Neuregulin-1, a member of the epidermal growth factor family, mitigates STING-mediated pyroptosis and necroptosis in ischaemic flaps.

Background: Ensuring the survival of the distal end of a random flap during hypoperfusion (ischaemia) is difficult in clinical practice. Effective prevention of programmed cell death is a potential strategy for inhibiting ischaemic flap necrosis. The activation of stimulator of interferon genes (STING) pathway promotes inflammation and leads to cell death. The epidermal growth factor family member neuregulin-1 (NRG1) reduces cell death by activating the protein kinase B (AKT) signalling pathway. Moreover, AKT signalling negatively regulates STING activity. We aimed to verify the efficacy of NRG1 injection in protecting against flap necrosis. Additionally, we investigated whether NRG1 effectively enhances ischemic flap survival by inhibiting pyroptosis and necroptosis through STING suppression. Methods: A random-pattern skin flap model was generated on the backs of C57BL/6 mice. The skin flap survival area was determined. The blood supply and vascular network of the flap was assessed by laser Doppler blood flow analysis. Cluster of differentiation 34 immunohistochemistry (IHC) and haematoxylin and eosin (H&E) staining of the flap sections revealed microvessels. Transcriptome sequencing analysis revealed the mechanism by which NRG1 promotes the survival of ischaemic flaps. The levels of angiogenesis, oxidative stress, necroptosis, pyroptosis and indicators associated with signalling pathways in flaps were examined by IHC, immunofluorescence and Western blotting. Packaging adeno-associated virus (AAV) was used to activate STING in flaps. Results: NRG1 promoted the survival of ischaemic flaps. An increased subcutaneous vascular network and neovascularization were found in ischaemic flaps after the application of NRG1. Transcriptomic gene ontology enrichment analysis and protein level detection indicated that necroptosis, pyroptosis and STING activity were reduced in the NRG1 group. The phosphorylation of AKT and forkhead box O3a (FOXO3a) were increased after NRG1 treatment. The increased expression of STING in flaps induced by AAV reversed the therapeutic effect of NRG1. The ability of NRG1 to phosphorylate AKT-FOXO3a, inhibit STING and promote flap survival was abolished after the application of the AKT inhibitor MK2206. Conclusions: NRG1 inhibits pyroptosis and necroptosis by activating the AKT-FOXO3a signalling pathway to suppress STING activation and promote ischaemic flap survival.

Evaluating the pro-survival potential of apoptotic bodies derived from 2D- and 3D- cultured adipose stem cells in ischaemic flaps.

In the realm of large-area trauma flap transplantation, averting ischaemic necrosis emerges as a pivotal concern. Several key mechanisms, including the promotion of angiogenesis, the inhibition of oxidative stress, the suppression of cell death, and the mitigation of inflammation, are crucial for enhancing skin flap survival. Apoptotic bodies (ABs), arising from cell apoptosis, have recently emerged as significant contributors to these functions. This study engineered three-dimensional (3D)-ABs using tissue-like mouse adipose-derived stem cells (mADSCs) cultured in a 3D environment to compare their superior biological effects against 2D-ABs in bolstering skin flap survival. The findings reveal that 3D-ABs (85.74 ± 4.51) % outperform 2D-ABs (76.48 ± 5.04) % in enhancing the survival rate of ischaemic skin flaps (60.45 ± 8.95) % (all p < 0.05). Mechanistically, they stimulated angiogenesis, mitigated oxidative stress, suppressed apoptosis, and facilitated the transition of macrophages from M1 to M2 polarization (all p < 0.05). A comparative analysis of microRNA (miRNA) profiles in 3D- and 2D-ABs identified several specific miRNAs (miR-423-5p-up, miR30b-5p-down, etc.) with pertinent roles. In summary, ABs derived from mADSCs cultured in a 3D spheroid-like arrangement exhibit heightened biological activity compared to those from 2D-cultured mADSCs and are more effective in promoting ischaemic skin flap survival. These effects are attributed to their influence on specific miRNAs.

Apoptotic Bodies Derived from Fibroblast-Like Cells in Subcutaneous Connective Tissue Inhibit Ferroptosis in Ischaemic Flaps via the miR-339-5p/KEAP1/Nrf2 Axis.

Preventing and treating avascular necrosis at the distal end of the flaps are critical to surgery success, but current treatments are not ideal. A recent study shows that apoptotic bodies (ABs) generated near the site of apoptosis can be taken up and promote cell proliferation. The study reveals that ABs derived from fibroblast-like cells in the subcutaneous connective tissue (FSCT cells) of skin flaps promoted ischaemic flap survival. It is also found that ABs inhibited cell death and oxidative stress and promoted M1-to-M2 polarization in macrophages. Transcriptome sequencing and protein level testing demonstrated that ABs promoted ischaemic flap survival in endothelial cells and macrophages by inhibiting ferroptosis via the KEAP1-Nrf2 axis. Furthermore, microRNA (miR) sequencing data and in vitro and in vivo experiments demonstrated that ABs inhibited KEAP1 by delivering miR-339-5p to exert therapeutic effects. In conclusion, FSCT cell-derived ABs inhibited ferroptosis, promoted the macrophage M1-to-M2 transition via the miR-339-5p/KEAP1/Nrf2 axis and promoted ischaemic flap survival. These results provide a potential therapeutic strategy to promote ischaemic flap survival by administering ABs.

Dihydrocapsaicin suppresses the STING-mediated accumulation of ROS and NLRP3 inflammasome and alleviates apoptosis after ischemia-reperfusion injury of perforator skin flap.

Avascular necrosis frequently occurs as a complication following surgery involving the distal perforator flap. Dihydrocapsaicin (DHC) can protect tissue from ischemia-reperfusion (I/R) injury, but its specific role in multizone perforator flaps remains unclear. In this study, the prospective target of DHC in the context of I/R injury was predicted using network pharmacology analysis. Flap viability was determined through survival area analysis, laser Doppler blood flow, angiograms, and histological examination. The expressions of angiogenesis, apoptosis, NLR family pyrin domain containing 3 (NLRP3) inflammasome, oxidative stress, and molecules related to cyclic guanosine monophosphate (GMP)-adenosine monophosphate synthase (cGAS)-interferon gene stimulant (STING) pathway were assessed using western blotting, immunofluorescence, TUNEL staining, and dihydroethidium (DHE) staining. Our finding revealed that DHC promoted the perforator flap survival, which involves the cGAS-STING pathway, oxidative stress, NLRP3 inflammasome, apoptosis, and angiogenesis. DHC induced oxidative stress resistance and suppressed the NLRP3 inflammasome, preventing apoptosis in vascular endothelial cells. Through regulation of STING pathway, DHC controlled oxidative stress in endothelial cells and NLRP3 levels in ischemic flaps. However, activation of the cGAS-STING pathway led to the accumulation of reactive oxygen species (ROS) and NLRP3 inflammasome, thereby diminishing the protective role of DHC. DHC enhanced the survival of multidomain perforator flaps by suppressing the cGAS-STING pathway, oxidative stress, and the formation of NLRP3 inflammasome. These findings unveil a potentially novel mechanism with clinical significance for promoting the survival of multidomain perforator flaps.

FGF-18 Protects the Injured Spinal cord in mice by Suppressing Pyroptosis and Promoting Autophagy via the AKT-mTOR-TRPML1 axis.

Spinal cord injury (SCI) is a severe medical condition with lasting effects. The efficacy of numerous clinical treatments is hampered by the intricate pathophysiological mechanism of SCI. Fibroblast growth factor 18 (FGF-18) has been found to exert neuroprotective effects after brain ischaemia, but its effect after SCI has not been well explored. The aim of the present study was to explore the therapeutic effect of FGF-18 on SCI and the related mechanism. In the present study, a mouse model of SCI was used, and the results showed that FGF-18 may significantly affect functional recovery. The present findings demonstrated that FGF-18 directly promoted functional recovery by increasing autophagy and decreasing pyroptosis. In addition, FGF-18 increased autophagy, and the well-known autophagy inhibitor 3-methyladenine (3MA) reversed the therapeutic benefits of FGF-18 after SCI, suggesting that autophagy mediates the therapeutic effects of FGF-18 on SCI. A mechanistic study revealed that after stimulation of the protein kinase B (AKT)-transient receptor potential mucolipin 1 (TRPML1)-calcineurin signalling pathway, the FGF-18-induced increase in autophagy was mediated by the dephosphorylation and nuclear translocation of transcription factor E3 (TFE3). Together, these findings indicated that FGF-18 is a robust autophagy modulator capable of accelerating functional recovery after SCI, suggesting that it may be a promising treatment for SCI in the clinic.

A snake cathelicidin enhances transcription factor EB-mediated autophagy and alleviates ROS-induced pyroptosis after ischaemia-reperfusion injury of island skin flaps.

BACKGROUND AND PURPOSE: Ischaemia-reperfusion (I/R) injury is a major contributor to skin flap necrosis, which presents a challenge in achieving satisfactory therapeutic outcomes. Previous studies showed that cathelicidin-BF (BF-30) protects tissues from I/R injury. In this investigation, BF-30 was synthesized and its role and mechanism in promoting survival of I/R-injured skin flaps explored. EXPERIMENTAL APPROACH: Survival rate analysis and laser Doppler blood flow analysis were used to evaluate I/R-injured flap viability. Western blotting, immunofluorescence, TdT-mediated dUTP nick end labelling (TUNEL) and dihydroethidium were utilized to examine the levels of apoptosis, pyroptosis, oxidative stress, transcription factor EB (TFEB)-mediated autophagy and molecules related to the adenosine 5'-monophosphate-activated protein kinase (AMPK)-transient receptor potential mucolipin 1 (TRPML1)-calcineurin signalling pathway. KEY RESULTS: The outcomes revealed that BF-30 enhanced I/R-injured island skin flap viability. Autophagy, oxidative stress, pyroptosis and apoptosis were related to the BF-30 capability to enhance I/R-injured flap survival. Improved autophagy flux and tolerance to oxidative stress promoted the inhibition of apoptosis and pyroptosis in vascular endothelial cells. Activation of TFEB increased autophagy and inhibited endothelial cell oxidative stress in I/R-injured flaps. A reduction in TFEB level led to a loss of the protective effect of BF-30, by reducing autophagy flux and increasing the accumulation of reactive oxygen species (ROS) in endothelial cells. Additionally, BF-30 modulated TFEB activity via the AMPK-TRPML1-calcineurin signalling pathway. CONCLUSION AND IMPLICATIONS: BF-30 promotes I/R-injured skin flap survival by TFEB-mediated up-regulation of autophagy and inhibition of oxidative stress, which may have possible clinical applications.

Baicalin promotes random-pattern skin flap survival by inducing autophagy via AMPK-regulated TFEB nuclear transcription.

The random-pattern skin flap is a generally used technique to cover the soft tissue defect, while its application is often constrained by complications after the flap transplant. Necrosis of the flap remains a principal obstacle. The purpose of this study was to investigate the effect of Baicalin on skin flap survival and its mechanism. First of all, we discovered that administering Baicalin stimulated cell migration and boosted the formation of capillary tubes in human umbilical vein endothelial cells. Then, we detected that Baicalin reduced apoptosis-induced oxidative stress by using western blot and oxidative stress test kit. After that, we observed that Baicalin increased autophagy and utilized 3MA to block autophagy augmentation substantially reversing the effects of Baicalin therapy. Furthermore, we uncovered the underlying mechanisms of Baicalin-induced autophagy via AMPK-regulated TFEB nuclear transcription. Finally, our in vivo experiment findings showed that Baicalin reduces oxidative stress, inhibits apoptosis, promotes angiogenesis, and boosts the levels of autophagy. After autophagy was blocked, substantially reversing the effects of Baicalin therapy. Our study indicated that Baicalin-induced autophagy via AMPK regulated TFEB nuclear transcription and then promotes angiogenesis and against oxidative stress and apoptotic promotes skin flap survival. These findings highlight the therapeutic potential for the clinical application of Baicalin in the future.

3,4-Dimethoxychalcone, a caloric restriction mimetic, enhances TFEB-mediated autophagy and alleviates pyroptosis and necroptosis after spinal cord injury.

Background: Caloric restriction mimetics (CRMs) mimic the favourable effects of caloric restriction (CR) and have been shown to have therapeutic effects in neuroinflammatory disease. However, whether CRMs improve the functional recovery from spinal cord injury (SCI) and the underlying mechanism of action remain unclear. In this study, we used the CRMs 3,4-dimethoxychalcone (3,4-DC) to evaluate the therapeutic value of CRMs for SCI. Methods: HE, Masson and Nissl staining; footprint analysis; and the Basso mouse scale were used to determine the functional recovery from SCI after 3,4-DC treatment. RNA sequencing was used to identify the mechanisms of 3,4-DC in SCI. Western blotting, qPCR and immunofluorescence were used to detect the levels of pyroptosis, necroptosis, autophagy and the AMPK-TRPML1-calcineurin signalling pathway. We employed a dual-luciferase reporter assay in vitro and applied AAV vectors to inhibit TFEB in vivo to explore the mechanism of 3,4-DC. Results: 3,4-DC reduced glial scar area and motor neuron death and improved functional recovery after SCI. RNA-sequencing results indicated that oxidative stress, pyroptosis, necroptosis, and autophagy may be involved in the ability of 3,4-DC to improve functional recovery. Furthermore, 3,4-DC inhibited pyroptosis and necroptosis by enhancing autophagy. We also found that 3,4-DC enhances autophagy by promoting TFEB activity. A decrease in the TFEB level abolished the protective effect of 3,4-DC. In addition, 3,4-DC partially regulated TFEB activity through the AMPK-TRPML1-calcineurin signalling pathway. Conclusions: 3,4-DC promotes functional recovery by upregulating TFEB-mediated autophagy and inhibiting pyroptosis and necroptosis after SCI, which may have potential clinical application value.

Diallyl Trisulfide Enhances the Survival of Multiterritory Perforator Skin Flaps.

The multiterritory perforator flap is one of the widest flap patterns used to repair tissue defects. However, flap necrosis of the distal part is still a challenging issue for plastic surgeons. Diallyl trisulfide (DATS) is an efficient ingredient extracted from garlic, exerting many important effects on different diseases. Our experiment aims to reveal whether DATS has a beneficial effect on the survival of perforator flaps and to explore its mechanism of action. The results showed that DATS enhanced angiogenesis and autophagy and reduced cell apoptosis and oxidative stress, thereby improving the survival rate of skin flaps. After co-administration with autophagy inhibitor 3-methyladenine (3MA), perforator flap survival was further improved. Mechanistically, we showed that PI3K/Akt and AMPK-HIF-1α signaling pathways in flap were activated under DATS treatment. All in all, DATS promoted the survival of multiterritory perforator flaps via the synergistic regulation of PI3K/Akt and AMPK-HIF-1α signaling pathways, and inhibition of DATS-induced autophagy further improves flap survival.

Inhibition of PLA2G4E/cPLA2 promotes survival of random skin flaps by alleviating Lysosomal membrane permeabilization-Induced necroptosis.

Necrosis that appears at the ischemic distal end of random-pattern skin flaps increases the pain and economic burden of patients. Necroptosis is thought to contribute to flap necrosis. Lysosomal membrane permeabilization (LMP) plays an indispensable role in the regulation of necroptosis. Nonetheless, the mechanisms by which lysosomal membranes become leaky and the relationship between necroptosis and lysosomes are still unclear in ischemic flaps. Based on Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, and liquid chromatography-mass spectrometry (LC-MS) analysis results, we found that LMP was presented in the ischemic distal portion of random-pattern skin flaps, which leads to disruption of lysosomal function and macroautophagic/autophagic flux, increased necroptosis, and aggravated necrosis of the ischemic flaps. Moreover, bioinformatics analysis of the LC-MS results enabled us to focus on the role of PLA2G4E/cPLA2 (phospholipase A2, group IVE) in LMP of the ischemic flaps. In vivo inhibition of PLA2G4E with an adeno-associated virus vector attenuated LMP and necroptosis, and promoted flap survival. In addition, microRNA-seq helped us determine that Mir504-5p was differentially expressed in ischemic flaps. A string of in vitro and in vivo tests was employed to verify the inhibitory effect of Mir504-5p on PLA2G4E, LMP and necroptosis. Finally, we concluded that the inhibition of PLA2G4E by Mir504-5p reduced LMP-induced necroptosis, thereby promoting the survival of random-pattern skin flaps.Abbreviations: AAV: adeno-associated virus; ACTA2/α;-SMA: actin alpha 2, smooth muscle, aorta; ALOX15/12/15-LOX: arachidonate 15- lipoxygenase; c-CASP8: cleaved caspase; c-CASP3: cleaved caspase 3; CTSD: cathepsin D; CTSB: cathepsin B; CTSL: cathepsin L; DMECs: primary mouse dermal microvascular endothelial cells; ELISA: enzyme-linked immunosorbent assay; F-CHP: 5-FAM-conjugated collagen hybridizing peptide; FISH: fluorescence in situ hybridization; HUVECs: human umbilical vein endothelial cells; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LC-MS: liquid chromatography-mass spectrometry; LDBF: laser doppler blood flow; LMP: lysosomal membrane permeabilization; LPE: lysophosphatidylethanolamine; LPC: lysophosphatidylcholine; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MLKL: mixed lineage kinase domain-like; NDI: N-dodecylimidazole; PECAM1/CD31: platelet/endothelial cell adhesion molecule 1; PLA2G4A/cPLA2: phospholipase A2, group IVA (cytosolic, calcium-dependent); PLA2G4E/cPLA2: phospholipase A2, group IVE; qPCR: quantitative real-time polymerase chain reaction; RIPK1: receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3: receptor-interacting serine-threonine kinase 3; RISC: RNA-induced silencing complex; ROS: reactive oxygen species; shRNA: short hairpin RNA; SQSTM1: sequestosome 1; TBHP: tert-butyl hydroperoxide; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.

Liraglutide, a TFEB-Mediated Autophagy Agonist, Promotes the Viability of Random-Pattern Skin Flaps.

Random skin flaps are commonly used in reconstruction surgery. However, distal necrosis of the skin flap remains a difficult problem in plastic surgery. Many studies have shown that activation of autophagy is an important means of maintaining cell homeostasis and can improve the survival rate of flaps. In the current study, we investigated whether liraglutide can promote the survival of random flaps by stimulating autophagy. Our results show that liraglutide can significantly improve flap viability, increase blood flow, and reduce tissue oedema. In addition, we demonstrated that liraglutide can stimulate angiogenesis and reduce pyroptosis and oxidative stress. Through immunohistochemistry analysis and Western blotting, we verified that liraglutide can enhance autophagy, while the 3-methylladenine- (3MA-) mediated inhibition of autophagy enhancement can significantly reduce the benefits of liraglutide described above. Mechanistically, we showed that the ability of liraglutide to enhance autophagy is mediated by the activation of transcription factor EB (TFEB) and its subsequent entry into the nucleus to activate autophagy genes, a phenomenon that may result from AMPK-MCOLN1-calcineurin signalling pathway activation. Taken together, our results show that liraglutide is an effective drug that can significantly improve the survival rate of random flaps by enhancing autophagy, inhibiting oxidative stress in tissues, reducing pyroptosis, and promoting angiogenesis, which may be due to the activation of TFEB via the AMPK-MCOLN1-calcineurin signalling pathway.

Targeting TFE3 Protects Against Lysosomal Malfunction-Induced Pyroptosis in Random Skin Flaps via ROS Elimination.

Increasing evidence indicates that pyroptosis, a new type of programmed cell death, may participate in random flap necrosis and play an important role. ROS-induced lysosome malfunction is an important inducement of pyroptosis. Transcription factor E3 (TFE3) exerts a decisive effect in oxidative metabolism and lysosomal homeostasis. We explored the effect of pyroptosis in random flap necrosis and discussed the effect of TFE3 in modulating pyroptosis. Histological analysis via hematoxylin-eosin staining, immunohistochemistry, general evaluation of flaps, evaluation of tissue edema, and laser Doppler blood flow were employed to determine the survival of the skin flaps. Western blotting, immunofluorescence, and enzyme-linked immunosorbent assays were used to calculate the expressions of pyroptosis, oxidative stress, lysosome function, and the AMPK-MCOLN1 signaling pathway. In cell experiments, HUVEC cells were utilized to ensure the relationship between TFE3, reactive oxygen species (ROS)-induced lysosome malfunction and cell pyroptosis. Our results indicate that pyroptosis exists in the random skin flap model and oxygen and glucose deprivation/reperfusion cell model. In addition, NLRP3-mediated pyroptosis leads to necrosis of the flaps. Moreover, we also found that ischemic flaps can augment the accumulation of ROS, thereby inducing lysosomal malfunction and finally initiating pyroptosis. Meanwhile, we observed that TFE3 levels are interrelated with ROS levels, and overexpression and low expression of TFE3 levels can, respectively, inhibit and promote ROS-induced lysosomal dysfunction and pyroptosis during in vivo and in vitro experiments. In conclusion, we found the activation of TFE3 in random flaps is partially regulated by the AMPK-MCOLN1 signal pathway. Taken together, TFE3 is a key regulator of ROS-induced pyroptosis in random skin flaps, and TFE3 may be a promising therapeutic target for improving random flap survival.

Numerical analysis of an ultra-wideband metamaterial absorber with high absorptivity from visible light to near-infrared.

In this study, we designed a novel ultra-wideband (UWB) absorber and numerically analyzed it to demonstrate that its light absorptivity was greater than 90% in the wavelength range of visible light and near-infrared (405-1505 nm). The structure of proposed novel UWB absorber consisted of four layers of films, including silica, titanium, magnesium fluoride, and aluminium, and the upper silica and titanium layers had rectangular cubes in them. For that, the excitations of propagating surface plasmon resonance (PSPR), local surface plasmon resonance (LSPR), and the resonance of Fabry-Perot (FP) cavity were generated at the same time and combined to reach the effect of perfect absorption and ultra-wideband. The proposed absorber had an average absorptivity of 95.14% in the wavelength range of 405 ∼ 1505 nm when the light was under normal incidence. In addition, the UWB absorber was large incident angle insensitive and polarization-independent. The absorber proposed in the paper had great prospects in the fields of thermal electronic equipment, solar power generation, and perfect cloaking.