Spermidine promotes nucleus pulposus autophagy as a protective mechanism against apoptosis and ameliorates disc degeneration.

Spermidine has therapeutic effects in many diseases including as heart diastolic function, myopathic defects and neurodegenerative disorders via autophagy activation. Autophagy has been found to mitigate cell apoptosis in intervertebral disc degeneration (IDD). Accordingly, we theorize that spermidine may have beneficial effects on IDD via autophagy stimulation. In this study, spermidine's effect on IDD was evaluated in tert-butyl hydroperoxide (TBHP)-treated nucleus pulposus cells of SD rats in vitro as well as in a puncture-induced rat IDD model. We found that autophagy was actuated by spermidine in nucleus pulposus cells. In addition, spermidine treatment weakened the apoptotic effects of TBHP in nucleus pulposus cells. Spermidine increased the expression of anabolic proteins including Collagen-II and aggrecan and decreased the expression of catabolic proteins including MMP13 and Adamts-5. Additionally, autophagy blockade using 3-MA reversed the beneficial impact of spermidine against nucleus pulposus cell apoptosis. Autophagy was thus important for spermidine's therapeutic effect on IDD. Spermidine-treated rats had an accentuated T2-weighted signal and a diminished histological degenerative grade than vehicle-treated rats, showing that spermidine inhibited intervertebral disc degeneration in vivo. Thus, spermidine protects nucleus pulposus cells against apoptosis through autophagy activation and improves disc, which may be beneficial for the treatment of IDD.

Spatiotemporal multi-omics: exploring molecular landscapes in aging and regenerative medicine.

Aging and regeneration represent complex biological phenomena that have long captivated the scientific community. To fully comprehend these processes, it is essential to investigate molecular dynamics through a lens that encompasses both spatial and temporal dimensions. Conventional omics methodologies, such as genomics and transcriptomics, have been instrumental in identifying critical molecular facets of aging and regeneration. However, these methods are somewhat limited, constrained by their spatial resolution and their lack of capacity to dynamically represent tissue alterations. The advent of emerging spatiotemporal multi-omics approaches, encompassing transcriptomics, proteomics, metabolomics, and epigenomics, furnishes comprehensive insights into these intricate molecular dynamics. These sophisticated techniques facilitate accurate delineation of molecular patterns across an array of cells, tissues, and organs, thereby offering an in-depth understanding of the fundamental mechanisms at play. This review meticulously examines the significance of spatiotemporal multi-omics in the realms of aging and regeneration research. It underscores how these methodologies augment our comprehension of molecular dynamics, cellular interactions, and signaling pathways. Initially, the review delineates the foundational principles underpinning these methods, followed by an evaluation of their recent applications within the field. The review ultimately concludes by addressing the prevailing challenges and projecting future advancements in the field. Indubitably, spatiotemporal multi-omics are instrumental in deciphering the complexities inherent in aging and regeneration, thus charting a course toward potential therapeutic innovations.

Mesenchymal stem cell-derived extracellular vesicles in skin wound healing: roles, opportunities and challenges.

Skin wounds are characterized by injury to the skin due to trauma, tearing, cuts, or contusions. As such injuries are common to all human groups, they may at times represent a serious socioeconomic burden. Currently, increasing numbers of studies have focused on the role of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) in skin wound repair. As a cell-free therapy, MSC-derived EVs have shown significant application potential in the field of wound repair as a more stable and safer option than conventional cell therapy. Treatment based on MSC-derived EVs can significantly promote the repair of damaged substructures, including the regeneration of vessels, nerves, and hair follicles. In addition, MSC-derived EVs can inhibit scar formation by affecting angiogenesis-related and antifibrotic pathways in promoting macrophage polarization, wound angiogenesis, cell proliferation, and cell migration, and by inhibiting excessive extracellular matrix production. Additionally, these structures can serve as a scaffold for components used in wound repair, and they can be developed into bioengineered EVs to support trauma repair. Through the formulation of standardized culture, isolation, purification, and drug delivery strategies, exploration of the detailed mechanism of EVs will allow them to be used as clinical treatments for wound repair. In conclusion, MSC-derived EVs-based therapies have important application prospects in wound repair. Here we provide a comprehensive overview of their current status, application potential, and associated drawbacks.

Spatial transcriptomics: recent developments and insights in respiratory research.

The respiratory system's complex cellular heterogeneity presents unique challenges to researchers in this field. Although bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) have provided insights into cell types and heterogeneity in the respiratory system, the relevant specific spatial localization and cellular interactions have not been clearly elucidated. Spatial transcriptomics (ST) has filled this gap and has been widely used in respiratory studies. This review focuses on the latest iterative technology of ST in recent years, summarizing how ST can be applied to the physiological and pathological processes of the respiratory system, with emphasis on the lungs. Finally, the current challenges and potential development directions are proposed, including high-throughput full-length transcriptome, integration of multi-omics, temporal and spatial omics, bioinformatics analysis, etc. These viewpoints are expected to advance the study of systematic mechanisms, including respiratory studies.

The marriage of immunomodulatory, angiogenic, and osteogenic capabilities in a piezoelectric hydrogel tissue engineering scaffold for military medicine.

BACKGROUND: Most bone-related injuries to grassroots troops are caused by training or accidental injuries. To establish preventive measures to reduce all kinds of trauma and improve the combat effectiveness of grassroots troops, it is imperative to develop new strategies and scaffolds to promote bone regeneration. METHODS: In this study, a porous piezoelectric hydrogel bone scaffold was fabricated by incorporating polydopamine (PDA)-modified ceramic hydroxyapatite (PDA-hydroxyapatite, PHA) and PDA-modified barium titanate (PDA-BaTiO3, PBT) nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. The physical and chemical properties of the Cs/Gel/PHA scaffold with 0-10 wt% PBT were analyzed. Cell and animal experiments were performed to characterize the immunomodulatory, angiogenic, and osteogenic capabilities of the piezoelectric hydrogel scaffold in vitro and in vivo. RESULTS: The incorporation of BaTiO3 into the scaffold improved its mechanical properties and increased self-generated electricity. Due to their endogenous piezoelectric stimulation and bioactive constituents, the as-prepared Cs/Gel/PHA/PBT hydrogels exhibited cytocompatibility as well as immunomodulatory, angiogenic, and osteogenic capabilities; they not only effectively induced macrophage polarization to M2 phenotype but also promoted the migration, tube formation, and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) and facilitated the migration, osteo-differentiation, and extracellular matrix (ECM) mineralization of MC3T3-E1 cells. The in vivo evaluations showed that these piezoelectric hydrogels with versatile capabilities significantly facilitated new bone formation in a rat large-sized cranial injury model. The underlying molecular mechanism can be partly attributed to the immunomodulation of the Cs/Gel/PHA/PBT hydrogels as shown via transcriptome sequencing analysis, and the PI3K/Akt signaling axis plays an important role in regulating macrophage M2 polarization. CONCLUSION: The piezoelectric Cs/Gel/PHA/PBT hydrogels developed here with favorable immunomodulation, angiogenesis, and osteogenesis functions may be used as a substitute in periosteum injuries, thereby offering the novel strategy of applying piezoelectric stimulation in bone tissue engineering for the enhancement of combat effectiveness in grassroots troops.

Nerve growth factor-induced Akt/mTOR activation protects the ischemic heart via restoring autophagic flux and attenuating ubiquitinated protein accumulation.

The dysregulation of autophagy is related to a variety of cardiovascular diseases, such as myocardial ischemia/reperfusion (I/R). Nerve growth factor (NGF) has been shown to have therapeutic potential in ischaemic heart injury. In this study, we demonstrate that NGF administration can accelerate autophagic flux and attenuate protein ubiquitination in myocardial I/R heart. Our results showed that NGF could restored heart function and decreased the apoptosis of cardiomyocytes which induced by myocardial I/R injury. The protective effect of NGF is associated with the inhibition of autophagy related proteins. On another hand, NGF enhances the clearance of ubiquitinated protein and increases the survival of myocardial cell in vivo and in vitro. Additionally, NGF could activate the PI3K/AKT and mTOR signaling pathways. These results suggested that the cardioprotective effect of NGF is related to the restoration of autophagic flux and attenuation of protein ubiquitination via the activation of PI3K/AKT and mTOR pathway.

bFGF Protects Against Blood-Brain Barrier Damage Through Junction Protein Regulation via PI3K-Akt-Rac1 Pathway Following Traumatic Brain Injury.

Many traumatic brain injury (TBI) survivors sustain neurological disability and cognitive impairments due to the lack of defined therapies to reduce TBI-induced blood-brain barrier (BBB) breakdown. Exogenous basic fibroblast growth factor (bFGF) has been shown to have neuroprotective function in brain injury. The present study therefore investigates the beneficial effects of bFGF on the BBB after TBI and the underlying mechanisms. In this study, we demonstrate that bFGF reduces neurofunctional deficits and preserves BBB integrity in a mouse model of TBI. bFGF suppresses RhoA and upregulates tight junction proteins, thereby mitigating BBB breakdown. In vitro, bFGF exerts a protective effect on BBB by upregulating tight junction proteins claudin-5, occludin, zonula occludens-1, p120-catenin, and ß-catenin under oxygen glucose deprivation/reoxygenation (OGD) in human brain microvascular endothelial cells (HBMECs). Both the in vivo and in vitro effects are related to the activation of the downstream signaling pathway, PI3K/Akt/Rac-1. Inhibition of the PI3K/Akt or Rac-1 by specific inhibitors LY294002 or si-Rac-1, respectively, partially reduces the protective effect of bFGF on BBB integrity. Overall, our results indicate that the protective role of bFGF on BBB involves the regulation of tight junction proteins and RhoA in the TBI model and OGD-induced HBMECs injury, and that activation of the PI3K/Akt /Rac-1 signaling pathway underlies these effects.

Gelatin Nanostructured Lipid Carriers Incorporating Nerve Growth Factor Inhibit Endoplasmic Reticulum Stress-Induced Apoptosis and Improve Recovery in Spinal Cord Injury.

Clinical translation of growth factor therapies faces multiple challenges; the most significant one is the short half-life of the naked protein. Gelatin nanostructured lipid carriers (GNLs) had previously been used to encapsulate the basic fibroblast growth factor to enhance the functional recovery in hemiparkinsonian rats. In this research, we comparatively study the enhanced therapy between nerve growth factor (NGF) loaded GNLs (NGF-GNLs) and NGF only in spinal cord injury (SCI). The effects of NGF-GNLs and NGF only were tested by the Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test, and footprint analysis. Western blot analysis and immunofluorescent staining were further performed to identify the expression of ER stress-related proteins, neuron-specific marker neuronal nuclei (NeuN), and growth-associated protein 43 (GAP43). Correlated downstream signals Akt/GSK-3ß and ERK1/2 were also analyzed with or without inhibitors. Results showed that NGF-GNLs, compared to NGF only, enhanced the neuroprotection effect in SCI rats. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 inhibited by NGF-GNL treatment were more obvious. Meanwhile, NGF-GNLs in the recovery of SCI are related to the inhibition of ER stress-induced cell death via the activation of downstream signals PI3K/Akt/GSK-3ß and ERK1/2.

Endoplasmic reticulum stress: relevance and therapeutics in central nervous system diseases.

Endoplasmic reticulum (ER) stress plays an important role in a range of neurological disorders, such as neurodegenation diseases, cerebral ischemia, spinal cord injury, sclerosis, and diabetic neuropathy. Protein misfolding and accumulation in the ER lumen initiate unfolded protein response in energy-starved neurons which are relevant to toxic effects. In neurological disorders, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, ER dysfunction is well recognized, but the mechanisms remain unclear. In stroke and ischemia, spinal cord injury, and amyotrophic lateral sclerosis, chronic activation of ER stress is considered as main pathogeny which causes neuronal disorders. By targeting components of these ER signaling responses, to explore clinical treatment strategies or new drugs in CNS neurological diseases might become possible and valuable in the future.

bFGF regulates autophagy and ubiquitinated protein accumulation induced by myocardial ischemia/reperfusion via the activation of the PI3K/Akt/mTOR pathway.

Autophagy is involved in the development and/or progression of many diseases, including myocardial ischemia/reperfusion (I/R). In this study, we hypothesized a protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the myocardial I/R model. Our results showed that bFGF improved heart function recovery and increased the survival of cardiomyocytes in myocardial I/R model. The protective effect of bFGF is related to the inhibition of LC3II levels. Additionally, bFGF enhances the clearance of Ub by p62 and increases the survival of H9C2 cells. Moreover, silencing of p62 partially blocks the clearance of Ub and abolishes the anti-apoptosis effect of bFGF. An shRNA against the autophagic machinery Atg7 increased the survival of H9C2 cells co-treated with bFGF and rapamycin. bFGF activates the downstream signaling of the PI3K/Akt/mTOR pathway. These results indicate that the role of bFGF in myocardial I/R recovery is related to the inhibition of excessive autophagy and increased ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new direction for bFGF drug development for heart disease and identifies protein signaling pathways involved in bFGF action.

[Effects of endoplasmic reticulum stress related proteins and their mediated apoptosis in the formation of deep tissue injury of pressure ulcer in rats].

OBJECTIVE: To explore the effects of endoplasmic reticulum stress (ERS) related proteins and their mediated apoptosis in the formation of deep tissue injury of pressure ulcer in rats. METHODS: Forty male Sprague-Dawley rats were divided into normal control group and groups A, B, C, D according to the random number table, with 8 rats in each group. Rats in group A were loaded with 22.47 kPa pressure with a special pressure apparatus for 2.0 h in the region over gracilis, and then unloaded for 0.5 h. Rats in group B were treated with the same manoeuvre as that in group A for 3 times in one day. Rats in groups C and D were treated with the same manoeuvre as that in group B for 2 and 3 days. Rats in normal control group were free from pressure loading. Rats in groups A, B, C, and D were sacrificed after pressure loading, and then the central part of pressure loaded muscular tissues were harvested for observation of histomorphological change with HE staining; apoptotic nucleoli per millimeter pressure loaded muscular tissue were counted with Hoechst 33258 staining; the levels of binding protein (BIP), protein disulfide isomerase (PDI), C/EBP homologous protein (CHOP), and caspase-12 were assessed with Western blotting (denoted as gray level ratio of target protein to GAPDH). The same parts of gracilis of rats in normal control group were harvested for determination of all the indexes as above. Data were processed with one-way analysis of variance, LSD-t test was applied for paired comparison. RESULTS: (1) Histomorphological observation. Some pathological changes, including inflammatory cell infiltration, myofibers lysis, and vacuolar degeneration, etc. were observed in pressure loaded muscular tissue of rats in groups A, B, C, and D, but not in the same parts of gracilis muscle of rats in normal control group. Compared with those in normal control group [(2.7 ± 1.4) per millimeter muscular tissue], the number of apoptotic nuclei was significantly increased in pressure loaded muscular tissue of rats in groups A, B, C, and D [(14.5 ± 4.4), (11.0 ± 2.9) , (13.8 ± 5.1), (21.3 ± 6.0) per millimeter pressure loaded muscular tissue, with t values from 4.223 to 6.000, P values all below 0.01). (2) Western blotting. The protein expressions of BIP and PDI in rats of normal control group and groups A, B, C, D were respectively 0.64 ± 0.12, 1.20 ± 0.34, 1.59 ± 0.24, 1.17 ± 0.28, 1.44 ± 0.33; 0.48 ± 0.15, 0.61 ± 0.19, 1.23 ± 0.38, 0.37 ± 0.19, 0.29 ± 0.15, and they showed significant statistical difference (with F values respectively 5.32, 7.95, P < 0.05 or P < 0.01). The protein expressions of CHOP and caspase-12 in rats of normal control group and groups A, B, C, D were respectively 0.58 ± 0.18, 1.48 ± 0.27, 1.03 ± 0.21, 0.95 ± 0.30, 1.69 ± 0.34; 0.55 ± 0.12, 1.08 ± 0.31, 0.69 ± 0.24, 1.79 ± 0.20, 2.06 ± 0.47, with significant statistical difference (with F values respectively 8.17, 15.48, P values all below 0.01). CONCLUSIONS: ERS related proteins and their apoptotic pathway may play an important role in the formation of deep tissue injury of pressure ulcer in rats.

Regulation of autophagy and ubiquitinated protein accumulation by bFGF promotes functional recovery and neural protection in a rat model of spinal cord injury.

The role of autophagy in the recovery of spinal cord injury remains controversial; in particular, the mechanism of autophagy regulated degradation of ubiquitinated proteins has not been discussed to date. In this study, we investigated the protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the rat model of trauma. bFGF administration improved recovery and increased the survival of neurons in spinal cord lesions in the rat model. The protective effect of bFGF is related to the inhibition of autophagic protein LC3II levels; bFGF treatment also enhances clearance of ubiquitinated proteins by p62, which also increases the survival of neuronal PC-12 cells. The activation of the downstream signals of the PI3K/Akt/mTOR pathway by bFGF treatment was detected both in vivo and in vitro. Combination therapy including the autophagy activator rapamycin partially abolished the protective effect of bFGF. The present study illustrates that the role of bFGF in SCI recovery is related to the inhibition of excessive autophagy and enhancement of ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new trend for bFGF drug development for central nervous system injuries and sheds light on protein signaling involved in bFGF action.

Exogenous basic fibroblast growth factor inhibits ER stress-induced apoptosis and improves recovery from spinal cord injury.

AIM: To investigate the mechanism of endoplasmic reticulum (ER) stress-induced apoptosis as well as the protective action of basic fibroblast growth factor (bFGF) both in vivo and in vitro. METHODS AND RESULTS: ER stress-induced apoptosis was involved in the injuries of spinal cord injury (SCI) model rat. bFGF administration improved the recovery and increased the survival of neurons in spinal cord lesions in model rat. The protective effect of bFGF is related to the inhibition of CHOP, GRP78 and caspase-12, which are ER stress-induced apoptosis response proteins. bFGF administration also increased the survival of neurons and the expression of growth-associated protein 43 (GAP43), which is related to neural regeneration. The protective effect of bFGF is related to the activation of downstream signals, PI3K/Akt/GSK-3ß and ERK1/2, especially in the ER stress cell model. CONCLUSIONS: This is the first study to illustrate that the role of bFGF in SCI recovery is related to the inhibition of ER stress-induced cell death via the activation of downstream signals. Our work also suggested a new trend for bFGF drug development in central neural system injuries, which are involved in chronic ER stress-induced apoptosis.

The anti-scar effects of basic fibroblast growth factor on the wound repair in vitro and in vivo.

Hypertrophic scars (HTS) and keloids are challenging problems. Their pathogenesis results from an overproduction of fibroblasts and excessive deposition of collagen. Studies suggest a possible anti-scarring effect of basic fibroblast growth factor (bFGF) during wound healing, but the precise mechanisms of bFGF are still unclear. In view of this, we investigated the therapeutic effects of bFGF on HTS animal model as well as human scar fibroblasts (HSF) model. We show that bFGF promoted wound healing and reduced the area of flattened non-pathological scars in rat skin wounds and HTS in the rabbit ear. We provide evidence of a new therapeutic strategy: bFGF administration for the treatment of HTS. The scar elevation index (SEI) and epidermal thickness index (ETI) was also significantly reduced. Histological reveal that bFGF exhibited significant amelioration of the collagen tissue. bFGF regulated extracellular matrix (ECM) synthesis and degradation via interference in the collagen distribution, the α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-ß1) expression. In addition, bFGF reduced scarring and promoted wound healing by inhibiting TGFß1/SMAD-dependent pathway. The levels of fibronectin (FN), tissue inhibitor of metalloproteinase-1 (TIMP-1) collagen I, and collagen III were evidently decreased, and matrix metalloproteinase-1 (MMP-1) and apoptosis cells were markedly increased. These results suggest that bFGF possesses favorable therapeutic effects on hypertrophic scars in vitro and in vivo, which may be an effective cure for human hypertrophic scars.

Electron holographic mapping of two-dimensional doping areas in cross-sectional device specimens prepared by the lift-out technique based on a focused ion beam.

Recently, electron holography has been successfully applied to analyze two-dimensional (2D) dopant distribution in semiconductor devices with high resolution and high sensitivity. The preparation of proper specimens is a fundamental step for the practical application of electron holography in the semiconductor industry. Therefore, it is important to explore a reliable and quick specimen preparation method. In our current work, we have tried to use the lift-out technique based on a focused ion beam, to fabricate cross-sectional CMOS device specimens for electron holographic observation. Using the lift-out technique, specimens with a large area and uniform thickness can be prepared directly from integrated circuit wafers in a very short time. Specimens with a complex and unknown dopant distribution were examined using off-axis electron holography. In the reconstructed phase images, the different 2D doping areas in a CMOS device, such as source, drain, well and substrate, were revealed successfully. The advantages and disadvantages of the technique are discussed.