Journal of advanced research最新文献

Introduction: Disruption of the circadian rhythm (CR) and autophagy in intervertebral discs contributes to intervertebral disc degeneration (IDD) progression. However, the circadian regulation of autophagy requires further investigation.

Objectives: We observed the expression of circadian proteins and autophagic markers of nucleus pulposus (NP) cells followed a diurnal rhythmic pattern in vivo and in vitro.

Methods: NP tissues were collected from light/dark cycle-shifted rats and IDD patients of varying severity. CR and ECM-related proteins were analyzed by immunohistochemistry and western blotting. Primary rat NP cells were treated with hypoxia or CoCl₂, followed by western blotting for CR proteins, HIF-1α, and autophagy markers. siRNA knockdown of PER2 or HIF-1α was performed to assess their roles in regulating autophagy, ECM, and CR-associated proteins.

Results: The silencing of clock gene PER2 disrupted the rhythmic expression of autophagic markers, by contrast PER2 overexpression inhibited mTOR pathway and enhanced autophagic levels. Co-IP analysis demonstrated the PER2-mTOR interaction, linking CR and autophagy rhythm. The inflammatory stimulator dampened the CR and autophagy rhythm, however intermittent hypoxia and cobalt chloride (CoCl₂) re-synchronized the rhythm. Mechanistically, HIF-1α-mediated regulation of PER2 by hypoxia was involved in the re-synchronization, which was further demonstrated by the loss of CR and autophagy rhythm after silencing of PER2 or HIF-1α under hypoxia. Furthermore, the rhythm of oxygen level and HIF-1α was proved in living healthy NP tissue, confirming the hypoxia as a Zeitgeber. CR disruption and autophagy dysfunction led to catabolism of extracellular matrix (ECM), but the hypoxia, CoCl₂ and autophagic stimulator could promote the rebalance of ECM metabolism.

Conclusion: Our study demonstrates that hypoxia maintains the intrinsic CR and autophagy rhythm through the HIF-1α/PER2/mTOR pathway to prevent IDD.

Artificial intelligence (AI) has played an excellent supporting role in novel drug discovery and development. This study introduces a reinforcement learning (RL) model based on the Soft Actor-Critic (SAC) algorithm for AI-driven de novo molecular generation targeting tyrosinase. The model facilitates forward molecular generation design by integrating a chemical reaction template and a molecular building block library, concurrently performing molecular docking and assessing drug-likeness. Through sequential decision-making, signal feedback, and a dynamic learning process, the model generates molecules exhibiting potent target affinity, optimal drug-like properties, and good synthetic feasibility. The AI-generated molecules undergo rigorous manual screening, synthesis, and biological evaluation, culminating in the identification of a prioritized lead compound V. Subsequent structural optimization of compound V reveals a series of compounds with significantly enhanced activity, shifting inhibitory potency from the micromolar to the nanomolar range. The optimized compound, V-24, demonstrates low cytotoxicity and significant anti-melanogenic activity both in cell melanogenesis inhibition and zebrafish anti-pigmentation models. Notably, it effectively reduces melanin content in an ultraviolet light-induced human 3D skin pigmentation model, exhibiting the potential to serve as a promising tyrosinase inhibitor for the treatment of skin pigmentation. More importantly, this "AI de novo Molecular Generation + Expert-Guided Structural Optimization" work demonstrates that integrating an AI algorithm with traditional medicinal chemistry experience is a novel approach and efficiency-redefined strategy for drug discovery.

Introduction: Wound healing impairment is highly prevalent in diabetes and frequently progresses to serious complications, including refractory ulcers and necessitated amputations. RNA sequencing in methylglyoxal (MGO)-injured HaCaT cells implicated fructose-1,6-bisphosphatase 1 (FBP1) in suppressing keratinocyte proliferation and migration, identifying it as a potential therapeutic target.

Objectives: This study aimed to validate FBP1 as a therapeutic target for diabetic wounds and evaluate asiatic acid (AA) and its novel hydrogen sulfide (H₂S)-donor derivatives, designed to enhance efficacy, as FBP1-targeted interventions.

Methods: Target discovery was performed via transcriptomics in MGO-injured HaCaT cells, identifying FBP1 as a key regulator. Virtual screening of compound libraries was combined with experimental screening to discover AA as a potent FBP1 inhibitor. Based on AA's structure, novel H₂S-donor derivatives were rationally designed and synthesized to enhance therapeutic properties. A topical AA4 gel was formulated and tested for its therapeutic impact on diabetic wound repair in mouse models.

Results: AA was identified as a potent FBP1 inhibitor (IC₅₀ = 2.5 μM). AA4, a synthesized H₂S-donor derivative, exhibited dual mechanisms: direct FBP1 enzymatic inhibition and H₂S-mediated FBP1 downregulation. This synergistically restored proliferation pathways (AKT/mTOR/HIF-1α/uPAR) and reduced apoptosis (Bcl-2/Bax/Caspase-3). Topical AA4 gel markedly enhanced wound closure rates in diabetic mice, primarily through promoting epidermal regeneration and collagen deposition.

Conclusion: This study validates FBP1 targeting as a feasible strategy to address diabetic wound healing. It establishes AA-H₂S donor derivatives, particularly AA4 acting via dual FBP1 targeting, as an encouraging precision therapy for diabetic wound healing.

Introduction: Berberine (BBR), the predominant isoquinoline alkaloid in Coptidis Rhizoma, exhibits remarkable anti- colorectal cancer (CRC) activity. However, whether BBR triggers CRC cell death through ferroptosis-associated disruption of energy metabolism remains to be elucidated.

Objective: To investigate if BBR induces mitochondrial energy metabolism disorder in CRC cells by regulating the ferroptosis signaling pathway.

Methods: BBR's effects on malignant phenotypes were evaluated in vitro (human cell line HCT116, murine cell line CT26 cells at 10, 20, 40 μM) and in vivo (80 mg/kg). Target engagement and mechanistic pathways were interrogated through RNA-sequence combined with convolutional neural network-based pathway prediction, corroborated by surface plasmon resonance, cellular thermal shift assay. Downstream validation mainly included quantification of Gli1, STAT3, GPX4, SLC7A11, and FTH1 expression via RT-qPCR, Western blot, immunofluorescence, and other molecular expression and functional confirmation experiments.

Results: BBR inhibited CRC cell proliferation with IC₅₀ value for HCT116 cells for 48 h at 19.86 ± 2.31 μM, and for CT26 cells for 48 h at 21.35 ± 2.63 μM. Concurrently, it elevated ferroptosis markers such as malondialdehyde, lactate dehydrogenase, Fe²⁺, and 4-hydroxynonenal, while suppressing ATP levels, superoxide dismutase activity, and energy metabolism-related enzymes. Graph convolutional network-based drug "on-target" pathway algorithm predicted Gli1 as top-9 target, and surface plasmon resonance confirmed direct BBR-Gli1 binding with KD value at 0.652 μM, cellular stability thermal assessment showed BBR stabilized Gli1 with thermal shift with ΔT = 2.3 °C. Mechanistically, BBR exerted its anti-CRC effects by inhibiting the Gli1/STAT3-ferroptosis negative regulation (Gli1/STAT3-FNR) axis, a novel regulatory pathway. Notably, BBR exhibited no significant organ or hematological toxicity in vivo at the experimental doses.

Conclusion: BBR triggers ferroptosis-mediated energy metabolism disorder by inhibiting Gli1/STAT3-FNR axis. This work provides a mechanistic support for BBR anti-CRC indications, and suggests an encouraging approach for treating CRC.

Introduction: Understanding of cerebral ischemic tolerance could provide important insight into viable neuroprotective strategies against ischemic stroke.

Objectives: To elucidate the function of superoxide dismutase 1 (SOD1) in cellular tolerance to ischemia and its underlying molecular mechanism.

Methods: Two-vessel occlusion of the carotid arteries and oxygen-glucose deprivation were used for modelling global ischemic preconditioning (IPC) and in vitro ischemic preconditioning models, respectively. Cleavage-Under-Targets-And-Tagmentation (CUT&Tag)-sequencing analysis was performed for mapping SOD1-whole genome-binding sites in cultured cortical neurons in the in vitro ischemic tolerance model. Cell fractionation and immunofluorescent staining were used for analyzing the subcellular localization of SOD1 in tissue and cultured neuronal cells.

Results: Here, we showed that cortex, striatum and thalamus, but not hippocampus, exhibited ischemic tolerance phenotypes after global IPC. Intriguingly, we found that SOD1 was accumulated in the cellular nucleus after IPC. Notably, cortex was the most responsive brain region in terms of neuronal SOD1 nuclear translocation in response to IPC. We further confirmed these observations in the cultured cortical neurons in the in vitro ischemic tolerance model. CUT&Tag-sequencing analysis revealed that SOD1 bound to promoter regions in cortical neurons, and its binding was significantly altered after IPC stimulus. Notably, SOD1 was most prominently accumulated at the gene promoter of CXCR4 after IPC, which was correlated with transcriptional activation of CXCR4. Furthermore, we found that upregulation of CXCR4 expression was accompanied by modulation of gene expression resulting in anti-apoptosis. Moreover, we showed that pharmacological manipulation of CXCR4 activity could regulate cell viability through STAT3 in cortical neurons. Finally, we found that SOD1 was required for CXCR4-STAT3-mediated apoptosis in the IPC-induced ischemic tolerance.

Conclusion: Therefore, these results demonstrate a non-canonical transcription-regulatory function of SOD1 which regulates CXCR4-STAT3 pathway to confer ischemic tolerance in cortical neurons.

Introduction: Adipose tissue (AT) immune cells regulate metabolic functions in obesity through both inflammatory and non-inflammatory pathways. However, the specific roles and mechanisms of individual AT immune cell types in glycemic control remain poorly understood.

Objective: This study investigates the function of myeloid-derived Zbtb46⁺ cells, a major subset of peripheral dendritic cells (DCs), in established obesity.

Methods: Chimeric Zbtb46-DTR mice were generated by transplanting bone marrow from Zbtb46-DTR donors into wild-type recipients with distinct congenic markers. Obesity was induced with a high-fat diet (HFD; 60% kcal from fat), and myeloid-derived Zbtb46⁺ cells were selectively depleted in obese mice via diphtheria toxin (DT) injection. DC-specific Dpp4 knockout (DC-Dpp4KO) mice were generated using the Cre-loxP system and subsequently challenged with the HFD.

Results: Inducible depletion of myeloid-derived Zbtb46⁺ cells improves glucose homeostasis and reduces body weight in obese mice. Notably, these effects were observed even in weight-matched mice and under conditions of increased ATM accumulation, suggesting benefits independent of weight loss or AT inflammation. The improvement in glucose homeostasis was primarily mediated by elevated GLP-1 levels, which enhanced insulin secretion and decreased food intake. Increased GLP-1 was associated with decreased DPP4 activity, attributed to the depletion of ATDCs, a key contributor to circulating DPP4. Consistently, DC-specific Dpp4 deficiency confirmed that ATDC-derived DPP4 regulates GLP-1-induced insulin secretion in obesity.

Conclusions: These findings uncover a novel, non-inflammatory role for ATDCs in glucose regulation via the DPP4/GLP-1/GLP-1R axis, positioning them as promising therapeutic targets for obesity and related metabolic diseases.

Introduction: Optimal ovarian stimulation (OS) selection is critical for IVF success, but expert-based decisions often lack consistency in outcomes, cost-efficiency, and personalization, highlighting the need for more individualized and data-driven approaches.

Objectives: This study propose an artificial intelligence (AI) system that analyzes extensive IVF-ET cycles to uncover OS-pregnancy outcome relationships, enabling personalized treatment recommendations while improving success rates and minimizing unnecessary costs.

Methods: This study analyzed anonymized data from 17,791 patients undergoing OS and IVF/ICSI at Tongji Hospital between May 2015 and May 2019. An adaptive AI model was developed to predict key indicators-including progesterone (P), number of oocytes retrieved (NOR), estradiol (E2), and endometrial thickness (EMT) on the hCG day-by integrating personal characteristics, ovarian reserve, and etiological factors. This model facilitated personalized OS selection, pregnancy outcome grading, and the development of an AI-driven clinical decision support system (CDSS).

Results: The key indicators-progesterone (P), number of oocytes retrieved (NOR), estradiol (E2), and endometrial thickness (EMT) on the hCG day-were used to establish a pregnancy grading system. Pregnancy rates are stratified as follows: Level IV (Total Score 15-16), 0.55; Level III (Total Score 13-14), 0.44; Level II (Total Score 11-12), 0.24; and Level I (Total Score 4-10), 0.07. After OS optimization, 1,355 patients who were initially at level I were elevated to a better level. Of the 2,341 patients initially in level II, 2,290 improved, and of the 3,839 initially in level III, 1,448 improved. Patients elevated to level IV accounted for 80 percent of all cases. The CDSS prioritized a GnRH antagonist regimen for 54.64 % of patients, resulting in per-patient time savings of 15.39-33.48 days and cost reductions of ¥989-¥2,623 compared to non-optimal to antagonist. Scaled to China's > 1 million ART cycles annually, this corresponds to projected direct savings of approximately ¥0.54-1.43 billion per year. In the new evaluation datasets (n = 4,251), implementation of CDSS recommendations increased the clinical pregnancy rate from 0.452 to 0.512 (p < 0.001) and reduced mean per-cycle cost from ¥7,385 to ¥7,242 (p = 0.018), demonstarting cost-effectiveness dominance with ICER saving of ¥2,383 per additional clinical pregnancy.

Conclusion: This AI-assisted CDSS streamlines clinicians' decision-making by enabling efficient and accurate initial judgments on OS, standardizing and personalizing recommendations, and optimizing OS for effectiveness and cost-efficiency.

Introduction: As an emerging natural nanomedicine, traditional Chinese medicine-derived exosome-like nanovesicles have shown unique potential in the treatment of chronic non-healing wounds due to their advantages such as good biocompatibility, high safety and large-scale production. However, at present, there are relatively few studies on different states and extraction processes of traditional Chinese medicine, which restricts its clinical transformation process.

Objective: This study aims to systematically compare the properties and biological activities of Portulaca oleracea L. (Purslane) derived exosome-like nanovesicles (Po-DENs) extracted from fresh and dried Purslane by using ultracentrifugation and polyethylene glycol extraction methods. The key functional metabolites and proteins were identified through multi-omics analysis, and their therapeutic potential in diabetic wound healing was verified both in vivo and in vitro.

Method: Fresh and dried Portulaca oleracea L. samples were treated by ultracentrifugation and polyethylene glycol precipitation methods, and four kinds of plant nanovesicles were separated. Their physical properties such as particle size were characterized. The composition of the vesicles was determined through metabolomics and proteomics. The proliferation, antioxidant, anti-apoptotic and anti-inflammatory effects of four types of vesicles on skin keratinocytes and fibroblasts were investigated, and the mechanism was analyzed through proteomics. And it was finally verified in the wound healing model of diabetic mice to confirm its therapeutic activity and biological safety.

Result: The results demonstrated that fresh Purslane-derived nanovesicles isolated by ultracentrifugation (F-Po-UC) exhibited superior bioactivity compared to other extraction methods and dried material preparations. Omics analyses further revealed that F-Po-UC is enriched with a wide range of metabolites and proteins, which act through signaling pathways related to tissue regeneration. Subsequent vivo studies demonstrated that dressings based on Purslane-derived vesicles significantly accelerated wound healing in a diabetic mouse model, confirming their potential as an innovative therapeutic strategy.

Conclusion: Fresh Purslane-derived nanovesicles demonstrate exceptional therapeutic potential for diabetic wound healing through their potent proliferative, antioxidant, and anti-inflammatory effects, supported by multi-omics characterization and robust in vivo validation.

Introduction: Plant-microbe collaborative remediation is a highly promising heavy metal remediation strategy. However, its remediation efficiency for manganese (Mn) tailings and its underlying mechanism remain inadequately explored.

Objectives: This study aimed to screen microbial strains capable of remediating Mn contamination and evaluate the potential of these strains to enhance the remediation of heavy metal-contaminated soils through plant-microbe interactions.

Methods: Bacterial strains with both Mn resistance and plant growth-promoting characteristics were isolated from the rhizosphere of plants in Mn tailings. Two selected strains were inoculated to the rhizosphere of 2-year-old Koelreuteria paniculata seedlings in non-polluted soil or in Mn tailings. The effectiveness of the strains in remediating Mn tailings was evaluated by measuring the growth, photosynthetic characteristics, antioxidant enzyme activity, Mn accumulation capacity of K. paniculata, and overall soil remediation effect.

Results: This study selected two Mn resistance and plant growth-promoting bacteria, including Bacillus cereus (EA-1) and Priestia megaterium (BN-4). Functioanl groups identified by Fourier transform infrared spectroscopy (FTIR), such as hydroxyl and phosphate groups, are essential for Mn adsorption on the bacterial surface. In Mn tailings, inoculation with the BN-4 strain significantly increased superoxide dismutase (SOD) and catalase (CAT) activities in the leaves of K. paniculata by 201.57% and 34.48%, respectively, compared to the uninoculated treatment. However, inoculation with the EA-1 strain enhanced the root, stem, and leaf biomass, net photosynthetic rate, stomatal conductance, Mn accumulation, and bioconcentration factor (BCF) of K. paniculata by 72.70%, 47.82%, 51.32%, 81.38 %, 86.05%, 113.09% and 76.19%, respectively, compared to the uninoculated treatment.

Conclusion: Considering the growth of K. paniculata and its capacity for Mn accumulation and BCF, inoculation with the EA-1 strain is a more favorable approach for remediation of Mn tailings.