High Glucose-induced Cell Apoptosis Research Articles

PSAT1 is upregulated by METTL3 to attenuate high glucose-induced retinal pigment epithelial cell apoptosis and oxidative stress

BackgroundDiabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, and a significant cause of visual impairment and blindness in adults. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme participating in serine synthesis, which might improve insulin signaling and insulin sensitivity. Furthermore, it has been reported that the m6A methylation in mRNA controls gene expression under many physiological and pathological conditions. Nevertheless, the influences of m6A methylation on PSAT1 expression and DR progression at the molecular level have not been reported.MethodsHigh-glucose (HG) was used to treat human retinal pigment epithelial cells (ARPE-19) to construct a cell injury model. PSAT1 and Methyltransferase-like 3 (METTL3) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). PSAT1, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and METTL3 protein levels were examined by western blot assay. Cell viability and apoptosis were detected by Cell Counting Kit-8 (CCK-8) and TUNEL assays. Reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidase (GSH-Px) levels were examined using special assay kits. Interaction between METTL3 and PSAT1 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assay.ResultsPSAT1 and METTL3 levels were decreased in DR patients and HG-treated ARPE-19 cells. Upregulation of PSAT1 might attenuate HG-induced cell viability inhibition and apoptosis and oxidative stress promotion in ARPE-19 cells. Moreover, PSAT1 was identified as a downstream target of METTL3-mediated m6A modification. METTL3 might improve the stability of PSAT1 mRNA via m6A methylation.ConclusionMETTL3 might mitigate HG-induced ARPE-19 cell damage partly by regulating the stability of PSAT1 mRNA, providing a promising therapeutic target for DR.

Hyperglycemia-depleted glutamine contributes to the pathogenesis of diabetic corneal endothelial dysfunction

Diabetic mellitus causes various complications, including the corneal endothelial dysfunction (CED) that leads to corneal edema and vision loss, especially in the DM patients with intraocular surgeries. However, the pathogenic mechanism of hyperglycemia-caused CED remains incomplete understood. Here we firstly screened and identified the glutamine contents of anterior humor were significantly reduced in both the type 2 diabetic patients and streptozotocin-induced type 1 diabetic mice. To explore the potential therapeutic effects of glutamine supplement on the protection of diabetic corneal endothelial dysfunction, we performed the anterior chamber perfusion with the addition of L-alanyl-L-glutamine (Ala-Gln), and confirmed that Ala-Gln supplement not only accelerated the resolution of corneal edema and recovery of corneal thickness, but also preserved the regular arrangement and barrier-pomp function of corneal. Mechanistically, we revealed that the supplements of Ala-Gln protect corneal endothelial cells from the deleterious effects of high glucose-induced oxidative stress, mitochondrial dysfunction, and cell apoptosis. Overall, these results indicate the glutamine depletion plays an important role in the diabetic corneal endothelial dysfunction, while the Ala-Gln supplement during intraocular surgery provide an effective prevention strategy through regulating the redox homeostasis and mitochondrial function of corneal endothelial cells.

Retraction: Resveratrol attenuates high glucose-induced nucleus pulposus cell apoptosis and senescence through activating the ROS-mediated PI3K/Akt pathway.

Retraction: Resveratrol attenuates high glucose-induced nucleus pulposus cell apoptosis and senescence through activating the ROS-mediated PI3K/Akt pathway.

High CD133 expression in proximal tubular cells in diabetic kidney disease: good or bad?

BackgroundProximal tubular cells (PTCs) play a critical role in the progression of diabetic kidney disease (DKD). As one of important progenitor markers, CD133 was reported to indicate the regeneration of dedifferentiated PTCs in acute kidney disease. However, its role in chronic DKD is unclear. Therefore, we aimed to investigate the expression patterns and elucidate its functional significance of CD133 in DKD.MethodsData mining was employed to illustrate the expression and molecular function of CD133 in PTCs in human DKD. Subsequently, rat models representing various stages of DKD progression were established. The expression of CD133 was confirmed in DKD rats, as well as in human PTCs (HK-2 cells) and rat PTCs (NRK-52E cells) exposed to high glucose. The immunofluorescence and flow cytometry techniques were utilized to determine the expression patterns of CD133, utilizing proliferative and injury indicators. After overexpression or knockdown of CD133 in HK-2 cells, the cell proliferation and apoptosis were detected by EdU assay, real-time cell analysis and flow analysis. Additionally, the evaluation of epithelial, progenitor cell, and apoptotic indices was performed through western blot and quantitative RT-PCR analyses.ResultsThe expression of CD133 was notably elevated in both human and rat PTCs in DKD, and this expression increased as DKD progressed. CD133 was found to be co-expressed with CD24, KIM-1, SOX9, and PCNA, suggesting that CD133+ cells were damaged and associated with proliferation. In terms of functionality, the knockdown of CD133 resulted in a significant reduction in proliferation and an increase in apoptosis in HK-2 cells compared to the high glucose stimulus group. Conversely, the overexpression of CD133 significantly mitigated high glucose-induced cell apoptosis, but had no impact on cellular proliferation. Furthermore, the Nephroseq database provided additional evidence to support the correlation between CD133 expression and the progression of DKD. Analysis of single-cell RNA-sequencing data revealed that CD133+ PTCs potentially play a role in the advancement of DKD through multiple mechanisms, including heat damage, cell microtubule stabilization, cell growth inhibition and tumor necrosis factor-mediated signaling pathway.ConclusionOur study demonstrates that the upregulation of CD133 is linked to cellular proliferation and protects PTC from apoptosis in DKD and high glucose induced PTC injury. We propose that heightened CD133 expression may facilitate cellular self-protective responses during the initial stages of high glucose exposure. However, its sustained increase is associated with the pathological progression of DKD. In conclusion, CD133 exhibits dual roles in the advancement of DKD, necessitating further investigation.

Integrin β5 subunit regulates hyperglycemia-induced vascular endothelial cell apoptosis through FoxO1-mediated macroautophagy.

Hyperglycemia frequently induces apoptosis in endothelial cells and ultimately contributes to microvascular dysfunction in patients with diabetes mellitus (DM). Previous research reported that the expression of integrins as well as their ligands was elevated in the diseased vessels of DM patients. However, the association between integrins and hyperglycemia-induced cell death is still unclear. This research was designed to investigate the role played by integrin subunit β5 (ITGB5) in hyperglycemia-induced endothelial cell apoptosis. We used leptin receptor knockout (Lepr-KO) ( db / db ) mice as spontaneous diabetes animal model. Selective deletion of ITGB5 in endothelial cell was achieved by injecting vascular targeted adeno-associated virus via tail vein. Besides, we also applied small interfering RNA in vitro to study the mechanism of ITGB5 in regulating high glucose-induced cell apoptosis. ITGB5 and its ligand, fibronectin, were both upregulated after exposure to high glucose in vivo and in vitro . ITGB5 knockdown alleviated hyperglycemia-induced vascular endothelial cell apoptosis and microvascular rarefaction in vivo.In vitro analysis revealed that knockdown of either ITGB5 or fibronectin ameliorated high glucose-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). In addition, knockdown of ITGB5 inhibited fibronectin-induced HUVEC apoptosis, which indicated that the fibronectin-ITGB5 interaction participated in high glucose-induced endothelial cell apoptosis. By using RNA-sequencing technology and bioinformatic analysis, we identified Forkhead Box Protein O1 (FoxO1) as an important downstream target regulated by ITGB5. Moreover, we demonstrated that the excessive macroautophagy induced by high glucose can contribute to HUVEC apoptosis, which was regulated by the ITGB5-FoxO1 axis. The study revealed that high glucose-induced endothelial cell apoptosis was positively regulated by ITGB5, which suggested that ITGB5 could potentially be used to predict and treat DM-related vascular complications.

Homoplantaginin attenuates high glucose-induced vascular endothelial cell apoptosis through promoting autophagy via the AMPK/TFEB pathway.

Vascular endothelial cell (VEC) injury is a key factor in the development of diabetic vascular complications. Homoplantaginin (Hom), one of the main flavonoids from Salvia plebeia R. Br. has been reported to protect VEC. However, its effects and mechanisms against diabetic vascular endothelium remain unclear. Here, the effect of Hom on VEC was assessed using high glucose (HG)-treated human umbilical vein endothelial cells and db/db mice. In vitro, Hom significantly inhibited apoptosis and promoted autophagosome formation and lysosomal function such as lysosomal membrane permeability and the expression of LAMP1 and cathepsin B. The antiapoptosis effect of Hom was reversed by autophagy inhibitor chloroquine phosphate or bafilomycin A1. Furthermore, Hom promoted gene expression and nuclear translocation of transcription factor EB (TFEB). TFEB gene knockdown attenuated the effect of Hom on upregulating lysosomal function and autophagy. Moreover, Hom activated adenosine monophosphate-dependent protein kinase (AMPK) and inhibited the phosphorylation of mTOR, p70S6K, and TFEB. These effects were attenuated by AMPK inhibitor Compound C. Molecular docking showed a good interaction between Hom and AMPK protein. Animal studies indicated that Hom effectively upregulated the protein expression of p-AMPK and TFEB, enhanced autophagy, reduced apoptosis, and alleviated vascular injury. These findings revealed that Hom ameliorated HG-mediated VEC apoptosis by enhancing autophagy via the AMPK/mTORC1/TFEB pathway.

Exosomes derived from mesenchymal stem cells attenuate diabetic kidney disease by inhibiting cell apoptosis and epithelial-to-mesenchymal transition via miR-424-5p.

Diabetic kidney disease (DKD) is well-acknowledged as one of the most common complications in diabetes mellitus. Recent studies have demonstrated the promising role of mesenchymal stem cell-derived exosomes (MSC-exos) as a cell-free treatment strategy for DKD. The present study sought to investigate the therapeutic potential and the underlying mechanisms of MSC-exos in DKD. The authentication of MSC-exos was validated by western blot, transmission electron microscope (TEM), and nanosight tracking analysis (NTA). Apoptosis was detected by western blot, TUNEL staining, and flow cytometry. Epithelial-to-mesenchymal transition (EMT) was evaluated by western blot and immunofluorescence. The relationship between miR-424-5p and Yes-associated protein 1 (YAP1) was revealed by dual luciferase reporter assay. We observed that MSC-exos could attenuate DKD by decreasing cell apoptosis and inhibiting epithelial-to-mesenchymal transition (EMT) in diabetic kidneys in db/db mice. Besides, we documented that MSC-exos could reverse high glucose-induced apoptosis and EMT in HK2 cells. Interestingly, miR-424-5p derived from MSC-exos could inhibit YAP1 activation in HK2 cells, resulting in alleviation of high glucose-induced cell apoptosis and EMT. Our study provides novel insights into MSC-exos-mediated protective effect in DKD. MSC-exos could inhibit high glucose-induced apoptosis and EMT through miR-424-5p targeting of YAP1.

High Glucose-Induced Human Kidney Cell Apoptosis and Inflammatory Injury Are Alleviated by Circ_0008529 Knockdown via Circ_0008529-Mediated miR-485-5p/WNT2B Signaling.

Human kidney cell injury is a representative characteristic of diabetic nephropathy (DN), and its development has been shown to be associated with the dysregulation of some circular RNAs (circRNAs). We thus explored the role of circ_0008529 in DN-conditioned human kidney cell injury.Human kidney cells (HK-2) were treated with high glucose (HG) to construct DN models in vitro. Quantitative real-time PCR (qPCR) assay and western blot assay were used for expression detection of circ_0008529, miR-485-5p, and Wnt family member 2B (WNT2B). Cell viability was ascertained by CCK-8 assay. Cell apoptosis was assessed by flow cytometry assay and the expression levels of apoptosis-related markers. The release of inflammatory factors was examined by ELISA. The putative binding relationship between miR-485-5p and circ_0008529 or WNT2B was further verified by dual-luciferase reporter experiment, RIP assay, and pull-down assay.Circ_0008529 was highly expressed in serum of DN patients and HG-treated HK-2 cells. HG largely impaired cell viability and promoted cell apoptosis and inflammation production, while circ_0008529 knockdown attenuated the effects of HG. Circ_0008529 targeted miR-485-5p, and miR-485-5p inhibition recovered HK-2 cell injury that was blocked by circ_0008529 knockdown. In addition, miR-485-5p bound to WNT2B whose expression was positively modulated by circ_0008529. WNT2B overexpression recovered the inhibition of HK-2 cell injury caused by miR-485-5p upregulation.Circ_0008529 targeted the miR-485-5p/WNT2B pathway to regulate HG-induced HK-2 cell apoptosis and inflammatory injury, suggesting that circ_0008529 was a vital regulator in DN development.

Circular RNA circHIPK3 is downregulated in diabetic cardiomyopathy and overexpression of circHIPK3 suppresses PTEN to protect cardiomyocytes from high glucose-induced cell apoptosis

ABSTRACT It has been reported that circHIPK3 can be downregulated by high glucose, suggesting its potential involvement in diabetes and diabetic complications. This study aimed to explore the role of circHIPK3 in diabetic cardiomyopathy (DC). PTEN is a kind of tumor suppressor gene, which is very commonly lost in human cancer. We detected the expression of circHIPK3 and PTEN in plasma samples from DC patients, diabetic patients without complications diabetes mellitus (DM) and health controls by RT-qPCR and ELISA. In vitro cell experiment, AC16 cells (cardiomyocytes) were treated with high glucose, followed by expression analysis of circHIPK3 and PTEN mRNA by RT-qPCR. CircHIPK3 or PTEN expression vector were used to overexpress circHIPK3 and PTEN in AC16 cells to explore the relationship between them. The role of circHIPK3 and PTEN in regulating the apoptosis of AC16 cells was analyzed by cell apoptosis assay. The result showed that CircHIPK3 was downregulated in diabetes and further downregulated in DC. In AC16 cells, high glucose treatment decreased the expression levels of circHIPK3. Across DC samples, the expression of circHIPK3 was inversely correlated with PTEN. In AC16 cells, overexpression of circHIPK3 decreased the expression levels of PTEN. CircHIPK3 may suppress AC16 cell apoptosis induced by high glucose and inhibited the role of PTEN in cell apoptosis. Therefore, circHIPK3 may downregulate PTEN to protect cardiomyocytes from high glucose-induced cell apoptosis.

CircPAG1 interacts with miR-211-5p to promote the E2F3 expression and inhibit the high glucose-induced cell apoptosis and oxidative stress in diabetic cataract

ABSTRACT Circular RNAs (circRNAs) are regulatory endogenous RNAs in human diseases by sponging microRNAs (miRNAs) to affect the gene expression. However, little research focused on the circRNA/miRNA/mRNA axis in diabetic cataract. This study was performed for the exploration of circRNA phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (circPAG1) in diabetic cataract. Human lens epithelial cells were treated with high glucose. The quantitative real-time polymerase chain reaction was used for the expression detection of circPAG1, microRNA-211-5p (miR-211-5p), and E2F transcription factor 3 (E2F3). Cell viability and proliferation were detected using Cell Counting Kit-8 assay and EdU assay. Cell apoptosis was analyzed by flow cytometry. The protein levels were measured by Western blot. Oxidative stress was assessed by malondialdehyde, reactive oxygen species, and superoxide dismutase via the corresponding detection kits. The target interaction was validated using the dual-luciferase reporter assay and RNA immunoprecipitation assay. The expression of circPAG1 was downregulated in diabetic cataract patients. The upregulation of circPAG1 could attenuate the high glucose-induced inhibition of cell viability and proliferation but promotion of cell apoptosis and oxidative stress. CircPAG1 served as a miR-211-5p sponge, and the protective role of circPAG1 was partly achieved by sponging miR-211-5p. MiR-211-5p targeted E2F3 and circPAG1 upregulated the E2F3 level by absorbing miR-211-5p. Inhibition of miR-211-5p repressed the high glucose-mediated cell dysfunction by increasing the expression of E2F3. This study clarified that circPAG1 protected human lens epithelial cells from the high glucose-induced cell damages by the mediation of miR-211-5p/E2F3 axis.

TFPI2 suppresses the interaction of TGF-β2 pathway regulators to promote endothelial–mesenchymal transition in diabetic nephropathy

Endothelial–mesenchymal transition (EndMT) is an important source of myofibroblasts, but also contributes to the progression of diabetic nephropathy (DN). By several differential gene expression analyses from the Gene Expression Omnibus (GEO) database, the tissue factor pathway inhibitor 2 (TFPI2) gene, known as a tumor suppressor, was shown to be dysregulated in DN; however, the potential role and regulatory mechanism of TFPI2 in DN are unclear. Here, we found abnormal upregulation of TFPI2 in the renal cortex of diabetic mice, accompanied by impaired renal function. We also injected a single dose of adeno-associated virus (AAV)2 carrying shRNA targeting TFPI2 intravenously into these mice and found that knockdown of TFPI2 improved renal function and reduced renal fibrosis and cell apoptosis in experimental DN. Furthermore, hyperglycemia-induced EndMT was inhibited in the absence of TFPI2, as evidenced by increased expression of endothelial markers (VE-cadherin and CD31) and decreased expression of mesenchymal markers (α-SMA, desmin, and FSP-1). To further explore the mechanism in vitro, human renal glomerular endothelial cells (hRGECs) were incubated in the presence of high glucose or transforming growth factor beta (TGF-β)2. TFPI2 deficiency inhibited high glucose-induced cell apoptosis and TGF-β2-induced EndMT in hRGECs, while overexpression of TFPI2 had the opposite effects. Importantly, TGF-β2 is a crucial driver of EndMT, and we found that TFPI2 promoted TGF-β2/Smad signaling activation by interferring the interaction of TGF-β pathway regulators (SMURF2 with SMAD7). Our results show that TFPI2 regulates EndMT and the TGF-β2 signaling pathway and is a potential promoter of DN pathogenesis.

A Novel MIR503HG/miR-497-5p/CCL19 Axis Regulates High Glucose-Induced Cell Apoptosis, Inflammation, and Fibrosis in Human HK-2 Cells.

Long non-coding RNAs (lncRNAs) play crucial roles in the development of diabetic nephropathy (DN). Here, we explored the activity and mechanism of MIR503 host gene (MIR503HG) in high glucose (HG)-evoked cytotoxicity in HK-2 cells. MIR503HG, microRNA (miR)-497-5p, and C-C motif chemokine ligand 19 (CCL19) were quantified by quantitative real-time PCR (qRT-PCR) and western blot. The direct relationship between miR-497-5p and MIR503HG or CCL19 was confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Cell viability and apoptosis were evaluated by XTT assay and flow cytometry, respectively. Our data showed that MIR503HG was overexpressed in HG-stimulated HK-2 cells. Knockdown of MIR503HG alleviated HG-evoked cell apoptosis, inflammation, and fibrosis in HK-2 cells. Mechanistically, MIR503HG regulated miR-497-5p expression via a binding site. MIR503HG depletion reduced HG-evoked cell apoptosis, inflammation, and fibrosis in HK-2 cells by up-regulating miR-497-5p. Moreover, miR-497-5p directly targeted and suppressed CCL19. MiR-497-5p-mediated suppression of CCL19 relieved HG-induced cell apoptosis, inflammation, and fibrosis in HK-2 cells. Furthermore, MIR503HG regulated CCL19 expression via miR-497-5p competition. Our findings identify a new MIR503HG/miR-497-5p/CCL19 network in the regulating HG-evoked cell apoptosis, inflammation, and fibrosis in HK-2 cells.

Ginsenoside Rb1 alleviates diabetic kidney podocyte injury by inhibiting aldose reductase activity.

Panax notoginseng, a traditional Chinese medicine, exerts beneficial effect on diabetic kidney disease (DKD), but its mechanism is not well clarified. In this study we investigated the effects of ginsenoside Rb1 (Rb1), the main active ingredients of Panax notoginseng, in alleviating podocyte injury in diabetic nephropathy and the underlying mechanisms. In cultured mouse podocyte cells, Rb1 (10 μM) significantly inhibited high glucose-induced cell apoptosis and mitochondrial injury. Furthermore, Rb1 treatment reversed high glucose-induced increases in Cyto c, Caspase 9 and mitochondrial regulatory protein NOX4, but did not affect the upregulated expression of aldose reductase (AR). Molecular docking analysis revealed that Rb1 could combine with AR and inhibited its activity. We compared the effects of Rb1 with eparestat, a known aldose reductase inhibitor, in high glucose-treated podocytes, and found that both alleviated high glucose-induced cell apoptosis and mitochondrial damage, and Rb1 was more effective in inhibiting apoptosis. In AR-overexpressing podocytes, Rb1 (10 μM) inhibited AR-mediated ROS overproduction and protected against high glucose-induced mitochondrial injury. In streptozotocin-induced DKD mice, administration of Rb1 (40 mg·kg-1·d-1, ig, for 7 weeks) significantly mitigated diabetic-induced glomerular injuries, such as glomerular hypertrophy and mesangial matrix expansion, and reduced the expression of apoptotic proteins. Collectively, Rb1 combines with AR to alleviate high glucose-induced podocyte apoptosis and mitochondrial damage, and effectively mitigates the progression of diabetic kidney disease.

Plumbagin attenuates high glucose-induced trophoblast cell apoptosis and insulin resistance via activating AKT/mTOR pathway

Plumbagin, a bioactive phytoconstituent, is isolated from the root of Plumbago zeylanica L. Plumbagin possesses antidiabetic effect to mediate glucose homeostasis, wound healing and diabetic nephropathy. However, the involvement of plumbagin in gestational diabetes mellitus (GDM) has not been reported yet. Trophoblast cell line (HTR8/SVneo) was incubated with high glucose to establish cell model of GDM. Cell viability and proliferation were detected by MTT and EdU staining. Flow cytometry was used to investigate cell apoptosis. Cell viability of HTR8/SVneo was reduced by high glucose or incubation of plumbagin. Plumbagin restored reduced cell viability and proliferation of HTR8/SVneo induced by high glucose. Plumbagin attenuated high glucose-induced cell apoptosis in HTR8/SVneo cells through upregulation of Bcl-2 and down-regulation of Bax, cleaved caspase-3 and cleaved caspase-9. Protein expression of glucose transporter type 4 (GLUT-4), insulin receptor (INSR)-B and INSR substrate (IRS1) was decreased in high glucose-induced HTR8/SVneo but increased by plumbagin. The suppressive effects of high glucose on phosphorylation of AKT and mTOR in HTR8/SVneo were reversed by plumbagin. Plumbagin improved high glucose-induced cell apoptosis and insulin resistance of HTR8/SVneo through activation of AKT/mTOR pathway, suggesting that plumbagin might be used as a potential strategy for the prevention of GDM.

Circ_0000615 promotes high glucose-induced human retinal pigment epithelium cell apoptosis, inflammation and oxidative stress via miR-646/YAP1 axis in diabetic retinopathy.

Diabetic retinopathy (DR), a common complication of diabetes mellitus, is the major cause of visual impairment and blindness. Circ_0000615 was found to be elevated in retina samples of diabetic patients. Hence, the detailed effects and molecular mechanisms of circ_0000615 in DN progression were explored. The levels of circ_0000615, microRNA (miR)-646 and YAP1 (yes-associated protein 1) were detected using quantitative real-time polymerase chain reaction and Western blot assays. Cell viability, apoptosis, inflammation and reactive oxygen species (ROS) generation were determined using cell counting kit-8 assay, flow cytometry, caspase3 activity analysis, Western blot, enzyme-linked immunosorbent assay (ELISA) and Dichlorofluorescein diacetate (DCFH-DA) assay, respectively. The binding interaction between miR-646 and circ_0000615 or YAP1 was determined using dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. Circ_0000615 was elevated in high glucose (HG)-induced human retinal pigment epithelium (HRPE) cells. Knockdown of circ_0000615 attenuated HG-triggered HRPE cell apoptosis, inflammation, and ROS generation. Mechanistically, miR-646 was confirmed to be a target of circ_0000615, inhibition of miR-646 reversed the protective effects of circ_0000615 knockdown on HG-evoked HRPE cell dysfunction. MiR-646 was verified to target YAP1, overexpression of YAP1 abolished the impairment induced by miR-646 on HG-induced HRPE cell damage. Besides that, we confirmed that circ_0000615 could regulate YAP1 expression via miR-646. Circ_0000615 contributed to HG-induced HRPE cell dysfunction via miR-646/YAP1 axis, suggesting a novel insight into the pathogenesis of DR and a potential candidate for DR treatment.

Role of microRNA-25 in high glucose cultured Müller glia.

To investigate the role of microRNA-25 (miR-25) in proliferation and apoptosis of retinal Müller glia (MG) under high glucose condition. The purity of the cultured cells was verified by immunocytochemistry and flow cytometry using antibodies that specifically recognized MG. The expression level of miR-25 under normal and high glucose conditions were validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). miR-25 mimics and negative control were transfected into MG and multiple functional experiments including cell counting kit-8 assay, EDU assay, and flow cytometry were conducted to explore the effects of miR-25 on the proliferation and apoptosis of high glucose cultured MG (HGMG). Immunocytochemistry and flow cytometry confirmed the high purity of primary cultured MG. RT-PCR results showed that the expression level of miR-25 was significantly repressed in HGMG, while over-expression of miR-25 by miR-25 mimic markedly inhibited the high glucose induced cell apoptosis and promoted the proliferation of MG. The expression level of miR-25 is significantly downregulated in HGMG and its overexpression could attenuate the high glucose damages on MG by promoting proliferation and reducing apoptosis.

Erythropoietin inhibits apoptosis of retinal ganglion cells induced by high glucose through JNK signaling pathway

The aim of this study was to investigate the effect of erythropoietin (EPO) on the apoptosis of retinal ganglion cells (RGCs) induced by high glucose and its mechanism. Rat primary RGCs were extracted to establish high glucose-induced apoptosis models using a 30 mM high-glucose medium. Then flow cytometry, cell counting kit-8 (CCK-8) assay and Western blotting assay were performed to detect the effects of high-, medium- and low-dose EPO on the apoptosis of RGCs induced by high glucose. Next, the molecular mechanism by which EPO suppressed the high glucose-induced apoptosis of RGCs was explored via gene array assay and bioinformatics analysis. The results and mechanism of bioinformatics analysis were verified by Western blotting assay. Finally, the small interfering ribonucleic acid (siRNA) experiment was applied to knock down tyrosine-protein phosphatase non-receptor type 1 (PTPN1) and PTPN11 to verify their roles in the inhibition of EPO on the apoptosis of RGCs triggered by high glucose. Flow cytometry-Annexin V/propidium iodide (PI) staining and CCK-8 assay confirmed that the high-, medium- and low-dose EPO inhibited the apoptosis of RGCs induced by high glucose in a dose-dependent manner (P<0.05). Subsequently, Western blotting assay results manifested that the high-, medium- and low-dose EPO reduced the expression levels of apoptosis-related proteins active-cysteinyl aspartate specific proteinase 3 (Caspase 3) and active- Caspase 9 in a dose-dependent manner (P<0.05). Moreover, according to gene array assay and bioinformatics analysis results, the c-Jun N-terminal kinase (JNK) signaling pathway, PTPN1 and PTPN11 might exert crucial effects in the inhibition of EPO on the apoptosis of RGCs induced by high glucose. Western blotting assay results also demonstrated that, compared with the high-glucose treatment, the high-dose EPO treatment decreased the protein expression level of phosphorylated (p)-JNK1/JNK but increased the protein expression levels of PTPN1 and PTPN11 (P<0.05). Moreover, flow cytometry-Annexin V/PI staining and CCK-8 assay results revealed that in EPO-treated cells, knocking down PTPN1 and PTPN11 significantly reversed the protective effect of EPO against high glucose-induced retinal ganglion cell apoptosis (P<0.05). Lastly, Western blotting assay illustrated that knocking down PTPN1 and PTPN11 significantly abolished the inhibition of high-dose EPO on the JNK signaling pathway. EPO may suppress the JNK signaling pathway by raising the expression levels of PTPN1 and PTPN11, so as to inhibit the apoptosis of RGCs triggered by high glucose.

Upregulation of miRNA-23a-3p rescues high glucose-induced cell apoptosis and proliferation inhibition in cardiomyocytes

Maternal hyperglycemia potentially inhibits the development of the fetal heart by suppressing cardiomyocyte proliferation and promoting apoptosis. Different studies have indicated that miRNAs are key regulators of cardiomyocyte proliferation, differentiation, and apoptosis and play a protective role in a variety of cardiovascular diseases. However, the biological function of miRNA-23a in hyperglycemia-related cardiomyocyte injury is not fully understood. The present study investigated the effect of miRNA-23a-3p on cell proliferation and apoptosis in a myocardial injury model induced by high glucose. H9c2 cardiomyocytes were exposed to high glucose to establish an in vitro myocardial injury model and then transfected with miRNA-23a-3p mimics. After miRNA-23a-3p transfection, lens-free microscopy was used to dynamically monitor cell numbers and confluence and calculate the cell cycle duration. CCK-8 and EdU incorporation assays were performed to detect cell proliferation. Flow cytometry was used to measured cell apoptosis. Upregulation of miRNA-23a-3p significantly alleviated high glucose-induced cell apoptosis and cell proliferation inhibition (p < 0.01 and p < 0.0001, respectively). The cell cycle of the miRNA-23a-3p mimics group was significantly shorter than that of the negative control group (p < 0.01). The expression of cell cycle–activating and apoptosis inhibition-associated factors Ccna2, Ccne1, and Bcl-2 was downregulated by high glucose and upregulated by miRNA-23a-3p overexpression in high glucose-injured H9c2 cells. miRNA-23a-3p mimics transfection before high glucose treatment had a significantly greater benefit than transfection after high glucose treatment (p < 0.0001), and the rescue effect of miRNA-23a-3p increased as the concentration increased. This study suggests that miRNA-23a-3p exerted a dose- and time-dependent protective effect on high glucose-induced H9c2 cardiomyocyte injury.

Effects of icariin and quercetin on high glucose-induced neuronal cell apoptosis

Purpose: To study the effects of icariin and quercetin on cell apoptotic changes in neurons induced by elevated glucose condition, and the mechanism involved.&#x0D; Methods: Neonatal male Sprague Dawley rats (n = 48) weighing 5 – 7 g were used. Neuronal cells were isolated from rat hippocampus and cultured after purification. The cells were randomly assigned to six groups: control, high glucose, icariin, quercetin, serine/threonine-specific protein kinase (Akt) inhibitor, and Akt agonist groups. The Akt inhibitor and agonist used in this study were MK-22062hci and SC79, respectively. The influence of icariin and quercetin on neuronal apoptotic changes were determined flow cytometrically, while their effects on levels of expression of Akt, p-Akt, bax and bcl-2 were determined with Western blotting.&#x0D; Results: Treatment with icariin or quercetin significantly inhibited apoptosis induced by high glucose. The concentrations of Akt, p-Akt, and bcl-2 proteins were markedly upregulated in high glucose group, relative to control (p &lt; 0.05). The corresponding expression of bax was significantly down-regulated in high glucose group, relative to control (p &lt; 0.05). Treatment with icariin or quercetin, or their agonists reversed high glucose-mediated alterations in these protein levels (p &lt; 0.05).&#x0D; Conclusion: Icariin and quercetin inhibit neuronal cell apoptosis induced by high glucose through upregulation of bcl-2 expression and down- regulations of bax expression and Akt-induced protein phosphorylation. Thus, Icariin and quercetin possess potential benefits for the treatment of neurological diseases.&#x0D; Keywords: Apoptosis, High glucose condition, Hippocampal neurons, Icariin, Quercetin

Circ_LARP4 regulates high glucose-induced cell proliferation, apoptosis, and fibrosis in mouse mesangial cells.

The current study aimed to investigate the role and underlying mechanisms of circ_LARP4 in diabetic nephropathy (DN). Here, mouse mesangial cells (SV40-MES13) were cultured with 30mM glucose to establish a DN cellular model. The qRT-PCR results indicated that circ_LARP4 expression was downregulated in the DN cellular model compared to that in the control cells. As determined by an MTT assay, circ_LARP4 overexpression via the circ_LARP4 overexpression (OE) plasmids inhibited the cell proliferation rate. As determined by an Annexin V/PI kit and flow cytometry, circ_LARP4 overexpression increased the cell apoptosis rate. As measured by Western blot, circ_LARP4 overexpression enhanced BAX expression but reduced Bcl-2 expression, also suggesting an enhancement of cell apoptosis. Moreover, regarding cell fibrosis, circ_LARP4 overexpression reduced the mRNA levels of fibrosis markers, including fibronectin, collagen I and collagen IV. Interestingly, miR-424 was found to be reduced in the DN cellular model after transfection with the circ_LARP4 OE plasmids. In addition, restoration of miR-424 expression with the miR-424 mimics reversed the negative effects of circ_LARP4 overexpression on cell proliferation and fibrosis. In conclusion, circ_LARP4 was lower in the DN cellular model than in normal cells, and circ_LARP4 overexpression resulted in decreased cell proliferation and cell fibrosis but increased cell apoptosis in the DN cellular model by sponging miR-424.