Haowen Xu’s research while affiliated with Wenzhou Medical University and other places

Background Myocardial ischemia/reperfusion injury (MI/RI) restricts the effect of myocardial reperfusion therapy and lacks effective prevention and treatment methods. Deubiquitinating enzymes (DUBs), especially members of the ubiquitin‐specific protease (USP) family of DUBs, are key proteins in the ubiquitination modification process and play a vital role in MI/RI. Therefore, we aimed to investigate the role of USP25, as a member of the USP family, in MI/RI and its molecular mechanism. Methods Transcriptome sequencing was applied to evaluate the differential expression of USP families during hypoxia/reoxygenation (H/R) and validated in human and mouse heart samples and cardiomyocytes by performing quantitative polymerase chain reaction. Wild‐type or USP25−/− mice were used to develop the MI/RI model. Co‐immunoprecipitation (Co‐IP) combined with liquid chromatography–tandem mass spectrometry analysis was used to screen the potential substrate protein of USP25 in H/R‐induced cardiomyocyte injury. TUNEL and Hoechst/propidium iodide staining and western blot were used to detect the level of pyroptosis. In addition, cardiomyocyte‐specific USP25 overexpression in NLRP3−/− mice with AAV9 vectors was used to validate the biological function of USP25 and NLRP3 interaction. Results We found that the expression level of USP25 was significantly decreased in I/R‐induced mouse heart tissues and primary cardiomyocytes in a time‐dependent manner. USP25 deficiency exacerbated MI/RI and aggravated I/R‐induced cardiac remodelling in mice. Mechanistically, USP25 directly binds to NLRP3 protein and K63‐linkedly deubiquitinates NLRP3 at residue K243 via its active site C178, thus hindering NLRP3–ASC interaction and ASC oligomerization to inhibit NLRP3 activation and pyroptosis in cardiomyocytes. We further showed that the overexpression of USP25 in cardiomyocytes ameliorated MI/RI in mice, whereas this protective effect disappeared when NLRP3 is knocked out. Conclusions Our study demonstrated that USP25 ameliorates MI/RI by regulating NLRP3 activation and its mediated pyroptosis. This finding extends the protective role of USP25 in cardiovascular disease and provides an experimental basis for future USP25‐based drug development for the treatment of MI/RI. Key points The deubiquitinating enzyme USP25 was down‐regulated both in myocardial ischemia/reperfusion injury (MI/RI) myocardium tissues. The deficiency of USP25 worsened exacerbated MI/RI in mice, whereas the overexpression of USP25 in cardiomyocytes mitigated this pathological phenotype. USP25 directly interacts with the NLRP3 protein and deubiquitinates it via K63 linkage at residue K243 through its active site C178, thus affecting NLRP3‐ASC interaction and ASC oligomerization to inhibit NLRP3 activation and pyroptosis in cardiomyocytes.

Background: NLR family pyrin domain-containing 3 (NLRP3)-mediated pyroptosis plays a key role in various acute and chronic inflammatory diseases. Targeted inhibition of NLRP3-mediated pyroptosis may be a potential therapeutic strategy for various inflammatory diseases. Ergolide (ERG) is a sesquiterpene lactone natural product derived from the traditional Chinese medicinal herb, Inula britannica. ERG has been shown to have anti-inflammatory and anti-cancer activities, but the target is remains unknown. Hypothesis/purpose: This study performed an in-depth investigation of the anti-inflammatory mechanism of ERG in NLRP3-mediated pyroptosis and NLPR3 inflammasome related sepsis and acute lung injury model. Methods: ELISA and Western blot were used to determine the IL-1β and P20 levels. Co-immunoprecipitation assays were used to detect the interaction between proteins. Drug affinity response target stability (DARTS) assays were used to explore the potential target of ERG. C57BL/6J mice were intraperitoneally injected with E. coli DH5α (2 × 109 CFU/mouse) to establish a sepsis model. Acute lung injury was induced by intratracheal administrationof lipopolysaccharide in wild-type mice and NLRP3 knockout mice with or without ERG treatment. Results: We showed that ERG is an efficient inhibitor of NLRP3-mediated pyroptosis in the first and second signals of NLRP3 inflammasome activation. Furthermore, we demonstrated that ERG irreversibly bound to the NACHT domain of NLRP3 to prevent the assembly and activation of the NLRP3 inflammasome. ERG remarkably improved the survival rate of wild-type septic mice. In lipopolysaccharide-induced acute lung injury model, ERG alleviated acute lung injury of wild-type mice but not NLRP3 knockout mice. Conclusion: Our results revealed that the anti-pyroptosis effect of ERG are dependent on NLRP3 and NLRP3 NACHT domain is ERG's direct target. Therefore, ERG can serve as a precursor drug for the development of novel NLRP3 inhibitors to treat NLRP3 inflammasome mediated inflammatory diseases.

Background: Acute tubular necrosis (ATN) is a common type of acute renal failure. Recent studies have shown that NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-mediated pyroptosis in macrophages plays a crucial role in the progression of ATN. Previously, we synthesized an anti-inflammatory compound 15a based on Tanshinone IIA (Tan IIA). In the present study, we found that compound 15a exhibited a greater inhibitory effect on NLRP3-mediated pyroptosis than Tan IIA in vitro. Methods: C57BL/6 and NLRP3-knockout (NLRP3-KO) mice were intraperitoneally injected with LPS or folic acid (FA) to develop ATN. In vitro, bone marrow-derived macrophages (BMDMs) were treated with LPS for 3 h and then treated with ATP for 0.5 h. Results: We explored the mechanism by which compound 15a inhibited NLRP3 inflammasome in BMDMs as well as its renal protective effect against ATN in mice. We found that compound 15a exhibited a protective effect on mitochondria and reduced the production of mitochondrial reactive oxygen species (mtROS). Moreover, we revealed that compound 15a remarkably reduced the production of mtROS by promoting mitophagy, which resulted in the inhibition of NLRP3 inflammasome to alleviates ATN in mice. Conclusion: In summary, compound 15a inhibited NLRP3-mediated inflammation by activating mitophagy in macrophages to alleviate ATN. Our results identified compound 15a as a promising candidate for the treatment of NLRP3-driven ATN.

Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein kinase, is involved in neurogenesis, and its levels are elevated in various human cancers. Recent studies suggest that DCLK1 may relate to inflammatory responses in the mouse model of colitis. However, cellular pathways engaged by DCLK1, and potential substrates of the kinase remain undefined. To understand how DCLK1 regulates inflammatory responses, we utilized the well-established lipopolysaccharide (LPS)-stimulated macrophages and mouse model. Through a range of macrophage-based and cell-free platforms, we discovered that DCLK1 binds directly with the inhibitor of κB kinase β (IKKβ) and induces IKKβ phosphorylation on Ser177/181 to initiate nuclear factor-κB (NF-κB) pathway. Deficiency in DCLK1, achieved by silencing or through pharmacological inhibition, prevented LPS-induced NF-κB activation and cytokine production in macrophages. We further show that mice with myeloid-specific DCLK1 knockout or DCLK1 inhibitor treatment are protected against LPS-induced acute lung injury and septic death. Our studies report a novel functional role of macrophage DCLK1 as a direct IKKβ regulator in inflammatory signaling and suggest targeted therapy against DCLK1 for inflammatory diseases.

Background and Purpose The NLR family pyrin domain‐containing 3 (NLRP3) inflammasome is activated in many inflammatory conditions. So far, no low MW compounds inhibiting NLRP3 have entered clinical use. Identification of naturally occurring NLRP3 inhibitors may be beneficial to the design and development of compounds targeting NLRP3. Alantolactone is a phytochemical from a traditional Chinese medicinal plant with anti‐inflammatory activity, but its precise target remains unclear. Experimental Approach A bank of phytochemicals was screened for inhibitors of NLRP3‐driven production of IL‐1β in cultures of bone‐marrow‐derived macrophages from female C57BL/6 mice. Models of gouty arthritis and acute lung injury in male C57BL/6J mice were used to determine the in vivo effects of the most potent compound. Key Results Among the 150 compounds screened in vitro, alantolactone exhibited the highest inhibitory activity against LPS + ATP‐induced production of IL‐1β in macrophages, suppressing IL‐1β secretion, caspase‐1 activation and pyroptosis. Alantolactone directly bound to the NACHT domain of NLRP3 to inhibit activation and assembly of NLRP3 inflammasomes. Molecular simulation analysis suggested that Arg335 in NLRP3 was a critical residue for alantolactone binding, leading to suppression of NLRP3–NEK7 interaction. In vivo studies confirmed significant alleviation by alantolactone of two NLRP3‐driven inflammatory conditions, acute lung injury and gouty arthritis. Conclusion and Implications The phytochemical alantolactone inhibited activity of NLRP3 inflammasomes by directly targeting the NACHT domain of NLRP3. Alantolactone shows great potential in the treatment of NLRP3‐driven diseases and could lead to the development of novel NLRP3 inhibitors.

The NLRP3 inflammasome’s core and most specific protein, NLRP3, has a variety of functions in inflammation-driven diseases. Costunolide (COS) is the major active ingredient of the traditional Chinese medicinal herb Saussurea lappa and has anti-inflammatory activity, but the principal mechanism and molecular target of COS remain unclear. Here, we show that COS covalently binds to cysteine 598 in NACHT domain of NLRP3, altering the ATPase activity and assembly of NLRP3 inflammasome. We declare COS’s great anti-inflammasome efficacy in macrophages and disease models of gouty arthritis and ulcerative colitis via inhibiting NLRP3 inflammasome activation. We also reveal that the α-methylene-γ-butyrolactone motif in sesquiterpene lactone is the certain active group in inhibiting NLRP3 activation. Taken together, NLRP3 is identified as a direct target of COS for its anti-inflammasome activity. COS, especially the α-methylene-γ-butyrolactone motif in COS structure, might be used to design and produce novel NLRP3 inhibitors as a lead compound.