Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

Effect of Helichrysum italicum in Promoting Collagen Deposition and Skin Regeneration in a New Dynamic Model of Skin Wound Healing.

Natural products have many healing effects on the skin with minimal or no adverse effects. In this study, we analyzed the regenerative properties of a waste product (hydrolate) derived from Helichrysum italicum (HH) on scratch-tested skin cell populations seeded on a fluidic culture system. Helichrysum italicum has always been recognized in the traditional medicine of Mediterranean countries for its wide pharmacological activities. We recreated skin physiology with a bioreactor that mimics skin stem cell (SSCs) and fibroblast (HFF1) communication as in vivo skin layers. Dynamic culture models represent an essential instrument for recreating and preserving the complex multicellular organization and interactions of the cellular microenvironment. Both cell types were exposed to two different concentrations of HH after the scratch assay and were compared to untreated control cells. Collagen is the constituent of many wound care products that act directly on the damaged wound environment. We analyzed the role played by HH in stimulating collagen production during tissue repair, both in static and dynamic culture conditions, by a confocal microscopic analysis. In addition, we performed a gene expression analysis that revealed the activation of a molecular program of stemness in treated skin stem cells. Altogether, our results indicate a future translational application of this natural extract to support skin regeneration and define a new protocol to recreate a dynamic process of healing.

Impact of Thermo-Responsive N-Acetylcysteine Hydrogel on Dermal Wound Healing and Oral Ulcer Regeneration.

This study investigates the efficacy of a thermo-responsive N-acetylcysteine (NAC) hydrogel on wound healing and oral ulcer recovery. Formulated by combining NAC with methylcellulose, the hydrogel's properties were assessed for temperature-induced gelation and cell viability using human fibroblast cells. In vivo experiments on Sprague Dawley rats compared the hydrogel's effects against saline, NAC solution, and a commercial NAC product. Results show that a 5% NAC and 1% methylcellulose solution exhibited optimal outcomes. While modest improvements in wound healing were observed, significant enhancements were noted in oral ulcer recovery, with histological analyses indicating fully regenerated mucosal tissue. The study concludes that modifying viscosity enhances NAC retention, facilitating tissue regeneration. These findings support previous research on the beneficial effects of antioxidant application on damaged tissues, suggesting the potential of NAC hydrogels in improving wound care and oral ulcer treatment.

Macrophages modulate fibrosis during newt lens regeneration.

BACKGROUND: Previous studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated. METHODS: Here we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery. RESULTS: Macrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process. CONCLUSIONS: Together, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

Novel approach to soft tissue regeneration: in vitro study of compound hyaluronic acid and horizontal platelet-rich fibrin combination.

OBJECTIVE: This study aims to develop a compound biomaterial to achieve effective soft tissue regeneration. METHODOLOGY: Compound hyaluronic acid (CHA) and liquid horizontal-platelet-rich fibrin (H-PRF) were mixed at a ratio of 1:1 to form a CHA-PRF gel. Human gingival fibroblasts (HGFs) were used in this study. The effect of CHA, H-PRF, and the CHA-PRF gel on cell viability was evaluated by CCK-8 assays. Then, the effect of CHA, H-PRF, and the CHA-PRF gel on collagen formation and deposition was evaluated by qRT‒PCR and immunofluorescence analysis. Finally, qRT‒PCR, immunofluorescence analysis, Transwell assays, and scratch wound-healing assays were performed to determine how CHA, H-PRF, and the CHA-PRF gel affect the migration of HGFs. RESULTS: The combination of CHA and H-PRF shortened the coagulation time of liquid H-PRF. Compared to the pure CHA and H-PRF group, the CHA-PRF group exhibited the highest cell proliferation at all time points, as shown by the CCK-8 assay. Col1a and FAK were expressed at the highest levels in the CHA-PRF group, as shown by qRT‒PCR. CHA and PRF could stimulate collagen formation and HGF migration, as observed by fluorescence microscopy analysis of COL1 and F-actin and Transwell and scratch healing assays. CONCLUSION: The CHA-PRF group exhibited greater potential to promote soft tissue regeneration by inducing cell proliferation, collagen synthesis, and migration in HGFs than the pure CHA or H-PRF group. CHA-PRF can serve as a great candidate for use alone or in combination with autografts in periodontal or peri-implant soft tissue regeneration.

Establishment of human hematopoietic organoids for evaluation of hematopoietic injury and regeneration effect.

BACKGROUND: Human hematopoietic organoids have a wide application value for modeling human bone marrow diseases, such as acute hematopoietic radiation injury. However, the manufacturing of human hematopoietic organoids is an unaddressed challenge because of the complexity of hematopoietic tissues. METHODS: To manufacture hematopoietic organoids, we obtained CD34+ hematopoietic stem and progenitor cells (HSPCs) from human embryonic stem cells (hESCs) using stepwise induction and immunomagnetic bead-sorting. We then mixed these CD34+ HSPCs with niche-related cells in Gelatin-methacryloyl (GelMA) to form a three-dimensional (3D) hematopoietic organoid. Additionally, we investigated the effects of radiation damage and response to granulocyte colony-stimulating factor (G-CSF) in hematopoietic organoids. RESULTS: The GelMA hydrogel maintained the undifferentiated state of hESCs-derived HSPCs by reducing intracellular reactive oxygen species (ROS) levels. The established hematopoietic organoids in GelMA with niche-related cells were composed of HSPCs and multilineage blood cells and demonstrated the adherence of hematopoietic cells to niche cells. Notably, these hematopoietic organoids exhibited radiation-induced hematopoietic cell injury effect, including increased intracellular ROS levels, γ-H2AX positive cell percentages, and hematopoietic cell apoptosis percentages. Moreover, G-CSF supplementation in the culture medium significantly improved the survival of HSPCs and enhanced myeloid cell regeneration in these hematopoietic organoids after radiation. CONCLUSIONS: These findings substantiate the successful manufacture of a preliminary 3D hematopoietic organoid from hESCs-derived HSPCs, which was utilized for modeling hematopoietic radiation injury and assessing the radiation-mitigating effects of G-CSF in vitro. Our study provides opportunities to further aid in the standard and scalable production of hematopoietic organoids for disease modeling and drug testing.

Comparison of Muscle Regeneration Effects at Different Melittin Concentrations in Rabbit Atrophied Muscle.

This research aimed to explore the healing impacts of Melittin treatment on gastrocnemius muscle wasting caused by immobilization with a cast in rabbits. Twenty-four rabbits were randomly allocated to four groups. The procedures included different injections: 0.2 mL of normal saline to Group 1 (G1-NS); 4 µg/kg of Melittin to Group 2 (G2-4 µg/kg Melittin); 20 µg/kg of Melittin to Group 3 (G3-20 µg/kg Melittin); and 100 µg/kg of Melittin to Group 4 (G4-100 µg/kg Melittin). Ultrasound was used to guide the injections into the rabbits' atrophied calf muscles following two weeks of immobilization via casting. Clinical measurements, including the length of the calf, the compound muscle action potential (CMAP) of the tibial nerve, and the gastrocnemius muscle thickness, were assessed. Additionally, cross-sectional slices of gastrocnemius muscle fibers were examined, and immunohistochemistry and Western blot analyses were performed following two weeks of therapy. The mean regenerative changes, as indicated by clinical parameters, in Group 4 were significantly more pronounced than in the other groups (p < 0.05). Furthermore, the cross-sectional area of the gastrocnemius muscle fibers and immunohistochemical indicators in Group 4 exceeded those in the remaining groups (p < 0.05). Western blot analysis also showed a more significant presence of anti-inflammatory and angiogenic cytokines in Group 4 compared to the others (p < 0.05). Melittin therapy at a higher dosage can more efficiently activate regeneration in atrophied gastrocnemius muscle compared to lower doses of Melittin or normal saline.

Application of Deferoxamine in Tissue Regeneration Attributed to Promoted Angiogenesis.

Deferoxamine, an iron chelator used to treat diseases caused by excess iron, has had a Food and Drug Administration-approved status for many years. A large number of studies have confirmed that deferoxamine can reduce inflammatory response and promote angiogenesis. Blood vessels play a crucial role in sustaining vital life by facilitating the delivery of immune cells, oxygen, and nutrients, as well as eliminating waste products generated during cellular metabolism. Dysfunction in blood vessels may contribute significantly to the development of life-threatening diseases. Anti-angiogenesis therapy and pro-angiogenesis/angiogenesis strategies have been frequently recommended for various diseases. Herein, we describe the mechanism by which deferoxamine promotes angiogenesis and summarize its application in chronic wounds, bone repair, and diseases of the respiratory system. Furthermore, we discuss the drug delivery system of deferoxamine for treating various diseases, providing constructive ideas and inspiration for the development of new treatment strategies.

Exploring the potential of bovine colostrum as a bioactive agent in human tissue regeneration: A comprehensive analysis of mechanisms of action and challenges to be overcome.

The study examines bovine colostrum as a potent source of bioactive compounds, particularly growth factors, for tissue regeneration in humans. While previous research has hinted at therapeutic benefits, a comprehensive understanding of its mechanisms remains elusive, necessitating further investigation. This review analyzes nine selected scientific articles on bovine colostrum's bioactive potential in tissue regeneration. In vitro studies highlight its positive impact on cell behavior, including reduced proliferation and induced differentiation. Notably, optimal concentrations and specific colostrum components, such as extracellular vesicles and insoluble milk fat, show more favorable outcomes. In vivo studies underscore bovine colostrum as a promising natural resource for wound healing, despite some studies failing to identify associated benefits. Further research is crucial to unravel the intricate mechanisms, grasp the full potential in regenerative medicine, and develop more effective wound healing therapies. This refined understanding will pave the way for harnessing the complete regenerative potential of bovine colostrum in clinical applications.

Mesenchymal stromal/stem cells promote intestinal epithelium regeneration after chemotherapy-induced damage.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for leukemia and a range of non-malignant disorders. The success of the therapy is hampered by occurrence of acute graft-versus-host disease (aGvHD); an inflammatory response damaging recipient organs, with gut, liver, and skin being the most susceptible. Intestinal GvHD injury is often a life-threatening complication in patients unresponsive to steroid treatment. Allogeneic mesenchymal stromal/stem cell (MSC) infusions are a promising potential treatment for steroid-resistant aGvHD. Data from our institution and others demonstrate rescue of approximately 40-50% of aGvHD patients with MSCs in Phase I, II studies and minor side effects. Although promising, better understanding of MSC mode of action and patient response to MSC-based therapy is essential to improve this lifesaving treatment. METHODS: Single cell human small intestine organoids were embedded in Matrigel, grown for 5 days and treated with busulfan for 48 h. Organoids damaged by treatment with busulfan or control organoids were co-cultured with 5000, 10,000, and 50,000 MSCs for 24 h, 48 h or 7 days and the analyses such as surface area determination, proliferation and apoptosis assessment, RNA sequencing and proteomics were performed. RESULTS: Here, we developed a 3D co-culture model of human small intestinal organoids and MSCs, which allows to study the regenerative effects of MSCs on intestinal epithelium in a more physiologically relevant setting than existing in vitro systems. Using this model we mimicked chemotherapy-mediated damage of the intestinal epithelium. The treatment with busulfan, the chemotherapeutic commonly used as conditioning regiment before the HSCT, affected pathways regulating epithelial to mesenchymal transition, proliferation, and apoptosis in small intestinal organoids, as shown by transcriptomic and proteomic analysis. The co-culture of busulfan-treated intestinal organoids with MSCs reversed the effects of busulfan on the transcriptome and proteome of intestinal epithelium, which we also confirmed by functional evaluation of proliferation and apoptosis. CONCLUSIONS: Collectively, we demonstrate that our in vitro co-culture system is a new valuable tool to facilitate the investigation of the molecular mechanisms behind the therapeutic effects of MSCs on damaged intestinal epithelium. This could benefit further optimization of the use of MSCs in HSCT patients.

Viscoelastic hydrogels regulate adipose-derived mesenchymal stem cells for nucleus pulposus regeneration.

Low back pain is a leading cause of disability worldwide, often attributed to intervertebral disc (IVD) degeneration with loss of the functional nucleus pulposus (NP). Regenerative strategies utilizing biomaterials and stem cells are promising for NP repair. Human NP tissue is highly viscoelastic, relaxing stress rapidly under deformation. However, the impact of tissue-specific viscoelasticity on the activities of adipose-derived stem cells (ASC) remains largely unexplored. Here, we investigated the role of matrix viscoelasticity in regulating ASC differentiation for IVD regeneration. Viscoelastic alginate hydrogels with stress relaxation time scales ranging from 100 s to 1000s were developed and used to culture human ASCs for 21 days. Our results demonstrated that the fast-relaxing hydrogel significantly enhanced ASCs long-term cell survival and NP-like extracellular matrix secretion of aggrecan and type-II collagen. Moreover, gene expression analysis revealed a substantial upregulation of the mechanosensitive ion channel marker TRPV4 and NP-specific markers such as SOX9, HIF-1α, KRT18, CDH2 and CD24 in ASCs cultured within the fast-relaxing hydrogel, compared to slower-relaxing hydrogels. These findings highlight the critical role of matrix viscoelasticity in regulating ASC behavior and suggest that viscoelasticity is a key parameter for novel biomaterials design to improve the efficacy of stem cell therapy for IVD regeneration. STATEMENT OF SIGNIFICANCE: Systematically characterized the influence of tissue-mimetic viscoelasticity on ASC. NP-mimetic hydrogels with tunable viscoelasticity and tissue-matched stiffness. Long-term survival and metabolic activity of ASCs are substantially improved in the fast-relaxing hydrogel. The fast-relaxing hydrogel allows higher rate of cell protrusions formation and matrix remodeling. ASC differentiation towards an NP-like cell phenotype is promoted in the fast-relaxing hydrogel, with more CD24 positive expression indicating NP committed cell fate. The expression of TRPV4, a molecular sensor of matrix viscoelasticity, is significantly enhanced in the fast-relaxing hydrogel, indicating ASC sensing matrix viscoelasticity during cell development. The NP-specific ECM secretion of ASC is considerably influenced by matrix viscoelasticity, where the deposition of aggrecan and type-II collagen are significantly enhanced in the fast-relaxing hydrogel.

Harnessing Nanomedicine for Cartilage Repair: Design Considerations and Recent Advances in Biomaterials.

Cartilage injuries are escalating worldwide, particularly in aging society. Given its limited self-healing ability, the repair and regeneration of damaged articular cartilage remain formidable challenges. To address this issue, nanomaterials are leveraged to achieve desirable repair outcomes by enhancing mechanical properties, optimizing drug loading and bioavailability, enabling site-specific and targeted delivery, and orchestrating cell activities at the nanoscale. This review presents a comprehensive survey of recent research in nanomedicine for cartilage repair, with a primary focus on biomaterial design considerations and recent advances. The review commences with an introductory overview of the intricate cartilage microenvironment and further delves into key biomaterial design parameters crucial for treating cartilage damage, including microstructure, surface charge, and active targeting. The focal point of this review lies in recent advances in nano drug delivery systems and nanotechnology-enabled 3D matrices for cartilage repair. We discuss the compositions and properties of these nanomaterials and elucidate how these materials impact the regeneration of damaged cartilage. This review underscores the pivotal role of nanotechnology in improving the efficacy of biomaterials utilized for the treatment of cartilage damage.

Vitamin C regulates skeletal muscle post-injury regeneration by promoting myoblast proliferation through its direct interaction with the Pax7 protein.

Previous studies have shown that vitamin C (VC), an essential vitamin for the human body, can promote the differentiation of muscle satellite cells (MuSCs) in vitro and play an important role in skeletal muscle post-injury regeneration. However, the molecular mechanism of VC regulating MuSC proliferation has not been elucidated. In this study, the role of VC in promoting MuSC proliferation and its molecular mechanism were explored using cell molecular biology and animal experiments. The results showed that VC accelerates the progress of skeletal muscle post-injury regeneration by promoting MuSC proliferation in vivo. VC can also promote skeletal muscle regeneration in the case of atrophy. Using the C2C12 myoblast murine cell line, we observed that VC also stimulated cell proliferation. In addition, after an in vitro study establishing the occurrence of a physical interaction between VC and Pax7, we observed that VC also upregulated the total and nuclear Pax7 protein levels. This mechanism increased the expression of Myf5 (Myogenic Factor 5), a Pax7 target gene. This study establishes a theoretical foundation for understanding the regulatory mechanisms underlying VC-mediated MuSC proliferation and skeletal muscle regeneration. Moreover, it develops the application of VC in animal muscle nutritional supplements and treatment of skeletal muscle-related diseases.

A highly biocompatible CE-crosslinked collagen implant with exceptional anti-calcification and collagen regeneration capabilities for aging skin rejuvenation.

Skin aging, a complex and inevitable biological process, results in wrinkles, dermal laxity, and skin cancer, profoundly influencing appearance and overall health. Collagen serves as the fundamental element of the dermal matrix; nevertheless, collagen is susceptible to enzymatic degradation within the body. Crosslinking is employed to enhance the physicochemical properties of collagen. However, conventional crosslinking agents may harbor potential issues such as cytotoxicity and calcification risks, constraining their application in the biomedical field. Therefore, we have for the first time developed a highly biocompatible CE-crosslinked collagen implant with exceptional anti-calcification and collagen regeneration capabilities for aging skin rejuvenation. A novel collagen crosslinking agent (CE) was synthesized through a reaction involving chitosan quaternary ammonium salt with 1,4-butanediol diglycidyl ether. Compared to collagen crosslinked with glutaraldehyde (GA), the CE-crosslinked collagen implant exhibited notable stability and durability. The implant demonstrated excellent injectability and viscosity, resisting displacement after implantation. Additionally, the CE-crosslinked collagen implant displayed superior biocompatibility, effectively promoting the proliferation and adhesion of HFF-1 cells compared with the GA-crosslinked collagen. The CE-crosslinked collagen represented a safer and more biologically active implant material. In vivo experiments further substantiated that the implant significantly facilitated collagen regeneration without inducing calcification. The innovative collagen implant has made substantial strides in enhancing aesthetics and reducing wrinkles, presenting the potential for revolutionary progress in the fields of skin rejuvenation and collagen regeneration.

Pilose antler extract promotes hair growth in androgenic alopecia mice by promoting the initial anagen phase.

Androgenetic alopecia (AGA) is a prevalent disease in worldwide, local application or oral are often used to treat AGA, however, effective treatments for AGA are currently limited. In this work, we observed the promoting the initial anagen phase effect of pilose antler extract (PAE) on hair regeneration in AGA mice. We found that PAE accelerated hair growth and increased the degree of skin blackness by non-invasive in vivo methods including camera, optical coherence tomography and dermoscopy. Meanwhile, HE staining of sagittal and coronal skin sections revealed that PAE augmented the quantity and length of hair follicles, while also enhancing skin thickness and hair papilla diameter. Furthermore, PAE facilitated the shift of the growth cycle from the telogen to the anagen phase and expedited the proliferation of hair follicle stem cells and matrix cells in mice with AGA. This acceleration enabled the hair follicles to enter the growth phase at an earlier stage. PAE upregulated the expression of the sonic hedgehog (SHH), smoothened receptor, glioma-associated hemolog1 (GLI1), and downregulated the expression of bone morphogenetic protein 4 (BMP4), recombinant mothers against decapentaplegic homolog (Smad) 1 and 5 phosphorylation. This evidence suggests that PAE fosters hair growth and facilitates the transition of the growth cycle from the telogen to the anagen phase in AGA mice. This effect is achieved by enhancing the proliferation of follicle stem cells and matrix cells through the activation of the SHH/GLI pathway and suppression of the BMP/Smad pathway.

Ellagic acid inhibits dihydrotestosterone-induced ferroptosis and promotes hair regeneration by activating the wnt/ß-catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Androgenic alopecia (AGA) is the most prevalent form of hair loss in clinical practice and affects the physical and psychological well-being of adolescents. Paeonia lactiflora Pallas (PL), which is widely used in traditional Chinese medicine, enhances blood function and promotes hair growth, and ellagic acid (EA), a polyphenol in PL extract, shows strong antioxidant, anti-aging, and anti-inflammatory properties and also plays a role in the treatment of various skin conditions. However, its role and mechanism of action in AGA remain unclear. AIM OF THE STUDY: To determine whether EA can rescue slow hair regeneration by regulating dihydrotestosterone (DHT)-induced ferroptosis in AGA mice and clarify the effect of EA on DHT-induced ferroptosis in dermal papilla cells (DPCs). MATERIALS AND METHODS: Male C57BL/6 mice were used to establish a DHT-induced AGA mouse model, whereas DPCs were used to establish a DHT-induced cellular model. Thereafter, we investigated the therapeutic mechanism of action of EA via immunofluorescence, western blot analysis, immunohistochemistry, electron microscopy, and molecular docking. RESULTS: EA stimulated hair regeneration in mice and reversed DHT-induced increases in iron content, lipid peroxidation, and DHT-induced mitochondrial dysfunction by activating the Wnt/ß-catenin signaling pathway. Further, ß-catenin knockdown suppressed the inhibitory effect of EA on DHT-induced ferroptosis in DPCs. CONCLUSION: EA inhibits DHT-induced ferroptosis and promotes hair regrowth in mice by activating the Wnt/ß-catenin signaling pathway. Thus, it has potential for use as a treatment option for AGA.

Melatonin pretreatment improves endometrial regenerative cell-mediated therapeutic effects in experimental colitis.

BACKGROUND: Endometrial regenerative cells (ERCs) have been proven to be an effective strategy for attenuating experimental colitis, but the complex in vivo microenvironment such as oxidative stress may largely limit and weaken ERC efficacy. Melatonin (MT) works as an anti-oxidative agent in a variety of preclinical diseases, and has been identified to promote mesenchymal stem cell-mediated therapeutic effects in different diseases. However, the ability of MT to enhance ERC-mediated effects in colitis is currently poorly understood. METHODS: Menstrual blood was collected from healthy female volunteers to obtain ERCs and identified. In vitro, H2O2-induced oxidative stress was introduced to test if MT could prevent ERCs from damage through detection of intracellular reactive oxidative species (ROS) and apoptosis assay. In vivo, dextran sodium sulfate (DSS)-induced acute colitis was treated by ERCs and MT-primed ERCs, therapeutic effects were assayed by the disease activity index (DAI), histological features, and macrophage and CD4+ T cell in the spleen and colon, and cytokine profiles in the sera and colon were also measured. RESULTS: In vitro, ERCs that underwent MT-precondition were found to possess more anti-oxidative potency in comparison to naïve ERCs, which were characterized by decreased apoptosis rate and intracellular ROS under H2O2 stimulation. In vivo, MT pretreatment can significantly enhance the therapeutic effects of ERCs in the attenuation of experimental colitis, including decreased DAI index and damage score. In addition, MT pretreatment was found to promote ERC-mediated inhibition of Th1, Th17, and M1 macrophage and pro-inflammatory cytokines, increase of Treg, and immunomodulation of cytokines in the spleen and colon. CONCLUSIONS: MT pretreatment facilitates the promotion of cell viability under oxidative stress in vitro, while also enhancing ERC-mediated therapeutic effects in experimental colitis.

Empagliflozin and pirfenidone confer renoprotection through suppression of glycogen synthase kinase-3ß and promotion of tubular regeneration in rats with induced metabolic syndrome.

Metabolic syndrome (MetS) is largely coupled with chronic kidney disease (CKD). Glycogen synthase kinase-3ß (GSK-3ß) pathway drives tubular injury in animal models of acute kidney injury; but its contribution in CKD is still elusive. This study investigated the effect empagliflozin and/or pirfenidone against MetS-induced kidney dysfunction, and to clarify additional underpinning mechanisms particularly the GSK-3ß signaling pathway. Adult male rats received 10%w/v fructose in drinking water for 20 weeks to develop MetS, then treated with either drug vehicle, empagliflozin (30 mg/kg/day) and/or pirfenidone (100 mg/kg/day) via oral gavage for subsequent 4 weeks, concurrently with the high dietary fructose. Age-matched rats receiving normal drinking water were used as controls. After 24 weeks, blood and kidneys were harvested for subsequent analyses. Rats with MetS showed signs of kidney dysfunction, structural changes and interstitial fibrosis. Activation of GSK-3ß, decreased cyclinD1 expression and enhanced apoptotic signaling were found in kidneys of MetS rats. There was abundant alpha-smooth muscle actin (α-SMA) expression along with up-regulation of TGF-ß1/Smad3 in kidneys of MetS rats. These derangements were almost alleviated by empagliflozin or pirfenidone, with evidence that the combined therapy was more effective than either individual drug. This study emphasizes a novel mechanism underpinning the beneficial effects of empagliflozin and pirfenidone on kidney dysfunction associated with MetS through targeting GSK-3ß signaling which can mediate the regenerative capacity, anti-apoptotic effects and anti-fibrotic properties of such drugs. These findings recommend the possibility of using empagliflozin and pirfenidone as promising therapies for management of CKD in patients with MetS.

High-Throughput Methods for the Discovery of Small Molecule Modulators of Pancreatic Beta-Cell Function and Regeneration.

The progression of type II diabetes (T2D) is characterized by a complex and highly variable loss of beta-cell mass, resulting in impaired insulin secretion. Many T2D drug discovery efforts aimed at discovering molecules that can protect or restore beta-cell mass and function have been developed using limited beta-cell lines and primary rodent/human pancreatic islets. Various high-throughput screening methods have been used in the context of drug discovery, including luciferase-based reporter assays, glucose-stimulated insulin secretion, and high-content screening. In this context, a cornerstone of small molecule discovery has been the use of immortalized rodent beta-cell lines. Although insightful, this usage has led to a more comprehensive understanding of rodent beta-cell proliferation pathways rather than their human counterparts. Advantages gained in enhanced physiological relevance are offered by three-dimensional (3D) primary islets and pseudoislets in contrast to monolayer cultures, but these approaches have been limited to use in low-throughput experiments. Emerging methods, such as high-throughput 3D islet imaging coupled with machine learning, aim to increase the feasibility of integrating 3D microtissue structures into high-throughput screening. This review explores the current methods used in high-throughput screening for small molecule modulators of beta-cell mass and function, a potentially pivotal strategy for diabetes drug discovery.

"Keep on ROCKIn": Repurposed ROCK inhibitors to boost corneal endothelial regeneration.

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.