macrophage depletion was observed to markedly abolish protective effects of NGR1, and reduced PGAM5, NEK7, NLRP3, Caspase-1, C-Caspase-1 levels. In vitro , NGR1 inhibited inflammatory factor secretion and NLRP3 inflammasome activation in LPS/ATP-stimulated BMDMs, these effects were partially blocked after PGAM5 or NEK7 gene silencing. Co-IP assays confirmed endogenous interactions between PGAM5, NEK7 and NLRP3. And the molecular docking showed NGR1 could bind to PGAM5, and NGR1 intervention significantly suppressed the expression of the ternary complex formed by the three proteins. Conclusion This study revealed that NGR1 disrupted the PGAM5-NEK7 interaction, restrained NLRP3 inflammasome assembly in macrophages, and relieved colonic damage triggered by DSS, providing a novel target for UC treatment.
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JOURNAL OF HAZARDOUS MATERIALS
·
2021
·
IF 10.588
Caiying Zhang,Zhisheng Hu,Ruiming Hu,Shaoxing Pi,Zejing Wei,Chang Wang,Fan Yang,Chenghong Xing,Gaohui Nie,Guoliang Hu
Pyroptosis and autophagy are two different biological processes that determine cell fates. Our previous studies revealed that pyroptosis and autophagy were involved in cytotoxicity co-induced by molybdenum (Mo) and cadmium (Cd) in duck renal tubular epithelial cells, but crosstalk between them is unclear. Hence, the cells were treated with 500.0 μM Mo, 4.0 μM Cd, 10.0 μM Z-YVAD-fluoromethylketone (YVAD), 2.5 μM 3-methyladenine (3-MA) and 10.0 μM chloroquine (CQ) alone or in combination for 12 h (CQ for the last 4 h). Under Mo and Cd co-stress, data evidenced that YVAD addition decreased the number of autophagosomes, LC3 puncta, and AMPKα-1, Atg5, Beclin-1, LC3A, LC3B mRNA levels and LC3-II/LC3-I, Beclin-1 protein levels, and increased p62 expression levels. Besides, both 3-MA and CQ addition increased NLRP3, Caspase-1, NEK7, ASC, GSDMA, GSDME, IL-1β, IL-18 mRNA levels, NLRP3, Caspase-1 p20, ASC, GSDMD protein and ROS levels, and NO, LDH, IL-1β, IL-18 releases. Collectively, our results revealed that pyroptosis and autophagy co-induced by Mo and Cd were interrelated in duck renal tubular epithelial cells, and inhibiting pyroptosis might attenuate Mo and Cd co-induced autophagy, but inhibiting autophagy might promote Mo and Cd co-induced pyroptosis.
研究领域:
细胞生物学
·
毒理学
·
分子生物学
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