NEMO-Binding Domain/IKKγ Inhibitory Peptide Alleviates Neuronal Pyroptosis in Spinal Cord Injury by Inhibiting ASMase-Induced Lysosome Membrane Permeabilization.

A short peptide termed NEMO-binding domain (NBD) peptide has an inhibitory effect on nuclear factor kappa-B (NF-κB). Despite its efficacy in inhibiting inflammatory responses, the precise neuroprotective mechanisms of NBD peptide in spinal cord injury (SCI) remain unclear. This study aims to determine whether the pyroptosis-related aspects involved in the neuroprotective effects of NBD peptide post-SCI.Using RNA sequencing, the molecular mechanisms of NBD peptide in SCI are explored. The evaluation of functional recovery is performed using the Basso mouse scale, Nissl staining, footprint analysis, Masson's trichrome staining, and HE staining. Western blotting, enzyme-linked immunosorbent assays, and immunofluorescence assays are used to examine pyroptosis, autophagy, lysosomal membrane permeabilization (LMP), acid sphingomyelinase (ASMase), and the NF-κB/p38-MAPK related signaling pathway.NBD peptide mitigated glial scar formation, reduced motor neuron death, and enhanced functional recovery in SCI mice. Additionally, NBD peptide inhibits pyroptosis, ameliorate LMP-induced autophagy flux disorder in neuron post-SCI. Mechanistically, NBD peptide alleviates LMP and subsequently enhances autophagy by inhibiting ASMase through the NF-κB/p38-MAPK/Elk-1/Egr-1 signaling cascade, thereby mitigating neuronal death. NBD peptide contributes to functional restoration by suppressing ASMase-mediated LMP and autophagy depression, and inhibiting pyroptosis in neuron following SCI, which may have potential clinical application value.

TRIM56 Modulates YBX1 Degradation to Ameliorate ZBP1-Mediated Neuronal PANoptosis in Spinal Cord Injury.

Spinal cord injury (SCI) is a severe injury to the central nervous system, and its treatment is always a major medical challenge. Proinflammatory cell death is considered an important factor affecting neuroinflammation and the prognosis after injury. PANoptosis, a newly discovered type of proinflammatory cell death, regulates the activation of executioner molecules of apoptosis, pyroptosis and necroptosis through the PANoptosome, providing a new target for therapeutic intervention after SCI. However, its role and regulatory mechanism in SCI are not yet elucidated. Here, based on proteomic data, YBX1 expression is significantly increased in neurons after SCI. Guided by RIP-seq, subsequent experiments reveal that YBX1 promotes ZBP1 expression by stabilizing the Zbp1 mRNA, thereby aggravating ZBP1-mediated PANoptosis. Furthermore, the E3 ubiquitin ligase TRIM56 is identified as an endogenous inhibitor of YBX1 via molecular docking and IP/MS analysis. Mechanistically, TRIM56 bound to YBX1 and promoted its ubiquitination, thereby accelerating its degradation. Taken together, these findings reveal a novel function of YBX1 in regulating ZBP1-mediated PANoptosis in the pathogenesis of SCI and verified that TRIM56 functions as an endogenous inhibitor to promote the ubiquitin-proteasomal degradation of YBX1, providing new insights into SCI treatment strategies.

Isoquercetin Ameliorates Osteoarthritis via Nrf2/NF-κB Axis: An In Vitro and In Vivo Study.

Osteoarthritis (OA) is a progressive joint disease characterized by extracellular matrix (ECM) degradation and inflammation, which is involved with pathological microenvironmental alterations induced by damaged chondrocytes. However, current therapies are not effective in alleviating the progression of OA. Isoquercetin is a natural flavonoid glycoside compound that has various pharmacological effects including anticancer, anti-diabetes and blood lipid regulation. Previous evidence suggests that isoquercetin has anti-inflammatory properties in various diseases, but its effect on OA has not been investigated yet. In this study, through western bolt, qRT-PCR and ELISA, it was found that isoquercetin could reduce the increase of ADAMTS5, MMP13, COX-2, iNOS and IL-6 induced by IL-1ß, suggesting that isoquercetin could inhibit the inflammation and ECM degradation of chondrocytes. Through nuclear-plasma separation technique, western blot and immunocytochemistry, it can be found that Nrf2 and NF-κB pathways are activated in this process, and isoquercetin may rely on this process to play its protective role. In vivo, the results of X-ray and SO staining show that intra-articular injection of isoquercetin reduces the degradation of cartilage in the mouse OA model. In conclusion, the present work suggests that isoquercetin may benefit chondrocytes by regulating the Nrf2/NF-κB signaling axis, which supports isoquercetin as a potential drug for the treatment of OA.

How to Set Working Cannula in Endoscopic-Assisted Transforaminal Lumbar Interbody Fusion: A Morphometric Analysis Based on Computed Tomography.

OBJECTIVES: There is a high risk of nerve root injury during endoscopic-assisted transforaminal lumbar interbody fusion (Endo-TLIF). This study used computed tomography (CT) imaging to assess the relationship between the exiting nerve root and its surroundings, and the corresponding intervertebral disc. We also measured the approximate position and angle for the placement of the working cannula to reduce the risk of nerve root injury during Endo-TLIF procedures in the Chinese population. METHODS: This retrospective study was conducted at our institution between December 2021 and December 2022. A total of 115 patients suffering from low back pain were recruited for the study. For each participant, three-dimensional (3D) vertebral models of the lumbar segments from L3 to S1 were constructed based on their CT images. The nerve root-disc distance, cannula insertion bypass distance and angle, foraminal height and width, exiting nerve root height, and nerve root-pedicle distance were measured. A paired t-test was used to compare measurements between the left and right sides, while inter- and intraobserver reproducibility was assessed using the intraclass correlation coefficient (ICC). RESULTS: From L3/4 to L5/S1 segments, the ideal cannula insertion distance range was 37.51 ± 4.91-120.38 ± 37.71 mm at L3/4; 42.38 ± 5.29-116.25 ± 27.22 mm at L4/5; and 37.78 ± 4.86-69.26 ± 12.64 mm at L5/S1. The appropriate cannula insertion angle range was 30.86° ± 5.05°-62.59° ± 6.66° at L3/4; 34.30° ± 4.73°-60.88° ± 7.34° at L4/5; and 35.89° ± 4.18°-47.65° ± 7.38° at L5/S1. The height of the intervertebral foramen (IVF) gradually decreased, and the width steadily increased. The exiting nerve root height and the nerve root-pedicle distance slightly decreased caudally. CONCLUSION: From L3/4 to L5/S1, the range of working cannula insertion distance and angle gradually decreased, and the exiting nerve root height occupying the IVF gradually increased. Our measurement can reduce the risk of nerve root injury caused by inserting the working cannula during Endo-TLIF.

Underlying Mechanism of Lysosomal Membrane Permeabilization in CNS Injury: A Literature Review.

Lysosomes play a crucial role in various intracellular pathways as their final destination. Various stressors, whether mild or severe, can induce lysosomal membrane permeabilization (LMP), resulting in the release of lysosomal enzymes into the cytoplasm. LMP not only plays a pivotal role in various cellular events but also significantly contributes to programmed cell death (PCD). Previous research has demonstrated the participation of LMP in central nervous system (CNS) injuries, including traumatic brain injury (TBI), spinal cord injury (SCI), subarachnoid hemorrhage (SAH), and hypoxic-ischemic encephalopathy (HIE). However, the mechanisms underlying LMP in CNS injuries are poorly understood. The occurrence of LMP leads to the activation of inflammatory pathways, increased levels of oxidative stress, and PCD. Herein, we present a comprehensive overview of the latest findings regarding LMP and highlight its functions in cellular events and PCDs (lysosome-dependent cell death, apoptosis, pyroptosis, ferroptosis, and autophagy). In addition, we consolidate the most recent insights into LMP in CNS injury by summarizing and exploring the latest advances. We also review potential therapeutic strategies that aim to preserve LMP or inhibit the release of enzymes from lysosomes to alleviate the consequences of LMP in CNS injury. A better understanding of the role that LMP plays in CNS injury may facilitate the development of strategic treatment options for CNS injury.

UBC9-mediated SUMOylation of Lamin B1 enhances DNA-damage-induced nuclear DNA leakage and autophagy after spinal cord injury.

Recent studies have shown that nucleophagy can mitigate DNA damage by selectively degrading nuclear components protruding from the nucleus. However, little is known about the role of nucleophagy in neurons after spinal cord injury (SCI). Western blot analysis and immunofluorescence were performed to evaluate the nucleophagy after nuclear DNA damage and leakage in SCI neurons in vivo and NSC34 expression in primary neurons cultured with oxygen-glucose deprivation (OGD) in vitro, as well as the interaction and colocalization of autophagy protein LC3 with nuclear lamina protein Lamin B1. The effect of UBC9, a Small ubiquitin-related modifier (SUMO) E2 ligase, on Lamin B1 SUMOylation and nucleophagy was examined by siRNA transfection or 2-D08 (a small-molecule inhibitor of UBC9), immunoprecipitation, and immunofluorescence. In SCI and OGD injured NSC34 or primary cultured neurons, neuronal nuclear DNA damage induced the SUMOylation of Lamin B1, which was required by the nuclear Lamina accumulation of UBC9. Furthermore, LC3/Atg8, an autophagy-related protein, directly bound to SUMOylated Lamin B1, and delivered Lamin B1 to the lysosome. Knockdown or suppression of UBC9 with siRNA or 2-D08 inhibited SUMOylation of Lamin B1 and subsequent nucleophagy and protected against neuronal death. Upon neuronal DNA damage and leakage after SCI, SUMOylation of Lamin B1 is induced by nuclear Lamina accumulation of UBC9. Furthermore, it promotes LC3-Lamin B1 interaction to trigger nucleophagy that protects against neuronal DNA damage.

Naringenin reduces oxidative stress and necroptosis, apoptosis, and pyroptosis in random-pattern skin flaps by enhancing autophagy.

BACKGROUND: Random skin flap grafting is one of the most commonly used techniques in plastic and orthopedic surgery. However, necrosis resulting from ischemia and ischemia-reperfusion injury in the distal part of the flap can severely limit the clinical application of the flap. Studies have revealed that naringenin reduces pyroptosis, apoptosis, and necroptosis, inhibits oxidative stress, and promotes autophagy. In this study, the effects of Naringenin on flap viability and its underlying mechanism were evaluated. METHODS: Mice with random skin flaps were randomly allocated to control, Naringenin, and Naringenin + 3-methyladenine groups. On postoperative day 7, flap tissues were collected to estimate angiogenesis, necroptosis, apoptosis, pyroptosis, oxidative stress, and autophagy via hematoxylin and eosin staining, immunofluorescence, and immunohistochemistry. RESULTS: The results revealed that naringenin promoted the viability of the random flaps as well as angiogenesis, while inhibiting oxidative stress and decreasing pyroptosis, apoptosis, and necroptosis. These effects were reversed by the autophagy inhibitor 3-methyladenine. CONCLUSIONS: The findings indicated that naringenin treatment could promote flap survival by inhibiting pyroptosis, apoptosis, necroptosis, and alleviating oxidative stress, caused by the activation of autophagy.

Ezrin inhibition alleviates oxidative stress and pyroptosis via regulating TRPML1-calcineurin axis mediated enhancement of autophagy in spinal cord injury.

Spinal cord injury (SCI) presents profound ramifications for patients, leading to diminished motor and sensory capabilities distal to the lesion site. Once SCI occurs, it not only causes great physical and psychological problems for patients but also imposes a heavy economic burden. Ezrin is involved in various cellular processes, including signal transduction, cell death, inflammation, chemotherapy resistance and the stress response. However, whether Ezrin regulates functional repair after SCI and its underlying mechanism has not been elucidated. Here, our results showed that there is a marked augmentation of Ezrin levels within neurons and Ezrin inhibition markedly diminished glial scarring and bolstered functional recuperation after SCI. RNA sequencing indicated the potential involvement of pyroptosis, oxidative stress and autophagy in the enhancement of functional recovery upon reduced Ezrin expression. Moreover, the inhibition of Ezrin expression curtailed pyroptosis and oxidative stress by amplifying autophagy. Our studies further demonstrated that Ezrin inhibition promoted autophagy by increasing TFEB activity via the Akt-TRPML1-calcineurin pathway. Finally, we concluded that inhibiting Ezrin expression alleviates pyroptosis and oxidative stress by enhancing TFEB-driven autophagy, thereby promoting functional recovery after SCI, which may be a promising therapeutic target for SCI treatment.

Identification of autophagy-associated circRNAs in sepsis-induced cardiomyopathy of mice.

Circular RNAs (circRNAs) play a role in sepsis-related autophagy. However, the role of circRNAs in autophagy after sepsis-induced cardiomyopathy (SICM) is unknown, so we explored the circRNA expression profiles associated with autophagy in an acute sepsis mouse model. At a dose of 10 mg/kg, mice were intraperitoneally administered with lipopolysaccharides. The myocardial tissue was harvested after 6 h for microarray analysis, qRT-PCR, and western blotting. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and Gene Set Enrichment Analysis were evaluated, and a competing endogenous RNA network was constructed, to evaluate the role of circRNAs related to autophagy in SICM. In total, 1,735 differently expressed circRNAs were identified in the LPS-treated group, including 990 upregulated and 745 downregulated circRNAs. The expression level of the autophagy-specific protein p62 decreased, while the ratio of LC3 II to LC3 I increased. Additionally, 309 mRNAs and 187 circRNAs were correlated with autophagy in myocardial tissue after SICM. Of these, 179 circRNAs were predicted to function as "miRNA sponges". Some distinctive circRNAs and mRNAs found by ceRNA analysis might be involved in autophagy in SICM. These findings provide insights into circRNAs and identified new research targets that may be used to further explore the pathogenesis of SICM.

Pinocembrin alleviates pyroptosis and apoptosis through ROS elimination in random skin flaps via activation of SIRT3.

Random skin flap grafting is the most common skin grafting technique in reconstructive surgery. Despite progress in techniques, the incidence of distal flap necrosis still exceeds 3%, which limits its use in clinical practice. Current methods for treating distal flap necrosis are still lacking. Pinocembrin (Pino) can inhibit reactive oxygen species (ROS) and cell death in a variety of diseases, such as cardiovascular diseases, but the role of Pino in random flaps has not been explored. Therefore, we explore how Pino can enhance flap survival and its specific upstream mechanisms via macroscopic examination, Doppler, immunohistochemistry, and western blot. The results suggested that Pino can enhance the viability of random flaps by inhibiting ROS, pyroptosis and apoptosis. The above effects were reversed by co-administration of Pino with adeno-associated virus-silencing information regulator 2 homolog 3 (SIRT3) shRNA, proving the beneficial effect of Pino on the flaps relied on SIRT3. In addition, we also found that Pino up-regulates SIRT3 expression by activating the AMP-activated protein kinase (AMPK) pathway. This study proved that Pino can improve random flap viability by eliminating ROS, and ROS-induced cell death through the activation of SIRT3, which are triggered by the AMPK/PGC-1α signaling pathway.