Effects of Life-Long Exercise on Age-Related Inflammation, Apoptosis, Oxidative Stress, Ferroptosis Markers, and NRF2/KAEP 1/Klotho Pathway in Rat Kidneys.

Xi-Kun Yuan Pin-Shi Ni Zhen-Hao Yan Zhi Yu Zhuang-Zhi Wang Chen-Kai Zhang Fang-Hui Li Xiao-Ming Yu 1Sports Department, Nanjing University of Science and Technology ZiJin College, Nanjing, China, 2School of Sport Sciences, Nanjing Normal University, Nanjing, China, 3Shanghai Seventh People's Hospital, Shanghai, China To investigate the effects of life-long exercise (LLE) on age-related inflammatory cytokines, apoptosis, oxidative stress, ferroptosis markers, and the NRF2/KAEP 1/Klotho pathway in rats. Eight-month-old female Sprague-Dawley rats were divided into four groups: 1) LLE: 18-month LLE training starting at 8 months of age, 2) Old moderate-intensity continuous training (OMICT): 8 months of moderate-intensity continuous training starting at 18 months of age, 3) Adult sedentary (ASED): 8 month-old adult sedentary control group, and 4) Old sedentary (OSED): a 26-month-old sedentary control group. Hematoxylin eosin staining was performed to observe the pathological changes of kidney tissue injury in rats; Masson's staining to observe the deposition of collagen fibers in rat kidney tissues; and western blotting to detect the expression levels of IL-6, IL 1beta, p53, p21, TNF-alpha, GPX4, KAEP 1, NRF2, SLC7A11, and other proteins in kidney tissues. Results: Compared with the ASED group, the OSED group showed significant morphological changes in renal tubules and glomeruli, which were swollen and deformed, with a small number of inflammatory cells infiltrated in the tubules. Compared with the OSED group, the expression levels of inflammation-related proteins such as IL-1beta, IL-6, TNF alpha, and MMP3 were significantly lower in the LLE group. Quantitative immunofluorescence analysis and western blotting revealed that compared with the ASED group, KAEP 1 protein fluorescence intensity and protein expression levels were significantly enhanced, while Klotho and NRF2 protein fluorescence intensity and protein expression levels were reduced in the OSED group. Compared with the OSED group, KAEP 1 protein fluorescence intensity and protein expression levels were reduced in the LLE and OMICT groups. Klotho and KAEP 1 protein expression levels and immunofluorescence intensity were higher in the LLE group than in the OSED group. The expression levels of GPX4 and SLC7A11, two negative marker proteins associated with ferroptosis, were significantly higher in the LLE group than in the OSED group, while the expression of p53 a cellular senescence-associated protein that negatively regulates SLC7A11, and the downstream protein p21 were significantly decreased. LLE may ameliorated aging-induced oxidative stress, inflammatory response, apoptosis, and ferroptosis by regulating Klotho and synergistically activating the NRF2/KAEP 1 pathway. Keywords: Life-long exercise, Moderate intensity continuous training, Aging, Kidney tissue, Ferroptosis.

Neutrophil membrane-coated multifunctional biomimetic nanoparticles for spinal cord injuries.

Spinal cord injury (SCI) is one of the most complex diseases. After SCI, severe secondary injuries can cause intense inflammatory storms and oxidative stress responses, leading to extensive neuronal apoptosis. Effective regulation of inflammation and oxidative stress after SCI remains an unresolved challenge. In this study, resveratrol-loaded nanoparticles coated with neutrophil membranes (NMR) were prepared using the emulsion-solvent evaporation method and membrane encapsulation technology. Multifunctional biomimetic nanoparticles retain neutrophil membrane-related receptors and possess a strong adsorption capacity for inflammatory factors. As a drug carrier, NMR can sustainably release resveratrol for >72 h. Moreover, co-culture studies in vitro show that the NMR help regulate macrophage polarization to relieve inflammatory response, reduce intracellular reactive oxygen species by approximately 50%, and improve mitochondrial membrane potential to alleviate oxidative stress. After injecting NMR into the injury site, it reduces early apoptosis, inhibit scar formation, and promote neural network recovery to improve motor function. This study demonstrates the anti-inflammatory, antioxidant, and neuroprotective effects of NMR, thus providing a novel therapeutic strategy for SCI.

Hydrogel Microsphere-Encapsulated Bimetallic Nanozyme for Promoting Diabetic Bone Regeneration via Glucose Consumption and ROS Scavenging.

The healing of bone defects among diabetic patients presents a critical challenge due to the pathological microenvironment, characterized by hyperglycemia, excessive reactive oxygen species (ROS) production, and inflammation. Herein, multifunctional composite microspheres, termed GMAP are developed, using a microfluidic technique by incorporating Au@Pt nanoparticles (NPs) and GelMA hydrogel to modulate the diabetic microenvironment for promoting bone regeneration. The GMAP enables the sustained release of Au@Pt NPs, which function as bimetallic nanozymes with dual enzyme-like activities involving glucose oxidase and catalase. The synergistic effect allows for efficient glucose consumption and ROS elimination concurrently. Thus, the GMAP effectively protects the proliferation of bone marrow mesenchymal stem cells (BMSCs) under adverse high-glucose conditions. Furthermore, it also promotes the osteogenic differentiation and paracrine capabilities of BMSCs, and subsequently inhibits inflammation and enhances angiogenesis. In vivo diabetic rats bone defect model, it is demonstrated that GMAP microspheres significantly improve bone regeneration, as verified by micro-computed tomography and histological examinations. This study provides a novel strategy for bone regeneration by modulating the diabetic microenvironment, presenting a promising approach for addressing the complex challenges associated with bone healing in diabetic patients.

Magnetically attracting hydrogel reshapes iron metabolism for tissue repair.

Ferroptosis, caused by disorders of iron metabolism, plays a critical role in various diseases, making the regulation of iron metabolism essential for tissue repair. In our analysis of degenerated intervertebral disc tissue, we observe a positive correlation between the concentration of extracellular iron ions (ex-iron) and the severity of ferroptosis in intervertebral disc degeneration (IVDD). Hence, inspired by magnets attracting metals, we combine polyether F127 diacrylate (FDA) with tannin (TA) to construct a magnetically attracting hydrogel (FDA-TA). This hydrogel demonstrates the capability to adsorb ex-iron and remodel the iron metabolism of cells. Furthermore, it exhibits good toughness and self-healing properties. Notably, it can activate the PI3K-AKT pathway to inhibit nuclear receptor coactivator 4-mediated ferritinophagy under ex-iron enrichment conditions. The curative effect and related mechanism are further confirmed in vivo. Consequently, on the basis of the pathological mechanism, a targeted hydrogel is designed to reshape iron metabolism, offering insights for tissue repair.

Clinical Analysis and Imaging Study of Lateral Lumbar Intervertebral Fusion in the Treatment of Degenerative Lumbar Scoliosis.

OBJECTIVE: As the population ages and technology advances, lateral lumbar intervertebral fusion (LLIF) is gaining popularity for the treatment of degenerative lumbar scoliosis (DLS). This study investigated the feasibility, minimally invasive concept, and benefits of LLIF for the treatment of DLS by observing and assessing the clinical efficacy, imaging changes, and complications following the procedure. METHODS: A retrospective analysis was performed for 52 DLS patients (12 men and 40 women, aged 65.84 ± 9.873 years) who underwent LLIF from January 2019 to January 2023. The operation time, blood loss, complications, clinical efficacy indicators (visual analogue scale [VAS], Oswestry disability index [ODI], and 36-Item Short Form Survey), and imaging indicators (coronal position: Cobb angle and center sacral vertical line-C7 plumbline [CSVL-C7PL]; and sagittal position: sagittal vertical axis [SVA], lumbar lordosis [LL], pelvic incidence angle [PI], and thoracic kyphosis angle [TK] were measured). All patients were followed up. The above clinical evaluation indexes and imaging outcomes of patients postoperatively and at last follow-up were compared to their preoperative results. RESULTS: Compared to the preoperative values, the Cobb angle and LL angle were significantly improved after surgery (p < 0.001). Meanwhile, CSVL-C7PL, SVA, and TK did not change much after surgery (p > 0.05) but improved significantly at follow-up (p < 0.001). There was no significant change in PI at either the postoperative or follow-up timepoint. The operation took 283.90 ± 81.62 min and resulted in a total blood loss of 257.27 ± 213.44 mL. No significant complications occurred. Patients were followed up for to 21.7 ± 9.8 months. VAS, ODI, and SF-36 scores improved considerably at postoperative and final follow-up compared to preoperative levels (p < 0.001). After surgery, the Cobb angle and LL angle had improved significantly compared to preoperative values (p < 0.001). CSVL-C7PL, SVA, and TK were stable after surgery (p > 0.05) but considerably improved during follow-up (p < 0.001). PI showed no significant change at either the postoperative or follow-up timepoints. CONCLUSION: Lateral lumbar intervertebral fusion treatment of DLS significantly improved sagittal and coronal balance of the lumbar spine, as well as compensatory thoracic scoliosis, with good clinical and radiological findings. Furthermore, there was less blood, less trauma, and quicker recovery from surgery.

Genetically engineered electrospinning contributes to spinal cord injury repair by regulating the immune microenvironment.

Introduction: Spinal cord injury (SCI) is associated with microenvironment imbalance, thereby resulting in poor regeneration and recovery of the spinal cord. Gene therapy can be used to balance the inflammatory response, however target genes cannot exist in localized injured areas. Methods: A genetically engineered electrospun scaffold (GEES) to achieve long-term immunoregulation and nerve repair was constructed. By combining the microfluidic and electrospinning techniques, interleukin-10 plasmid (pIL10) was loaded into lipid nanoparticles (LNPs) (pIL10-LNP), which was encapsulated to the nerve growth factor (NGF). Immunofluorescence staining, qRT-PCR, ELISA, flow cytometry, and other tests were employed to comprehensively assess the role of GEES in modulating macrophage polarization and facilitating neural repair. Results: The results showed that the scaffold released >70% of the pIL10-LNP within 10 d and continued slow release within 30 d. In vitro cell experiments have demonstrated that GEES effectively stimulates macrophages to secrete anti-inflammatory cytokines and facilitates the differentiation of neural stem cells into neuronal cells. In rat T9 SCI model, the GEES significantly inhibited the inflammatory response in the acute and chronic phases of SCI by transfecting local tissues with slow-release pIL10-LNP to promote the release of the anti-inflammatory factor IL10, thereby creating a favorable microenvironment. With the addition of NGF, the repair and regeneration of nerve tissues was effectively promoted, and the post-SCI motor function of rats improved. Discussion: GEES can regulate post-SCI immune responses through continuous and effective gene delivery, providing a new strategy for the construction of electrospun scaffolds for nerve repair in gene therapy.

Ac2-26 activated the AKT1/GSK3ß pathway to reduce cerebral neurons pyroptosis and improve cerebral function in rats after cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) results in brain injury, which is primarily caused by inflammation. Ac2-26 protects against ischemic or hemorrhage brain injury. The present study was to explore the effect and mechanism of Ac2-26 on brain injury in CPB rats. METHODS: Forty-eight rats were randomized into sham, CPB, Ac, Ac/AKT1, Ac/GSK3ßi and Ac/AKT1/GSK3ßa groups. Rats in sham group only received anesthesia and in the other groups received standard CPB surgery. Rats in the sham and CPB groups received saline, and rats in the Ac, Ac/AKT1, Ac/GSK3ßi and Ac/AKT1/GSK3ßa groups received Ac2-26 immediately after CPB. Rats in the Ac/AKT1, Ac/GSK3ßi and Ac/AKT1/GSK3ßa groups were injected with shRNA, inhibitor and agonist of GSK3ß respectively. The neurological function score, brain edema and histological score were evaluated. The neuronal survival and hippocampal pyroptosis were assessed. The cytokines, activity of NF-κB, S100 calcium-binding protein ß(S100ß) and neuron-specific enolase (NSE), and oxidative were tested. The NLRP3, cleaved-caspase-1 and cleaved-gadermin D (GSDMD) in the brain were also detected. RESULTS: Compared to the sham group, all indicators were aggravated in rats that underwent CPB. Compared to the CPB group, Ac2-26 significantly improved neurological scores and brain edema and ameliorated pathological injury. Ac2-26 reduced the local and systemic inflammation, oxidative stress response and promoted neuronal survival. Ac2-26 reduced hippocampal pyroptosis and decreased pyroptotic proteins in brain tissue. The protection of Ac2-26 was notably lessened by shRNA and inhibitor of GSK3ß. The agonist of GSK3ß recovered the protection of Ac2-26 in presence of shRNA. CONCLUSIONS: Ac2-26 significantly improved neurological function, reduced brain injury via regulating inflammation, oxidative stress response and pyroptosis after CPB. The protective effect of Ac2-26 primarily depended on AKT1/ GSK3ß pathway.

Reactive Oxygen Species-Responsive Composite Fibers Regulate Oxidative Metabolism through Internal and External Factors to Promote the Recovery of Nerve Function.

It is challenging to sufficiently regulate endogenous neuronal reactive oxygen species (ROS) production, reduce neuronal apoptosis, and reconstruct neural networks under spinal cord injury conditions. Here, hydrogel surface grafting and microsol electrospinning are used to construct a composite biomimetic scaffold with "external-endogenous" dual regulation of ROS. The outer hydrogel enhances local autophagy through responsive degradation and rapid release of rapamycin (≈80% within a week), neutralizing extracellular ROS and inhibiting endogenous ROS production, further reducing neuronal apoptosis. The inner directional fibers continuously supply brain-derived neurotrophic factors to guide axonal growth. The results of in vitro co-culturing show that the dual regulation of oxidative metabolism by the composite scaffold approximately doubles the neuronal autophagy level, reduces 60% of the apoptosis induced by oxidative stress, and increases the differentiation of neural stem cells into neuron-like cells by ≈2.5 times. The in vivo results show that the composite fibers reduce the ROS levels by ≈80% and decrease the formation of scar tissue. RNA sequencing results show that composite scaffolds upregulate autophagy-associated proteins, antioxidase genes, and axonal growth proteins. The developed composite biomimetic scaffold represents a therapeutic strategy to achieve neurofunctional recovery through programmed and accurate bidirectional regulation of the ROS cascade response.

Immune-defensive microspheres promote regeneration of the nucleus pulposus by targeted entrapment of the inflammatory cascade during intervertebral disc degeneration.

Sustained and intense inflammation is the pathological basis for intervertebral disc degeneration (IVDD). Effective antagonism or reduction of local inflammatory factors may help regulate the IVDD microenvironment and reshape the extracellular matrix of the disc. This study reports an immunomodulatory hydrogel microsphere system combining cell membrane-coated mimic technology and surface chemical modification methods by grafting neutrophil membrane-coated polylactic-glycolic acid copolymer nanoparticles loaded with transforming growth factor-beta 1 (TGF-ß1) (T-NNPs) onto the surface of methacrylic acid gelatin anhydride microspheres (GM) via amide bonds. The nanoparticle-microsphere complex (GM@T-NNPs) sustained the long-term release of T-NNPs with excellent cell-like functions, effectively bound to pro-inflammatory cytokines, and improved the release kinetics of TGF-ß1, maintaining a 36 day-acting release. GM@T-NNPs significantly inhibited lipopolysaccharide-induced inflammation in nucleus pulposus cells in vitro, downregulated the expression of inflammatory factors and matrix metalloproteinase, and upregulated the expression of collagen-II and aggrecan. GM@T-NNPs effectively restored intervertebral disc height and significantly improved the structure and biomechanical function of the nucleus pulposus in a rat IVDD model. The integration of biomimetic technology and nano-drug delivery systems expands the application of biomimetic cell membrane-coated materials and provides a new treatment strategy for IVDD.

A Genetically Engineered "Reinforced Concrete" Scaffold Regulates the N2 Neutrophil Innate Immune Cascade to Repair Bone Defects.

The innate immune response is crucial to inflammation, but how neutrophils and macrophages act in bone repair and tissue engineering treatment strategies await clarification. In this study, it is found that N2 neutrophils release stronger "eat me" signals to induce macrophage phagocytosis and polarize into the M2 anti-inflammatory phenotype. Guided by this biological mechanism, a mesoporous bioactive glass scaffold (MBG) is filled with hyaluronic acid methacryloyl (HAMA) hydrogel loaded with Transforming growth factor-ß1 (TGFß1) adenovirus (Ad@H), constructing a genetically engineered composite scaffold (Ad@H/M). The scaffold not only has good hydrophilicity and biocompatibility, but also provides mechanical stress support for bone repair. Adenovirus infection quickly induces N2 neutrophils, upregulating NF-κB and MAPK signaling pathways through Toll-like receptor 4 (TLR4) to promote the inflammatory response and macrophage phagocytosis. Macrophages perform phagocytosis and polarize towards the M2 phenotype, mediating the inflammatory response by inhibiting the PI3K-AKT-NF-κB pathway, maintaining homeostasis of the osteogenic microenvironment. The role of the Ad@H/M scaffold in regulating early inflammation and promoting long-term bone regeneration is further validated in vivo. In brief, this study focuses on the cascade of reactions between neutrophils and macrophage subtypes, and reports a composite scaffold that coordinates the innate immune response to promote bone repair.

Ligand-Selective Targeting of Macrophage Hydrogel Elicits Bone Immune-Stem Cell Endogenous Self-Healing Program to Promote Bone Regeneration.

Targeting macrophages can facilitate the site-specific repair of critical bone defects. Herein, a composite hydrogel, gelatin-Bletilla striata polysaccharide-mesoporous bioactive glass hydrogel (GBMgel), is constructed via the self-assembly of mesoporous bioactive glass on polysaccharide structures, through the Schiff base reaction. GBMgel can efficiently capture macrophages and drive the recruitment of seed stem cells and vascular budding required for regeneration in the early stages of bone injury, and the observed sustained release of inorganic silicon ions further enhances bone matrix deposition, mineralization, and vascular maturation. Moreover, the use of macrophage-depleted rat calvarial defect models further confirms that GBMgel, with ligand-selective macrophage targeting, increases the bone regeneration area and the proportion of mature bone. Mechanistic studies reveal that GBMgel upregulates the TLR4/NF-κB and MAPK macrophage pathways in the early stages and the JAK/STAT3 pathway in the later stages; thus initiating macrophage polarization at different time points. In conclusion, this study is based on the endogenous self-healing properties of bone macrophages, which enhances stem cell homing, and provides a research and theoretical basis upon which bone tissue can be reshaped and regenerated using the body's immune power, providing a new strategy for the treatment of critical bone defects.

Simultaneous determination of colistin sulfate and tigecycline in human plasma by liquid chromatography-tandem mass spectrometry method.

OBJECTIVE: To establish a high-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) to simultaneously determine colistin sulfate and tigecycline in human plasma. METHODS: Polymyxin B1 internal standard (20 µL) was added into 200 µL of plasma sample. The samples were treated with methanol-5% trichloroacetic acid (50:50, V/V) solution, and the protein precipitation method was adopted for post-injection analysis. The chromatographic column was a Dikma C18 (4.6 mm × 150 mm, 5 µm). For the mobile phase, 0.1% formic acid in aqueous solution was used for phase A, 0.1% formic acid in acetonitrile solution for phase B, and gradient elution was also applied. The flow rate was 0.8 mL/min, the column temperature was 40 °C, and the injection volume was 10 µL; Electrospray ionization and multiple reaction ion monitoring were adopted and scanned by the HPLC-MS/MS positive ion mode. RESULTS: The endogenous impurities in the plasma had no interference in the determination of the analytes. There existed a good linear relationship of colistin sulfate within the range of 0.1-10 µg/mL (R2 = 0.9986), with the lower limit of quantification (LLOQ) of 0.1 µg/mL. There existed a good linear relationship of tigecycline within the range of 0.05-5 µg/ mL (R2 = 0.9987), with the LLOQ of 0.05 µg/mL. The intra- and inter-day relative standard deviations of colistin sulfate and tigecycline were both less than 15%, and the accuracy was between 88.21% and 108.24%. The extraction had good stability, the extraction recovery rate was 87.75-91.22%, and the matrix effect was 99.40-105.26%. CONCLUSION: This study successfully established a method for simultaneously detecting colistin sulfate and tigecycline plasma concentrations. The method was simple, rapid, and highly sensitive and could be applied for therapeutic medication monitoring.

Osteoperiosteal Iliac Autograft Transplantation for Unreconstructable Tibial Plafond After Malunions of Pilon Fractures in Young Patients.

BACKGROUND: Malunion of tibial pilon fracture, especially with a large cartilage loss of the tibial plafond, is a tough clinical conundrum. This study describes a joint-preserving technique that mainly involves corrective intraarticular osteotomy and osteoperiosteal iliac autograft transplantation for treating these generally considered unreconstructable tibial plafond. METHODS: Sixteen patients with an average age of 33.6 years who were treated with this joint-preserving method between 2013 and 2020 were retrospectively analyzed. Ankle distraction was applied in all patients. Additional osteochondral autograft transplantation for talus was performed in 4 patients and supramalleolar osteotomy in 2 patients. The visual analog scale (VAS) score, the American Orthopaedic Foot & Ankle Society (AOFAS) ankle-hindfoot score, the 36-Item Short Form Health Survey (SF-36) score, and the ankle range of motion (ROM) were used for outcome analysis. Radiographic assessment was conducted, and the complications were recorded. RESULTS: At a mean follow-up of 41.1 months, the mean VAS, AOFAS, and SF-36 scores improved from 6.3, 47.6, and 38.0 to 1.7, 84.4, and 70.8, respectively (P < .001 for each). The ankle ROM improved from 27.5 to 32.2 degrees (P = .023). The mean area of ilium blocks was 3.5 cm2, and the mean external fixation time was 94.1 days. Radiographs showed that good osteointegration was found in all patients and no significant progression of osteoarthritis in 15 patients. The major complications included poor incision healing in 2 patients and severe ankle stiffness in 2 patients, with one of them developing considerable varus-type osteoarthritis but reporting no pain. No deep infection, nonunion, or malunion occurred, and no secondary arthrodesis was performed during the final follow-up. CONCLUSION: Osteoperiosteal iliac autograft transplantation might be an alternative surgical option for reconstructing unreconstructable malunited pilon fractures with a large cartilage loss of the tibial plafond in young patients. LEVEL OF EVIDENCE: Level IV, case series.

Ligand-Screened Cerium-Based MOF Microcapsules Promote Nerve Regeneration via Mitochondrial Energy Supply.

Although mitochondria are crucial for recovery after spinal cord injury (SCI), therapeutic strategies to modulate mitochondrial metabolic energy to coordinate the immune response and nerve regeneration are lacking. Here, a ligand-screened cerium-based metal-organic framework (MOF) with better ROS scavenging and drug-loading abilities is encapsulated with polydopamine after loading creatine to obtain microcapsules (Cr/Ce@PDA nanoparticles), which reverse the energy deficits in both macrophages and neuronal cells by combining ROS scavenging and energy supplementation. It reprogrames inflammatory macrophages to the proregenerative phenotype via the succinate/HIF-1α/IL-1ß signaling axis. It also promotes the regeneration and differentiation of neural cells by activating the mTOR pathway and paracrine function of macrophages. In vivo experiments further confirm the effect of the microcapsules in regulating early ROS-inflammation positive-feedback chain reactions and continuously promoting nerve regeneration. This study provides a new strategy for correcting mitochondrial energy deficiency in the immune response and nerve regeneration following SCI.

A responsive hydrogel modulates innate immune cascade fibrosis to promote ocular surface reconstruction after chemical injury.

Following an ocular chemical injury, the release of neutrophil extracellular traps (NETs) triggers an innate immune cascade fibrotic effect involving macrophages (Mø), which limits corneal repair. However, the interplay and mechanisms between NETs and macrophages, as well as the coordination between the innate immunity and corneal repair, remain challenging issues. Using a co-culture system, we report that chemical stimulation exacerbates the accumulation of reactive oxygen species (ROS) within the polymorphonuclear neutrophils, leading to NET formation and the activation of M2 macrophages, ultimately inducing pathological fibrosis of the ocular surface through the IL-10/STAT3/TGF-ß1/Smad2 axis. Inspired by the locally formed acidic microenvironment mediated by innate acute inflammatory stimulation, we further integrate sericin with oxidized chitosan nanoparticles loaded with black phosphorus quantum dots (BPQDs) using Schiff base chemistry to construct a functional pH-responsive hydrogel. Following corneal injury, the hydrogel selectively releases BPQDs in response to the acidic environment, inhibiting the innate immune cascade fibrosis triggered by the PMN-ROS-NETs. Thus, corneal pathological fibrosis is alleviated and reshaping of the ocular surface takes place. These results represent a refinement of the mechanism of inherent immune effector cell interactions, and provide new research ideas for the construction of nano biomaterials that regulate pathological fibrosis.

CK1ε drives osteogenic differentiation of bone marrow mesenchymal stem cells via activating Wnt/ß-catenin pathway.

The treatment of bone defects is a difficult problem in orthopedics. At present, the treatment mainly relies on autologous or allogeneic bone transplantation, which may lead to some complications such as foreign body rejection, local infection, pain, or numbness at the bone donor site. Local injection of conservative therapy to treat bone defects is one of the research hotspots at present. Bone marrow mesenchymal stem cells (BMSCs) can self-renew, significantly proliferate, and differentiate into various types of cells. Although it has been reported that CK1ε could mediate the Wnt/ß-catenin pathway, leading to the development of the diseases, whether CK1ε plays a role in bone regeneration through the Wnt/ß-catenin pathway has rarely been reported. The purpose of this study was to investigate whether CK1ε was involved in the osteogenic differentiation (OD) of BMSCs through the Wnt/ß-catenin pathway and explore the mechanism. We used quantitative reverse transcription-polymerase chain reaction (qRT-qPCR), Western blots, immunofluorescence, alkaline phosphatase, and alizarin red staining to detect the effect of CK1ε on the OD of BMSCs and the Wnt/ß-catenin signaling pathway. CK1ε was highly expressed in BMSCs with OD, and our study further demonstrated that CK1ε might promote the OD of BMSCs by activating DLV2 phosphorylation, initiating Wnt signaling downstream, and activating ß-catenin nuclear transfer. In addition, by locally injecting a CK1ε-carrying adeno-associated virus (AAV5- CK1ε) into a femoral condyle defect rat model, the overexpression of CK1ε significantly promoted bone repair. Our data show that CK1ε was involved in the regulation of OD by mediating Wnt/ß-catenin. This may provide a new strategy for the treatment of bone defects.

Expression and prognosis of inducible T-cell co-stimulator and its ligand in Chinese stage I-III lung adenocarcinoma patients.

BACKGROUND: Immunotherapy has become the fastest-adopting treatment paradigm for lung cancer with improved survival. By binding with its ligand (inducible T-cell co-stimulator and its ligand [ICOSL]), an inducible T-cell co-stimulator (ICOS) could contribute to reversing immunosuppression and improving immune response and thus be a potential target for cancer immunotherapy. METHODS: We selected 54 formalin-fixed, paraffin-embedded tumor tissues from cases with stage I-III lung adenocarcinoma cancer. Immunohistochemical expression of ICOS and ICOSL was evaluated. The correlation with clinical parameters in Chinese patients was also compared with TCGA results. RESULTS: The positive rates of ICOS and ICOSL were 68% and 81.5%, respectively, in lung tumor tissues. Of these, 9 cases had a low expression of ICOS, and 22 cases had a high expression of ICOS; ICOSL expression was low in 20 cases and high in 24 cases. According to the International Association for the Study of Lung Cancer (8th edition), phase I lesions were detected in 21 cases, phase II lesions in 15 cases, and phase III lesions in 18 cases. The median survival time of all patients was 44.5 months, and the median disease-free survival was 32 months. Univariate analysis showed that the factors significantly associated with overall survival were tumor size, regional lymph node involvement, stage, and expression level of ICOS/ICOSL. Survival analysis using log-rank test indicated that the lower ICOS+ cell infiltration may predict poor prognosis, whereas lower ICOSL protein expression may be associated with better prognosis, but ICOSL data need further validation in larger samples due to inconsistency in TCGA mRNA prediction. CONCLUSION: ICOS/ICOSL might be associated with prognosis of lung cancer, and ICOS and its ligand may be potential therapeutic targets in non-small cell lung cancer.

ECM-engineered electrospun fibers with an immune cascade effect for inhibiting tissue fibrosis.

Tissue regeneration/fibrosis after injury is intricately regulated by the immune cascade reaction and extracellular matrix (ECM). Dysregulated cascade signal could jeopardize tissue homeostasis leading to fibrosis. Bioactive scaffolds mimicking natural ECM microstructure and chemistry could regulate the cascade reaction to achieve tissue regeneration. The current study constructed an ECM-engineered micro/nanofibrous scaffold using self-assembled nanofibrous collagen and decorin (DCN)-loaded microfibers to regulate the immune cascade reaction. The ECM-engineered scaffold promoted anti-inflammatory and pro-regenerative effects, M2 polarization of macrophages, by nanofibrous collagen. The ECM-engineered scaffold could release DCN to inhibit inflammation-associated fibrous angiogenesis. Yet, to prevent excessive M2 activity leading to tissue fibrosis, controlled release of DCN was expected to elicit M1 activity and achieve M1/M2 balance in the repair process. Regulated cascade reaction guided favorable crosstalk between macrophages, endothelial cells and fibroblasts by proximity. Additionally, decorin could also antagonize TGF-ß1 via TGF-ß/Smad3 pathway to suppress fibrotic activity of fibroblasts. Hence, ECM-engineered scaffolds could exert effective regulation of the immune cascade reaction by microstructure and DCN release and achieve the balance between tissue fibrosis and regeneration. STATEMENT OF SIGNIFICANCE: With the incidence of up to 74.6%, failed back surgery syndrome (FBSS) has been a lingering issue in spine surgery, which poses a heavy socio-economic burden to society. Epidural fibrosis is believed to be responsible for the onset of FBSS. Current biomaterial-based strategies treating epidural fibrosis mainly rely on physical barriers and unidirectional suppression of inflammation. Regulation of the immune cascade reaction for inhibiting fibrosis has not been widely studied. Based on the simultaneous regulation of M1/M2 polarization and intercellular crosstalk, the ECM-engineered micro/nanofibrous scaffolds constructed in the current study could exert an immune cascade effect to coordinate tissue regeneration and inhibit fibrosis. This finding makes a significant contribution in the development of a treatment for epidural fibrosis and FBSS.

Composite Hydrogel Modulates Intrinsic Immune-Cascade Neovascularization for Ocular Surface Reconstruction after Corneal Chemical Injury.

Ocular alkali burns recruit neutrophils and triggers neutrophil extracellular trap (NET)-neovascularization cascade effects that limit ocular surface reconstruction and functional repair. However, effective inhibition of the release of neutrophil extracellular traps after a corneal chemical injury, coordination of intrinsic immunity with corneal repair, and exploration of more effective and non-invasive drug-delivery modes are still urgently needed. Using an in vitro coculture system, we found that an alkaline environment stimulates neutrophils to release NETs, which can be regulated by deoxyribonuclease I (DNase I). Inspired by this, we loaded DNase I, which effectively regulates NETs, onto chitosan nanoparticles and combined them with silk fibroin to construct a composite hydrogel that can sustainably regulate NETs. The hydrogel reduced neutrophil extracellular trap production by 50% and neovascularization by approximately 70% through sustained DNase I release after a corneal alkali burn. The complex hydrogel promotes ocular surface reconstruction by modulating the intrinsic immune-cascade neovascularization effect, providing a new research basis for the construction of nanobiomaterials that modulate pathological neovascularization.

Diagnosis of Early Bacterial Pneumonia and Sepsis After Cardiovascular Surgery: A Diagnostic Prediction Model Based on LASSO Logistic Regression.

Background: Early postoperative bacterial pneumonia and sepsis (ePOPS), which occurs within the first 48 hours after cardiovascular surgery, is a serious life-threatening complication. Diagnosis of ePOPS is extremely challenging, and the existing diagnostic tools are insufficient. The purpose of this study was to construct a novel diagnostic prediction model for ePOPS. Methods: Least Absolute Shrinkage and Selection Operator (LASSO) with logistic regression was used to construct a model to diagnose ePOPS based on patients' comorbidities, medical history, and laboratory findings. The area under the receiver operating characteristic curve (AUC) was used to evaluate the model discrimination. Results: A total of 1203 patients were recruited and randomly split into a training and validation set in a 7:3 ratio. By early morning on the 3rd postoperative day (POD3), 103 patients had experienced 133 episodes of bacterial pneumonia or sepsis (15 patients had both). LASSO logistic regression model showed that duration of mechanical ventilation (P=0.015), NYHA class ≥ III (P=0.001), diabetes (P<0.001), exudation on chest radiograph (P=0.011) and IL-6 on POD3 (P<0.001) were independent risk factors. Based on these factors, we created a nomogram named DICS-I with an AUC of 0.787 in the training set and 0.739 in the validation set. Conclusion: The DICS-I model may be used to predict the risk of ePOPS after cardiovascular surgery, and is also especially suitable for predicting the risk of IRAO. The DICS-I model could help clinicians to adjust antibiotics on the POD3.