Celastrol enhances the viability of random-pattern skin flaps by regulating autophagy through the AMPK-mTOR-TFEB axis.

In reconstructive and plastic surgery, random-pattern skin flaps (RPSF) are often used to correct defects. However, their clinical usefulness is limited due to their susceptibility to necrosis, especially on the distal side of the RPSF. This study validates the protective effect of celastrol (CEL) on flap viability and explores in terms of underlying mechanisms of action. The viability of different groups of RPSF was evaluated by survival zone analysis, laser doppler blood flow, and histological analysis. The effects of CEL on flap angiogenesis, apoptosis, oxidative stress, and autophagy were evaluated by Western blot, immunohistochemistry, and immunofluorescence assays. Finally, its mechanistic aspects were explored by autophagy inhibitor and Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) inhibitor. On the seventh day after surgery, the survival area size, blood supply, and microvessel count of RPSF were augmented following the administration of CEL. Additionally, CEL stimulated angiogenesis, suppressed apoptosis, and lowered oxidative stress levels immediately after elevated autophagy in ischemic regions; These effects can be reversed using the autophagy inhibitor chloroquine (CQ). Specifically, CQ has been observed to counteract the protective impact of CEL on the RPSF. Moreover, it has also been discovered that CEL triggers the AMPK-mTOR-TFEB axis activation in the area affected by ischemia. In CEL-treated skin flaps, AMPK inhibitors were demonstrated to suppress the AMPK-mTOR-TFEB axis and reduce autophagy levels. This investigation suggests that CEL benefits the survival of RPSF by augmenting angiogenesis and impeding oxidative stress and apoptosis. The results are credited to increased autophagy, made possible by the AMPK-mTOR-TFEB axis activation.

Erastin promotes random-pattern skin flaps survival by inducing mTORC1-TFEB mediated autophagy.

Random-pattern skin flaps are important method for skin reconstruction after defect; however, the distal end of flaps is not easily viable due to inadequate nutrient supply. Erastin is a well-established ferroptosis inducer, but our study found that low-dose of erastin (2 µM) may reduce nutrient deficiency induced cell death in human umbilical vein endothelial cells (HUVECs). RNA-seq analysis suggested that its role was related to autophagy regulation. Follow-up studies have shown that the use of autophagy inhibitors or the knockdown of TFEB in HUVECs can both reduce the anti-apoptotic effect of erastin in HUVECs. Mechanism study demonstrated that erastin can suppress mTORC1 and promote TFEB activity in HUVECs, suggesting that the effect of erastin on the survival of HUVECs under nutrient deprivation conditions is regulated by mTORC1/TFEB. Subsequently, we evaluated the effect of erastin on the survival of random-pattern skin flaps in mice in vivo. On the postoperative day 7, we observed a significant increase in flap survival area, blood perfusion, and microvascular density after erastin treatment; also, erastin treatment showed enhanced autophagy within the ischemic region. In summary, our study demonstrates that low-dose of erastin may suppress cell death in endothelial cells under nutrient deficiency condition, and its effects may relate to the mTORC1-TFEB medicated autophagy regulation, erastin treatment may be a potential therapy for random-pattern skin flaps.

20-Deoxyingenol Activates Mitophagy Through TFEB and Promotes Functional Recovery After Spinal Cord Injury.

Spinal cord injury (SCI) can lead to permanent paralysis and various motor, sensory and autonomic nervous system dysfunction. The complex pathophysiological processes limit the effectiveness of many clinical treatments. Mitochondria has been reported to play a key role in the pathogenesis of SCI; while mitophagy is a protective mechanism against mitochondrial dysfunction. However, there is recently little drugs that may targeted activate mitophagy to treat SCI. In this study, we evaluated the role of 20-Deoxyingenol (20-DOI) in SCI and explored its potential mechanisms. We used a SCI rat model and evaluated the functional outcomes after the injury. Western blotting and immunofluorescence techniques were used to analyze the levels of mitophagy, apoptosis, and TFEB-related signaling pathways. Our research results show that 20-DOI significantly improves the apoptosis of neural cells after TBHP stimulation and functional recovery after spinal cord injury. In addition, mitophagy, TFEB levels, and apoptosis are related to the mechanism of 20-DOI treatment for spinal cord injury. Specifically, our research results indicate that 20-DOI restored the autophagic flux after injury, thereby inducing mitophagy, eliminating the accumulation of Cyto C, and inhibiting apoptosis. Further mechanism research suggests that 20-DOI may regulate mitophagy by promoting TFEB nuclear translocation. These results indicate that 20-DOI can significantly promote recovery after spinal cord injury, which may be a promising treatment method for spinal cord injury.

Fisetin suppresses ferroptosis through Nrf2 and attenuates intervertebral disc degeneration in rats.

Low back pain, primarily caused by intervertebral disc degeneration (IVDD), lacks effective pharmacological treatments. Oxidative stress has been identified as a significant contributor to IVDD. This study aims to establish an in vitro model of IVDD induced by oxidative stress and identify potential therapeutic agents and their underlying mechanisms. By screening the natural product library, fisetin emerged as the most promising compound in suppressing cell death induced by oxidative stress in nucleus pulposus cells (NPCs). Furthermore, our investigation revealed that the cell death induced by oxidative stress was predominantly associated with ferroptosis, and fisetin demonstrated the ability to inhibit ferroptosis in NPCs. Mechanistic exploration suggested that the impact of fisetin on ferroptosis may be mediated through the Nrf2/HO-1 (Nuclear factor erythroid 2-related factor 2/heme oxygenase-1) axis. Notably, the in vivo study demonstrated that fisetin could alleviate IVDD in rats. These findings highlight fisetin as a potential therapeutic option for IVDD and implicate the involvement of the Nrf2/HO-1 pathway in its mechanism of action.

Acceleration of bone repairation by BMSCs overexpressing NGF combined with NSA and allograft bone scaffolds.

BACKGROUND: Repairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis. METHODS: The relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF+/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague-Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair. RESULTS: The pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF+/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects. CONCLUSION: NGF+/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF+/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs.