HG-induced Apoptosis Research Articles

Inhibition of NLRP3 inflammasome ameliorates podocyte damage by suppressing lipid accumulation in diabetic nephropathy

BackgroundNucleotide leukin-rich polypeptide 3 (NLRP3) inflammasome is documented as a potent target for treating metabolic diseases and inflammatory disorders. Our recent work demonstrated that inhibition of NLRP3 inflammasome activation inhibits renal inflammation and fibrosis in diabetic nephropathy. This study was to investigate the effect of NLRP3 inflammasome on podocyte injury and the underlying mechanism in diabetic nephropathy. MethodsIn vivo, db/db mice were treated with MCC950, a NLRP3 inflammasome specific inhibitor. NLRP3 knockout (NKO) mice were induced to diabetes by intraperitoneal injections of streptozotocin (STZ). We assessed renal function, albuminuria, podocyte injury and glomerular lipid accumulation in diabetic mice. In vitro, apoptosis, cytoskeleton change, lipid accumulation, NF-κB p65 activation and reactive oxygen species (ROS) generation were evaluated in podocytes interfered with NLRP3 siRNA or MCC950 under high glucose (HG) conditions. In addition, the effect and mechanism of IL-1β on lipid accumulation was explored in podocytes exposed to normal glucose (NG) or HG. ResultsMCC950 treatment improved renal function, attenuated albuminuria, mesangial expansion, podocyte loss, as well as glomerular lipid accumulation in db/db mice. The diabetes-induced podocyte loss and glomerular lipid accumulation were reversed in NLRP3 knockout mice. The increased expression of sterol regulatory element-binding protein1 (SREBP1) and SREBP2, and decreased expression of ATP-binding cassette A1 (ABCA1) in podocytes were reversed by MCC950 treatment or NLRP3 knockout in diabetic mice. In vitro, NLRP3 siRNA or MCC950 treatment markedly inhibited HG-induced apoptosis, cytoskeleton change, lipid accumulation, NF-κB p65 activation, and mitochondrial ROS production in cultured podocytes. In addition, BAY11–7082 or tempol treatment inhibited HG-induced lipid accumulation in podocytes. Moreover, exposure of IL-1β to podocytes induced lipid accumulation, NF-κB p65 activation and mitochondrial ROS generation. ConclusionInhibition of NLRP3 inflammasome protects against podocyte damage through suppression of lipid accumulation in diabetic nephropathy. IL-1β/ROS/NF-κB p65 mediates diabetes-associated lipid accumulation in podocytes. The suppression of NLRP3 inflammasome activation may be an effective therapeutic approach to diabetic nephropathy.

MicroRNA-301a-3p promotes diabetic retinopathy via regulation of six-transmembrane epithelial antigen of prostate 4.

Diabetic retinopathy (DR) is one of the most serious microvascular complications of diabetes mellitus (DM). MicroRNAs (miRNAs) have been discovered to play a crucial role in DR, but the mechanisms underlying the effects of miR-301a-3p on DR are poorly understood. This paper was designed to explore the possible role of miR-301a-3p in DR. The diabetic rat model was established by a single intraperitoneal injection of streptozotocin (STZ). The effects of miR-301a-3p on the biological functions of HRECs were determined through a series of experiments in vitro/vivo. The results revealed that interference with miR-301a-3p could decrease the expressions of inflammatory factors and apoptosis in the retinal tissue of DR. Furthermore, it can alleviate the oxidative stress in DR serum, reduce VEGF expression, increase endothelial cell marker expression, and inhibit (High Glucose) HG-induced apoptosis of HRECs. Six-transmembrane epithelial antigen of prostate 4 (STEAP4) was the target of miR-301a-3p. All the effects of miR-301a-3p in DR model were reversed by STEAP4 inhibitor. miR-301a-3p promotes diabetic retinopathy via regulation of STEAP4. The findings in this study may provide a vital reference for the drug research and development in DR treatment.

Elevated MST1 leads to apoptosis via depletion of YAP1 in cardiomyocytes exposed to high glucose

BackgroundGestational diabetes mellitus is a risk factor for congenital heart defects. The article aimed to investigate the expression and roles of MST1, YAP1, Last1/2 and Survivin in modulating HG-induced cardiomyocyte apoptosis and maternal diabetes-induced heart abnormality.MethodsDiabetes mellitus was induced in rats using streptozotocin. The protein expression and phosphorylation analysis in fetal heart tissue was assessed by western blot and immunohistochemical staining. Hoechst 33342 staining assay was performed to explore H9C2 apoptosis. The gene and protein expression in H9C2 cells was assessed by quantitative PCR and western blot. Knockdown of gene expression was assessed by RNA interference.ResultsOur results revealed that increased MST1 protein levels in the heart tissues of the offspring of diabetic rats in vivo and in H9C2 cardiomyocytes under HG treatment in vitro, respectively. Knockdown and overexpression experiments showed that MST1 played a key role in mediating HG-induced apoptosis in cardiomyocytes. Downregulation of YAP1 was associated with HG-induced, MST1-mediated cardiomyocyte apoptosis. Further study showed that MST1 downregulated the protein level of YAP1 through mediation of YAP1 phosphorylation on Ser127 and Ser397; this process also required LATS1/2 participation. MST1 overexpression increased the phosphorylation levels of LATS1/2, which were also shown to be increased in the heart tissues of diabetic offspring. We also found that YAP1 mediated the expression of Survivin during HG-induced apoptosis, and the Survivin-inhibitor YM155 partially inhibited the role of YAP1 in suppressing apoptosis induced by HG in cardiomyocytes.ConclusionThese findings reveal a regulatory mechanism of MST1/YAP1/Survivin signaling in modulating cardiomyocyte apoptosis in vitro and maternal diabetes-induced congenital heart defects in vivo.

Polygonatum sibiricum polysaccharide inhibits high glucose-induced oxidative stress, inflammatory response, and apoptosis in RPE cells

Diabetic retinopathy is one of the major diabetic complications and remains the most common cause of adult blindness among patients with diabetes mellitus. Polygonatum sibiricum polysaccharides (PSP) are a group important component of Polygonatum sibiricum (PS) with anti-diabetic activity. However, the effect and underlying mechanism of PSP on diabetic retinopathy remains unclear. We used high glucose (HG)-stimulated ARPE-19 cells to establish in vitro diabetic retinopathy model. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was performed to evaluate cell viability of ARPE-19 cells. The changes in the ROS production, malondialdehyde (MDA) content, and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were detected to indicate oxidative stress. The secretion levels of tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) were detected by ELISA. The protein levels of TNF-α, IL-8, bcl-2, bax, nuclear Nrf2, and anti-hemeoxygenase-1 (HO-1) were detected by western blot analysis. Our results showed that HG treatment caused a significant reduction in cell viability of ARPE-19 cells. PSP treatment improved the reduced cell viability of ARPE-19 cells. PSP also attenuated HG-induced oxidative stress with decreased ROS production and MDA content, as well as increased the activities of SOD and GPx. In addition, HG significantly increased bax expression and caspase-3 activity, and decreased bcl-2 expression. However, these changes were mitigated by PSP treatment. Furthermore, PSP markedly induced the activation of Nrf2/HO-1 pathway in HG-induced ARPE-19 cells. Knockdown of Nrf2 reversed the protective effects of PSP on HG-induced ARPE-19 cells. Taken together, these findings indicated that PSP protects ARPE-19 cells from HG-induced oxidative stress, inflammation, and cell apoptosis through regulation of Nrf2/HO-1 signaling pathway.

Perilipin 5 ameliorates high-glucose-induced podocyte injury via Akt/GSK-3β/Nrf2-mediated suppression of apoptosis, oxidative stress, and inflammation

Hyperglycemia-induced podocyte damage contributes to the onset of diabetic nephropathy, a severe complication of diabetes. Perilipin 5 (Plin5) exerts a vital role in numerous pathological conditions via affecting cell apoptosis, oxidative stress, and inflammation. However, whether Plin5 plays a role in regulating podocyte damage of diabetic nephropathy has not been fully determined. This work aimed to explore the role of Plin5 in mediating high glucose (HG)-induced injury of podocytes in vitro. Our results demonstrated that Plin5 expression was markedly decreased in mouse podocytes challenged with HG. Plin5 overexpression markedly suppressed HG-induced apoptosis, reactive oxygen species (ROS) production, and the pro-inflammatory response in podocytes. On the contrary, Plin5 silencing produced the opposite effects. Further mechanistic analysis demonstrated that Plin5 upregulation remarkably increased the levels of phospho-Akt and phospho-glycogen synthase kinase-3β (GSK-3β) in HG-exposed podocytes. Moreover, Plin5 overexpression increased the levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and enhanced the activation of Nrf2 signaling. Akt inhibition markedly blocked Plin5-mediated activation of Nrf2, while GSK-3β inhibition reversed Plin5-silencing-induced suppressive effects on Nrf2 activation. Notably, Nrf2 suppression significantly blocked Plin5-mediated protective effects against HG-induced podocyte injury. In summary, our work indicates a vital role for Plin5 in protecting against HG-induced apoptosis, oxidative stress, and inflammation in podocytes via modulation of Akt/GSK-3β/Nrf2 signaling. This study suggests that Plin5 may participate in modulating podocyte damage in diabetic nephropathy.

LncRNA XIST protects podocyte from high glucose-induced cell injury in diabetic nephropathy by sponging miR-30 and regulating AVEN expression

Diabetic nephropathy (DN) is one of the most important complications of diabetes mellitus. Thus, it is urgent to develop a novel diagnosis or therapeutic strategy that could suspend DN progression. Moreover, there is increasing evidence demonstrating that long non-coding RNA (lncRNA) acts as critical players in regulating autophagy and are involved in DN. We demonstrated that lncRNA X-inactive specific transcript (XIST) was downregulated in high glucose (HG) treated podocytes, accompanied by increased apoptosis of podocytes. Overexpression of XIST significantly reduced the apoptosis and promoted the number of viable cells of podocyte under HG treatment. Prediction by Targets can and dual-luciferase reporter assay revealed the interaction between miR-30 and XIST and AVEN. Further WB (Western Blot), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and flow cytometry confirmed that XIST could reverse the expression of AVEN and ameliorate HG-induced apoptosis. In conclusion, our research revealed that XIST plays a protective effect on podocyte injury induced by HG through miR-30/AVEN axis.

High-Glucose-Induced Rab20 Upregulation Disrupts Gap Junction Intercellular Communication and Promotes Apoptosis in Retinal Endothelial and Müller Cells: Implications for Diabetic Retinopathy.

To investigate whether high glucose (HG) alters Rab20 expression and compromises gap junction intercellular communication (GJIC) and cell survival, retinal cells were studied for altered intracellular trafficking of connexin 43 (Cx43). Retinal endothelial cells (RRECs) and retinal Müller cells (rMCs) were grown in normal (N; 5 mM glucose) or HG (30 mM glucose) medium for seven days. In parallel, cells grown in HG medium were transfected with either Rab20 siRNA or scrambled siRNA as a control. Rab20 and Cx43 expression and their localization and distribution were assessed using Western Blot and immunostaining, respectively. Changes in GJIC activity were assessed using scrape load dye transfer, and apoptosis was identified using differential dye staining assay. In RRECs or rMCs grown in HG medium, Rab20 expression was significantly increased concomitant with a decreased number of Cx43 plaques. Importantly, a significant increase in the number of Cx43 plaques and GJIC activity was observed in cells transfected with Rab20 siRNA. Additionally, Rab20 downregulation inhibited HG-induced apoptosis in RRECs and rMCs. Results indicate HG-mediated Rab20 upregulation decreases Cx43 localization at the cell surface, resulting in compromised GJIC activity. Reducing Rab20 expression could be a useful strategy in preventing HG-induced vascular and Müller cell death associated with diabetic retinopathy.

Downregulation of PHLPP1 ameliorates high glucose-evoked injury in retinal ganglion cells by attenuating apoptosis and oxidative stress through enhancement of Nrf2 activation

High glucose (HG)-induced oxidative stress contributes significantly to the pathogenesis of diabetic retinopathy. Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) has emerged as a key regulator of oxidative stress implicated in various pathological processes. However, whether PHLPP1 participates in the regulation of HG-induced oxidative stress injury of retinal ganglion cells (RGCs) in diabetic retinopathy is undetermined. The purpose of this study was to explore the potential role and molecular mechanism of PHLPP1 in regulating HG-induced injury of RGCs. Our data showed that PHLPP1 expression was markedly elevated in RGCs from diabetic rats and HG-exposed RGCs. Our functional assay elucidated that knockdown of PHLPP1 improved cell viability and decreased cell apoptosis and reactive oxygen species (ROS) production in HG-exposed RGCs. Additionally, upregulation of PHLPP1 lowered cell viability and increased cell apoptosis and ROS production in HG-exposed RGCs. Mechanistically, knockdown of PHLPP1 resulted in an increase in nuclear factor erythroid-2 related factor 2 (Nrf2) nuclear expression and Nrf2/antioxidant response element (ARE)-mediated transcription associated with upregulation of glycogen synthase kinase-3β (GSK-3β) phosphorylation. Moreover, inhibition of GSK-3β significantly reversed the suppressive effect of PHLPP1 overexpression on Nrf2/ARE activation. Notably, the protective effect of PHLPP1 knockdown on HG-induced injury in RGCs was markedly abolished by Nrf2 inhibition. In conclusion, Our findings demonstrate that downregulation of PHLPP1 activates Nrf2/ARE signaling to protect RGCs from HG-induced apoptosis and oxidative stress. This study indicates a potential role of PHLPP1 in regulating HG-induced injury of RGCs during the development and progression of diabetic retinopathy.

SLC30A7 has anti-oxidant stress effects in high glucose-induced apoptosis via the NFE2L2/HMOX1 signal transduction pathway

AimsApoptosis and oxidant stress are known to be involved in the pathogenesis of diabetic kidney disease (DKD). We have previously reported that zinc transporter 7 in SLC30 family (SLC30A7) inhibits apoptosis in rat peritoneal mesothelial cells under high glucose (HG) conditions. In the current study, we aimed to investigate whether SLC30A7 had effect for anti-oxidant stress in renal tubular epithelial cells under HG. MethodsSLC30A7 in HG-induced apoptosis in a normal rat kidney tubular epithelial cell line (NRK-52E cells)/kidneys of STZ-induced diabetic mice was examined and the activity of nuclear factor erythroid 2-related factor 2 (NFE2L2) was further analyzed by using real time RT-PCR, siRNA and Western blot protocols. ResultsSLC30A7 was found to be up-regulated, while NFE2L2 was activated in kidneys of STZ-induced diabetic mice and HG-induced apoptosis of NRK-52E cells. Knock-down of SLC30A7 with siRNA protocol resulted in lower intracellular free zinc levels in the cells and decreased zinc distribution in the Golgi apparatus. Furthermore, knock-down of NFE2L2 down-regulated its target HMOX1 gene expression, decreased SLC30A7 activity but increased HG-induced apoptosis. ConclusionThe current study provides new evidence that SLC30A7 has anti-oxidant stress effects in HG-induced apoptosis via the NFE2L2/HMOX1 signal transduction pathway.

Regulation of the miR-19b-mediated SOCS6-JAK2/STAT3 pathway by lncRNA MEG3 is involved in high glucose-induced apoptosis in hRMECs.

Objective: Diabetic retinopathy (DR) is one of the most severe and common complications of diabetes mellitus. The present study aimed to investigate the molecular mechanism of MEG3, miR-19b and SOCS6 in human retinal microvascular endothelial cells (hRMECs) under high glucose conditions. Methods: HRMECs were cultured in 5 or 30 mM D-glucose medium. qRT-PCR and Western blotting were used to determine the mRNA expression and protein levels. MTT assay and flow cytometry analysis were performed to detect the viability and apoptosis of hRMECs, respectively. TNF-α, IL-6 and IL-1β levels in cell supernatants were detected by ELISA. The activity of caspase-3/7 was also determined. A luciferase reporter assay was performed to confirm the targeting relationship between miR-19b and SOCS6, as well as MEG3 and miR-19b. Results: Our study demonstrated that miR-19b was increased and SOCS6 was decreased in HG-induced hRMECs. Knockdown of SOCS6 inhibited cell viability and reversed the promotion of cell viability induced by knockdown of miR-19b. Additionally, miR-19b directly targeted and negatively regulated SOCS6. Moreover, miR-19b promoted the cell apoptosis rate and caspase-3/7 activity and increased inflammatory factors through the SOCS6-mediated JAK2/STAT3 signalling pathway. In addition, MEG3 attenuated HG-induced apoptosis of hRMECs by targeting the miR-19b/SOCS6 axis. Conclusion: These findings indicate that MEG3 inhibited HG-induced apoptosis and inflammation by regulating the miR-19b/SOCS6 axis through the JAK2/STAT3 signalling pathway in hRMECs. Thus, these findings might provide a new target for the treatment of DR.

Spiraeoside protects human cardiomyocytes against high glucose-induced injury, oxidative stress, and apoptosis by activation of PI3K/Akt/Nrf2 pathway.

The present study aimed to explore the effect of spiraeoside, an active quercetin glucoside, on diabetic cardiomyopathy in vitro. Our results showed that spiraeoside attenuated high glucose (HG)-induced reduction of cell viability and increased myocardial enzymes lactate dehydrogenase and aspartate aminotransferase in AC16 cells. Spiraeoside exerted antioxidant activity in HG-induced AC16 cells as spiraeoside inhibited reactive oxygen species and malondialdehyde production and increased activities of superoxide dismutase, glutathione peroxidase, and catalase. Spiraeoside prevented HG-induced apoptosis of AC16 cells. HG stimulation-caused the decrease in the expression levels of p-Akt, nuclear Nrf2, and HO-1 was elevated after spiraeoside treatment in AC16 cells. However, the effects of spiraeoside were reversed by LY294002. In conclusion, spiraeoside protected AC16 cells against HG-induced oxidative stress, cell injury, and apoptosis. The protective effect of spiraeoside was regulated by the PI3K/Akt/Nrf2 signaling pathway.

Inhibition of long noncoding RNA MALAT1 suppresses high glucose-induced apoptosis and inflammation in human umbilical vein endothelial cells by suppressing the NF-κB signaling pathway.

The study investigated the expression of long noncoding RNA (lncRNA) MALAT1 in high glucose (HG)-induced human vascular endothelial cells (HUVECs) and the role of MALAT1 in the apoptosis of HG-induced HUVECs. The HUVECs were cultured and induced with 25 mmol/L HG. After that, the HUVECs were transfected with MALAT1 siRNA. The expression levels of MALAT1 were detected with qPCR, whereas the expression levels of Bax, Bcl-2, cleaved-caspase-3, cleaved-caspase-9, p-65, and p-p65 were detected using Western blot. The roles of MALAT1 in cell activities, including apoptosis, were evaluated using the CCK-8 assay, TUNEL staining, and flow cytometry. The expression levels of inflammatory factors (TNF-α and IL-6) were measured using ELISA. The expression levels of MALAT1, TNF-α, and IL-6 in HUVECs were increased in the HG environment; however, when MALAT1 was silenced in the HUVECs, cell proliferation increased significantly, the expression levels of TNF-α, IL-6, Bax, cleaved-caspase-3, and cleaved-caspase-9 decreased, and the rate of apoptosis also decreased. Silencing MALAT1 inhibited the expression of p-p65 in HG-induced HUVECs. In conclusion, our study demonstrated that MALAT1 is upregulated in HG-induced HUVECs, and inhibition of MALAT1 inhibits HG-induced apoptosis and inflammation in HUVECs by suppression of the NF-κB signaling pathway.

Topical GDF11 accelerates skin wound healing in both type 1 and 2 diabetic mouse models

This study aimed to investigate the role of truncated growth differentiation factor 11 (GDF11), in which the recognition site of Furin from wild-type GDF11 was deleted to enhance the cellular stability, in skin wound healing in the setting of diabetes mellitus (DM) and the underlying mechanisms. Our study found that both truncated and natural GDF11s effectively accelerated wound healing processes in both T1DM and T2DM mice with a potency compatible to PDGF, bFGF, and EGF, but being much higher than GDF8. At the cellular level, GDF11 stimulated the proliferation and suppressed HG-induced apoptosis of HSFs. Further study revealed that GDF11 activated the YAP—Smad2/3—CTGF fibrotic signaling pathway by reversing HG-induced upregulation of phosphorylated form of YAP (p-YAP), increases p-Smad2/3 levels, and restoring HG-induced repression of CTGF expression by GDF11. Overall, the study shows that both natural and truncated GDF11s promote the healing process of skin wound in mice of both T1DM and T2DM partly via stimulating dermal fibrosis via the YAP—Smad2/3—CTGF pathway, suggesting it a potential agent for treating skin wound in diabetic population.

488-P: Isoliquiritigenin Protects Renal Tubular Epithelial Cells from Inflammatory and Oxidative Injuries via Directly Activating SIRT1 in Type 1 Diabetic Nephropathy

Emerging evidence shows that increasing fibrosis and apoptosis have been observed in the tubular epithelia in the progression of type 1 diabetic nephropathy (DN). Excessive inflammatory response and oxidative stress contribute to the pathogenesis of progression. Isoliquiritigenin (ISL), a natural chalcone compound, has been reported to exert anti-inflammatory and anti-oxidative properties. However, little is known about the effects of ISL on diabetic nephropathy and its mechanism. In this study, we investigated the potential protective effects and pharmacological mechanisms of ISL on DN in vivo and in vitro. We test beneficial activities of ISL on streptozotocin (STZ)-induced type 1 diabetes mellitus mice and renal tubular epithelial NRK-52E cells. The study showed that ISL attenuated diabetic nephropathy by decreasing both pro-fibrotic and pro-apoptotic biomarkers. Diabetes or high glucose (HG) induced inflammatory response and oxidative stress were eliminated by ISL administration. The molecular docking using the multiple ligand simultaneous docking (MLSD) software indicated that three molecules of ISL might bound to the binding site of SIRT1 and formation of a stable complex. We also found ISL could neutralize HG-mediated inactivation of SIRT1. Activation of SIRT1 could attenuate HG-induced inflammation, oxidative stress, fibrosis and apoptosis in NRK-52E cells, paralleled with the effects of ISL. While inhibition of SIRT1 could significantly abrogate the protective effects of ISL. Our study implicated that ISL might have great pharmacological actions on attenuating tubular damage of diabetic nephropathy. The beneficial effects of ISL are likely associated with inhibiting excessive inflammatory response and oxidative stress via directly activating SIRT1. Disclosure X. Chen: None. F. Shen: None. Funding Wenzhou Science and Technology Bureau (Y20190125); First Affiliated Hospital of Wenzhou Medical University (FHY2019015)

Long Noncoding RNA MEG3 Inhibits Apoptosis of Retinal Pigment Epithelium Cells Induced by High Glucose via the miR-93/Nrf2 Axis

Diabetic retinopathy (DR) is the leading cause of visual impairment in developed nations. Though plasma microRNA-93 (miR-93) is associated with the risk of DR, the function and regulatory mechanism of miR-93 during DR remains unclear. Blood samples were collected from 12 DR patients and 12 healthy controls. Primary human retinal pigment epithelium (RPE) cells and ARPE-19 cells were cultured in 5 mmol/L or 33 mmol/L d-glucose medium. Long noncoding (lnc) RNA MEG3 and miR-93 expression was detected by real-time quantitative PCR. The effect of MEG3 and miR-93 on high glucose (HG)-induced apoptosis was detected by MTT and flow cytometry. IL-6 and tumor necrosis factor-α levels were detected by enzyme-linked immunosorbent assay. The relationships among MEG3, miR-93, and Nrf2 (also known as NFE2L2) were explored via dual-luciferase reporter assay. lncRNA MEG3 and Nrf2 were decreased and miR-93 was increased in blood samples of DR patients and HG-treated human RPE and ARPE-19 cells. Overexpression of miR-93 inhibited cell proliferation and promoted apoptosis, whereas overexpression of Nrf2 or MEG3 promoted proliferation and suppressed apoptosis and inflammation. In addition, MEG3 targeted miR-93 and down-regulated miR-93. Moreover, miR-93 directly targeted Nrf2and negatively regulated Nrf2. This study suggests that lncRNA MEG3 depresses HG-induced apoptosis and inflammation of RPE via miR-93/Nrf2 axis, providing a novel perspective on the genesis and development of DR.

Knockdown of NEAT1 exerts suppressive effects on diabetic retinopathy progression via inactivating TGF-β1 and VEGF signaling pathways.

Diabetic retinopathy (DR) is complication resulted from Type 2 diabetes mellitus. Accumulating evidence has proved the functions of long noncoding RNAs (lncRNAs) in the progression of DR. Recent reports exert the numerous regulatory functions of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in various diseases. However, its implications in DR remain barely known. Therefore, this study was carried out to explore the role of NEAT1 in high-glucose (HG)-triggered injury of human retinal endothelial cells (hRECs). Here, we found the NEAT1 level was significantly elevated in patients with DR, in the retina of diabetic rats and mice. Meanwhile, hRECs under HG stimuli also exhibited an increase of NEAT1. Moreover, the loss of NEAT1 enhanced hRECs proliferation and repressed HG-induced apoptosis, which was accompanied by an upregulation of Bcl-2 and a downregulation of Bax. Subsequently, the knockdown of NEAT1 obviously reduced HG-triggered oxidative stress injury in hRECs. It was reflected that intracellular reactive oxygen species and malondialdehyde level induced by HG were repressed by NEAT1 downregulation, while superoxide dismutase activity was increased. In addition, decreased NEAT1 repressed the inflammatory processes effectively as indicated by the inactivation of inflammatory cytokines Cox-2, interleukin-6, and tumor necrosis factor-α. Furthermore, vascular endothelial growth factor A (VEGF) and transforming growth factor-β1 (TGF-β1) expression in patients with DR, DR rats, and HG-incubated hRECs was obviously increased. The silence of NEAT1 could reduce the enhanced expression of VEGF and TGF-β1 induced by HG. Hence, we concluded NEAT1 might contribute to the development of DR through activating TGF-β1 and VEGF.

Tumor Necrosis Factor Receptor-Associated Protein 1 Protects against Mitochondrial Injury by Preventing High Glucose-Induced mPTP Opening in Diabetes.

Diabetic kidney disease (DKD) has become the leading cause of end-stage renal disease worldwide. Renal tubular epithelial cell apoptosis and tubular atrophy have been recognized as indicators of the severity and progression of DKD, while the mechanism remains elusive. Tumor necrosis factor receptor-associated protein 1 (TRAP1) plays critical roles in apoptosis. The aim of this study was to investigate the protective role TRAP1 plays in DKD and to study the potential underlying mechanisms. TRAP1 expression was decreased, and mitochondria were injured in NRK-52e cells under high-glucose (HG) conditions. The overexpression of TRAP1 ameliorated HG-induced apoptosis, increased cell viability, maintained mitochondrial morphology, adenosine triphosphate (ATP) levels, and mitochondrial membrane potential (MMP), and buffered oxidative stress, whereas TRAP1 knockdown aggravated these effects. The protective effects of TRAP1 may be exerted via the inhibition of mitochondrial permeability transition pore (mPTP) opening, and the damage caused by TRAP1 knockdown can be partially reversed by treatment with the mPTP opening inhibitor cyclosporin A (CsA). In vivo, TRAP1 expression upregulation by AAV2/9 injection prevented renal dysfunction, ameliorated histopathological changes, maintained mitochondrial morphology and function, and reduced apoptosis and reactive oxygen species (ROS) in STZ-treated DKD rats. Thus, our results suggest that TRAP1 ameliorates diabetes-induced renal injury by preventing abnormal mPTP opening and maintaining mitochondrial structure and function, which may be treated as a potential target for DKD treatment.

Upregulation of heme oxygenase-1 by Brahma-related gene 1 through Nrf2 signaling confers protective effect against high glucose-induced oxidative damage of retinal ganglion cells

High glucose (HG)-induced oxidative damage of retinal ganglion cells (RGCs) contributes to the pathogenesis of diabetic retinopathy, a severe complication of diabetes mellitus. Brahma-related gene 1 (Brg1) has currently emerged as a cytoprotective protein that alleviates oxidative damage induced by various stress. However, whether Brg1 is involved in the regulation of HG-induced oxidative damage of RGCs remains unknown. In this study, we aimed to investigate the potential role and underlying mechanism of Brg1 in regulating HG-induced damage of RGCs. We found that Brg1 expression was significantly downregulated in RGCs in response to HG treatment. Functional experiments showed that Brg1 knockdown enhanced HG-induced apoptosis and production of reactive oxygen species, while Brg1 overexpression suppressed HG-induced apoptosis and reactive oxygen species production, showing a protective effect. Moreover, Brg1 overexpression resulted in an increase in nuclear expression of nuclear factor-erythroid-2-related factor-2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) in RGCs. Notably, inhibition of Nrf2 or HO-1 significantly blocked Brg1-mediated protection against HG-induced damage. Overall, these findings demonstrate that Brg1 protects RGCs from HG-induced oxidative damage through promotion of Nrf2/HO-1 signaling, indicating a potential role of Brg1 in the pathogenesis of diabetic retinopathy.

Rhein-8-O-β-D-glucopyranoside inhibited high glucose-induced apoptosis of human mesangial cells by regulating the lincRNA ANRIL/let-7a/TGF-β1/Smad signaling pathway.

Diabetic nephropathy is one of most frequent complications of diabetes, and is the major cause of end-stage disease in diabetic patients. The present study investigated the roles and mechanisms of Rhein-8-O-β-D-glucopyranoside (Rg) protecting human mesangial cells (HMCs) from high glucose (HG)-induced apoptosis. Using a Cell Counting Kit-8 assay the proliferation of HMCs was analyzed, and flow cytometry was applied to detect apoptosis. The apoptosis-associated protein Bcl-2, caspase-3 and members of the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway were analyzed using a western blotting assay. HG significantly induced HMC apoptosis, and Rg markedly attenuated the HG-induced apoptosis. HG decreased the Bcl-2 expression and increased the caspase-3 expression, and Rg treatment recovered the expressions of Bcl-2 and caspase-3 affected by HG. The underlying mechanisms were further analyzed, and it was demonstrated that HG significantly upregulated the long intervening non-coding RNA (lincRNA) ANRIL expression level, downregulated let-7a expression and activated the TGF-β1/Smad signaling pathway; Rg treatment recovered the expressions of lincRNA ANRIL and let-7a, and inhibited the TGF-β1/Smad signaling pathway in the condition of HG. In conclusion, the present results suggested that Rg attenuated HG-induced apoptosis of HMCs by regulating the lincRNA ANRIL/let-7a/TGF-β1/Smad signaling pathway.

Puerarin Attenuates Diabetic Nephropathy by Promoting Autophagy in Podocytes.

Puerarin, an active compound of radix puerariae, is a major compound used in Chinese herbal medicines to treat patients with diabetic nephropathy (DN). In the previous studies, we showed that puerarin exerts renoprotective effects in Streptozocin (STZ)-induced diabetic mice through activation of Sirt1 and anti-oxidative effects. Here, we further investigated the underlying mechanism mediating the renal protective effects of puerarin in DN. We studied the effects and mechanism of puerarin in STZ-induced diabetic mice and in cultured immortalized mouse podocytes treated with high glucose. We confirmed that puerarin ameliorated urinary albumin creatinine ratio and kidney injury in STZ-induced DN mice. We found that expression of heme oxygenase 1 (HMOX-1) and Sirt1 was suppressed in diabetic glomeruli but restored by puerarin treatment at both mRNA and protein levels. Additionally, we found that puerarin induced autophagy in the kidney of DN mice. In conditionally immortalized mouse podocytes, puerarin inhibited HG-induced apoptosis and restored the mRNA and protein levels of HMOX-1 and Sirt1. Interestingly, we showed that puerarin decreased liver kinase B1 (LKB1) acetylation, thereby promoting adenosine 5′-monophosphate-activated protein kinase-dependent autophagy. Knockdown of HMOX-1 and Sirt1 expression or treatment with the autophagy inhibitor 3-methyladenine abolished the protective effects of puerarin in HG-treated podocytes. Taken together, these results suggest that puerarin protects podocytes from diabetes-induced injury through HMOX1 and Sirt1-mediated upregulation of autophagy, a novel mechanism explaining its renal protective effects in DN.