Growth differentiation factor 11 regulates high glucose-induced cardiomyocyte pyroptosis and diabetic cardiomyopathy by inhibiting inflammasome activation.

BACKGROUND: Diabetic cardiomyopathy (DCM) is a crucial complication of long-term chronic diabetes that can lead to myocardial hypertrophy, myocardial fibrosis, and heart failure. There is increasing evidence that DCM is associated with pyroptosis, a form of inflammation-related programmed cell death. Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor ß superfamily, which regulates oxidative stress, inflammation, and cell survival to mitigate myocardial hypertrophy, myocardial infarction, and vascular injury. However, the role of GDF11 in regulating pyroptosis in DCM remains to be elucidated. This research aims to investigate the role of GDF11 in regulating pyroptosis in DCM and the related mechanism. METHODS AND RESULTS: Mice were injected with streptozotocin (STZ) to induce a diabetes model. H9c2 cardiomyocytes were cultured in high glucose (50 mM) to establish an in vitro model of diabetes. C57BL/6J mice were preinjected with adeno-associated virus 9 (AAV9) intravenously via the tail vein to specifically overexpress myocardial GDF11. GDF11 attenuated pyroptosis in H9c2 cardiomyocytes after high-glucose treatment. In diabetic mice, GDF11 alleviated cardiomyocyte pyroptosis, reduced myocardial fibrosis, and improved cardiac function. Mechanistically, GDF11 inhibited pyroptosis by preventing inflammasome activation. GDF11 achieved this by specifically binding to apoptosis-associated speck-like protein containing a CARD (ASC) and preventing the assembly and activation of the inflammasome. Additionally, the expression of GDF11 during pyroptosis was regulated by peroxisome proliferator-activated receptor α (PPARα). CONCLUSION: These findings demonstrate that GDF11 can treat diabetic cardiomyopathy by alleviating pyroptosis and reveal the role of the PPARα-GDF11-ASC pathway in DCM, providing ideas for new strategies for cardioprotection.

Apigenin suppresses epithelial-mesenchymal transition in high glucose-induced retinal pigment epithelial cell by inhibiting CBP/p300-mediated histone acetylation.

Epithelial mesenchymal transition (EMT) is a critical process implicated in the pathogenesis of retinal fibrosis and the exacerbation of diabetic retinopathy (DR) within retinal pigment epithelium (RPE) cells. Apigenin (AP), a potential dietary supplement for managing diabetes and its associated complications, has demonstrated inhibitory effects on EMT in various diseases. However, the specific impact and underlying mechanisms of AP on EMT in RPE cells remain poorly understood. In this study, we have successfully validated the inhibitory effects of AP on high glucose-induced EMT in ARPE-19 cells and diabetic db/db mice. Notably, our findings have identified CBP/p300 as a potential therapeutic target for EMT in RPE cells and have further substantiated that AP effectively downregulates the expression of EMT-related genes by attenuating the activity of CBP/p300, consequently reducing histone acetylation alterations within the promoter region of these genes. Taken together, our results provide novel evidence supporting the inhibitory effect of AP on EMT in RPE cells, and highlight the potential of specifically targeting CBP/p300 as a strategy for inhibiting retinal fibrosis in the context of DR.

Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

BACKGROUND: Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. METHODS AND RESULTS: In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-ß, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-ß protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. CONCLUSIONS: In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

Role of Hsa_circ_0000880 in the Regulation of High Glucose-Induced Apoptosis of Retinal Microvascular Endothelial Cells.

Purpose: Circular RNAs (circRNAs) have been verified to participate in multiple biological processes and disease progression. Yet, the role of circRNAs in the pathogenesis of diabetic retinopathy (DR) is still poorly understood and deserves further study. This study aimed to investigate the role of circRNAs in the regulation of high glucose (HG)-induced apoptosis of retinal microvascular endothelial cells (RMECs). Methods: Epiretinal membranes from patients with DR and nondiabetic patients with idiopathic macular epiretinal membrane were collected for this study. The circRNA microarrays were performed using high-throughput sequencing. Hierarchical clustering, functional enrichment, and network regulation analyses were used to analyze the data generated by high-throughput sequencing. Next, RMECs were subjected to HG (25 mM) conditions to induce RMECs apoptosis in vitro. A series of experiments, such as Transwell, the Scratch wound, and tube formation, were conducted to explore the regulatory effect of circRNA on RMECs. Fluorescence in situ hybridization (FISH), immunofluorescence staining, and Western blot were used to study the mechanism underlying circRNA-mediated regulation. Results: A total of 53 differentially expressed circRNAs were found in patients with DR. Among these, hsa_circ_0000880 was significantly upregulated in both the diabetic epiretinal membranes and in an in vitro DR model of HG-treated RMECs. Hsa_circ_0000880 knockout facilitated RMECs vitality and decreased the paracellular permeability of RMECs under hyperglycemia. More importantly, silencing of hsa_circ_0000880 significantly inhibited HG-induced ROS production and RMECs apoptosis. Hsa_circ_0000880 acted as an endogenous sponge for eukaryotic initiation factor 4A-III (EIF4A3). Knockout of hsa_circ_0000880 reversed HG-induced decrease in EIF4A3 protein level. Conclusions: Our findings suggest that hsa_circ_0000880 is a novel circRNA can induce RMECs apoptosis in response to HG conditions by sponging EIF4A3, offering an innovative treatment approach against DR. Translational Relevance: The circRNAs participate in the dysregulation of microvascular endothelial function induced by HG conditions, indicating a promising therapeutic target for DR.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium.

BACKGROUND: Histone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium. METHODS: We studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2-) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice. RESULTS: Incubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2- generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice. CONCLUSIONS: EZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Curcumin prevents high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation via mitigating intracellular free radicals and TGF-ß secretion.

Diabetic kidney disease (DKD) is a leading cause of kidney failure. However, the involvement of renal fibroblasts and their communications with renal epithelial cells during DKD remain poorly understood. We investigated the potential role of renal proximal tubular epithelial cells (PTECs) in renal fibroblast activation that might lead to DKD. Additionally, the protective effects of curcumin, a known antioxidant, against renal fibroblast activation induced by high glucose-treated PTECs were investigated. Secretome was collected from HK-2 PTECs under normal glucose, high glucose, high glucose pretreated/cotreated with curcumin, or osmotic control condition for 24 h. Such secretome was then used to treat BHK-21 renal fibroblasts for 24 h. BHK-21 cells treated with high glucose-induced secretome had increased levels of fibroblast activation markers, including spindle index, F-actin, α-smooth muscle actin (α-SMA), fibronectin, collagen I, matrix metalloproteinase-2 (MMP-2) and MMP-9, as compared with normal glucose and osmotic control conditions. However, all these increases were successfully mitigated by curcumin. In addition, high glucose markedly increased intracellular reactive oxygen species (ROS) and transforming growth factor-ß (TGF-ß) secretion, but did not affect the secretion of platelet-derived growth factor A (PDGFA) and interleukin-1ß (IL-1ß), in HK-2 renal cells as compared with normal glucose and osmotic control conditions. Both intracellular ROS and secreted TGF-ß levels were successfully mitigated by curcumin. Therefore, curcumin prevents the high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation, at least in part, via mitigating intracellular ROS and TGF-ß secretion.

Beneficial Effects of Fibroblast Growth Factor-1 on Retinal Pigment Epithelial Cells Exposed to High Glucose-Induced Damage: Alleviation of Oxidative Stress, Endoplasmic Reticulum Stress, and Enhancement of Autophagy.

Diabetic retinopathy (DR) severely affects vision in individuals with diabetes. High glucose (HG) induces oxidative stress in retinal cells, a key contributor to DR development. Previous studies suggest that fibroblast growth factor-1 (FGF-1) can mitigate hyperglycemia and protect tissues from HG-induced damage. However, the specific effects and mechanisms of FGF-1 on DR remain unclear. In our study, FGF-1-pretreated adult retinal pigment epithelial (ARPE)-19 cells were employed to investigate. Results indicate that FGF-1 significantly attenuated HG-induced oxidative stress, including reactive oxygen species, DNA damage, protein carbonyl content, and lipid peroxidation. FGF-1 also modulated the expression of oxidative and antioxidative enzymes. Mechanistic investigations showed that HG induced high endoplasmic reticulum (ER) stress and upregulated specific proteins associated with apoptosis. FGF-1 effectively alleviated ER stress, reduced apoptosis, and restored autophagy through the adenosine monophosphate-activated protein kinase/mammalian target of the rapamycin signaling pathway. We observed that the changes induced by HG were dose-dependently reversed by FGF-1. Higher concentrations of FGF-1 (5 and 10 ng/mL) exhibited increased effectiveness in mitigating HG-induced damage, reaching statistical significance (p < 0.05). In conclusion, our study underscores the promising potential of FGF-1 as a safeguard against DR. FGF-1 emerges as a formidable intervention, attenuating oxidative stress, ER stress, and apoptosis, while concurrently promoting autophagy. This multifaceted impact positions FGF-1 as a compelling candidate for alleviating retinal cell damage in the complex pathogenesis of DR.

Sirt3 Protects Retinal Pigment Epithelial Cells From High Glucose-Induced Injury by Promoting Mitophagy Through the AMPK/mTOR/ULK1 Pathway.

Purpose: The regulation of mitophagy by Sirt3 has rarely been studied in ocular diseases. In the present study, we determined the effects of Sirt3 on AMPK/mTOR/ULK1 signaling pathway-mediated mitophagy in retinal pigment epithelial (RPE) cells in a high glucose environment. Methods: The mRNA expression levels of Sirt3, AMPK, mTOR, ULK1, and LC3B in RPE cells under varying glucose conditions were measured by real-time polymerase chain reaction (RT-PCR). The expressions of Sirt3, mitophagy protein, and AMPK/mTOR/ULK1 signaling pathway-related proteins were detected by Western blotting. Lentivirus (LV) transfection mediated the stable overexpression of Sirt3 in cell lines. The experimental groups were NG (5.5 mM glucose), hypertonic, HG (30 mM glucose), HG + LV-GFP, and HG + LV-Sirt3. Western blotting was performed to detect the expressions of mitophagy proteins and AMPK/mTOR/ULK1-related proteins in a high glucose environment during the overexpression of Sirt3. Reactive oxygen species (ROS) production in a high glucose environment was measured by DCFH-DA staining. Mitophagy was detected by labeling mitochondria and lysosomes with MitoTracker and LysoTracker probes, respectively. Apoptosis was detected by flow cytometry. Results: Sirt3 expression was reduced in the high glucose group, inhibiting the AMPK/mTOR/ULK1 pathway, with diminished mitophagy and increased intracellular ROS production. The overexpression of Sirt3, increased expression of p-AMPK/AMPK and p-ULK1/ULK1, and decreased expression of p-mTOR/mTOR inhibited cell apoptosis and enhanced mitophagy. Conclusions: Sirt3 protected RPE cells from high glucose-induced injury by activating the AMPK/mTOR/ULK1 signaling pathway. Translational Relevance: By identifying new targets of action, we aimed to establish effective therapeutic targets for diabetic retinopathy treatment.

Kinase PIM1 governs ferroptosis to reduce retinal microvascular endothelial cell dysfunction triggered by high glucose.

Previous studies have implicated targeting Pim-1 proto-oncogene, serine/threonine kinase (PIM1) as a preventive measure against high glucose-induced cellular stress and apoptosis. This study aimed to reveal the potential role and regulatory mechanism of PIM1 in diabetic retinopathy. Human retinal microvascular endothelial cells (hRMECs) underwent high glucose induction, and fluctuations in PIM1 levels were assessed. By overexpressing PIM1, its effects on the levels of inflammatory factors, oxidative stress indicators, migration and tube formation abilities, tight junction protein expression levels, and ferroptosis in hRMECs were identified. Afterwards, hRMECs were treated with the ferroptosis-inducing agent erastin, and the effect of erastin on the above PIM1 regulatory functions was focused on. PIM1 was downregulated upon high glucose, and its overexpression inhibited the inflammatory response, oxidative stress, cell migration, and tube formation potential in hRMECs, whereas elevated tight junction protein levels. Furthermore, PIM1 overexpression reduced intracellular iron ion levels, lipid peroxidation, and levels of proteins actively involved in ferroptosis. Erastin treatment reversed the impacts of PIM1 on hRMECs, suggesting the mediation of ferroptosis in PIM1 regulation. The current study has yielded critical insights into the role of PIM1 in ameliorating high glucose-induced hRMEC dysfunction through the inhibition of ferroptosis.

Effect of Brassica rapa var. rapifera on Insulin Resistance and Inflammatory Analysis of Visceral Adipose Tissue of Obese Wistar Rats Under Glucolipotoxicity Condition / Efecto de Brassica rapa var. rapifera sobre la Resistencia a la Insulina y el Análisis Inflamatorio del Tejido Adiposo Visceral de Ratas Wistar Obesas en Condiciones de Glucolipotoxicidad

SUMMARY: Obesity-related pathophysiologies such as insulin resistance and the metabolic syndrome show a markedly increased risk for type 2 diabetes and atherosclerotic cardiovascular disease. This risk appears to be linked to alterations in adipose tissue function, leading to chronic inflammation and the dysregulation of adipocyte-derived factors. Brassica rapa have been used in traditional medicine for the treatment of several diseases, including diabetes. This study aimed to investigate the effect of nutritional stress induced by a high-fat and high-sucrose diet on the pathophysiology of visceral adipose tissue and the therapeutic effect of Brassica rapa in male Wistar rats. We subjected experimental rats to a high-fat (10 %) high-sucrose (20 %)/per day for 11 months and treated them for 20 days with aqueous extract Br (AEBr) at 200 mg/kg at the end of the experiment. At the time of sacrifice, we monitored plasma and tissue biochemical parameters as well as the morpho-histopathology of visceral adipose tissue. We found AEBr corrected metabolic parameters and inflammatory markers in homogenized visceral adipose tissue and reduced hypertrophy, hyperplasia, and lipid droplets. These results suggest that AEBr enhances anti-diabetic, anti-inflammatory and a protective effect on adipose tissue morphology in type 2 diabetes and obesity.

La fisiopatología relacionadas con la obesidad, como la resistencia a la insulina y el síndrome metabólico, muestran un riesgo notablemente mayor de diabetes tipo 2 y enfermedad cardiovascular aterosclerótica. Este riesgo parece estar relacionado con alteraciones en la función del tejido adiposo, lo que lleva a una inflamación crónica y a la desregulación de los factores derivados de los adipocitos. Brassica rapa se ha utilizado en la medicina tradicional para el tratamiento de varias enfermedades, incluida la diabetes. Este estudio tuvo como objetivo investigar el efecto del estrés nutricional inducido por una dieta rica en grasas y sacarosa sobre la fisiopatología del tejido adiposo visceral y el efecto terapéutico de Brassica rapa en ratas Wistar macho. Sometimos a ratas experimentales a una dieta rica en grasas (10 %) y alta en sacarosa (20 %)/por día durante 11 meses y las tratamos durante 20 días con extracto acuoso de Br (AEBr) a 200 mg/kg al final del experimento. En el momento del sacrificio, monitoreamos los parámetros bioquímicos plasmáticos y tisulares, así como la morfohistopatología del tejido adiposo visceral. Encontramos parámetros metabólicos corregidos por AEBr y marcadores inflamatorios en tejido adiposo visceral homogeneizado y reducción de hipertrofia, hiperplasia y gotitas de lípidos. Estos resultados sugieren que AEBr mejora el efecto antidiabético, antiinflamatorio y protector sobre la morfología del tejido adiposo en la diabetes tipo 2 y la obesidad.

A novel lncRNA TCONS_00071187 upregulated by activated GSK3ß promotes high glucose-induced mesangial cell proliferation in the diabetic nephropathy.

Inhibiting mesangial cell proliferation is one of the strategies to control the early progression of diabetic nephropathy (DN). GSK3ß is closely related to cell apoptosis as well as the development of DN, but whether it acts on the proliferation of mesangial cells is unclear. This study aimed to elucidate the role and mechanism of GSK3ß-mediated lncRNA in high glucose-induced mesangial cell proliferation. HBZY-1 cells were used to establish the cell model of DN. The automatic cell counter was applied to assess cell proliferation. Flow cytometry was used to detect cell apoptosis and intracellular ROS levels. High-throughput transcriptomics sequencing was performed to detect the different expressions of long noncoding RNAs (lncRNAs) in the cell model of DN after knocking down the expression of GSK3ß by the transfection of siRNA. The expression of RNA was detected by real-time PCR. In the cell model of DN using HBZY-1 cells, cell proliferation was enhanced accompanied by GSK3ß activation and elevated apoptosis rate and reactive oxygen species (ROS) levels. A panel of novel lncRNAs, which were differentially expressed after GSK3ß knockdown in the cell model of DN, were identified by high-throughput transcriptomics sequencing. Among them, the expression of TCONS_00071187 was upregulated under high glucose conditions while the knockdown of the GSK3ß expression led to the downregulation of TCONS_00071187. The knockdown of TCONS_00071187 resulted in reduced mesangial cell proliferation, and decreased apoptosis rates and ROS levels. In conclusion, GSK3ß promoted mesangial cell proliferation by upregulating TCONS_00071187, which led to enhanced ROS production under high glucose conditions in the cell model of DN. This study revealed the role of GSK3ß medicated lncRNAs in the development of DN.

Salvianolic Acid B Alleviates High Glucose-Induced Vascular Smooth Muscle Cell Inflammation by Upregulating the miR-486a-5p Expression.

The macrovascular complications of diabetes cause high mortality and disability in patients with type 2 diabetes mellitus (T2DM). The inflammatory response of vascular smooth muscle cell (VSMC) runs through its pathophysiological process. Salvianolic acid B (Sal B) exhibits beneficial effects on the cardiovascular system. However, its role and mechanism in diabetic vascular inflammatory response remain unclear. In this study, we found that Sal B reduced vascular inflammation in diabetic mice and high glucose- (HG-) induced VSMC inflammation. Subsequently, we found that Sal B reduced HG-induced VSMC inflammation by downregulating FOXO1. Furthermore, miR-486a-5p expression was obviously reduced in HG-treated VSMC. Sal B attenuated HG-induced VSMC inflammation by upregulating miR-486a-5p. Loss- and gain-of-function experiments had proven that the transfection of the miR-486a-5p mimic inhibited HG-induced VSMC inflammation whereas that of the miR-486a-5p inhibitor promoted HG-induced VSMC inflammation, thereby leading to the amelioration of vascular inflammation in the diabetic mice. Furthermore, studies had shown that miR-486a-5p inhibited FOXO1 expression by directly targeting its 3'-UTR. In conclusion, Sal B alleviates the inflammatory response of VSMC by upregulating miR-486a-5p and aggravating its inhibition of FOXO1 expression. Sal B exerts a significant anti-inflammatory effect in HG-induced VSMC inflammation by modulating the miR-486a-5p/FOXO1 axis.

Silencing of METTL3 prevents the proliferation, migration, epithelial-mesenchymal transition, and renal fibrosis of high glucose-induced HK2 cells by mediating WISP1 in m6A-dependent manner.

Diabetic nephropathy (DN) is one of the most serious complications in diabetic patients. And m6A modifications mediated by METTL3 are involved multiple biological processes. However, the specific function and mechanism of METTL3 in DN remains unclear. DN model mice were first established with streptozotocin, and WISP1 expression was confirmed by qRT-PCR. Then the influences of WISP1 or/and METTL3 on the proliferation, migration, and epithelial-mesenchymal transition (EMT) and fibrosis-related proteins of high glucose (HG)-induced HK2 cells or HK2 cells were tested through CCK-8, wound healing, and western blot. We first revealed that WISP1 was highly expressed in renal tissues of DN model mice and HG-induced HK2 cells. Functionally, WISP1 or METTL3 silencing could weaken the proliferation, migration, EMT, and fibrosis of HG-treated HK2 cells, and WISP1 or METTL3 overexpression could induce the proliferation, migration, EMT, and fibrosis of HK2 cells. Additionally, METTL3 silencing could decrease WISP1 m6A modification, and silencing of METTL3 also could notably suppress the biological functions of HG-induced HK2 cells by downregulating WISP1. Silencing of METTL3 prevents DN development process by decreasing WISP1 with m6A modification pattern. Therefore, we suggest that METTL3/WISP1 axis might be a novel therapeutic target for DN.

Metformin inhibits high glucose-induced apoptosis of renal podocyte through regulating miR-34a/SIRT1 axis.

BACKGROUND: Previous studies have reported SIRT1 was inversely modulated by miR-34a, However, mechanism of metformin (MFN)'s renal podocyte protection under high glucose (HG) conditions and the connection between miR-34a and SIRT1 expression in diabetic nephropathy (DN) remain unclear. METHOD: We aimed to further elucidate the role of miR-34a in HG-treated podocytes in DN. A conditionally immortalized human podocyte cell line was cultivated in d-glucose (30 mM). RESULTS: Microarray and RT-qPCR revealed that miR-34a was downregulated in HG-treated podocytes. Additionally, miR-34a levels increased in MFN-treated HG-induced podocytes. CCK-8 assay, colony formation assay, flow cytometry, and Western blot detection showed that HG treatment reduced cell viability and promoted via HG treatment, and MFN treatment reversed this phenotypic change. MiR-34a upregulation caused restored cell viability and suppressed cell apoptosis in HG-treated podocytes, and miR-34a downregulation led to damaged cell survival and induced apoptosis in MFN-administered and HG-treated podocytes. The dual luciferase reporter assay showed that SIRT1 3'-UTR was a direct miR-34a target. Further studies demonstrated an elevation in SIRT1 levels in HG-exposed podocytes, whereas MFN treatment decreased SIRT1 levels. In addition, miR-34a upregulation led to reduced SIRT1 expression, whereas miR-34a inhibition increased SIRT1 levels in cells. MFN-induced miR-34a suppresses podocyte apoptosis under HG conditions by acting on SIRT1. CONCLUSION: This study proposes a promising approach to interpret the mechanisms of action of the MFN-miR-34a axis involved in DN.

SB431542 partially inhibits high glucose-induced EMT by restoring mitochondrial homeostasis in RPE cells.

BACKGROUND: The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells participated in the development of retinal fibrosis. SB431542 is a small molecule inhibitor with inhibitory effects on the ALK4, ALK5 and ALK7. Our study aimed to explore the effect of SB431542 on the EMT of RPE cells and to provide new ideas for the treatment of retinal fibrosis. METHODS: We performed fundus fluorescein angiography, optical coherence tomography and hematoxylin-eosin staining in vivo to observe the effect of SB431542 on choroidal neovascularization (CNV)-induced retinopathy. The proliferation, migration, cytoskeleton, adhesion, reactive oxygen species (ROS), mitochondrial morphology and membrane potential of RPE cells were observed in vitro through fluorescein diacetate staining, Cell Counting Kit-8 experiment, wound healing assay, phalloidin staining, immunofluorescence, MitoSOX, DCFH-DA, MitoTracker and JC-10 staining. Western blot, reverse transcription quantitative and immunofluorescence were used to detect the expression of EMT-related markers, pERK1/2, pGSK3ß and ß-catenin. RESULTS: SB431542 significantly alleviated retinopathy in the CNV model. The proliferation, migration and adhesion in RPE cells decreased to a certain extent in SB431542 treatment. SB431542 partially normalized the structure of RPE cells. The expression levels of E-cadherin increased, while the expression levels of laminin and N-cadherin decreased with SB431542 treatment. SB431542 reduced the production of total ROS, mitochondrial SOX and recovered the mitochondrial membrane potential to a certain degree. In addition, our study showed that SB431542 downregulated the phosphorylation of ERK1/2, GSK3ß and the expression of ß-catenin. CONCLUSION: SB431542 improved EMT in RPE cells by maintaining mitochondrial homeostasis via the ERK1/2 and GSK3ß/ß-catenin pathways. Video Abstract SB431542 inhibits EMT in RPE cells under high glucose conditions.

Upregulation of TRPC1 protects against high glucose-induced HUVECs dysfunction by inhibiting oxidative stress.

-To explore the effect of TRPC1 on endothelial cell function damage under a high glucose environment and its downstream molecular mechanism, and provide new theory and strategy for improving diabetic endothelial cell function and promoting vascular injury repair. In vitro, we use high glucose to treat human umbilical vein endothelial cells (HUVECs) and upregulated TRPC1 with adenovirus infection. HUVECs were split into 4 groups: (i) NG Group: Treated with normal glucose; (ii) HG Group: Treated with high glucose; (iii) HG + adGFP Group: High glucose + the control adenovirus (adGFP); (iv) HG + adTRPC1 Group: High glucose + recombinant adenovirus encoding TRPC1. We found that high glucose significantly decreased the expression level of TRPC1 protein, and impaired the proliferation and migration of HUVECs, which could be reversed by overexpression of TRPC1. In addition, high glucose induced an increase in ROS and MDA and a decrease in SOD activity, whereas TRPC1 overexpression could inhibit the growth of oxidative stress level. These findings suggest that overexpression of TRPC1 prevents HUVECs proliferation and migration dysfunction induced by high glucose via inhibiting oxidative stress injuries.

Tauroursodeoxycholic acid protects Schwann cells from high glucose-induced cytotoxicity by targeting NLRP3 to regulate cell migration and pyroptosis.

Diabetic peripheral neuropathy (DPN) is the most prevalent complication of type 2 diabetes mellitus (T2DM), and it seriously affects the quality of life of patients. Tauroursodeoxycholic acid (TUDCA) is a bile acid that plays a protective role against various diseases. However, the function of TUDCA in DPN progression needs to be elucidated. Hence, this study clarified the action of TUDCA on DPN development and explored its mechanism of action. Fecal samples were collected from 50 patients with T2DM or DPN. Schwann cells induced by high levels were constructed to simulate an uncontrolled diabetic state. Cell viability and migration were measured using the CCK-8 and wound-healing assays, respectively. Reactive oxygen species and pyroptosis were detected using flow cytometry. Parabacteroides goldsteinii and Parabacteroides distasonis levels were decreased in the feces of patients with DPN. TUDCA enhanced the viability and migration ability of high glucose-stimulated Schwann cells. In addition, Schwann cell pyroptosis stimulated by high glucose levels was inhibited by TUDCA. Furthermore, the protective roles of TUDCA in cell viability, migration ability, and pyroptosis of Schwann cells stimulated by high glucose were suppressed by the overexpression of NLRP3. TUDCA enhanced cell viability and migration and suppressed pyroptosis in Schwann cells stimulated by high glucose levels by modulating NLRP3 expression. Thus, TUDCA may be a promising drug for DPN therapy.

Gigantol protects retinal pigment epithelial cells against high glucose-induced apoptosis, oxidative stress and inflammation by inhibiting MTDH-mediated NF-kB signaling pathway.

OBJECTIVE: As a frequent complication of diabetes mellitus (DM), diabetic retinopathy (DR) is now one of the major causes of blindness. Recent reports have shown that retinal pigment epithelial cell (RPEC) damage plays an essential part in DR development and progression. This work intended to explore the potential effects of Gigantol on high glucose (HG)-stimulated RPEC damage and identify potential mechanisms. METHODS: Cell viability, cell damage, and cell apoptosis were evaluated by CCK-8, lactate dehydrogenase (LDH) and flow cytometry assays. The levels of oxidative stress biomarkers and pro-inflammatory cytokines were assessed using corresponding commercial kits and ELISA. Additionally, the levels of MTDH and NF-kB signaling pathway-related proteins were detected by western blotting. RESULTS: Gigantol dose-dependently enhanced cell viability and decreased apoptosis in HG-challenged ARPE-19 cells. Also, Gigantol notably relieved oxidative stress and inflammatory responses in ARPE-19 cells under HG conditions. Gigantol dose-dependently suppressed MTDH expression. In addition, MTDH restoration partially counteracted the protective effects of Gigantol on ARPE-19 cells subject to HG treatment. Mechanically, Gigantol inactivated the NF-kB signaling pathway, which was partly restored after MTDH overexpression. CONCLUSION: Our findings suggested that Gigantol protected against HG-induced RPEC damage by inactivating the NF-kB signaling via MTDH inhibition, offering a potent therapeutic drug for DR treatment.

Metformin protects human lens epithelial cells from high glucose-induced senescence and autophagy inhibition by upregulating SIRT1.

PURPOSE: The aim of this study is to explore whether metformin (MET) protects the human lens epithelial cells (HLECs) from high glucose-induced senescence and to identify the underlying mechanisms. METHODS: A cellular senescence model was established by treating HLE-B3 cells with D-glucose and then intervened with MET. Concentrations of high glucose (HG) and MET were detected using CCK-8 and western blot. qRT-PCR, western blot, and senescence-associated ß-galactosidase (SA-ß-gal) were performed to verify the protective effect of MET on senescent HLE-B3 cells. Additionally, western blot and qRT-PCR were conducted to detect the effects of MET on autophagy-related markers p62 and LC3, as well as SIRT1. RESULTS: In vitro, we observed apparent senescence in human lens epithelial cells (HLECs) under high glucose conditions. This was characterized by increased senescence-associated genes p21 and p53. However, the addition of MET significantly reduced the occurrence of HLECs senescence. We also observed that high glucose inhibited both autophagy and SIRT1, which could be restored by MET. Moreover, we verified that the anti-senescence effect of MET was mediated by SIRT1 using SIRT1 activators and inhibitors. CONCLUSION: We have demonstrated that autophagy and SIRT1 activity are inhibited in HLE-B3 cells using the HG induced senescence model. Furthermore, our results showed that MET can delay senescence by activating SIRT1 and autophagy. These findings suggest that MET may be a promising candidate for alleviating cataract development and provide a direction for further investigation into the underlying molecular mechanisms.