Sirui Shen’s research while affiliated with Wenzhou Medical University and other places

Atherosclerosis has an urgent need for new therapeutic targets. Protein kinases orchestrate multiple cellular events in atherosclerosis and may provide new therapeutic targets for atherosclerosis. Here, a protein kinase, WEE1 G2 checkpoint kinase (WEE1), promoting inflammation in atherosclerosis is identified. Kinase enrichment analysis and experimental evidences reveal macrophage WEE1 phosphorylation at S642 in human and mouse atherosclerotic tissues. RNA‐seq analysis, combined with experiment studies using mutant WEE1 plasmids, shows that WEE1 phosphorylation, rather than WEE1 expression, mediated oxLDL‐induced inflammation in macrophages. Macrophage‐specific deletion of WEE1 or pharmacological inhibition of WEE1 kinase activity attenuates atherosclerosis by reducing inflammation in mice. Mechanistically, RNA‐seq and co‐immunoprecipitation followed by proteomics analysis are used to explore the mechanism and substrate of WEE1. p‐WEE1 promoted inflammatory response through activating NF‐κB shown and further revealed that WEE1 can directly bind to the p65 subunit. It is confirmed that p‐WEE1 directly interacts with the RHD domain of p65 and phosphorylates p65 at S536, thereby facilitating subsequent NF‐κB activation and inflammatory response in macrophages. The findings demonstrate that macrophage WEE1 drives NF‐κB activation and atherosclerosis by directly phosphorylating p65 at S536. This study identifies WEE1 as a new upstream kinase of p65 and a potential therapeutic target for atherosclerosis.

Background The severe cardiotoxicity of doxorubicin (Dox) significantly restricts its clinical application. Deubiquitinating enzymes (DUBs) play pivotal roles in cardiac pathophysiology because of their precise regulation of protein function, localization and degradation. Objectives The objective of this study was to investigate the role and molecular mechanism of ubiquitin-specific peptidase 20 (USP20), a DUB, in doxorubicin-induced cardiotoxicity. Methods Cardiomyocyte-specific USP20-knockout (USP20-CKO) mice were utilized to assess the role of USP20 in doxorubicin-induced cardiomyopathy (DIC). Coimmunoprecipitation (co-IP) combined with liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) analysis was employed to screen the substrate protein of USP20. Furthermore, mutant plasmids of USP20 were constructed to elucidate the molecular mechanism underlying the regulation of human antigen R (HuR) by USP20. Finally, an AAV9 vector was used to overexpress USP20 in the hearts of cardiac-specific HuR-knockout mice to assess the interaction between USP20 and HuR. Results The results revealed a decrease in USP20 expression in Dox-stimulated mouse cardiomyocytes. Cardiomyocyte-specific USP20 knockout resulted in increased cardiomyocyte ferroptosis and led to DIC. Mechanistically, USP20 directly interacted with HuR through its ubiquitin-specific protease structural domain. Deubiquitination at position 154 was crucial for maintaining HuR protein stability by cleaving K48 ubiquitin chains and inhibiting proteasomal degradation. Additionally, HuR bound to GPX4 mRNA to suppress its degradation, thereby mitigating ferroptosis and contributing to alleviating DIC. Furthermore, targeted USP20 overexpression via AAV9 in cardiomyocytes significantly alleviated DIC. However, in mice with cardiomyocyte-specific HuR knockout, USP20 no longer had an anti-DIC effect, indicating that HuR, as a downstream target protein of USP20, plays an irreplaceable role in DIC. Conclusions Our findings indicate that USP20 enhances the stability of the HuR protein through deubiquitination, thereby inhibiting ferroptosis and mitigating DIC.

Atherosclerosis is a common chronic inflammatory disease. Recent studies have highlighted the key role of macrophages and inflammation in process of atherosclerotic lesion formation. A natural product, tussilagone (TUS), has previously exhibited anti-inflammatory activities in other diseases. In this study, we explored the potential effects and mechanisms of TUS on the inflammatory atherosclerosis. Atherosclerosis was induced in ApoE-/- mice by feeding them with a high-fat diet (HFD) for 8 weeks, followed by administration of TUS (10, 20 mg ·kg-1·d-1, i.g.) for 8 weeks. We demonstrated that TUS alleviated inflammatory response and reduced atherosclerotic plaque areas in HFD-fed ApoE-/- mice. Pro-inflammatory factor and adhesion factors were inhibited by TUS treatment. In vitro, TUS suppressed foam cell formation and oxLDL-induced inflammatory response in MPMs. RNA-sequencing analysis indicated that MAPK pathway was related to the anti-inflammation and anti-atherosclerosis effects of TUS. We further confirmed that TUS inhibited MAPKs phosphorylation in plaque lesion of aortas and cultured macrophages. MAPK inhibition blocked oxLDL-induced inflammatory response and prevented the innately pharmacological effects of TUS. Our findings present a mechanistic explanation for the pharmacological effect of TUS against atherosclerosis and indicate TUS as a potentially therapeutic candidate for atherosclerosis.

Costunolide (Cos) is a naturally occurring sesquiterpene lactone that exhibits antioxidative properties. In this study, we demonstrate the protective mechanism of Cos against ischemia/reperfusion (I/R)-induced myocardial injury. Cos significantly decreased levels of reactive oxygen species (ROS) and ameliorated apoptosis of I/R cardiomyocytes both in vitro and in vivo. Further investigation revealed that Cos increased expression of the antioxidant proteins HO-1 and NQO-1 and decreased the Bax/Bcl-2 ratio, thus protecting cardiac cells. NF-E2-related factor 2 (Nrf2) silencing significantly attenuated the protective effects of Cos in tert-butyl hydroperoxide (TBHP)-treated H9C2 cells. Additionally, Cos significantly intensified the I/R- or TBHP-induced dissociation of the Kelch-like ECH-associated protein 1 (Keap1) / Nrf2 complex both in vitro and in vivo. These results suggest that activation of Nrf2/Keap1 using Cos may be a therapeutic strategy for myocardial I/R injury.

Atherosclerosis is a chronic inflammatory disease with high morbidity and mortality rates worldwide. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein kinase, is involved in neurogenesis and human cancers. However, the role of DCLK1 in atherosclerosis remains undefined. In this study, we identified upregulated DCLK1 in macrophages in atherosclerotic lesions of ApoE-/- mice fed an HFD and determined that macrophage-specific DCLK1 deletion attenuates atherosclerosis by reducing inflammation in mice. Mechanistically, RNA sequencing analysis indicated that DCLK1 mediates oxLDL-induced inflammation via NF-κB signaling pathway in primary macrophages. Coimmunoprecipitation followed by LC-MS/MS analysis identified IKKβ as a binding protein of DCLK1. We confirmed that DCLK1 directly interacts with IKKβ and phosphorylates IKKβ at S177/181, thereby facilitating subsequent NF-κB activation and inflammatory gene expression in macrophages. Finally, a pharmacological inhibitor of DCLK1 prevents atherosclerotic progression and inflammation both in vitro and in vivo. Our findings demonstrated that macrophage DCLK1 promotes inflammatory atherosclerosis by binding to IKKβ and activating IKKβ/NF-κB. This study reports DCLK1 as a new IKKβ regulator in inflammation and a potential therapeutic target for inflammatory atherosclerosis.

Understanding the molecular mechanisms of pathological vascular remodeling is important for treating cardiovascular diseases and complications. Recent studies have highlighted a role of deubiquitinases in vascular pathophysiology. Here, we investigate the role of a deubiquitinase, OTUD1, in angiotensin II (Ang II)-induced vascular remodeling. We detect upregulated OTUD1 in the vascular endothelium of Ang II-challenged mice and show that OTUD1 deletion attenuates vascular remodeling, collagen deposition, and EndMT. Conversely, OTUD1 overexpression aggravates these pathological changes both in vivo and in vitro. Mechanistically, SMAD3 is identified as a substrate of OTUD1 using co-immunoprecipitation followed by LC-MS/MS. We find that OTUD1 stabilizes SMAD3 and facilitates SMAD3/SMAD4 complex formation and subsequent nuclear translocation through both K48- and K63-linked deubiquitination. OTUD1-mediated SMAD3 activation regulates transcription of genes involved in vascular EndMT and remodeling in HUVECs. Finally, SMAD3 inhibition reverses OTUD1-promoted vascular remodeling. Our findings demonstrate that endothelial OTUD1 promotes Ang II-induced vascular remodeling by deubiquitinating SMAD3. We identify SMAD3 as a target of OTUD1 and propose OTUD1 as a potential therapeutic target for diseases related to vascular remodeling.

Purpose Costunolide (Cos) is a naturally occurring sesquiterpene lactone that exhibits anti-oxidative properties. In this study, we demonstrated the protective mechanism of Cos against ischemia/reperfusion (I/R)-induced heart injury. Methods C57BL/6 mice were pretreated with Cos (10 mg/kg/day) or 1% CMC-Na for 1 week. The left anterior descending coronary artery (LAD) was ligated for 30 minutes for ischemia, and followed by ligation release for 4 hours for reperfusion. H9c2 cells challenged with tert-butyl hydroperoxide (TBHP) were used for in vitro studies. Results Pretreatment of Cos significantly reduced myocardial infarct size, serum CK-MB and LDH level in I/R injured heart. Cos administration significantly attenuated the amount of reactive oxygen species (ROS) and ameliorated the apoptosis both in in vitro and in vivo studies. Further investigation revealed that Cos significantly increased the expression of heme oxygenase 1 (HO-1) and NAD(P)H [quinone] dehydrogenase 1 (NQO-1). Meanwhile Cos increased B-cell lymphoma-2(Bcl-2) and decreased the BCL2-Associated X Protein (Bax) protein level. Silence of nuclear factor erythroid 2-related factor (Nrf2) significantly reverses the protective effect of Cos in TBHP-challenged H9C2 cells. Conclusion Our data clearly showed that Cos reduced TBHP challenged H9c2 cells and attenuated myocardial I/R injury through Nrf2 activation. These results indicate that Cos might be benefit in the therapy of myocardial I/R injury.