Liquid-liquid phase separation (LLPS) has emerged as a flexible intracellular compartment that modulates various pathological processes. Hypoxia-inducible factor-1α (HIF-1α) has been shown to play an essential role in inflammation after ischemic injury. However, the mechanisms underlying HIF-1α-induced inflammation in ischemic diseases have not been defined. This study found that HIF-1α mediated the progression of ischemia-induced muscle injury. After ischemic injury, SUMO1 is upregulated and rapidly activates NLRP3 inflammasome through the upregulation of HIF-1α, leading to enhanced inflammation and pyroptosis. Co-IP revealed an interaction between SUMO1 and HIF-1α and SUMOylation of HIF-1α at K477. Moreover, we demonstrated the important role of dynamic phase separation of Nucleolin (Ncl) in regulating HIF-1α mRNA stability through fluorescence recovery after photobleach (FRAP) analysis. The stability of HIF-1α is regulated by Ncl liquid-liquid phase separation and SUMOylation in ischemia-induced hindlimb injury. HIF-1α can promote the expression of NLRP3 and other inflammation-related molecules, leading to pyroptosis, suggesting that Ncl/LLPS/HIF-1α or SUMO1/HIF-1α pathway may be a new target for the treatment of inflammation in ischemic diseases. Although previous studies have found that HIF-1α is able to promote the expression of target genes after hypoxia, and these genes are used to maintain the stability of the intracellular environment to adapt to hypoxia. We found that HIF-1α is involved in the activation process of NLRP3 inflammasomes after hind limb ischemia, which enriches our understanding of the biological role of HIF-1α.
1. Introduction
Limb ischemia is a serious clinical condition affecting many patients worldwide and there is no effective therapy . Ischemia activates the NOD-like receptor protein 3 (NLRP3) inflammasome, which induces tissue damage by releasing inflammatory cytokines including interleukin-1β (IL-1β) and IL-18 or Gasdermin-D (GSDMD) . However, the molecular mechanisms underlying activation of the NLRP3 inflammasome remain largely unknown.
Previous studies have shown that many inflammatory factors are released from damaged tissues after ischemic injury . Our prior research found that the increased expression of NLRP3 inflammasomes led to the activation of Caspase-1 (Casp-1), IL-1β and IL-18 and pyroptosis . However, the specific regulatory mechanism of NLRP3 activation in limb ischemic disease is unclear.
Hypoxia-inducible factor-1α (HIF-1α) is an essential hypoxic response molecule in the body . Under hypoxic conditions, HIF-1α and HIF-1β form a dimer, which promotes the transcription of hypoxia stress-related genes . Therefore, HIF-1α is vital in inflammation and pyroptosis in ischemic diseases .
Specifically, during the initial phases of atherosclerosis, HIF-1α has been shown to stimulate the production of reactive oxygen species (ROS) and activate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, both of which are instrumental in triggering endothelial cell dysfunction . This dysfunction is a key event in the pathogenesis of atherosclerosis, which can ultimately cause myocardial infarction. In this context, HIF-1α further exerts its influence on the inflammatory cascade by regulating the NLRP3 inflammasome, a process that can result in the pyroptotic death of cardiomyocytes . Despite these insights, the specific contribution of HIF-1α to the pathophysiology of lower limb ischemic diseases remains understudied. Our study aims to address this gap by investigating the potential regulatory mechanisms of HIF-1α in these conditions, which could provide novel therapeutic targets for the treatment of peripheral arterial disease.
2. Results
2.1. Ischemia triggers the activation of NLRP3 inflammasome in vivo
To explore whether ischemia can cause the activation of NLRP3 in muscle tissue, we constructed a mouse hindlimb ischemia model and examined the gastrocnemius muscles three days after the ischemic surgery. We found that the mRNA levels of NLRP3, Casp-1, GSDMD, IL-18, and IL-1β were increased in the ischemic left limb compared with the non-ischemic right limb . Western blot analysis further showed that the protein expression of NLRP3, Casp-1, and GSDMD was increased in the ischemic tissues compared to the non-ischemic tissues. The release of IL-18 and IL-1β in the serum of ischemic mice was significantly higher than that of non-ischemic mice measured by ELISA.
(E-H) Western blot analysis showed increased NLRP3, Pro-Casp-1, Cleaved-Casp-1 and GSDMD protein expression in muscle after ischemic surgery (n = 4 or 3). (I) ELISA of serum IL-1β and IL-18 levels from control and ischemia groups (n = 3). Data are presented as Mean ± SEM and analyzed using a two-tailed t-test. Expression of mRNA and protein is expressed as fold changes relative to the CTRL, which is set to 1.
2.2. Hypoxia induces the activation of NLRP3 inflammasome and promotes cell pyroptosis in vitro
The C2C12 cell line, a commonly used murine myoblast model in biomedical research, serves as a valuable in vitro system for investigating various physiological and pathological aspects of muscle. Its widespread application stems from its exceptional ability to simulate muscle injury and repair processes. To verify whether hypoxia could promote inflammatory response in C2C12 cells, we divided C2C12 cells into normoxic and hypoxic groups. To induce hypoxia, the cells were treated at 37 °C, 5 % CO2 and 1 % O2 for 24 h, and we found that the mRNA levels of NLRP3, Casp-1, GSDMD, IL-18, and IL-1β were increased in hypoxic cells compared with control cells by qPCR . Western Blot also showed that the protein levels of NLRP3 and GSDMD were upregulated in the hypoxic group, and the levels of Pro-Casp-1 and Cleaved-Casp-1 were increased compared with the control group . These findings indicate that hypoxia induces Casp-1 activation by promoting NLRP3 expression. Subsequently, we found that IL-1β and IL-18 in the hypoxic cells were significantly increased by ELISA . We detected pyroptosis by PI staining. The results showed that the positive PI staining rate in hypoxic cells was significantly higher than that in control cells, proving that hypoxia caused cell membrane rupture and induced pyroptosis. All these results indicate that hypoxia can promote NLRP3-mediated inflammatory response and pyroptosis.
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