High Glucose Treatment Research Articles

GLUT1 and prorenin receptor mediate differential regulation of TGF-β and CTGF in renal inner medullary collecting duct cells during high glucose conditions.

During diabetes, prorenin is highly produced by the renal collecting ducts. The binding of prorenin to (pro)renin receptor (PRR) on the apical plasma membrane triggers intracellular profibrotic genes, including TGF-β and CTGF. However, the underlying mechanisms contributing to the stimulation of these pathways remain unclear. Hence, we hypothesize that the glucose transporter-1 (GLUT1) favors the PRR-dependent stimulation of TGF-β and CTGF in the distal nephron segments during high glucose (HG) conditions. To test this hypothesis, primary cultured renal inner medullary collecting duct (IMCD) cells were treated with normal glucose (NG, 5mM) or high glucose (HG, 25mM) for 48h in the presence or absence of the GLUT1-specific inhibitor BAY 876 (2nM). Additionally, IMCD cells were treated with the PRR antagonist PRO20. The expression of TGF-β and CTGF was quantified by immunoblot and qRT-PCR. HG increased GLUT1 mRNA and protein abundance, while BAY 876 inhibited these responses. HG treatment upregulated PRR, but the concomitant treatment with BAY 876 partially prevented this effect. TGF-β and CTGF expressions were augmented in IMCD cells treated with HG. However, PRO20 prevented the increases in TGF-β but not those of CTGF. GLUT1 inhibition partially prevented the increases in reactive oxygen species (ROS) during HG while PRO20 did not. ROS scavenging impaired CTGF upregulation during HG conditions. Additionally, long-term exposure to HG increases lipid peroxidation and reduced cell viability. The data indicate that glucose transportation via GLUT1 is implicated in the PRR-dependent upregulation of TGF-β while CTGF is mediated mainly via a mechanism depending on ROS formation in renal medullary collecting duct cells.

Monocytes perturbation implicated in the association of stress hyperglycemia with postoperative poor prognosis in non-diabetic patients with Stanford type-A acute aortic dissection

ObjectivesThe study aimed to investigate the interaction of intraoperative stress hyperglycemia with monocyte functions and their impact on major adverse events (MAEs) in acute aortic dissection (AAD) patients who underwent open repair surgery.MethodsA total of 321 adults who underwent open surgery for AAD at two tertiary medical centers in China were enrolled in the study. The primary endpoint was defined as the incidence and characteristics of perioperative stress hyperglycemia. The secondary endpoints included the incidence of postoperative MAEs, postoperative monocyte counts and inflammatory cytokine expression. Multi-logistic, linear regression and receiver operating characteristic (ROC) curve analyses were used to establish relationships between intraoperative time-weighted average glucose (TWAG), day-one postoperative monocyte counts, serum inflammatory cytokines and postoperative outcomes. In addition, in vitro experiments were conducted to evaluate changes in the inflammatory features of monocytes under high glucose conditions.ResultsIntraoperative hyperglycemia, as indicated by a TWAG level over 142 mg/dL, was associated with elevated postoperative monocyte counts and inflammatory cytokines, which correlated with extended intensive care unit (ICU) stays and worsened outcomes. In vitro, high glucose treatment induced mitochondrial impairment in monocytes, increased the release of inflammatory cytokines and the proportion of classical monocytes from AAD patients.ConclusionsIntraoperative stress hyperglycemia, in combination with day-one postoperative monocyte counts, were clinically significant for predicting adverse outcomes in AAD patients undergoing open repair surgery. Elevated glucose concentrations shaped the inflammatory features of monocytes in AAD by impairing mitochondrial functions.Graphical abstractHigh glucose-driven mitochondrial functional impairment mediates a more pronounced monocytic inflammatory state in Stanford type-A AAD.

β-Sitosterol Mitigates Apoptosis, Oxidative Stress and Inflammatory Response by Inactivating TLR4/NF-кB Pathway in Cell Models of Diabetic Nephropathy.

Podocyte injury plays a pivotal role in the pathogenesis of diabetic nephropathy (DN), leading to proteinuria formation. β-Sitosterol is a natural compound with anti-inflammatory, anti-diabetic, nephroprotective and antioxidant properties. The studyaimed to explore whether and how β-Sitosterol protected podocytes against high glucose (HG)-induced inflammatory andoxidative injury. DN cell models were established by stimulating podocytes or renal tubular epithelial cells (HK-2) cells with 25 mM glucose. Cell viability and apoptosis were evaluated using cell counting kit-8 assays and flow cytometry analyses. Westernblotting was used to quantify protein levels of genes related to podocyte injury, HK-2 cell damage, inflammation, and TLR4/NF-кB pathway. Contents of oxidative stress biomarkers were evaluated by corresponding commercial kits while proinflammatorycytokine levels were determined by enzyme-linked immunosorbent assay. Immunofluorescence staining was performed todetect intracellular levels of reactive oxygen species (ROS) and Nrf2 nuclear translocation. Experimental results revealed that HG treatment induced podocyte dysfunction by impairing cell viability while accelerating theapoptosis, and the changes were reversed by β-sitosterol treatment. Moreover, β-sitosterol repressed HG-evoked oxidative stressby reducing ROS and malondialdehyde (MDA) levels while increasing activities of antioxidant enzymes. The reduction ofproinflammatory cytokines mediated by β-sitosterol in HG-stimulated podocytes suggested the anti-inflammatory role of β-sitosterol. Additionally, the activation of the TLR4/NF-кB signaling induced by HG was inhibited by β-sitosterol in podocytes.Inactivation of the TLR4 using TAK-242 enhanced the protective effects of β-sitosterol against HG-mediated oxidative stressand inflammation. Similarly, β-sitosterol also protected HK-2 cells from HG-induced oxidative stress, inflammation, andapoptosis. In summary, β-sitosterol exerts anti-inflammatory, anti-oxidative, and anti-apoptotic activities in HG-induced podocytes or HK-2 cells by inhibiting TLR4/NF-кB signaling.

FTO mediates the diabetic kidney disease progression through regulating the m6A modification of NLRP3

BackgroundThe objective of our research was to investigate the specific mechanism of FTO in diabetic kidney disease (DKD) progression.MethodsThe DKD model was established with renal tubular epithelial HK-2 cells and mice in vitro and in vivo. The N6-methyladenosine (m6A) content in cells was detected using dot plot assay and the m6A levels of NLRP3 was detected with the MeRIP assay. The mRNA and protein levels were tested with real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot. The IL-1β and IL-18 levels were assessed with enzyme-linked immunosorbent assay (ELISA). The cell viability was measured by cell counting kit (CCK)-8 assay and cell pyroptosis was determined with Annexin V and propidium iodide (PI) double staining followed by flow cytometry analysis. RNA-binding protein immunoprecipitation (RIP) and dual luciferase reporter assays were conducted to detect the interaction between FTO and NLRP3. m6A levels were detected by Me-RIP assay. The renal injury was measured by observing the renal morphology and urine and blood levels of relevant indicators.ResultsThe results indicated that high glucose treatment induced HK-2 cell pyroptosis. m6A levels were prominently elevated in high glucose treated HK-2 cells while FTO expression were significantly down-regulated. FTO over-expression promoted cell viability but inhibited pyroptosis of HK-2 cells under high glucose (HG) treatment. Moreover, FTO could inhibit NLRP3 expression. RIP and Me-RIP assays indicated that FTO could bind with NLRP3 and regulate its m6A modification level. Further luciferase assay confirmed that FTO binds with the 233–237 bp region of NLRP3. NLRP3 neutralized the function of FTO in the HG stimulated HK-2 cells. In vivo, the H&E staining showed that FTO over-expression alleviated the kidney injury and suppressed the pyroptosis induced by DKD.ConclusionWe found that FTO could inhibit the DKD progression in vivo and in vitro by regulated the m6A modification of NLRP3.

CLEC14A facilitates angiogenesis and alleviates inflammation in diabetic wound healing

BackgroundDelayed wound healing is a serious complication of diabetic wounds, posing a significant challenge to the treatment of patients with diabetes. Diabetic wound healing is a complex dynamic process involving angiogenesis and inflammatory responses. Currently, there are limited targeted therapies to promote diabetic wound healing. This study aimed to reveal the role of CLEC14A in the process of diabetic wound healing, with the hope of identifying new therapeutic targets to accelerate the healing of diabetic wounds. MethodsIn vivo, diabetic mice were generated by combined streptozotocin (STZ) and high-fat diet treatment. The wound healing model was established in wild-type and Clec14a−/− diabetic mice. The degree of wound healing, as well as angiogenesis and inflammation during the healing process, were evaluated through Hematoxylin and Eosin (H&E) staining, immunohistochemical staining, and immunofluorescence staining. In vitro, the angiogenic activities of Human Umbilical Vein Endothelial Cells (HUVECs) were assessed following treatment with high glucose and adenoviruses overexpressing CLEC14A, using scratch assays and tube formation assays. Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) were utilized to evaluate the levels of inflammation in HUVECs. ResultsCLEC14A expression was suppressed in diabetic wounds. Deletion of the Clec14a inhibited angiogenesis and activated inflammatory responses in vivo. High-glucose treatment led to decreased CLEC14A expression, impaired angiogenic capacity, and elevated inflammatory levels in vitro. However, adenoviral-mediated overexpression of CLEC14A reversed the response induced by high glucose. ConclusionCLEC14A accelerates diabetic wound healing by promoting angiogenesis and reducing wound inflammation.

High glucose promotes lipopolysaccharide-induced macrophage pyroptosis through GSDME O-GlcNAcylation.

The high glucose (HG) environment in diabetic periodontitis aggravates the damage of periodontal tissue. Pyroptosis has been shown to be positively correlated with the severity of periodontitis, including macrophage pyroptosis. O-GlcNAcylation is a posttranslational modification that is involved in the pathogenesis of periodontitis. However, whether HG regulates macrophage pyroptosis through O-GlcNAcylation remains uncertain. This study aimed to investigate the effect of HG on the O-GlcNAcylation level of a pyroptosis regulator GSDME in macrophages to further probe the mechanisms of diabetic periodontitis. Blood samples were collected from patients with diabetic periodontitis. THP-1 monocytes were induced to differentiate into macrophages by phorbol 12-myristate 13-acetate and then treated with HG to simulate periodontitis invitro. GSDME expression of blood samples and macrophages was measured by quantitative real-time PCR. Pyroptosis was assessed by propidium iodide staining, measurement of cell viability, cytotoxicity, protein levels of inflammation factors, and pyroptosis-related proteins. O-GlcNAcylation of GSDME was analyzed using co-immunoprecipitation (co-IP), IP, and western blot. The results showed that GSDME expression was elevated in patients with periodontitis and HG-treated macrophages. HG inhibited cell viability but increased LDH content, levels of IL-1β, IL-18, TNF-α, NLRP3, GSDMD, and Caspase-1, indicating that HG promoted pyroptosis of macrophages, which was reversed by GSDME knockdown. HG treatment increased O-GlcNAcylation in macrophages. Mechanically, GSDME interacted with OGT, and OGT knockdown suppressed O-GlcNAcylation of GSDME at Ser (S)339 site. Knockdown of OGT inhibited pyroptosis in HG-treated macrophages, while GSDME overexpression partially reversed this inhibition. HG treatment enhanced OGT-mediated GSDME O-GlcNAcylation, thereby augmenting pyroptosis in LPS-induced macrophages. These results may provide a novel sight for the treatment of periodontitis.

Cytoarchitecture of Breast Cancer Cells under Diabetic Conditions: Role of Regulatory Kinases-Rho Kinase and Focal Adhesion Kinase.

Diabetes greatly reduces the survival rates in breast cancer patients due to chemoresistance and metastasis. Reorganization of the cytoskeleton is crucial to cell migration and metastasis. Regulatory cytoskeletal protein kinases such as the Rho kinase (ROCK) and focal adhesion kinase (FAK) play a key role in cell mobility and have been shown to be affected in cancer. It is hypothesized that diabetes/high-glucose conditions alter the cytoskeletal structure and, thus, the elasticity of breast cancer cells through the ROCK and FAK pathway, which can cause rapid metastasis of cancer. The aim of the study was to investigate the role of potential mediators that affect the morphology of cancer cells in diabetes, thus leading to aggressive cancer. Breast cancer cells (MDA-MB-231 and MCF-7) were treated with 5 mM glucose (low glucose) or 25 mM glucose (high glucose) in the presence of Rho kinase inhibitor (Y-27632, 10 mM) or FAK inhibitor (10 mM). Cell morphology and elasticity were monitored using atomic force microscopy (AFM), and actin staining was performed by fluorescence microscopy. For comparative study, normal mammary breast epithelial cells (MCF-10A) were used. It was observed that high-glucose treatments modified the cytoskeleton of the cells, as observed through AFM and fluorescence microscopy, and significantly reduced the elasticity of the cells. Blocking the ROCK or FAK pathway diminished the high-glucose effects. These changes were more evident in the breast cancer cells as compared to the normal cells. This study improves the knowledge on the cytoarchitecture properties of diabetic breast cancer cells and provides potential pathways that can be targeted to prevent such effects.

Endoplasmic reticulum stress induces hepatic steatosis through interaction between PPARα and FoxO6 in vivo and in vitro

Endoplasmic reticulum (ER) stress is a major cause of hepatic steatosis through increasing de novo lipogenesis. Forkhead box O6 (FoxO6) is a transcription factor mediating insulin signaling to glucose and lipid metabolism. Therefore, dysregulated FoxO6 is involved in hepatic lipogenesis. This study elucidated the role of FoxO6 in ER stress–induced hepatic steatosis in vivo and in vitro. Hepatic ER stress responses and β-oxidation were monitored in mice overexpressed with constitutively active FoxO6 allele and FoxO6-null mice. For the in vitro study, liver cells overexpressing constitutively active FoxO6 and FoxO6-siRNA were treated with high glucose, and lipid metabolism alterations were measured. ER stress–induced FoxO6 activation suppressed hepatic β-oxidation in vivo. The expression and transcriptional activity of peroxisome proliferator-activated receptor α (PPARα) were significantly decreased in the constitutively active FoxO6 allele. Otherwise, inhibiting β-oxidation genes were reduced in the FoxO6-siRNA and FoxO6-KO mice. Our data showed that the FoxO6-induced hepatic lipid accumulation was negatively regulated by insulin signaling. High glucose treatment as a hyperglycemia condition caused the expression of ER stress–inducible genes, which was deteriorated by FoxO6 activation in liver cells. However, high glucose-mediated ER stress suppressed β-oxidation gene expression through interactions between PPARα and FoxO6 corresponding to findings in the in vivo study—lipid catabolism is also regulated by FoxO6. Furthermore, insulin resistance suppressed b-oxidation through the interaction between FoxO6 and PPARα promotes hepatic steatosis, which, due to hyperglycemia-induced ER stress, impairs insulin signaling.Key messagesOur original aims were to delineate the interrelation between the regulation of PPARα and the transcription factor FoxO6 pathway in relation to lipid metabolism at molecular levels.Evidence on high glucose promoted FoxO6 activation induced lipid accumulation in liver cells.The effect of PPARα activation of the insulin signaling.FoxO6 plays a pivotal role in hepatic lipid accumulation through inactivation of PPARα in FoxO6-overexpression mice.

MiR-454-3p regulates high glucose-induced mesothelial-mesenchymal transition and glycolysis in peritoneal mesothelial cells by targeting STAT3

Background The quality of life of patients receiving long-term peritoneal dialysis (PD) is significantly impacted by the onset of peritoneal fibrosis (PF), and one of the pathological changes is mesothelial-mesenchymal transition (MMT). In this study, we investigated the potential roles of miR-454-3p and signal transducer and activator of transcription 3 (STAT3) in the progression of peritoneal MMT and the underlying mechanisms. Methods Peritoneums were collected to detect morphology via hematoxylin-eosin staining and differentially expressed miRNAs were detected via RT-qPCR. PD effluent-derived cell populations in the peritoneal cavity were isolated from the effluents of 20 PD patients to determine miR-454-3p, STAT3, and MMT markers via Western blotting and RT-qPCR. The relationship between miR-454-3p and STAT3 was examined via a dual-luciferase reporter assay. Western blotting and RT-qPCR were utilized to evaluate the expression of STAT3, MMT markers, and glycolytic enzymes. Immunofluorescence staining revealed the localization and expression of MMT markers and STAT3. Results MiR-454-3p was downregulated in the peritoneums and PD effluent-derived cell populations of long-term PD patients. High glucose (HG) treatment promoted HMrSV5 cell MMT and glycolysis. MiR-454-3p overexpression alleviated HG-induced MMT and suppressed the expression of STAT3 and glycolytic enzymes. In contrast, the miR-454-3p inhibitor exacerbated HG-induced MMT and promoted the expression of glycolytic enzymes and STAT3. Moreover, STAT3 was the target of miR-454-3p. Conclusions This study demonstrated the protective role of miR-454-3p in HG-induced MMT and glycolysis in HMrSv5 cells, suggesting that miR-454-3p may prevent MMT by suppressing glycolytic enzymes via the STAT3/PFKFB3 pathway in the HG environment.

The Expression of Circ-Astn1 Inhibits High Glucose Induced Endothelial Progenitor Cell Dysfunction by Activating Autophagy

ABSTRACT Background Diabetes mellitus (DM) and complications such as chronic kidney disease and cardiovascular symptoms pose a substantial public health burden. Increasing studies have shown that circular RNAs (circRNAs) regulate many gene expressions that are essential in diverse pathological and biological procedures. However, the roles of particular circRNAs in DM are unclear. Methods In the current investigation, endothelial progenitor cells (EPCs) were used to search for abnormal expression of circRNAs by using high-throughput sequencing under high glucose (HG) conditions. The regulatory mechanisms and targets were then studied through bioinformatics analysis, luciferase reporter analysis, angiogenic differentiation experiments, flow cytometry detection of apoptosis and RT-qPCR analysis. Results The circ-Astn1 expression in EPCs decreased after HG treatment. Overexpression or circ-Astn1 suppressed HG induced endothelial cell damage. MicroRNA (miR)-138-5p and SIRT5 were found to be the downstream targets of circ-Astn1 through luciferase reporter analysis. SIRT5 downregulation or miR-138-5p overexpression reversed circ-Astn1’s protective effect against HG induced endothelial cell dysfunction, including apoptosis and abnormal vascular differentiation. Furthermore, circ-Astn1 overexpression promoted autophagy activation by increasing SIRT5 expression under HG conditions. Our findings suggest that circ-Astn1 mediated promotion of SIRT5 facilitates autophagy by sponging miR-138-5p. Conlusion Together, our findings show that the overexpression of circ-Astn1 suppresses HG induced endothelial cell damage by targeting miR-138-5p/SIRT5 axis.

FTMT-dependent mitophagy is crucial for ferroptosis resistance in cardiac fibroblast

Metabolic responses to cellular stress are pivotal in cell ferroptosis, with mitophagy serving as a crucial mechanism in both metabolic processes and ferroptosis. This study aims to elucidate the effects of high glucose on cardiomyocytes (CMs) and cardiac fibroblasts (CFs) regarding ferroptosis and to uncover the underlying mechanisms involved. We examined alterations in glycolysis, mitochondrial oxidative phosphorylation (OXPHOS), and mitophagy, which are essential for metabolic adaptations and ferroptosis. High glucose exposure induced ferroptosis specifically in CMs, while CFs exhibited resistance to ferroptosis, increased glycolytic activity, and no change in OXPHOS. Moreover, high glucose treatment enhanced mitophagy and upregulated mitochondrial ferritin (FTMT). Notably, the combination of FTMT and the autophagy-related protein nuclear receptor coactivator 4 (NCOA4) increased under high glucose conditions. Silencing FTMT significantly impeded mitophagy and eliminated ferroptosis resistance in CFs cultured under high glucose conditions. The transcription factor forkhead box A1 (FOXA1) was upregulated in CFs upon high glucose exposure, playing a crucial role in the increased expression of FTMT. Within the 5′-flanking sequence of the FTMT mRNA, approximately −500 nt from the transcription initiation site, three putative FOXA1 binding sites were identified. High glucose augmented the binding affinity between FOXA1 and these sequences, thereby promoting FTMT transcription. In summary, high glucose upregulated FOXA1 expression and stimulated FTMT promoter activity in CFs, thereby promoting FTMT-dependent mitophagy and conferring ferroptosis resistance in CFs.

CircRNA Arf3 suppresses glomerular mesangial cell proliferation and fibrosis in diabetic nephropathy via miR-107-3p/Tmbim6 axis

Diabetic nephropathy (DN) is one of microvascular complication associated with diabetes. Circular RNAs (circRNAs) have been shown to be involved in DN pathogenesis. Hence, this work aimed to explore the role and mechanism of circ_Arf3 in DN. Mouse mesangial cells (MCs) cultured in high glucose (HG) condition were used for functional analysis. Cell proliferation was determined using 5-ethynyl-2’-deoxyuridine (EdU) and cell counting kit-8 assays. Western blotting was used to measure the levels of proliferation indicator PCNA and fibrosis-related proteins α-smooth muscle actin (α-SMA), collagen I (Col I), fibronectin (FN), and collagen IV (Col IV). The binding interaction between miR-107-3p and circ_Arf3 or Tmbim6 (transmembrane BAX inhibitor motif containing 6) was confirmed using dual-luciferase reporter and pull-down assays. Circ_Arf3 is a stable circRNA, and the expression of circ_Arf3 was decreased after HG treatment in MCs. Functionally, ectopic overexpression of circ_Arf3 protected against HG-induced proliferation and elevation of fibrosis-related proteins in MCs. Mechanistically, circ_Arf3 directly bound to miR-107-3p, and Tmbim6 was a target of miR-107-3p. Further rescue assay showed miR-107-3p reversed the protective action of circ_Arf3 on MCs function under HG condition. Moreover, inhibition of miR-107-3p suppressed HG-induced proliferation and fibrosis, which were attenuated by Tmbim6 knockdown in MCs. CircRNA Arf3 could suppress HG-evoked mesangial cell proliferation and fibrosis via miR-107-3p/Tmbim6 axis, indicating the potential involvement of this axis in DN progression.

MicroRNA-2861 regulates the proliferation and apoptosis of human retinal vascular endothelial cells treated with high glucose by targeting NDUFB7

ObjectivesAlthough anti-VEGF and retinal laser photocoagulation are two therapeutic modalities that have been used in the clinical treatment of diabetic retinopathy (DR), it is unknown how these modalities target vascular endothelial function in DR. MethodsWe first downloaded and analyzed the differential genes in two DR-related datasets, GSE60436 and GSE53257. The differential gene expression was then verified using RT-qPCR, and the most upregulated gene, NDUFB7, was selected for subsequent experiments. Subsequently, the role of NDUFB7 silencing and enforced expression on the proliferation and apoptosis of HRVECs was explored using CCK-8 assay, EDU proliferation assay and apoptotic TUNEL staining. In addition, the upstream potential miRNAs of NDUFB7 were predicted online using the Targetscan website. RT-qPCR, Western blotting (WB), and dual luciferase gene reporter assay were used to confirm the targeting connection between miR-2861 and NDUFB7. Finally, miR-2861 expression after high glucose (HG) treatment and its effect on proliferation and apoptosis of HRVECs under HG were investigated. ResultsIn this study, we first downloaded and analyzed the differential genes in two DR-related datasets, GSE60436 and GSE53257. We found that TUFM, PRELID1, MRPL32, NDUFB7, MRPL4, MRPL40, HSD17B10 and SLC25A13 were upregulated in DR, and RT-qPCR showed that NDUFB7 was most upregulated. Subsequent CCK-8 assay, EDU proliferation assay and TUNEL staining showed that up-picked NDUFB7 promotes proliferation and inhibits apoptosis of HRVECs. In addition, the upstream potential miRNAs of NDUFB7 were predicted online using the Targetscan website. RT-qPCR, Western blotting (WB), and dual luciferase gene reporter assay confirmed the targeting connection between miR-2861 and NDUFB7. Finally, it was observed that miR-2861 can inhibit the proliferation and promote the apoptosis of HRVECs by targeting NDUFB7. ConclusionsOur findings showed that upregulated NDUFB7 in DR promotes proliferation and inhibits apoptosis of HRVECs, and miR-2861 can rescue the pathogenic effect of NDUFB7 upregulation by targeting NDUFB7.

Hyperglycemia induces microglial pyroptosis by increasing oxygen extraction rate: Implication in neurological impairment during ischemic stroke.

Elevated levels of blood glucose in patients with ischemic stroke are associated with a worse prognosis. The present study aimed to explore whether hyperglycemia promotes microglial pyroptosis by increasing the oxygen extraction rate in an acute ischemic stroke model. C57BL/6 mice that underwent middle cerebral artery occlusion were used for assessment of blood glucose level and neurological function. The cerebral oxygen extraction ratio (CERO2), oxygen consumption rate (OCR) and partial pressure of brain tissue oxygen (PbtO2) were measured. To investigate the significance of the NOD‑like receptor protein 3 (NLRP3) inflammasome, NLRP3‑/‑ mice were used, and the expression levels of NLRP3, caspase‑1, full‑length gasdermin D (GSDMD‑FL), GSDMD‑N domain (GSDMD‑N), IL‑1β and IL‑18 were evaluated. In addition, Z‑YVAD‑FMK, a caspase‑1 inhibitor, was used to treat microglia to determine whether activation of the NLRP3 inflammasome was required for the enhancing effect of hyperglycemia on pyroptosis. It was revealed that hyperglycemia accelerated cerebral injury in the acute ischemic stroke model, as evidenced by decreased latency to fall and the percentage of foot fault. Hyperglycemia aggravated hypoxia by increasing the oxygen extraction rate, as evidenced by increased CERO2 and OCR, and decreased PbtO2 in response to high glucose treatment. Furthermore, hyperglycemia‑induced microglial pyroptosis was confirmed by detection of increased levels of caspase‑1, GSDMD‑N, IL‑1β and IL‑18 and a decreased level of GSDMD‑FL. However, the knockout of NLRP3 attenuated these effects. Pharmacological inhibition of caspase‑1 also reduced the expression levels of GSDMD‑N, IL‑1β and IL‑18 in microglial cells. These results suggested that hyperglycemia stimulated NLRP3 inflammasome activation by increasing the oxygen extraction rate, thus leading to the aggravation of pyroptosis following ischemic stroke.

Obesity-induced upregulation of miR-483-5p impairs the function and identity of pancreatic β-cells.

To assess the expression and function of miR-483-5p in diabetic β cells. The expression of miR-483-5p was evaluated in the pancreatic islets of obesity mouse models by quantitative reverse transcription polymerase chain reaction. Dual-luciferase activity, and western blotting assays, were utilized for miR-483-5p target gene verification. Mice with β cell-specific miR-483-5p downregulation were studied under metabolic stress (i.e. a high-fat diet) condition. Lineage tracing was used to determine β-cell fate. miR-483-5p increased in the islets of obese mouse models. Expression levels of miR-483-5p were significantly upregulated with the treatment of high glucose and palmitate, in both MIN6 cells and mouse islets. Overexpression of miR-483-5p in β cells results in impaired insulin secretion and β-cell identity. Cell lineage-specific analyses revealed that miR-483-5p overexpression deactivated β-cell identity genes (insulin, Pdx1 and MafA) and derepressed β-cell dedifferentiation (Ngn3) genes. miR-483-5p downregulation in β cells of high-fat diet-fed mice alleviated diabetes and improved glucose intolerance by enhancing insulin secretory capacity. These detrimental effects of miR-483-5p relied on its seed sequence recognition and repressed expression of its target genes Pdx1 and MafA, two crucial markers of β-cell maturation. These findings indicate that the miR-483-5p-mediated reduction of mRNAs specifies β-cell identity as a contributor to β-cell dysfunction via the loss of cellular differentiation.

USP7-stabilised HIPK2 promotes high glucose-induced endothelial cell dysfunctions to accelerate diabetic foot ulcers

Background: This study aimed to explore the molecular mechanism of homeodomain-interacting protein kinase 2 (HIPK2) in diabetic foot ulcers (DFU). Methods: High glucose (HG)-induced human umbilical vein endothelial cells (HUVECs) were used to construct DFU cell models. Cell functions were determined using CCK8 assay, EdU assay, flow cytometry, transwell assay, wound healing assay and tube formation assay. Quantitative real-time PCR and western blot were applied to measure the gene expression. Results: HG treatment suppressed HUVECs proliferation, invasion, migration, and angiogenesis, while enhanced apoptosis. HIPK2 was overexpressed in DFU patients, and its knockdown alleviated HG-induced HUVECs dysfunctions. USP7 stabilised HIPK2 protein by reducing its ubiquitination. USP7 overexpression promoted HG-induced HUVECs dysfunctions, and HIPK2 upregulation also reversed the regulation of USP7 knockdown on HG-induced HUVECs dysfunctions. USP7/HIPK2 axis inhibited the activity of PI3K/AKT pathway. Conclusion: Our study revealed that USP7-stabilised HIPK2 contributed to HG-induced HUVECs dysfunctions, thus accelerating DFU process.

Smooth Muscle Cells-Expressed Bmal1 Regulates Vascular Calcification Independent of the Canonical Circadian Pathway.

Vascular calcification is a major cardiovascular issue that increases morbidity and mortality in diabetes patients. While dysregulation of the circadian master regulator Basic Helix-Loop-Helix ARNT-Like Protein 1 (Bmal1) in vascular smooth muscle cells (VSMC) under diabetic conditions has been suggested, its role in vascular calcification is unclear. In VSMC, Bmal1 was upregulated under high glucose treatment and in aortic tissues from a diabetic mouse model. RNA sequencing from isolated VSMC between Bmal1 deletion and wildtype mice indicated Bmal1's pro-calcification role. Indeed, reduced levels of the osteogenic master regulator, Runt-Related Transcription Factor 2 (Runx2), were found in Bmal1 deletion VSMC under diabetic conditions. Alizarin red staining showed reduced calcification in Bmal1 deletion VSMC in vitro and vascular rings ex vivo . Furthermore, in a diabetic mouse model, SMC-Bmal1 deletion showed reduced calcium deposition in aortas. Collectively, diabetes-upregulated circadian regulator Bmal1 in VSMC contributes to vascular calcification. Maintaining normal circadian regulation may improve vascular health in diabetes.

CircZNF532 promotes endothelial-to-mesenchymal transition in diabetic retinopathy by recruiting TAF15 to stabilize PIK3CD.

Endothelial-to-mesenchymal transition (EndMT) is a pivotal event in diabetic retinopathy (DR). This study explored the role of circRNA zinc finger protein 532 (circZNF532) in regulating EndMT in DR progression. Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to induce the DR cell model. Actinomycin D-treated HRMECs were used to confirm the mRNA stability of phosphoinositide-3 kinase catalytic subunit δ (PIK3CD). The interaction between TATA-box-binding protein-associated factor 15 (TAF15) and circZNF532/PIK3CD was subsequently analyzed using RNA immunoprecipitation (RIP), RNA pull-down. It was found that HG treatment accelerated EndMT process, facilitated cell migration and angiogenesis, and enhanced PIK3CD and p-AKT levels in HRMECs, whereas si-circZNF532 transfection neutralized these effects. Further data showed that circZNF532 recruited TAF15 to stabilize PIK3CD, thus elevating PIK3CD expression. Following rescue experiments suggested that PIK3CD overexpression partially negated the inhibitory effect of circZNF532 silencing on EndMT, migration, and angiogenesis of HG-treated HRMECs. In conclusion, our results suggest that circZNF532 recruits TAF15 to stabilize PIK3CD, thereby facilitating EndMT in DR.

SP1 promotes high glucose-induced lens epithelial cell viability, migration and epithelial-mesenchymal transition via regulating FGF7 and PI3K/AKT pathway.

Diabetic cataract (DC) is a common complication of diabetes and its etiology and progression are multi-factorial. In this study, the roles of specific protein 1 (SP1) and fibroblast growth factor 7 (FGF7) in DC development were explored. DC cell model was established by treating SRA01/04 cells with high glucose (HG). MTT assay was conducted to evaluate cell viability. Transwell assay and wound-healing assay were performed to assess cell migration and invasion. Western blot assay and qRT-PCR assay were conducted to measure the expression of N-cadherin, E-cadherin, Collagen I, Fibronectin, SP1 and FGF7 expression. CHIP assay and dual-luciferase reporter assay were conducted to analyze the combination between FGF7 and SP1. FGF7 was upregulated in DC patients and HG-induced SRA01/04 cells. HG treatment promoted SRA01/04 cell viability, migration, invasion and epithelial-mesenchymal transition (EMT), while FGF7 knockdown abated the effects. Transcription factor SP1 activated the transcription level of FGF7 and SP1 overexpression aggravated HG-induced SRA01/04 cell injury. SP1 silencing repressed HG-induced SRA01/04 cell viability, migration, invasion and EMT, but these effects were ameliorated by upregulating FGF7. Additionally, SP1 knockdown inhibited the PI3K/AKT pathway by regulating the transcription level of FGF7. Transcription factor SP1 activated the transcription level of FGF7 and the PI3K/AKT pathway to regulate HG-induced SRA01/04 cell viability, migration, invasion and EMT.

Integrating serum pharmacochemistry and network pharmacology to explore potential compounds and mechanisms of Alpiniae oxyphyllae fructus in the treatment of cellular senescence in diabetic kidney disease.

Diabetic kidney disease (DKD), one of the microvascular complications in patients with diabetes mellitus, is a common cause of end-stage renal disease. Cellular senescence is believed to be an essential participant in the pathogenesis of DKD. Although there is evidence that Alpiniae oxyphyllae fructus (AOF) can ameliorate DKD progression and organismal senescence, its ability to ameliorate renal cellular senescence in DKD as well as active components and molecular mechanisms remain to be explored. This study aimed to investigate the role of AOF in the treatment of cellular senescence in DKD and to explore its active components and potential molecular mechanisms. The pharmacological efficacy of AOF in ameliorating cellular senescence in DKD was assessed by establishing DKD mouse models and HK-2 cells under high glucose stress. UHPLC-QTOF-MS was used to screen the active compounds in AOF, which were used in conjunction with network pharmacology to predict the molecular mechanism of AOF in the treatment of cellular senescence in DKD. In vivo experiments showed that AOF reduced GLU, mAlb, Scr, BUN, MDA, SOD levels, and ameliorated renal pathological damage and renal cell senescence in DKD mice. In vitro experiments showed that AOF-containing serum improved the decline in HK-2 cell viability and alleviated cellular senescence under high glucose intervention. The results of the UHPLC-QTOF-MS screened 26 active compounds of AOF. The network pharmacological analyses revealed that Cubebin, 2',6'-dihydroxy-4'-methoxydihydrochalcone, Chalcone base + 3O,1Prenyl, Batatasin IV, and Lucidenolactone were the five core compounds and TP53, SRC, STAT3, PIK3CA, and AKT1 are the five core targets of AOF in the treatment of DKD. Molecular docking simulation results showed that the five core compounds had good binding ability to the five core targets. Western blot validated the network pharmacological prediction results and showed that AOF and AOF-containing serum down-regulate the expression of TP53, and phosphorylation of SRC, STAT3, PIK3CA, and AKT. Our study shows that AOF may delay the development of cellular senescence in DKD by down-regulating the levels of TP53, and phosphorylation of SRC, STAT3, PIK3CA, and AKT.