Diabetic Mouse Model Research Articles

Enhancing Skin Wound Healing in Diabetic Mice Using SIKVAV-Modified Chitosan Hydrogels

Diabetic foot ulcers (DFUs), a prevalent chronic disease caused by various factors, significantly impact patients’ quality of life due to prolonged healing times and increased infection risks. Current treatment modalities, including pharmacological, physical, and surgical interventions, often yield limited efficacy and adverse effects, highlighting the urgent need for novel therapeutic strategies. The objective of this research is to create SIKVAV-modified chitosan hydrogels with the intention of improving the process of skin wound healing in diabetic mice. We synthesized the hydrogels and established a diabetic mice model with skin wounding to evaluate its healing effects and underlying mechanisms. The results of our study indicate that the SIKVAV-modified chitosan hydrogels markedly enhance the wound healing process in diabetic mice. This effect may be attributed to several mechanisms, including differentiation of fibroblasts, proliferation of keratinocytes, the promotion of angiogenesis, stimulation of collagen synthesis, upregulation of growth factor expression, and possible involvement of the TGF-β1/Smad3 signaling pathway. This research not only provides a new biomaterial for the treatment of diabetic wounds but also elucidates the related molecular mechanisms involved in wound healing of DFUs, offering valuable insights for future clinical applications.

Prolonged Photobiomodulation with Deep Red Light Mitigates Incipient Retinal Deterioration in a Mouse Model of Type 2 Diabetes

Diabetic retinopathy is a prevalent complication of type 2 diabetes mellitus, characterized by progressive damage to the retinal structure and function. Photobiomodulation therapy, using red or near-infrared light, has been proposed as a non-invasive intervention to mitigate retinal damage, but has been tested generally with short-term stimuli. This study aimed to evaluate the effects of prolonged photobiomodulation with deep red light on retinal structure and function in a type 2 diabetes mouse model. Transgenic LepRdb/J (db/db) mice were exposed to photobiomodulation therapy via LED devices emitting 654 nm light for 12 h daily over ten weeks and compared to untreated mice. Retinal function was evaluated by flash electroretinography, while structural changes were assessed through histology and immunohistochemistry to detect astro- and microgliosis. At 33 weeks of age, db/db mice were obese and severely diabetic, but exhibited only incipient indicators of retinal deterioration. Electroretinogram b-wave peak latencies were prolonged at intermediate flash intensities, while the outer plexiform layer displayed significantly elevated IBA1 expression. Photobiomodulation therapy prevented these two markers of early retinal deterioration but had no effect on other morphological and functional parameters. Photobiomodulation is well-tolerated and maintains potential as a complementary treatment option for diabetic retinopathy but requires further optimization of therapeutic settings and combinatory treatment approaches.

Abstract 4139148: Mechanisms of Maternal Cardiovascular Disease Development in a Novel Mouse Model of Pregnancy-Induced Diabetes

Background: Women with gestational diabetes mellitus (GDM) are more likely to develop cardiovascular disease (CVD) within 3 to 10 years following pregnancy. Endothelial dysfunction is a mechanism implicated in both hyperglycemia and CVDs, and it may constitute the missing link in this interaction. One reason for the lack of progress into the cardiovascular complications of GDM is the need for appropriate preclinical models that mimic human GDM. Also, the exact mechanisms mediating increased future cardiovascular risk in maternal with pregnancy-induced diabetes is unclear. Aim: To determine the mechanisms underlying cardiovascular disease in hormonal model of GDM. Methods: Sixteen week-old C57BL/6J female mice were treated with insulin receptor antagonist (S961) or saline (n=4) from the second trimester of pregnancy until delivery to develop a model of GDM. Glucose tolerance test was determined by glucometer and fasting insulin by colorimetry. Cardiac structure and function were measured by echocardiography (VEVO 3100) and pulse wave velocity was measured by Pulse Wave Doppler. Exercise tolerance test was performed on treadmill. Plasma endothelin-1 and nitrite were measured by ELISA and colorimetric assay. Aortic SIRT-1 was determined by western blot and aortic vascular reactivity was determined by wire myograph. Results: S961-induced GDM mice exhibit hyperglycemia (169.2 ± 3.6 vs. 105.5 ± 10.8mg/dL, P<0.004 ) and hyperinsulinemia (1.9 ± 0.2 vs. 0.8 ± 0.1ng/ml , p<0.01 ) which mimics the condition observed in human GDM. Interestingly, S961-induced GDM mice went on to develop systolic and diastolic dysfunction, vascular stiffness (3.7 ± 0.1 vs. 3.1 ± 0.08m/s, p<0.005 ), exercise intolerance and increased thickness of the cardiac wall at 10 weeks post-delivery. Plasma ET-1 was elevated in our model during pregnancy (28.1 ± 1.7 vs. 5.2 ± 0.2pg/ml, p<0.002 ) and at 10 weeks post-delivery (19.9 ± 2.9 vs. 6.7 ± 0.7pg/ml, p<0.05 ). Circulating NO and aortic expression of sirtuin-1 (SIRT 1), that is involved in endothelial longevity is reduced in GDM mice. Additionally, vascular relaxation to acetylcholine was attenuated in S961-induced GDM mice. Conclusion: We have developed a novel mouse model of GDM similar to that observed in pregnant women by treatment with S961. Moreover, S961 treated mice develop cardiac dysfunction and vascular stiffness at 10 weeks post-delivery. Alterations in ET-1, SIRT 1, and NO levels may be responsible for the development of CVD in this model.

Astaxanthin has a beneficial influence on pain-related symptoms and opioid-induced hyperalgesia in mice with diabetic neuropathy-evidence from behavioral studies.

The treatment of painful diabetic neuropathy is still a clinical problem. The aim of this study was to determine whether astaxanthin, a substance that inhibits mitogen-activated protein kinases, activates nuclear factor erythroid 2-related factor 2 and influences N-methyl-D-aspartate receptor, affects nociceptive transmission in mice with diabetic neuropathy. The studies were performed on streptozotocin-induced mouse diabetic neuropathic pain model. Single intrathecal and intraperitoneal administrations of astaxanthin at various doses were conducted in both males and females. Additionally, repeated twice-daily treatment with astaxanthin (25mg/kg) and morphine (30mg/kg) were performed. Hypersensitivity was evaluated with von Frey and cold plate tests. This behavioral study provides the first evidence that in a mouse model of diabetic neuropathy, single injections of astaxanthin similarly reduce tactile and thermal hypersensitivity in both male and female mice, regardless of the route of administration. Moreover, repeated administration of astaxanthin slightly delays the development of morphine tolerance and significantly suppresses the occurrence of opioid-induced hyperalgesia, although it does not affect blood glucose levels, body weight, or motor coordination. Surprisingly, astaxanthin administered repeatedly produces a better analgesic effect when administered alone than in combination with morphine, and its potency becomes even more pronounced over time. These behavioral results provide a basis for further evaluation of the potential use of astaxanthin in the clinical treatment of diabetic neuropathy and suggest that the multidirectional action of this substance may have positive effects on relieving neuropathic pain in diabetes.

Abstract 4129523: MicroRNA-1282 Rescues Diabetic Limb Ischemia Via a SERF2-Protein Aggregation Pathway

Introduction: Patients with diabetes are at higher risk of chronic limb-threatening ischemia (CLTI), a severe form of peripheral artery disease (PAD) causing restricted blood flow to the lower limbs due, in part, to impaired angiogenesis. However, the role of microRNAs (miRNAs) in diabetic CLTI remains poorly understood. By integrating plasma miRNA sequencing data from PAD patients with diabetes with a diabetic CLTI mouse model, we have recently identified the conserved miRNA miR-1282 that is in cis-antisense orientation to SERF2, a gene associated with amyloid aggregation. Therefore, we hypothesize that miR-1282 orchestrates endothelial angiogenesis and proteostasis during diabetic CLTI. Methods: Using miR-1282 overexpression or SERF2 knockdown studies, we characterized mouse orthologs of human miR-1282 and SERF2 for angiogenesis, apoptosis, protein aggregation, and oxidative stress in diabetic mouse skeletal muscle endothelial cells (ECs). In vivo, miR-1282 mimics were delivered intramuscularly to assess their impact on blood flow recovery, angiogenesis, and protein aggregation. Mechanistic insights in ECs were gained via RNA-seq, predictive algorithms, and proteomic analyses. Results: miR-1282 inhibited the expression of its cis-antisense target SERF2 by 98%. miR-1282 is a hypoxia-induced endothelial-enriched miRNA, and its expression was inversely correlated with SERF2 expression. miR-1282 levels were markedly reduced after femoral artery ligation (FAL) in diabetic db/db mice. Overexpression of miR-1282 or SERF2 knockdown enhanced angiogenesis and reduced protein aggregation, apoptosis, and oxidative stress in both normal and hypoxic conditions in vitro. Delivery of miR-1282 mimics in db/db mice improved blood flow recovery by 108% and angiogenesis by 98%, and reduced protein aggregation by 48% and tissue necrosis. Coupling RNA-seq profiling and prediction algorithms of ECs upon miR-1282 overexpression or SERF2 knockdown revealed EREG, BAG5, CASP3, ARG1, and HSP90AA1 as potential downstream regulators. Pathway enrichment analysis implicated inhibition of endothelial apoptosis, ER stress, and protein stability among the most dysregulated processes. Conclusion: A novel mouse ortholog of human miR-1282 augments endothelial functions and diminishes protein aggregation in diabetic CLTI via suppression of its cis-antisense target SERF2. These findings uncover new potential therapeutic targets in treating diabetic CLTI.

A microenvironment-modulating dressing with proliferative degradants for the healing of diabetic wounds.

Diabetic wounds are usually entangled in a disorganized and self-perpetuating microenvironment and accompanied by a prolonged delay in tissue repair. Sustained and coordinated microenvironment regulation and tissue regeneration are key to the healing process of diabetic wounds, yet they continue to pose a formidable challenge. Here we report a rational double-layered dressing design based on chitosan and a degradable conjugated polymer polydiacetylene, poly(deca-4,6-diynedioic acid) (PDDA), that can meet this intricate requirement. With an alternating ene-yne backbone, PDDA degrades when reacting with various types of reactive oxygen species (ROS), and more importantly, generates proliferative succinic acid as a major degradant. Inheriting from PDDA, the developed PDDA-chitosan double layer dressing (PCD) can eliminate ROS in the microenvironment of diabetic wounds, alleviate inflammation, and downregulate gene expression of innate immune receptors. PCD degradation also triggers simultaneous release of succinic acid in a sustainable manner, enabling long-term promotion on tissue regeneration. We have validated the biocompatibility and excellent performance of PCD in expediting the wound healing on both diabetic mouse and porcine models, which underscores the significant translational potential of this microenvironment-modulating, growth-promoting wound dressing in diabetic wounds care.

Abstract 4122717: An Epigenetic Drug, GSK126 Mitigates Endothelial to Mesenchymal Transition Attenuating Atherosclerosis in Diabetes

Background: Endothelial to mesenchymal transition (EndMT) involves transformation of an endothelial to mesenchymal like cellular state. Recent studies implicate EndMT in atherosclerosis development. Diabetes is associated with both EndMT as well as accelerated atherosclerosis. Experimental evidence supports the use of epigenetic drugs that act on relevant epigenetic mechanisms to attenuate atherosclerosis. Recently, inhibition of a histone methyltransferase Enhancer of zest homolog 2 (EZH2) with GSK-126 attenuated atherosclerosis development. However, the role of EZH2 in induction of EndMT in diabetes induced atherosclerosis is not known. Methods: An in-vitro model of EndMT was generated using high glucose (HG) and TNF-α in human aortic endothelial cells (HAECs). EZH2 methyltransferase activity was inhibited using EZH2 specific inhibitor GSK-126. RNA sequencing was performed in this setting. EZH2 genetic knockdown (shRNA) in HAECs was also performed to validate role of EZH2 in EndMT. In addition, EZH2 mediated H3K27me3 and EndMT was assessed in atheroprone diabetic mouse model. Results: In HAECs, morphological changes as well as gene and protein expression confirmed TNF-α+HG induced EndMT, which was blunted by GSK-126. Elevated EZH2 mediated H3K27me3 activity by the TNF-α + HG stimulation was blunted with GSK-126 treatment. Furthermore, RNA sequencing identified several enriched pathways including AGE-RAGE signaling pathway in diabetic complications, focal adhesion, and leukocyte trans-endothelial migration directly relevant to EndMT and commonly deregulated in diabetic complications, which were rescued by EZH2 inhibition. Transcriptomic analysis showed that 242 genes including MMP2, NOS3 linked to EndMT were dysregulated. Interestingly, expression of 76 dysregulated genes in EndMT were rescued by GSK-126. EZH2 knockdown studies in HAECs also validated the role of EZH2 in EndMT. Furthermore, immunofluorescence staining identified elevated levels of H3K27me3 in the aortic endothelial layer of diabetic Apoe -/- mice. Co-localization of EndMT markers was observed in the endothelial layer of aortic sinus of diabetic Apoe -/- mice which was blunted with GSK-126 treatment. Conclusion: The study identified EZH2 inhibition as a novel therapeutic target in diabetes induced EndMT in atherosclerosis. These findings highlighted ongoing efforts to develop targeted therapies for diabetic patients who are at risk to develop cardiovascular disease.

Cysteine Leukotriene Receptor Antagonist-Montelukast Effects on Diabetic Retinal Microvascular Endothelial Cells Curtail Autophagy.

Diabetic macular edema (DME) is the primary cause of vision impairment in diabetic retinopathy (DR) patients. A previous study has shown the efficacy of montelukast, a cysteinyl leukotriene receptor (CysLTR)1 antagonist, in a diabetic mouse model. This study aims to understand the CysLTR1 signaling in retinal endothelial cells and the impact of montelukast. Primary human retinal microvascular endothelial cells (HRECs) challenged with 20 ng/mL TNF-α and 30mM D-glucose (D-glu) for six to 24 hours served as a model of endothelial activation. HRECs were incubated with L-glucose (L-glu) as an osmotic control. CysLTR1 knockdown and montelukast pretreatment assessed CysLTR1 antagonism. Gene expression, protein expression, and cell-permeable dyes were utilized to measure autophagy and inflammation. Transendothelial electrical resistance (TER) and transendothelial migration of mononuclear leukocytes across HRECs monolayer were measured as a functional assessment of vascular permeability. Endothelial activation induced by hyperglycemia and inflammation increased CysLTR1 expression, triggering autophagy within two to six hours, IL-1β production, loss of junction integrity, decreased TER, and increased leukocyte migration within six to 24 hours. Pretreatment with montelukast effectively alleviated these effects, demonstrating its dependence on CysLTR1. Dysfunctional retinal endothelium initiates a self-reinforcing loop of inflammation, autophagy, and compromised integrity associated with heightened CysLTR1 levels. The antagonistic effect of montelukast against CysLTR1 effectively mitigates these detrimental changes. This study reveals CysLTR1 as a potential therapeutic target in treating DME and offers a novel strategy to mitigate detrimental changes in DR.

Mammalian Ste-20-like Kinase 1/2 (MST1/2) Inhibitor XMU-MP-1: A Potential Compound to Improve Spermatogenesis in Mouse Model of Diabetes Mellitus

Background/Objectives: Spermatogenesis is a key process in male reproduction that, if it does not happen correctly, can lead to infertility, with diabetes being one of the most prevalent causes of spermatogenesis disruption. Currently, there is a lack of research examining the potential benefits of targeting cell proliferation to enhance spermatogenesis in this condition. XMU-MP1 has been identified as an inhibitor of MST1, a core component of the Hippo pathway, which is anticipated to promote proliferation and regeneration. This study aims to evaluate the effects of XMU-MP1 treatment on sperm and testicular characteristics in mice. Methods: We used the STZ-induced diabetic mouse model to investigate the impact of administering XMU-MP1 on testicular tissue and sperm parameters. This study compared the seminiferous tubules, specifically focusing on the diameter of the seminiferous tubule, the thickness of the seminiferous tubule epithelium, the ratio of the thickness of the seminiferous tubule epithelium to the diameter of the seminiferous tubules, and the lumen diameter of the seminiferous tubules. We also conducted a comparison of sperm parameters, including sperm concentration, progressive motility, total motility, total motility, and morphology. Results: XMU-MP1-treated mice had a larger spermatogenesis area and better sperm motility than control mice. Diabetic mice treated with XMU-MP1 also showed a trend toward improvements in the spermatogenesis area, sperm concentration, sperm motility, and sperm morphology, although these improvements were not statistically significant. Conclusions: XMU-MP1 serves as a potential compound to improve spermatogenesis in mice.

ASK-1 activation exacerbates kidney dysfunction via increment of glomerular permeability and accelerates cellular aging in diabetic kidney disease model mice

Diabetic kidney disease (DKD) is a major disease characterized by early albuminuria and heightened risk of renal deterioration. Increased reactive oxygen species (ROS) production, especially in glomeruli, plays an important role in the progression of DKD. ROS also cause activation of Apoptosis signal-regulating kinase 1 (ASK-1), which is implicated in various organ injuries. However, the detailed mechanisms remain unclear. This study investigates ASK-1 activation in advanced DKD and its underlying mechanisms using GS442172, an ASK-1 inhibitor. In the DKD mouse model, activation of ASK-1 was observed. Although inhibition of ASK-1 activation improved hyperpermeability in glomerular endothelial cells. ASK-1 inhibition significantly reduced glomerular injury and albuminuria, while also attenuating tubular damage and interstitial fibrosis. RNA-seq analysis revealed an aging phenotype associated with ASK-1 activation in DKD. In vitro experiments demonstrated ASK-1 activation-induced cellular senescence in tubular cells via redox signaling. These results suggested that the critical role of ASK-1 activation in DKD pathogenesis, implicating glomerular injury, tubular damage, and cellular senescence. ASK-1 inhibitors are promising therapeutic strategies to mitigate the progression of DKD.

Degree of sulfation of freeze-dried calcium alginate sulfate scaffolds dramatically influence healing rate of full-thickness diabetic wounds.

Diabetic foot ulcer (DFU) is a chronic and non-healing wound in all age categories with a high prevalence and mortality in the world. An ideal wound dressing for DFU should possess the ability of adsorbing high contents of exudate and actively promote wound healing. Here, we introduced the calcium alginate sulfate as a new biomaterial appropriate for use in wound dressing to promote the healing of full-thickness ulcers in a diabetic mouse model. In this regard, alginate sulfate (Alg-S) solutions with different degree of substitution (DS) of 0.2, 0.5, and 0.9 were synthesized, freeze-dried, crosslinked by calcium cations, purified by washing and refreeze-dried. Primary analyses including swelling ratio, porosity content and mechanical properties revealed that all Alg-S scaffolds possess necessities for use as a wound dressing. After confirming the cytocompatibility of both alginate and alginate sulfate-based scaffolds by MTT assay, they were used as wound dressing for healing of full-thickness ulcers in diabetic mice. The results of wound healing process confirmed that calcium alginate sulfate scaffolds can heal the wounds faster than both alginate-treated and non-treated wounds. Furthermore, the histological analyses of healed tissues reveled normal regeneration of the skin tissue layers and collagen deposition similar to the healthy tissue.

Role of the PGAM5-CypD mitochondrial pathway in methylglyoxal-induced bone loss in diabetic osteoporosis

Diabetic osteoporosis (DOP) is a skeletal complication with a high rate of disability. It results in a great burden to the patient's family and society. Methylglyoxal (MG) is a toxic by-product of the glycolytic process that occurs during diabetic conditions. It causes osteoblastic injury and con-tributes to the initiation and development of DOP. Disruption of mitochondrial homeostasis has been implicated as a cause of dysregulated osteo-blastogenesis, an essential step in bone formation. It is unclear whether mitochondrial dysfunction is involved in MG-induced osteoblast dysfunction. In this study, we showed that mitochondrial dysfunction contributes to MG-induced MC3T3-E1 cell apoptosis and impaired differentiation. A significant reduction of mitochondrial membrane potential (MMP) and ATP production occurred in MG-induced osteoblasts as well as increasing mitochondrial reactive oxygen species (mtROS) and intracellular Ca2+. Classical antioxidant N-Acetylcysteine (NAC) significantly attenuated mitochondrial dysfunction as well as osteoblast apoptosis and osteogenic differentiation damage induced by MG. More importantly, we found that activating phosphoglycerate mutase family member 5 (PGAM5) and cyclophilin D (CypD), which contributes to mitochondrial homeostasis, is involved in MG-induced osteoblast injury. Both PGAM5 and CypD knockdown effectively reversed osteoblast viability and function, whereas PGAM5 or CypD overexpression aggravated osteoblast injury caused by MG. Moreover, the result of co-transfection revealed that PGAM5 is an upstream signaling molecule of CypD. By constructing type I diabetes mouse models, we further found that the expression of PGAM5 and CypD were both increased in the femur along with a reduction of ATP and increased TUNEL-positive cells. These results, for the first time, suggest that MG-induced mitochondrial dysfunction induces osteoblast injury through the PGAM5-CypD pathway. This study provides insight into the prevention and treatment of DOP. Lay summaryThis study highlights the role of mitochondria in regulating osteoblast viability and function under conditions of diabetic osteoporosis (DOP). We found that the PGAM5-CypD mitochondrial pathway is activated following glycolytic by-product methylglyoxal (MG) treatment, which contributes to mitochondrial dysfunction and osteogenic dysfunction. This mechanism implicates mitochondria as a potential therapeutic target for osteoporosis.

Amino Acid Compound 2 (AAC2) Treatment Counteracts Insulin-Induced Synaptic Gene Expression and Seizure-Related Mortality in a Mouse Model of Alzheimer's Disease.

Diabetes is a major risk factor for Alzheimer's disease (AD). Amino acid compound 2 (AAC2) improves glycemic and cognitive functions in diabetic mouse models through mechanisms distinct from insulin. Our goal was to compare the effects of AAC2, insulin, and their nanofiber-forming combination on early asymptomatic AD pathogenesis in APP/PS1 mice. Insulin, but not AAC2 or the combination treatment (administered intraperitoneally every 48 h for 120 days), increased seizure-related mortality, altered the brain fat-to-lean mass ratio, and improved specific cognitive functions in APP/PS1 mice. NanoString and pathway analysis of cerebral gene expression revealed dysregulated synaptic mechanisms, with upregulation of Bdnf and downregulation of Slc1a6 in insulin-treated mice, correlating with insulin-induced seizures. In contrast, AAC2 promoted the expression of Syn2 and Syp synaptic genes, preserved brain composition, and improved survival. The combination of AAC2 and insulin counteracted free insulin's effects. None of the treatments influenced canonical amyloidogenic pathways. This study highlights AAC2's potential in regulating synaptic gene expression in AD and insulin-induced contexts related to seizure activity.

Protein O-GlcNAcylation coupled to Hippo signaling drives vascular dysfunction in diabetic retinopathy

Metabolic disorder significantly contributes to diabetic vascular complications, including diabetic retinopathy, the leading cause of blindness in the working-age population. However, the molecular mechanisms by which disturbed metabolic homeostasis causes vascular dysfunction in diabetic retinopathy remain unclear. O-GlcNAcylation modification acts as a nutrient sensor particularly sensitive to ambient glucose. Here, we observe pronounced O-GlcNAc elevation in retina endothelial cells of diabetic retinopathy patients and mouse models. Endothelial-specific depletion or pharmacological inhibition of O-GlcNAc transferase effectively mitigates vascular dysfunction. Mechanistically, we find that Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, are O-GlcNAcylated in diabetic retinopathy. We identify threonine 383 as an O-GlcNAc site on YAP, which inhibits its phosphorylation at serine 397, leading to its stabilization and activation, thereby promoting vascular dysfunction by inducing a pro-angiogenic and glucose metabolic transcriptional program. This work emphasizes the critical role of the O-GlcNAc-Hippo axis in the pathogenesis of diabetic retinopathy and suggests its potential as a therapeutic target.

Therapeutic effects of Mudan granules on diabetic retinopathy: Mitigating fibrogenesis caused by FBN2 deficiency and inflammation associated with TNF-α elevation

Ethnopharmacological relevanceMudan granules (MuD), a time-honored traditional Chinese patent medicine (TCPM), are widely utilized in the clinical treatment of diabetic peripheral neuropathy (DPN). In the field of biomedical diagnostics, both diabetic retinopathy (DR) and DPN are recognized as critical microvascular complications associated with diabetes. According to the principles of traditional Chinese medicine (TCM), these conditions are primarily attributed to a deficiency in Qi and the obstruction of collaterals. Despite this, the protective effects of MuD on DR and the underlying mechanisms remain to be comprehensively elucidated. Aims of the studyThe purpose of this study was to investigate the effect of MuD on DR and to further explore the promising therapeutic targets. MethodsA diabetic mouse model was established by administering 60 mg/kg of streptozotocin (STZ) via intraperitoneal injection for five consecutive days. The therapeutic efficacy of MuD was evaluated using a comprehensive approach, which included electroretinogram (ERG) analysis, histopathological examination, and assessment of serum biochemical markers. Then, the pharmacodynamic mechanisms of MuD were systematically analyzed using Tandem Mass Tags-based proteomics. Meanwhile, the candidate compounds of MuD were analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and molecular docking was applied to estimate the affinity of the active ingredient to their potential key targets. In addition, the functional mechanisms identified through bioinformatics analysis were confirmed by molecular biological methods. ResultsWe demonstrated that MuD provided significant protection to retinal function and effectively mitigated the reduction in retinal thickness observed in the animal model. Through proteomic analysis, we identified a substantial regulation by MuD of 70 biomarkers associated with diabetic retinal damage. These proteins were notably enriched in the tumor necrosis factor (TNF) signaling pathway, a critical mediator in inflammatory processes. A particularly intriguing finding was the significant downregulation of fibrillin-2 (FBN2) in the diabetic retina compared to the control group (0.36 times the level), and its most pronounced upregulation (3.26 times) in the MuD treatment group. This suggests that FBN2 may play a pivotal role in the protective effects of MuD. Molecular docking analyses have unveiled a robust interplay between the components of MuD and TNF-α. Further corroboration was provided by molecular biological methods, which confirmed that MuD could suppress TNF-mediated inflammation and prevent retinal neovascularization and fibrogenesis. ConclusionMuD have the potential to alleviate diabetic retinal dysfunction by effectively curbing the fibrogenesis-associated neoangiogenesis and mitigating the inflammatory response, thereby restoring retinal health and function.

Sacubitril/valsartan improves diastolic left ventricular stiffness with increased titin phosphorylation via cGMP-PKG activation in diabetic mice

Titin, a giant sarcomeric protein, regulates diastolic left ventricular (LV) passive stiffness as a molecular spring and could be a therapeutic target for diastolic dysfunction. Sacubitril/valsartan (Sac/Val), an angiotensin receptor neprilysin inhibitor, has been shown to benefit patients with heart failure with preserved ejection fraction. The effect of Sac/Val is thought to be due to the enhancement of the cGMP/PKG pathway via natriuretic peptide. In this study, the effects of Sac/Val on LV diastolic dysfunction are demonstrated in a mouse diabetic cardiomyopathy model focusing on titin phosphorylation. Sac/Val-treated diabetic mice showed a greater increase in myocardial levels of cGMP-PKG than Val-treated and control mice. Conductance catheter analysis showed a significant reduction in LV stiffness in diabetic mice, but not in non-diabetic mice. Notably, diastolic LV stiffness was significantly reduced in Sac/Val-treated diabetic hearts compared with Val-treated or vehicle-treated diabetic mice. The phosphorylation level of titin (N2B), which determines passive stiffness and modulates active contraction, was higher in Sac/Val-treated hearts compared with Val-treated hearts in diabetic mice. Given that alteration of titin phosphorylation through PKG contributes to myocardial stiffness, the beneficial effects of Sac/Val in heart failure might be partly attributed to the induction of titin phosphorylation.

Effect of aspirin on platelet-rich plasma of diabetes mellitus with lower extremity atherosclerosis.

Aim: Platelet-rich plasma (PRP), enriched with multiple growth factors, is a promising adjunctive therapy for diabetic foot ulcers (DFUs). As a classic anti-platelet drug for diabetic patients, the effects of aspirin on the content of growth factors in PRP remains unclear.Methods: Our study enrolled diabetic patients who were currently taking or not taking aspirin as the research subjects, with healthy volunteers as the control. PRP from these individuals was activated with glucose calcium and thrombin. Growth factors levels in PRP activated supernatant (PRP-AS) and wound healing ability of platelet gel (PG) in the full-thickness skin defect diabetic mouse model were compared.Results: We found the level of growth factors in PRP-AS derived from two groups of diabetic patients were not statistically different, whereas both lower than that from healthy volunteers. Similarly, we found better wound healing ability of PG from healthy volunteers than those from diabetic patients, but no difference between the two groups of diabetic patients in the mouse model.Discussion: Aspirin does not interfere with autologous PRP therapy when using calcium gluconate and thrombin as agonists. However considering the content of growth factors, PRP from healthy volunteers is a preferable option for promoting DFU repair.

Metformin Syncs CeO2 to Recover Intra- and Extra-Cellular ROS Homeostasis in Diabetic Wound Healing.

Excessive generation of reactive oxygen species (ROS) poses a huge obstacle to the healing process of diabetic wounds, resulting in chronic, non-healing wounds. While numerous anti-ROS therapeutics have been developed, satisfied intra- and extra- cellular ROS homeostasis is hard to be established in diabetic wounds. To address this issue, a nanoparticle via loading metformin and CeO2 into mesoporous silica (MSN@Met-CeO2) is designed and synthesized, which is then encapsulated within ROS-responsive hydrogel and shaped as microneedles (MNs) for better application in diabetic wounds. Interestingly, a unique metformin-cerium chelate (Ce· 3Metformin) is formed during the synthesis of MSN@Met-CeO2 MN, which significantly strengthened the inhibitory effect of metformin on mitochondrial complex I. With the presence of Ce· 3Metformin, MSN@Met-CeO2 MN performed a remarkable effect on intracellular mtROS reduction as well as extracellular ROS elimination, the latter is primarily accomplished through the dissociative CeO2 in MSN@Met-CeO2 MN. In the mouse diabetic wound model, MSN@Met-CeO2 MN exhibited a superior pro-healing effect with accelerated inflammation resolution and enhanced angiogenesis, thus highlighting its significant potential for clinical application.

Honokiol protects against diabetic retinal microvascular injury via sirtuin 3-mediated mitochondrial fusion

IntroductionMitochondrial dysfunction and oxidative stress play important roles in diabetic retinal vascular injuries. Honokiol (HKL) is a small-molecule polyphenol that exhibits antioxidant effects and has a beneficial effect in diabetes. This study aimed to explore the potential ability of HKL to ameliorate vascular injury in diabetic retinopathy (DR) and its possible mechanisms of action.MethodsThe effect of HKL was evaluated in vascular injury in an in vivo type 2 diabetic (db/db) mouse model. In vitro, retinal microvascular endothelial cells were treated with high glucose (HG) to simulate the pathological diabetic environment. Cell viability, expression of apoptosis-related proteins, cellular reactive oxygen species, mitochondrial membrane potential, and morphological changes in the mitochondria were examined.ResultsThe diabetic mice exhibited severe retinal vascular damage, including vascular leakage in vivo and capillary endothelial cell apoptosis in vitro. HKL reversed the retinal vascular leakage in the diabetic mice. In vitro, HKL improved retinal capillary endothelial cell viability, decreased apoptosis, and reversed the HG-induced increased cellular oxidative stress and mitochondrial fragmentation. The sirtuin 3 (SIRT3) inhibitor 3-TYP blocked all the in vivo and in vitro protective effects of HKL against diabetic retinal vascular leakage and capillary endothelium and eliminated the decrease in oxidative stress levels and reduction of mitochondrial fragmentation.DiscussionIn conclusion, these findings suggest that HKL inhibits vascular injury in DR, which was likely achieved through SIRT3-mediated mitochondrial fusion. This study provides a potential new strategy for the treatment of DR.

TGF-β-mediated crosstalk between TIGIT+ Tregs and CD226+CD8+ T cells in the progression and remission of type 1 diabetes

Type 1 diabetes (T1D) is a chronic autoimmune condition characterized by hyperglycemia resulting from the destruction of insulin-producing β-cells that is traditionally deemed irreversible, but partial remission (PR) with temporary reversal of hyperglycemia is sometimes observed. Here we use single-cell RNA sequencing to delineate the immune cell landscape across patients in different T1D stages. Together with cohort validation and functional assays, we observe dynamic changes in TIGIT+CCR7− Tregs and CD226+CCR7−CD8+ cytotoxic T cells during the peri-remission phase. Machine learning algorithms further identify TIGIT+CCR7− Tregs and CD226+CD8+ T cells as biomarkers for β-cell function decline in a predictive model, while cell communication analysis and in vitro assays suggest that TIGIT+CCR7− Tregs may inhibit CD226+CCR7−CD8+ T cells via TGF-β signaling. Lastly, in both cyclophosphamide-induced and streptozotocin (STZ)-induced mouse diabetes models, CD226 inhibition postpones insulitis onset and reduces hyperglycemia severity. Our results thus identify two interrelated immune cell subsets that may serve as biomarkers for monitoring disease progression and targets for therapeutic intervention of T1D.