The involvement of Sting in exacerbating acute lung injury in sepsis via the PARP-1/NLRP3 signaling pathway.

BACKGROUND: Interferon gene stimulator (Sting) is an indispensable adaptor protein that plays a crucial role in acute lung injury (ALI) induced by sepsis, and the PARP-1/NLRP3 signaling pathway may be an integral component of the inflammatory response mediated by Sting. However, the regulatory role of Sting in the PARP-1/NLRP3 pathway in ALI remains insufficiently elucidated. METHODS: Using lipopolysaccharide (LPS) to induce ALI in C57BL/6 mice and HUVEC cells, an in vivo and in vitro model was established. In vivo, Sting agonists and inhibitors were administered, while in vitro, Sting was knocked down using siRNA. ELISA was employed to quantify the levels of IL-1ß, IL-6, and TNF-α. TUNEL staining was conducted to assess cellular apoptosis, while co-immunoprecipitation was utilized to investigate the interaction between Sting and NLRP3. Expression levels of Sting, NLRP3, PARP-1, among others, were assessed via Western blotting and RT-qPCR. Lung HE staining and lung wet/dry ratio were evaluated in the in vivo mouse model. To validate the role of the PARP-1/NLRP3 signaling pathway, PARP-1 inhibitors were employed both in vivo and in vitro. RESULTS: In vitro experiments revealed that the Sting agonist group exacerbated LPS-induced pulmonary pathological damage, pulmonary edema, inflammatory response (increased levels of IL-6, TNF-α, and IL-1ß), and cellular injury, whereas the Sting inhibitor group significantly ameliorated the aforementioned injuries, with further improvement observed in the combination therapy of Sting inhibitor and PARP-1 inhibitor. Western blotting and RT-qPCR results demonstrated significant suppression of ICAM-1, VCAM-1, NLRP3, and PARP-1 expression in the Sting inhibitor group, with this reduction further enhanced in the Sting inhibitor + PARP-1 inhibitor treatment group, exhibiting opposite outcomes to the agonist. Furthermore, in vitro experiments using HUVEC cell lines validated these findings. CONCLUSIONS: Our study provides new insights into the roles of Sting and the PARP-1/NLRP3 signaling pathway in inflammatory responses, offering novel targets for the development of therapeutic interventions against inflammatory reactions.

Silencing miRNA-324-3p protects against cerebral ischemic injury via regulation of the GATA2/A1R axis.

Previous studies have suggested that miR-324-3p is related to the pathophysiology of cerebral ischemia, but the mechanism underlying this relationship is unclear. In this study, we found that miR-324-3p expression was decreased in patients with acute ischemic stroke and in in vitro and in vivo models of ischemic stroke. miR-324-3p agomir potentiated ischemic brain damage in rats subjected to middle cerebral artery occlusion, as indicated by increased infarct volumes and cell apoptosis rates and greater neurological deficits. In a PC12 cell oxygen-glucose deprivation/reoxygenation model, a miR-324-3p mimic decreased cell viability and expression of the anti-apoptotic protein BCL2 and increased expression of the pro-apoptotic protein BAX and rates of cell apoptosis, whereas treatment with a miR-324-3p inhibitor had the opposite effects. Silencing miR-324-3p increased adenosine A1 receptor (A1R) expression through regulation of GATA binding protein 2 (GATA2). These findings suggest that silencing miR-324-3p reduces ischemic brain damage via the GATA2/A1R axis.

Nicotinamide N-methyltransferase ameliorates renal fibrosis by its metabolite 1-methylnicotinamide inhibiting the TGF-ß1/Smad3 pathway.

Chronic kidney disease (CKD), a disease involving damage to the kidney structure and function, is a global public health problem. Tubulointerstitial fibrosis (TIF) is both an inevitable pathological change in individuals with CKD and a driving force in the progression of renal fibrosis. Nicotinamide N-methyltransferase (NNMT) and its metabolite 1-methylnicotinamide (MNAM) have been shown to protect against lipotoxicity-induced kidney tubular injury. However, the biological roles of NNMT and MNAM in regulating TIF remain elusive. This study aimed to investigate the protective effect of NNMT and MNAM on TIF and the mechanisms involved. We explored the functions and mechanisms of NNMT and MNAM in TIF, as well as the interaction between NNMT and MNAM, using unilateral ureteral obstruction (UUO) mice and cultured mouse tubular epithelial cells (mTECs) stimulated with transforming growth factor-ß1 (TGF-ß1). Several important findings were obtained as follows: (1) NNMT expression was upregulated in the kidneys of UUO mice and TGF-ß1-induced mTECs, and this upregulation was proposed to be a protective compensatory response to TIF. (2) MNAM was a potentially effective antifibrotic and anti-inflammatory medication in UUO mice. (3) The antifibrotic effect of NNMT overexpression was exerted by increasing the concentration of MNAM. (4) The renoprotective role of MNAM depended on the selective blockade of the interaction of Smad3 with TGFß receptor I. Overall, our study shows that NNMT is involved in the development and progression of CKD and that its metabolite MNAM may be a novel inhibitor of the TGF-ß1/Smad3 pathway with great therapeutic potential for CKD.

Electroacupuncture pre­conditioning protects from lung injury induced by limb ischemia/reperfusion through TLR4 and NF­κB in rats.

Limb ischemia/reperfusion (I/R) can induce inflammation, causing acute lung injury. The Toll­like receptor 4 (TLR4)/NF­κB pathway plays an important role in acute and chronic inflammatory disorders. Several studies have demonstrated the efficacy of acupuncture in lung inflammatory injury. The aim of the present study was to elucidate the mechanism underlying the protective effect of electroacupuncture (EA) against lung injury induced by limb I/R. EA applied at the Zusanli and Sanyinjiao acupoints attenuated lung injury and decreased the secretion of inflammatory factors such as tumor necrosis factor­α, interleukin (IL)­1, IL­6 and myeloperoxidase. Moreover, the expression levels of TLR4 and NF­κB were suppressed by EA. Thus, the present findings suggested that EA can reduce pulmonary inflammation induced by limb I/R injury, possibly via the inhibition of the TLR4/NF­κB pathway.

Astrocytic Yes-associated protein attenuates cerebral ischemia-induced brain injury by regulating signal transducer and activator of transcription 3 signaling.

Astrocytic Yes-associated protein (YAP) has been implicated in astrocytic proliferation and differentiation in the developing neocortex. However, the role of astrocytic YAP in diseases of the nervous system remains poorly understood. Here, we hypothesized that astrocytic YAP exerted a neuroprotective effect against cerebral ischemic injury in rats by regulating signal transducer and activator of transcription 3 (STAT3) signaling. In this study, we investigated whether the expression of nuclear YAP in the astrocytes of rats increased significantly after middle cerebral artery occlusion (MCAO) and its effect on cerebral ischemic injury. We used XMU-MP-1 to trigger localization of YAP into the nucleus and found that XMU-MP-1 treatment decreased ischemia/stroke-induced brain injury including reduced neuronal death and reactive astrogliosis, and extenuated release of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Mechanically, XMU-MP-1 treatment suppressed the expression of phospho-STAT3 (P-STAT3). We established an in-vitro oxygen-glucose deprivation/reperfusion (OGD/R) model to simulate an ischemic condition and further explore the function of astrocytic YAP. We found that nuclear translocation of astrocytic YAP in rats could improve cell vitality, decrease the release of inflammatory cytokines and reduce the expression of P-STAT3 in vitro. In contrast, we also found that inhibition of YAP by verteporfin further aggravated the injury induced by OGD/R via STAT3 signaling. In summary, our results showed that nuclear localization of astrocytic YAP exerted a neuroprotective effect after cerebral ischemic injury in rats via inhibition of the STAT3 signaling.

N6-methyladenosine demethylases Alkbh5/Fto regulate cerebral ischemia-reperfusion injury.

BACKGROUND: Although N6-methyladenosine (m6A) plays a very important role in different biological processes, its function in the brain has not been fully explored. Thus, we investigated the roles of the RNA demethylases Alkbh5/Fto in cerebral ischemia-reperfusion injury. METHODS: We used a rat model and primary neuronal cell culture to study the role of m6A and Alkbh5/Fto in the cerebral cortex ischemic penumbra after cerebral ischemia-reperfusion injury. We used Alkbh5-shRNA and Lv-Fto (in vitro) to regulate the expression of Alkbh5/Fto to study their regulation of m6A in the cerebral cortex and to study brain function after ischemia-reperfusion injury. RESULTS: We found that RNA m6A levels increased consecutive to the increase of Alkbh5 expression in both the cerebral cortex of rats after middle cerebral artery occlusion, and in primary neurons after oxygen deprivation/reoxygenation. In contrast, Fto expression decreased after these perturbations. Our results suggest that knocking down Alkbh5 can aggravate neuronal damage. This is due to the demethylation of Alkbh5 and Fto, which selectively demethylate the Bcl2 transcript, preventing Bcl2 transcript degradation and enhancing Bcl2 protein expression. CONCLUSION: Collectively, our results demonstrate that the demethylases Alkbh5/Fto co-regulate m6A demethylation, which plays a crucial role in cerebral ischemia-reperfusion injury. The results provide novel insights into potential therapeutic mechanisms for stroke.

Electroacupuncture pretreatment prevents ischemic stroke and inhibits Wnt signaling-mediated autophagy through the regulation of GSK-3ß phosphorylation.

Electroacupuncture (EA), a traditional Chinese replacement therapy, is widely accepted to treat ischemic stroke. Increasing evidence show that autophagy is involved in the process of cerebral ischemia injury and the Wnt/GSK3ß pathway, playing an important role in protecting central nervous system. In this study, rats were treated with EA prior to focal ischemia by middle cerebral artery occlusion (MCAO). Deficit score, infarct volumes and levels of autophagy markers, such as LC3I, LC3II and p62, were assessed with either PI3K inhibitor wortmannin or a GSK-3ß inhibitor LiCl. Oxygen-glucose deprivation/re-oxygenation (OGD/R) was made in the primitive neuron in vitro, and was respectively treated with autophagy inhibitors 3-MA, LiCl, GSK3ß siRNA, or mTOR inhibitor rapamycin. The results indicated that EA pretreatment increased the levels of autophagy marker LC3-II and reduced the levels of p62. Meanwhile, deficit outcome was improved, and infarct volumes were reduced by EA pretreatment. Furthermore, the beneficial effects of EA pretreatment were reversed by wortmannin. LiCl and GSK3ß siRNA can mimic the neuroprotective effects of EA pretreatment by downregulating autophagy, and increasing protein levels of p-mTOR, p-GSK3ß and ß-catenin in OGD/R neurons. However, the protective effects of GSK3ß siRNA were blocked by rapamycin. These results suggest that EA pretreatment induces tolerance to cerebral ischemia by inhibiting autophagy via the Wnt pathway through the inhibition of GSK3ß.

Electroacupuncture Attenuates CFA-Induced Inflammatory Pain by Regulating CaMKII.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine kinase that is ubiquitously distributed in the central and peripheral nervous systems. Moreover, its phosphorylated protein (P-CaMKII) is involved in memory, mood, and pain regulation in the anterior cingulate cortex (ACC). Electroacupuncture (EA) is a traditional Chinese therapeutic technique that can effectively treat chronic inflammatory pain. However, the CaMKII-GluA1 role in EA analgesia in the ACC remains unclear. This study investigated the role of P-CaMKII and P-GluA1 in a mouse model of inflammatory pain induced by complete Freund's adjuvant (CFA). There were increased P-CaMKII and P-GluA1 levels in the ACC. We found that intracerebroventricular injection of KN93, a CaMKII inhibitor, as well as EA stimulation, attenuated complete Freund's adjuvant-induced pain behavior. Further, EA increased pCaMKII-PICK1 complex (abbreviated as C-P complex) levels. Our findings demonstrate that EA inhibits inflammatory pain by inhibiting CaMKII-GluA1 phosphorylation. P-CaMKII is involved in EA analgesia as the pCaMKII-PICK1 complex.