A muscarinic receptor antagonist reverses multiple indices of diabetic peripheral neuropathy: preclinical and clinical studies using oxybutynin.

Preclinical studies indicate that diverse muscarinic receptor antagonists, acting via the M1 sub-type, promote neuritogenesis from sensory neurons in vitro and prevent and/or reverse both structural and functional indices of neuropathy in rodent models of diabetes. We sought to translate this as a potential therapeutic approach against structural and functional indices of diabetic neuropathy using oxybutynin, a muscarinic antagonist approved for clinical use against overactive bladder. Studies were performed using sensory neurons maintained in vitro, rodent models of type 1 or type 2 diabetes and human subjects with type 2 diabetes and confirmed neuropathy. Oxybutynin promoted significant neurite outgrowth in sensory neuron cultures derived from adult normal rats and STZ-diabetic mice, with maximal efficacy in the 1-100 nmol/l range. This was accompanied by a significantly enhanced mitochondrial energetic profile as reflected by increased basal and maximal respiration and spare respiratory capacity. Systemic (3-10 mg/kg/day s.c.) and topical (3% gel daily) oxybutynin reversed paw heat hypoalgesia in the STZ and db/db mouse models of diabetes and reversed paw tactile allodynia in STZ-diabetic rats. Loss of nerve profiles in the skin and cornea of db/db mice was also prevented by daily topical delivery of 3% oxybutynin for 8 weeks. A randomized, double-blind, placebo-controlled interventional trial was performed in subjects with type 2 diabetes and established peripheral neuropathy. Subjects received daily topical treatment with 3% oxybutynin gel or placebo for 6 months. The a priori designated primary endpoint, significant change in intra-epidermal nerve fibre density (IENFD) in skin biopsies taken before and after 20 weeks of treatments, was met by oxybutynin but not placebo. Secondary endpoints showing significant improvement with oxybutynin treatment included scores on clinical neuropathy, pain and quality of life scales. This proof-of-concept study indicates that muscarinic antagonists suitable for long-term use may offer a novel therapeutic opportunity for treatment of diabetic neuropathy. Trial registry number: NCT03050827.

Long noncoding RNA Glis2 regulates podocyte mitochondrial dysfunction and apoptosis in diabetic nephropathy via sponging miR-328-5p.

Podocyte apoptosis exerts a crucial role in the pathogenesis of DN. Recently, long noncoding RNAs (lncRNAs) have been gradually identified to be functional in a variety of different mechanisms associated with podocyte apoptosis. This study aimed to investigate whether lncRNA Glis2 could regulate podocyte apoptosis in DN and uncover the underlying mechanism. The apoptosis rate was detected by flow cytometry. Mitochondrial membrane potential (ΔΨM) was measured using JC-1 staining. Mitochondrial morphology was detected by MitoTracker Deep Red staining. Then, the histopathological and ultrastructure changes of renal tissues in diabetic mice were observed using periodic acid-Schiff (PAS) staining and transmission electron microscopy. We found that lncRNA Glis2 was significantly downregulated in high-glucose cultured podocytes and renal tissues of db/db mice. LncRNA Glis2 overexpression was found to alleviate podocyte mitochondrial dysfunction and apoptosis. The direct interaction between lncRNA Glis2 and miR-328-5p was confirmed by dual luciferase reporter assay. Furthermore, lncRNA Glis2 overexpression alleviated podocyte apoptosis in diabetic mice. Taken together, this study demonstrated that lncRNA Glis2, acting as a competing endogenous RNA (ceRNA) of miRNA-328-5p, regulated Sirt1-mediated mitochondrial dysfunction and podocyte apoptosis in DN.

CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Empagliflozin reduces the adverse effects of diabetes mellitus on testicular tissue in type 2 diabetic Rats: A stereological and biochemical study.

Empagliflozin as an antioxidant decreases blood glucose and insulin resistance in type 2 diabetes mellitus. Base on the empagliflozin antioxidant properties we decided to investigate the its effects on the testis histological changes through stereological techniques and biochemical evaluations in T2 diabetes mellitus rats. Rats were divided into: control, diabetes mellitus (DM, streptozotocin + nicotinamide) and diabetes mellitus + empagliflozin (DM + EMPA, 10 mg/kg/day) groups. 56 days after inducing diabetes mellitus testis histological changes and serum biochemical factors along with the level of Bax, Bcl2 and Nrf2 genes expression in the testicular tissue were assessed. A significant decrease in the mean total volume of testis and its components, the level of Bcl2 and Nrf2 gene expression (p < 0.001) along with a significant increase in the level of IL-6, TNF-α, MDA, Bax gene expression were observed in the DM group compared to the control group (p < 0.001). In the DM + EMPA group, the mean total volume of testis and its components, the level of Bcl2 gene expression (p< 0.01) and Nrf2 (p < 0.001) significantly increased whereas the mean level of IL-6 (p < 0.01), TNF-α (p < 0.001), MDA (p < 0.001), Bax (p < 0.001) gene expression significantly decreased compared to the DM group. Our results showed that empagliflozin, by improving the antioxidant defense system, can reduce testicular inflammation and apoptosis and partly prevent the adverse effects of diabetes mellitus on testicular tissue.

mm9_circ_014683 regulates microglia polarization through canonical NFκB signaling pathway in diabetic retinopathy.

Diabetic retinopathy (DR) is still the major cause of visual loss in working-aged people, one of the critical pathological processes are retinal microglia-mediated inflammation. Our previous study demonstrated that enhanced M1 microglial polarization was involved in retinal inflammation in DR, but the detailed mechanism needs further investigation. Circular RNAs (circRNAs) are important kind of noncoding RNAs involved in the regulation of various cell biological processes. Herein, the circRNA expression profiles of BV2 mouse microglia treated with or without glucose were detected, and a total of 347 differentially expressed circRNAs were identified in glucose-treated BV2 cells. The key circRNA mm9_circ_014683 increased after glucose stimulation. Inhibiting or overexpressing mm9_circ_014683 showed no effect on the proliferation and apoptosis of microglia. Inhibiting mm9_circ_014683 impeded M1 polarization and promoted M2 polarization, and overexpressing mm9_circ_014683 showed the opposite effect. A total of 216 differentially expressed genes were identified in mm9_circ_014683-knockdown BV2 cells, which were enriched in several signaling pathways, including the NFκB signaling pathway. Moreover, mm9_circ_014683 positively regulated the canonical, NFκB signaling pathway. Besides, mm9_circ_014683 was highly expressed in the retinal microglia of diabetic mice, and intraocular injection of Lv-circRNA inhibited M1 but enhanced M2 retinal microglial polarization. In conclusion, mm9_circ_014683 regulates microglial polarization through the canonical NFκB signaling pathway in diabetic retinopathy. This study may provide insight into the pathogenesis and treatment of DR.

VDR regulates mitochondrial function as a protective mechanism against renal tubular cell injury in diabetic rats.

PURPOSE: To investigate the regulatory effect and mechanism of Vitamin D receptor (VDR) on mitochondrial function in renal tubular epithelial cell under diabetic status. METHODS: The diabetic rats induced by streptozotocin (STZ) and HK-2 cells under high glocose(HG)/transforming growth factor beta (TGF-ß) stimulation were used in this study. Calcitriol was administered for 24 weeks. Renal tubulointerstitial injury and some parameters of mitochondrial function including mitophagy, mitochondrial fission, mitochondrial ROS, mitochondrial membrane potential (MMP), mitochondrial ATP, Complex V activity and mitochondria-associated ER membranes (MAMs) integrity were examined. Additionally, paricalcitol, 3-MA (an autophagy inhibitor), VDR over-expression plasmid, VDR siRNA and Mfn2 siRNA were applied in vitro. RESULTS: The expression of VDR, Pink1, Parkin, Fundc1, LC3II, Atg5, Mfn2, Mfn1 in renal tubular cell of diabetic rats were decreased significantly. Calcitriol treatment reduced the levels of urinary albumin, serum creatinine and attenuated renal tubulointerstitial fibrosis in STZ induced diabetic rats. In addition, VDR agonist relieved mitophagy dysfunction, MAMs integrity, and inhibited mitochondrial fission, mitochondrial ROS. Co-immunoprecipitation analysis demonstrated that VDR interacted directly with Mfn2. Mitochondrial function including mitophagy, mitochondrial membrane potential (MMP), mitochondrial Ca2+, mitochondrial ATP and Complex V activity were decreased dramatically in HK-2 cells under HG/TGF-ß ambience. In vitro pretreatment of HK-2 cells with autophagy inhibitor 3-MA, VDR siRNA or Mfn2 siRNA negated the activating effects of paricalcitol on mitochondrial function. Pricalcitol and VDR over-expression plasmid activated Mfn2 and then partially restored the MAMs integrity. Additionally, VDR restored mitophagy was partially associated with MAMs integrity through Fundc1. CONCLUSION: Activated VDR could contribute to restore mitophagy through Mfn2-MAMs-Fundc1 pathway in renal tubular cell. VDR could recover mitochondrial ATP, complex V activity and MAMs integrity, inhibit mitochondrial fission and mitochondrial ROS. It indicating that VDR agonists ameliorate renal tubulointerstitial fibrosis in diabetic rats partially via regulation of mitochondrial function.

Intervention treatment reducing cellular senescence inhibits tubulointerstitial fibrosis in diabetic mice following acute kidney injury.

Senescence of kidney tubules leads to tubulointerstitial fibrosis (TIF). Proximal tubular epithelial cells undergo stress-induced senescence during diabetes and episodes of acute kidney injury (AKI), and combining these injuries promotes the progression of diabetic kidney disease (DKD). Since TIF is crucial to progression of DKD, we examined the therapeutic potential of targeting senescence with a senolytic drug (HSP90 inhibitor) and/or a senostatic drug (ASK1 inhibitor) in a model of TIF in which AKI is superimposed on diabetes. After 8 weeks of streptozotocin-induced diabetes, mice underwent bilateral clamping of renal pedicles to induce mild AKI, followed by 28 days of reperfusion. Groups of mice (n=10-12) received either vehicle, HSP90 inhibitor (alvespimycin), ASK1 inhibitor (GS-444217), or both treatments. Vehicle-treated mice displayed tubular injury at day 3 and extensive tubular cell senescence at day 10, which remained unresolved at day 28. Markers of senescence (Cdkn1a and Cdkn2a), inflammation (Cd68, Tnf, and Ccl2), and TIF (Col1a1, Col4a3, α-Sma/Acta2, and Tgfb1) were elevated at day 28, coinciding with renal function impairment. Treatment with alvespimycin alone reduced kidney senescence and levels of Col1a1, Acta2, Tgfb1, and Cd68; however, further treatment with GS-444217 also reduced Col4a3, Tnf, Ccl2, and renal function impairment. Senolytic therapy can inhibit TIF during DKD, but its effectiveness can be improved by follow-up treatment with a senostatic inhibitor, which has important implications for treating progressive DKD.

Cross-Modal Imaging Reveals Nanoparticle Uptake Dynamics in Hematopoietic Bone Marrow during Inflammation.

Nanoparticles have been employed to elucidate the innate immune cell biology and trace cells accumulating at inflammation sites. Inflammation prompts innate immune cells, the initial responders, to undergo rapid turnover and replenishment within the hematopoietic bone marrow. Yet, we currently lack a precise understanding of how inflammation affects cellular nanoparticle uptake at the level of progenitors of innate immune cells in the hematopoietic marrow. To bridge this gap, we aimed to develop imaging tools to explore the uptake dynamics of fluorescently labeled cross-linked iron oxide nanoparticles in the bone marrow niche under varying degrees of inflammation. The inflammatory models included mice that received intramuscular lipopolysaccharide injections to induce moderate inflammation and streptozotocin-induced diabetic mice with additional intramuscular lipopolysaccharide injections to intensify inflammation. In vivo magnetic resonance imaging (MRI) and fluorescence imaging revealed an elevated level of nanoparticle uptake at the bone marrow as the levels of inflammation increased. The heightened uptake of nanoparticles within the inflamed marrow was attributed to enhanced permeability and retention with increased nanoparticle intake by hematopoietic progenitor cells. Moreover, intravital microscopy showed increased colocalization of nanoparticles within slowly patrolling monocytes in these inflamed hematopoietic marrow niches. Our discoveries unveil a previously unknown role of the inflamed hematopoietic marrow in enhanced storage and rapid deployment of nanoparticles, which can specifically target innate immune cells at their production site during inflammation. These insights underscore the critical function of the hematopoietic bone marrow in distributing iron nanoparticles to innate immune cells during inflammation. Our findings offer diagnostic and prognostic value, identifying the hematopoietic bone marrow as an imaging biomarker for early detection in inflammation imaging, advancing personalized clinical care.

HMGN1 down-regulation in the diabetic kidney attenuates tubular cells injury and protects against renal inflammation via suppressing MCP-1 and KIM-1 expression through TLR4.

BACKGROUND: Renal tubular injury, accompanied by damaging inflammation, has been identified to drive diabetic kidney disease (DKD) toward end-stage renal disease. However, it is unclear how damage-associated molecular patterns (DAMPs) activate innate immunity to mediate tubular epithelial cell (TEC) injury, which in turn causes with subsequent sterile inflammation in diabetic kidneys. High mobility group nucleosome-binding protein 1 (HMGN1) is a novel DAMP that contributes to generating the innate immune response. In this study, we focused on determining whether HMGN1 is involved in DKD progression. METHODS: Streptozotocin (STZ)-induced diabetic mice model was established. Then we downrergulated HMGN1 expression in kidney with or without HMGN1 administration. The renal dysfunction and morphological lesions in the kidneys were evaluated. The expressions of KIM-1, MCP-1, F4/80, CD68, and HMGN1/TLR4 signaling were examined in the renal tissue. In vitro, HK2 cells were exposed in the high glucose with or without HMGN1, and further pre-incubated with TAK242 was applied to elucidate the underlying mechanism. RESULTS: We demonstrated that HMGN1 was upregulated in the tubular epithelial cells of streptozotocin (STZ)-induced type 1 and type 2 diabetic mouse kidneys compared to controls, while being positively correlated with increased TLR4, KIM-1, and MCP-1. Down-regulation of renal HMGN1 attenuated diabetic kidney injury, decreased the TLR4, KIM-1, and MCP-1 expression levels, and reduced interstitial infiltrating macrophages. However, these phenotypes were reversed after administration of HMGN1. In HK-2 cells, HMGN1 promoted the expression of KIM-1 and MCP-1 via regulating MyD88/NF-κB pathway; inhibition of TLR4 effectively diminished the in vitro response to HMGN1. CONCLUSIONS: Our study provides novel insight into HMGN1 signaling mechanisms that contribute to tubular sterile injury and low-grade inflammation in DKD. The study findings may help to develop new HMGN1-targeted approaches as therapy for immune-mediated kidney damage rather than as an anti-infection treatments.

Morphological and biomechanical characterization of long bones and peri-implant bone repair in type 2 diabetic rats treated with resveratrol.

Type 2 diabetes interferes with bone remodeling mechanisms, requiring studies to reverse this damage, and resveratrol is a polyphenol with rich properties. This study aimed to characterize the long bone morphology and peri-implant biomechanics of normoglycemic and type 2 diabetic animals treated with resveratrol. Thirty-two male Wistar rats were used and divided into normoglycemic and diabetic with or without treatment. They had the installation of implants in the tibia and treatment with oral resveratrol within 45 days. Resveratrol was responsible for weight homeostasis and decreased glycemic levels in rats with type 2 diabetes. The three-point bending testing, resveratrol showed positive effects on the biomechanics of long bones, corroborating a more resistant bone in comparison to untreated diabetics. Micro-ct revealed how bone metabolism is affected by systemic disease, decreasing bone quality. The counter-torque of normoglycemic animals showed superior osseointegration to diabetes, with no differences in the administration of the polyphenol, showing the sovereignty of the deleterious effects of the disease when there is a tissue lesion and an inflammatory picture installed. Overall, resveratrol acted positively in the etiopathogenesis of type 2 diabetes and revealed positive effects on the strength of long bones.

Assessment of anti-hyperglycemic and anti-hyperlipidemic effects of thiazolidine-2,4-dione derivatives in HFD-STZ diabetic animal model.

Type 2 diabetes mellitus (T2DM) is a chronic endocrine/metabolic disorder characterized by elevated postprandial and fasting glycemic levels that result in disturbances in primary metabolism. In this study, we evaluated the metabolic effects of thiazolidine-2,4-dione derivatives in Wistar rats and Swiss mice that were fed a high-fat diet (HFD) for 4 weeks and received 90 mg/kg of streptozotocin (STZ) intraperitoneally as a T2DM model. The HFD consisted of 17% carbohydrate, 58% fat, and 25% protein, as a percentage of total kcal. The thiazolidine-2,4-dione derivatives treatments reduced fasting blood glucose (FBG) levels by an average of 23.98%-50.84%, which were also improved during the oral starch tolerance test (OSTT). Treatment with thiazolidine-2,4-dione derivatives also improved triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), and total cholesterol levels (P < 0.05). The treatment intake has also shown a significant effect to modulate the altered hepatic and renal biomarkers. Further treatment with thiazolidine-2,4-dione derivatives for 28 days significantly ameliorated changes in appearance and metabolic risk factors, including favorable changes in histopathology of the liver, kidney, and pancreas compared with the HFD/STZ-treated group, suggesting its potential role in the management of diabetes. Thiazolidine-2,4-dione derivatives are a class of drugs that act as insulin sensitizers by activating peroxisome proliferator-activated receptor-gamma (PPAR-γ), a nuclear receptor that regulates glucose and lipid metabolism. The results of this study suggest that thiazolidine-2,4-dione derivatives may be a promising treatment option for T2DM by improving glycemic control, lipid metabolism, and renal and hepatic function.

Fasting mimicking diet in diabetic mice partially preserves glomerular endothelial glycocalyx coverage, without changing the diabetic metabolic environment.

Intermittent fasting has become of interest for its possible metabolic benefits and reduction of inflammation and oxidative damage, all of which play a role in the pathophysiology of diabetic nephropathy. We tested in a streptozotocin (60 mg/kg)-induced diabetic apolipoprotein E knockout mouse model whether repeated fasting mimicking diet (FMD) prevents glomerular damage. Diabetic mice received 5 FMD cycles in 10 wk, and during cycles 1 and 5 caloric measurements were performed. After 10 wk, glomerular endothelial morphology was determined together with albuminuria, urinary heparanase-1 activity, and spatial mass spectrometry imaging to identify specific glomerular metabolic dysregulation. During FMD cycles, blood glucose levels dropped while a temporal metabolic switch was observed to increase fatty acid oxidation. Overall body weight at the end of the study was reduced together with albuminuria, although urine production was dramatically increased without affecting urinary heparanase-1 activity. Weight loss was found to be due to lean mass and water, not fat mass. Although capillary loop morphology and endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced together with the presence of UDP-glucuronic acid. Mass spectrometry imaging further revealed reduced protein catabolic breakdown products and increased oxidative stress, not different from diabetic mice. In conclusion, although FMD preserves partially glomerular endothelial glycocalyx, loss of lean mass and increased glomerular oxidative stress argue whether such diet regimes are safe in patients with diabetes.NEW & NOTEWORTHY Repeated fasting mimicking diet (FMD) partially prevents glomerular damage in a diabetic mouse model; however, although endothelial glycocalyx heparan sulfate contents were preserved, hyaluronan surface expression was reduced in the presence of UDP-glucuronic acid. The weight loss observed was of lean mass, not fat mass, and increased glomerular oxidative stress argue whether such a diet is safe in patients with diabetes.

Effects of conditioned media derived from human Wharton's jelly mesenchymal stem cells on diabetic nephropathy and hepatopathy via modulating TGF-ß and apelin signaling pathways in male rats.

BACKGROUND: Diabetic nephropathy and hepatopathy are health problems described by specific renal and hepatic structure and function disturbances. The protective effects of the stem cell secretome have been shown in several kidney and liver diseases. The current study aims to evaluate the capability of conditioned media derived from human Wharton's jelly mesenchymal stem cells (hWJ-MSCs-CM) to alleviate diabetic complications. METHODS: Twenty Sprague Dawley rats were made diabetic through injection of STZ (60 mg/kg, i.p.). At week 8, diabetic rats were divided into two groups: treated [DM + hWJ-MSCs-CM (500 µl/rat for three weeks, i.p.)] and not treated (DM). At the 11th week, three groups (control, DM, and DM + hWJ-MSCs-CM) were kept in metabolic cages, and urine was collected for 24 h. The serum samples were maintained for measuring fasting blood glucose (FBG) and kidney and liver functional analysis. The left kidney and liver parts were kept at -80 °C to assess apelin and transforming growth factor-beta (TGF-ß) expression. The right kidney, pancreas, and liver parts were used for histopathologic evaluation. RESULTS: DM was detected by higher FBG, microalbuminuria, increased albumin/creatinine ratio, and pancreas, renal, and hepatic structural disturbances. Diabetic hepatopathy was determined by increasing liver enzymes and decreasing total bilirubin. The TGF-ß gene expression was significantly upregulated in the diabetic kidney and liver tissues. Apelin gene expression was significantly downregulated in the diabetic liver tissue but did not change in kidney tissue. Administration of hWJ-MSCs-CM improved renal and hepatic functional and structural disturbances. Moreover, CM therapy significantly decreased TGF-ß expression and enhanced apelin expression in the kidney and liver tissues. CONCLUSION: Human WJ-MSCs-CM may have protective effects on diabetic renal and hepatic complications. These effects may happen through the regulation of TGF-ß and apelin signaling pathways.

Transthyretin-Regulated Diabetic Retinopathy Through the VEGFA/PI3K/AKT Pathway.

Purpose: Transthyretin (TTR) plays a regulatory role in a variety of diabetes-related diseases. The objective of this work was to probe whether TTR affects diabetic retinopathy (DR) through the VEGFA/PI3K/AKT pathway. Methods: High glucose (HG, 25 mM) was used to treat human retinal microvascular endothelial cells (hRMECs) and C57BL/6J mice were intraperitoneally injected with STZ (50 mg/kg) to construct a DR model. In vitro, the effect of TTR on DR was evaluated by measuring hRMEC proliferation, migration, and angiogenesis. The changes in retinal tissue were observed by hematoxylin and eosin staining in vivo. ELISA, immunohistochemistry, and immunofluorescence staining were used to measure VEGFA or CD31 levels. The levels of all proteins were evaluated through Western blot. Results: The increase of proliferation, migration, and angiogenesis and decrease of apoptosis in hRMECs caused by HG were notably reversed by TTR. TTR greatly impeded HG-raised VEGFA, PI3K p-p85, and p-AKT in hRMECs. Inhibition of TTR further exacerbated the effect of HG-induced hRMECs. Inhibition of VEGFA reversed the effect of HG-induced hRMECs. VEGFA neutralized the function of TTR on cell proliferation, apoptosis, migration, and angiogenesis in HG-triggered hRMECs. It was further confirmed in vivo that TTR can alleviate the occurrence of DR in diabetic mice models. Conclusions: TTR significantly restrained the progression of DR via molecular modulation of the VEGFA/PI3K/AKT axis.

TRIM25 inhibition attenuates inflammation, senescence, and oxidative stress in microvascular endothelial cells induced by hyperglycemia.

PURPOSES: This work aimed to assess the possible role of TRIM25 in regulating hyperglycemia-induced inflammation, senescence, and oxidative stress in retinal microvascular endothelial cells, all of which exert critical roles in the pathological process of diabetic retinopathy. METHODS: The effects of TRIM25 were investigated using streptozotocin-induced diabetic mice, human primary retinal microvascular endothelial cells cultured in high glucose, and adenoviruses for TRIM25 knockdown and overexpression. TRIM25 expression was evaluated by western blot and immunofluorescence staining. Inflammatory cytokines were detected by western blot and quantitative real-time PCR. Cellular senescence level was assessed by detecting senescent marker p21 and senescence-associated-ß-galactosidase activity. The oxidative stress state was accessed by detecting reactive oxygen species and mitochondrial superoxide dismutase. RESULTS: TRIM25 expression is elevated in the endothelial cells of the retinal fibrovascular membrane from diabetic patients compared with that of the macular epiretinal membrane from non-diabetic patients. Moreover, we have also observed a significant increase in TRIM25 expression in diabetic mouse retina and retinal microvascular endothelial cells under hyperglycemia. TRIM25 knockdown suppressed hyperglycemia-induced inflammation, senescence, and oxidative stress in human primary retinal microvascular endothelial cells while TRIM25 overexpression further aggregates those injuries. Further investigation revealed that TRIM25 promoted the inflammatory responses mediated by the TNF-α/NF-κB pathway and TRIM25 knockdown improved cellular senescence by increasing SIRT3. However, TRIM25 knockdown alleviated the oxidative stress independent of both SIRT3 and mitochondrial biogenesis. CONCLUSION: Our study proposed TRIM25 as a potential therapeutic target for the protection of microvascular function during the progression of diabetic retinopathy.

GMFB/AKT/TGF-ß3 in Müller cells mediated early retinal degeneration in a streptozotocin-induced rat diabetes model.

Retinal degeneration, characterized by Müller cell gliosis and photoreceptor apoptosis, is considered an early event in diabetic retinopathy (DR). Our previous study proposed that GMFB may mediate diabetic retinal degeneration. This study identified GMFB as a sensitive and functional gliosis marker for DR. Compared to the wild type (WT) group, Gmfb knockout (KO) significantly improved visual function, attenuated gliosis, reduced the apoptosis of neurons, and decreased the mRNA levels of tumor necrosis factor α (Tnf-α) and interleukin-1ß (Il-1ß) in diabetic retinas. Tgf-ß3 was enriched by hub genes using RNA sequencing in primary WT and KO Müller cells. Gmfb KO significantly upregulated the transforming growth factor (TGF)-ß3 protein level via the AKT pathway. The protective effect of TGF-ß3 in the vitreous resulted in significantly improved visual function and decreased the number of apoptotic cells in the diabetic retina. The protection of Gmfb KO in primary Müller cells against high glucose (HG)-induced photoreceptor apoptosis was partially counteracted by TGF-ß3 antibody and administration of TGFBR1/2 inhibitors. Nuclear receptor subfamily 3 group C member 1 (NR3C1) binds to the promoter region of Gmfb and regulates Gmfb mRNA at the transcriptional level. NR3C1 was increased in the retinas of early diabetic rats but decreased in the retinas of late diabetic rats. N'-[(1E)-(3-Methoxyphenyl)Methylene]-3-Methyl-1H-Pyrazole-5-Carbohydrazide (DS-5) was identified as an inhibitor of GMFB, having a protective role in DR. We demonstrated that GMFB/AKT/TGF-ß3 mediated early diabetic retinal degeneration in diabetic rats. This study provides a novel therapeutic strategy for treating retinal degeneration in patients with DR.

Stem cell therapy with CRISPR/Cas9-mediated MALAT1 delivery modulates miR-142 and rescues wound healing in rats with age-associated diabetic foot ulcers.

BACKGROUND: Diabetic foot ulcer (DFU) is a serious diabetes complication, significantly impacting the quality of life, particularly in the elderly. Age-associated DFUs pose additional challenges due to impaired healing mechanisms. Our study aims to explore the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a miR-142 sponge in repairing diabetic rat foot ulcer tissue under age-associated diabetes, offering a new theoretical basis and therapeutic target for preventing and treating diabetic vascular disease in the elderly. METHODS: Using qPCR, we analyzed MALAT1 and miR-142 expression in EPCs and hUC-MSCs. Targetscan predicted potential interaction targets for MALAT1 and miR-142, confirmed by dual luciferase reporter gene assay. An age-associated diabetic rat model was established using Streptozotocin (STZ) injection. Hypoxia, apoptosis, and angiogenesis-related proteins were assessed through Western Blot. In vitro, miR-142 inhibition and MALAT1 overexpression promoted foot ulcer healing in diabetic rats. RESULTS: MALAT1 acted as a miR-142 sponge, downregulated in hUC-MSCs under high glucose, relevant to age-associated diabetic foot ulcers. MiR-142 negatively regulated SIRT1 and Nrf2. In vitro experiments demonstrated potential significance for age-related DFU treatment. CONCLUSIONS: MALAT1 in human umbilical cord mesenchymal stem cells expedited foot ulcer healing in diabetic rats, particularly in age-associated diabetes, through miR-142 sponge activity. These findings offer insights for novel therapeutic strategies targeting elderly diabetic foot ulcers, emphasizing exogenous stem cell transplantation's potential in effective DFU treatment for the elderly.

Ferroptosis in the ageing retina: A malevolent fire of diabetic retinopathy.

Ageing retina is prone to ferroptosis due to the iron accumulation and impaired efficiency of intracellular antioxidant defense system. Ferroptosis acts as a cell death modality that is characterized by the iron-dependent accumulation of lipid peroxidation. Ferroptosis is distinctively different from other types of regulated cell death (RCD) at the morphological, biochemical, and genetic levels. Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Its prevalence and severity increase progressively with age. Recent reports have shown that ferroptosis is implicated in the pathophysiology of DR. Under hyperglycemia condition, the endothelial cell and retinal pigment epithelium (RPE) cell will undergo ferroptosis, which contributes to the increased vascular permeability and the disrupted blood retinal barrier (BRB). The underlying etiology of DR can be attributed to the impaired BRB integrity and subsequent damages of the neurovascular units. In the absence of timely intervention, the compromised BRB can ultimately cause profound visual impairments. In particular, the ageing retina is vulnerable to ferroptosis, and hyperglycemia will accelerate the progression of this pathological process. In this article, we discuss the contributory role of ferroptosis in DR pathogenesis, and summarize recent therapeutic trials that targeting the ferroptosis. Further study on the ferroptosis mediated damage would enrich our knowledge of DR pathology, and promote the development of clinical treatment for this degenerative retinopathy.

Genetic lineage tracing identifies adaptive mechanisms of pancreatic islet ß cells in various mouse models of diabetes with distinct age of initiation.

During the pathogenesis of type 1 diabetes (T1D) and type 2 diabetes (T2D), pancreatic islets, especially the ß cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet (HFD) and streptozotocin (STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of islet ß cells. Cre-LoxP systems were used to generate islet cell type-specific (α, ß, or δ) green fluorescent protein (GFP)-labeled mice for genetic lineage tracing, thereinto ß-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing (scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled ß cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of ß cells and some of which transdifferentiated into a or δ cells in both youth- and adulthood-initiated mice while this phenomenon was barely observed in HFD models. ß cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into ß cells in STZ-treated mice (both youth- and adulthood-initiated). In addition to the re-dedifferentiation of ß cells, it is also highly likely that these "α or δ" cells transdifferentiated from pre-existing ß cells could also re-trans-differentiate into insulin-producing ß cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of ß cells. Our findings shed light on how islet ß cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis.

IFN-γ enhances the therapeutic efficacy of MSCs-derived exosome via miR-126-3p in diabetic wound healing by targeting SPRED1.

BACKGROUND AND AIMS: The traditional treatment of diabetic wounds is unsatisfactory. Exosomes isolated from bone marrow mesenchymal stem cells (BMSCs) promote the healing of diabetic wounds. However, whether the exosomes secreted by interferon (IFN)-γ-pretreated BMSCs have an enhanced therapeutic effect on diabetic wound healing and the relevant mechanisms remain unclear. METHODS: In this study, we isolated exosomes from the corresponding supernatants of BMSCs with (IExos) or without IFN-γ treatment (NExos). Human umbilical vein endothelial cells (HUVECs) were used to investigate the proliferation, migration, and tube formation under different treatments in vitro. Diabetic mice were induced by intraperitoneal administration of streptozotocin, and a circular full-thickness dermal defect was then made on the back of each mouse, followed by a multisite subcutaneous injection of phosphate buffered saline or exosomes. Hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and histological analysis were performed to assess the speed and quality of wound healing. RESULTS: NExos treatment accelerated the healing of diabetic wounds by promoting angiogenesis in vivo and in vitro, and IExos exhibited superior therapeutic efficiency. MicroRNA (miR)-126-3p was significantly increased in IExos, and exosomal miR-126-3p promoted angiogenesis and diabetic wound healing via its transfer to HUVECs. miR-126-3p regulates SPRED1 by directly targeting the 3'-UTR. Mechanistically, IFN-γ-pretreated BMSCs secreted miR-126-3p-enriched exosomes, which enhanced the function of HUVECs and promoted angiogenesis via the SPRED1/Ras/Erk pathway. CONCLUSION: Exosomal miR-126-3p secreted from IFN-γ-pretreated BMSCs exhibited higher therapeutic efficacy than NExos in diabetic wound healing by promoting angiogenesis via the SPRED1/Ras/Erk axis.