High Glucose Group Research Articles

Silent Information Regulator 1 (SIRT1) Promotes Pancreatic β-Cell Mitochondrial Autophagy and Pyrin Domains-Containing Protein 3 (NLRP3) Activation Under High Glucose Environment

Mitochondrial autophagy and inflammatory response involves in diabetes. This study mainly explores the role of Silent Information Regulator (SIRT1) in pancreatic β-cells under high glucose conditions and related mechanism. Pancreatic β cells was cultured in a high-glucose environment with SRT1720 and EX527 respectively to define activation group and inhibition group followed by analysis of SIRT1, P-FOXO1, FOXO1, LC3, ATG5, PINK, Parkin, Mfn1, Mfn2, Fis1, IL-6, TNF-α, NLRP3 protein and mRNA expression by qRT-PCR, Western blot and fluorescent probe technology. Compared with control group, SIRT1 protein and mRNA expression in the high glucose group was significantly reduced. Activation group had highest protein and mRNA expression of SIRT1 P-FOXO1, FOXO1, Mfn1, Mfn2, Fis1, PINK, Parkin and mitochondrial membrane potential followed by blank group and inhibition group.SIRT1 secretion by pancreatic β-cells under high glucose environment is reduced. After activating SIRT1, mitochondrial autophagy decreased significantly and inflammatory response is significantly alleviated, indicating that SIRT1 might be used as a therapeutic target.

Dapagliflozin Reduces Apoptosis of Diabetic Retina and Human Retinal Microvascular Endothelial Cells Through ERK1/2/cPLA2/AA/ROS Pathway Independent of Hypoglycemic.

Introduction: It is known that the metabolic disorder caused by high glucose is one of pathogenesis in diabetic retinopathy (DR), the leading cause of blindness, due to the main pathological change of apoptosis of endothelial cells (ECs). In previous studies, the potential impact of sodium glucose cotransporter-2 (SGLT-2), whose inhibitors slow the progression of DR, has not been elucidated. The purpose of the presented study was to explore the effect of SGLT-2 inhibitors dapagliflozin (DAPA) on apoptosis of diabetic mice retina and human retinal microvascular endothelial cells (HRMECs), examine the effects of dapagliflozin on HRMECs metabolism, and explore the molecular processes that affect DR. Methods and Results: The eyeballs of male streptozotocin (STZ)-induced diabetic C57BL/6N mice were evaluated. C57BL/6N mice were divided into control group (CON), diabetic untreated group (DM), diabetic dapagliflozin treatment group (DM + DAPA) and diabetic insulin treatment group (DM + INS). Hematoxylin-Eosin (HE) staining was performed to observe the pathological structure of the mice retina, and TUNEL staining to detect apoptosis of mice retinal cells. In vitro, DCFH-DA and western blot (WB) were used to evaluate ROS, Bcl-2, BAX, cleaved-caspase 3 in HRMECs and metabolomics detected the effect of dapagliflozin on the metabolism of HRMECs. And then, we performed correlation analysis and verification functions for significantly different metabolites. In vivo, dapagliflozin reduced the apoptosis of diabetic mice retina independently of hypoglycemic. In vitro, SGLT-2 protein was expressed on HRMECs. Dapagliflozin reduced the level of ROS caused by high glucose, decreased the expression of cleaved-caspase3 and the ratio of BAX/Bcl-2. Metabolomics results showed that dapagliflozin did not affect the intracellular glucose level. Compared with the high glucose group, dapagliflozin reduced the production of arachidonic acid (AA) and inhibited the phosphorylation of ERK1/2, therefore, reducing the phosphorylation of cPLA2, which is a key enzyme for arachidonic acid release. Conclusion: Collectively, results unearthed for the first time that dapagliflozin reduced apoptosis of retina induced by DM whether in vivo or in vitro. Dapagliflozin did not affect the glucose uptake while mitigated intracellular arachidonic acid in HRMECs. Dapagliflozin alleviated HRMECs apoptosis induced by high glucose through ERK/1/2/cPLA2/AA/ROS pathway.

Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism

Effect of human decidua mesenchymal stem cells-derived exosomes on the function of high glucose-induced senescent human dermal fibroblasts and its possible mechanism

Magnesium supplementation in high carbohydrate diets: Implications on growth, muscle fiber development and flesh quality of Megalobrama amblycephala

This study was conducted to investigate the effects of magnesium (Mg) supplementation on the growth performance, muscle fiber development and flesh quality of Megalobrama amblycephala fed a high-carbohydrate (CHO) diet. In vivo, six experimental diets, a control diet (30% CHO, C), and five Mg supplementation levels (0 (HC1), 200 (HC2), 400 (HC3), 800 (HC4) and 1200 (HC5) mg kg−1, respectively) of high-CHO diet (45% CHO), were formulated and then fed to M. amblycephala (initial weight 43.15 ± 0.43 g) for 13 weeks. Results showed that the HC1 diets remarkably decreased weight gain rate (WGR), specific growth rate (SGR), water holding capacity (WHC), pH 24 h postmortem, collagen content, textural properties (hardness, tenderness, chewiness and resilience), muscle fiber density, succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activities, as well as transcriptions of ampkα2, pgc-1α, mrf-4, myf-5 and myf-6 compared with the C group, but muscle AMP/ATP ratio, ATPase activities and mstna and mstnb transcription showed an opposite trend. The HC3 group of fish obtained relatively high values of WGR, SGR, muscle lipid and protein content, collagen content, textural properties (hardness, tenderness, chewiness and resilience), fiber density, SDH and MDH activities, AMP/ATP ratio as well as transcriptions of ampkα1, pgc-1α, myod, myog and myf-5 compared with other HC groups. In vitro, the isolated primary hepatocytes from M. amblycephala were assigned into four experimental groups: a control group (5.5 mM glucose, Control), a high glucose group (30 mM glucose, HG), an AICAR group (30 mM glucose + 250 μM AICAR, HGA) and an MgSO4 group (30 mM glucose + 2 mM MgSO4, HGM). Results showed that MgSO4 treatment remarkably increased p-Ampk/t-Ampk ratio, Pgc-1α protein content and transcriptions of ampkα1, ampkα2 and pgc-1α compared with the HG group. Overall, based on regression analysis for SGR, the optimal Mg level of M. amblycephala fed high-CHO diets was 366.67 mg kg-1, which was consistent with the above results that fish fed HC3 diets displayed higher the growth performance, muscle fiber density and flesh quality of M. amblycephala.

POS-352 MiR-26a-5p PROTECTS PODOCYTE FROM APOPTOSIS VIA DOWNREGULATION OF TRPC6 IN TYPE 2 DIABETIC NEPHROPATHY

Podocyte injury is a major etiological factor in diabetic nephropathy (DN), while transient receptor potential cationic channel 6 protein (TRPC6) channels play a key role in podocyte injury. Our study is designed to investigate the regulatory effect of microRNA-26a-5p (miR-26a-5p) on DN podocyte injury induced by TRPC6 and the underlying mechanisms. 20 SPF, 8-week male Sprague-Dawley (SD) rats were used for establishing the type 2 diabetic mellitus (T2DM) group by feeding them with high fat and high sugar food for 8 weeks and intraperitoneal injection of low-dose streptozotocin (STZ, 30mg/kg). Another 10 normal male SD rats were selected as normal control group (NC). The 24h urinary albumin excretion rate (UAE), urinary creatinine (Ucr), creatinine clearance rate (CCr) and other major biochemical parameters including the fasting insulin level (FINS), serum creatinine (Scr), fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) were measured in both groups after establishing the DM models. Light and electron microscope were used to observe the pathology of kidneys and podocyte ultrastructures, respectively. Immunohistochemical method was used to show the distribution of nephrin and TRPC6 protein. To determine the expression of nephrin, TRPC6, apoptotic-associated proteins (cleaved-caspase-3 and bax/bcl-2), western blot was applied. qRT-PCR was used to detect miR-26a-5p and mRNAs of nephrin, TRPC6, cleaved-caspase-3 and bax/bcl-2. Podocyte apoptotic rate was measured using TUNEL method. Cultured immortalized mouse podocytes (MPC5) were divided into three groups: normal glucose group (NG), hypertonic group (HM), high glucose group (HG). Before and after miR-26a-5p mimics and inhibitor transfecting, the expression and mRNAs of nephrin, TRPC6, cleaved-caspase-3 and bax/bcl-2 were detected again using the same methods. Flow cytometry (FCM) was uesd to detect podocyte apoptosis. 1. Compared with NC group, rats in DM group exhibited disrupted biochemical conditions, for uAER, CCr, FINS, FBG, TC, TG and LDL were all significantly increased (all P <0.05) while HDL were decreased (P <0.05), along with significant insulin resistance. 2. Increased glomeruli volume, mesangial cell proliferation, thickened basement membrane and foot process fusion could be observed by light and electron microscope. 3. Immunochemistry results showed a increased expression of TRPC6 and a decreased of nephrin in DM rats than those in NC group (both P <0.05). 4. Both qRT-PCR and Western blot results in vivo and in vitro showed a notable increase in the expression of TRPC6, cleaved-caspase-3 and bax/bcl-2 and decrease in the expression of nephrin and miR-26a-5p (P <0.05). TUNEL and flow cytometry indicated higher podocyte apoptosis in DM rats and HG group. 5. The expression of TRPC6, cleaved-caspase-3 and bax/bcl-2 were down-regulated and nephrin expression was restored after miR-26a-5p mimics transfection (all P <0.05), while miR-26a-5p inhibitor intervention could aggravate all above changes as the effects of HG on podocytes (all P <0.05). High-glucose condition significantly decreased miR-26a-5p expression and increased TRPC6 expression and podocyte apoptosis. In DN, miR-26a-5p can protect podocyte from apoptosis via down-regulating the expression of TRPC6.

Integrated Analysis of Long Non-Coding RNA -mRNA Profile and Validation in Diabetic Cataract

ABSTRACTPurposeTo illustrate the expression profile of lncRNAs and mRNAs in diabetic cataract (DC) and explore their potential role in the pathogenesis of DC.MethodsLncRNA and mRNA microarray were conducted using the lens epithelium of DC patients (n = 3) and controls from eye bank (n = 3). Further, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were employed to unveil their underlying biological function and signaling pathway. The co-expression network was constructed to explore the relationship between lncRNAs and mRNAs. Then, lens epithelial cells (LECs) were cultured in vitro under high glucose (HG) and normal glucose (NG) groups to further verified the expression profiles of mRNAs. At last, quantitative RT-PCR (qRT-PCR) was performed to confirm the microarray results for the aberrantly expressed lncRNAs, and to unveil the expression prolife of mRNAs in LECs.ResultsEight thousand three hundred and twenty-six lncRNAs and 3303 mRNAs were dysregulated in DC. GO analysis unveiled that the upregulated mRNAs, highest enrichment score of the GO term belonged to oxidation-reduction process, while the highest for downregulated mRNAs went to protein lipidation. In the CC category, the most significant terms for upregulated and downregulated mRNAs appeared in intracellular part and intrinsic component of membrane, respectively. As to MF category, the most significant term for upregulated mRNAs was protein binding, and for downregulated mRNAs was catecholamine binding. KEGG pathways analysis demonstrated that the major enrichment score of pathways in upregulated mRNAs included Glycolysis/Gluconeogenesis, lysosome, carbon metabolism and AGE-RAGE signaling pathway. For the downregulated mRNAs, the glycosphingolipid biosynthesis-lacto and neolacto series pathway was included. Three lncRNAs (LINC01508, MAFA-AS1, MIAT) and the Sirtuin 2 are positively related in the co-expression network.ConclusionsThis study provided an overview of the lncRNAs and mRNAs expression, and suggesting that several aberrantly expressed lncRNAs might participate in the pathogenesis of DC.

Effect of dexmedetomidine on AKT/ERK signaling pathway and EMT-related proteins in high glucose-induced apoptosis in human renal tubular epithelial cells

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To study the effect of dexmedetomidine (Dex) on AKTERK signaling pathway and EMT- related proteins in high glucose-induced apoptosis in human kidney tubular epithelial cells.&#x0D; Methods: HK-2 cells were assigned to control, high-glucose and Dex groups. Levels of ROS were determined using live cell station. Flow cytometry was used to measure cell apoptosis and cell cycle while Western blot was applied to assay levels of PI3K, Akt, p-Akt, ERK and p-ERK.&#x0D; Results: The ROS concentrations were markedly reduced in Dex group, relative to high glucose group (p &lt; 0.05). Apoptosis was reduced in Dex group, relative to high glucose group, while P13K protein levels were significantly lower in high glucose and Dex groups than their corresponding control levels. In the high glucose-treated cells, AKT protein expression was downregulated, relative to control group, and p-AKT expression was markedly reduced in Dex group (p &lt; 0.05). Protein expressions of ERK and p-ERK in high glucose group were lower than control values, but were significantly accentuated in Dex group, relative to high glucose group (p &lt; 0.05).&#x0D; Conclusion: Dex mitigates high glucose-induced apoptosis of HK-2 cells, increases the proportion of cells in G1 phase, and reduces their EMT via a mechanism related to regulation of AKTERK signaling pathway-associated proteins. AKTERK signaling pathway-associated proteins provide insights into the development of drugs for the treatment of diabetic nephropathy.&#x0D; &#x0D; &#x0D; &#x0D;

PFKP Activation Ameliorates Foot Process Fusion in Podocytes in Diabetic Kidney Disease.

BackgroundGlycolysis dysfunction is an important pathogenesis of podocyte injury in diabetic kidney disease (DKD). Foot process fusion of podocytes and increased albuminuria are markers of early DKD. Moreover, cytoskeletal remodeling has been found to be involved in the foot process fusion of podocytes. However, the connections between cytoskeletal remodeling and alterations of glycolysis in podocytes in DKD have not been clarified.MethodsmRNA sequencing of glomeruli obtained from db/db and db/m mice with albuminuria was performed to analyze the expression profiling of genes in glucose metabolism. Expressions of phosphofructokinase platelet type (PFKP) in the glomeruli of DKD patients were detected. Clotrimazole (CTZ) was used to explore the renal effects of PFKP inhibition in diabetic mice. Using Pfkp siRNA or recombinant plasmid to manipulate PFKP expression, the effects of PFKP on high glucose (HG) induced podocyte damage were assessed in vitro. The levels of fructose-1,6-bisphosphate (FBP) were measured. Targeted metabolomics was performed to observe the alterations of the metabolites in glucose metabolism after HG stimulation. Furthermore, aldolase type b (Aldob) siRNA or recombinant plasmid were applied to evaluate the influence of FBP level alteration on podocytes. FBP was directly added to podocyte culture media. Db/db mice were treated with FBP to investigate its effects on their kidney.ResultsmRNA sequencing showed that glycolysis enzyme genes were altered, characterized by upregulation of upstream genes (Hk1, and Pfkp) and down-regulation of downstream genes of glycolysis (Pkm, and Ldha). Moreover, the expression of PFKP was increased in glomeruli of DKD patients. The CTZ group presented more severe renal damage. In vitro, the Pfkp siRNA group and ALDOB overexpression group showed much more induced cytoskeletal remodeling in podocytes, while overexpression of PFKP and suppression of ALDOB in vitro rescued podocytes from cytoskeletal remodeling through regulation of FBP levels and inhibition of the RhoA/ROCK1 pathway. Furthermore, targeted metabolomics showed FBP level was significantly increased in HG group compared with the control group. Exogenous FBP addition reduced podocyte cytoskeletal remodeling and renal damage of db/db mice.ConclusionsThese findings provide evidence that PFKP may be a potential target for podocyte injury in DN and provide a rationale for applying podocyte glycolysis enhancing agents in patients with DKD.

Effects of acteoside on the expressions of MCP-1 and TGF-β1 in the diabetic nephropathy mice

Objective: Immune inflammatory cells and cytokines play an important role in the occurrence and development of diabetic nephropathy (DN). Acteoside has been reported to regulate the inflammation and immune response. The study aims to investigate the effects of acteoside on the expressions of MCP-1 and TGF-β1 on nephropathy in diabetic mice. Methods: C57BL/6J mice in the model group were given a single intraperitoneal injection of STZ (150 mg/kg). Model mice were divided randomly into two groups: 5 without treatment, 5 treated with acteoside. After continuous administration for 8 weeks, serum, urine, and kidney tissue were collected, then, ralated biochemical parameters, pathological characteristics and MCP-1 and TGF-β1 mRNA or protein were detected. The NRK-52E cells were divided into three groups as follows: the normal control group (NC group), the high glucose group (HS group), the high glucose+acteoside group (HS+ACT group). The expressions of MCP-1 and TGF-β1 in the mRNA and protein levels were assessed with RT-PCR, western blot and ELISA. Results: The expressions of MCP-1 and TGF-β1 were significantly enhanced in DN group and dramatically reduced after acteoside treatment. Compared with those in NC group, the expressions of MCP-1 and TGF-β1 in NRK-52E cell of HS group were significantly enhanced, while both were significantly decreased in HS+ACT group compared with HS group. Conclusion: Our findings indicate that Acteoside has protective effects on DN via inhibiting the expressions of MCP-1 and TGF-β1.

The role of vitamin D receptor agonist on podocyte injury induced by high glucose

Abstract Background The effects of vitamin D receptor (VDR) agonist paricalcitol on the podocyte injury induced by high glucose (HG) were investigated in conditioned immortalized mouse podocytes (MPC-5). Methods (1) Grouped according to different glucose concentrations: normal group (NG): 5.6 mmol/L glucose; HG stimulation group: 25 mmol/L glucose (25HG); high osmotic control group (NG+M): 5.6 mmol/L glucose + 19.4 mmol/L D-mannitol. The expression levels of VDR, podocyte marker proteins podocin, nephrin and mesenchymal marker proteins α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMP9) in MPC-5 were measured, respectively. (2) Effect of VDR agonist-paricalcitol on podocyte epithelial-mesenchymal transition (EMT) induced by HG: cultured podocytes are divided into NG group, NG with dimethylsulfoxide (DMSO) group (NG+D), NG with paricalcitol (0.1 μmol/L) group (NG+P), HG group, HG with DMSO group (HG+D), and HG with paricalcitol (0.1 μmol/L) group (HG+P). The expression levels of VDR, podocyte marker proteins, marker proteins of mesenchymal cells, and the albumin flow in each group were then detected. Results (1) Under HG conditions, the expressions of VDR, podocin, and nephrin were decreased, while the expressions of α-SMA and MMP9 were increased (all P &lt; 0.05). After administration of paricalcitol, the levels of VDR, podocin, and nephrin were increased, while the expressions of α-SMA and MMP9 were decreased compared with the control groups (all P &lt; 0.05). (2) The results of albumin flow showed that the albumin flow of podocytes increased under the condition of HG, while it decreased after the treatment of paricalcitol. Conclusion The podocyte injury induced by HG could be partly rescued by Paricalcitol.

Danggui Buxue Decoction Protects Retinal Vascular Endothelial Cells from High Glucose-Induced Apoptotic and Oxidative Injury by Decreasing Forkhead Box O4

To explore the effect and mechanism of Danggui Buxue decoction on high glucose-induced apoptotic and oxidative injury in human retinal vascular endothelial cells. Human retinal vascular endothelial cells were treated with high glucose to mimic the diabetic retinopathy condition in vitro. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry were used to investigate cell viability and apoptosis. Western blot was applied to detect the proteins of apoptosis-related markers and forkhead box O4. The oxidative injury was assessed using enzyme-linked immunosorbent assay analysis. Human retinal vascular endothelial cells in high glucose group showed the decrease of cell viability and superoxide dismutase activity, as well as the increase of cell apoptosis rate, reactive oxygen species level and malondialdehyde content, which were reversed by Danggui Buxue decoction treatment in a dose-dependent manner. Subsequently, we found that forkhead box O4 level was elevated in high glucose-induced human retinal vascular endothelial cells, while Danggui Buxue decoction dose-dependently down-regulated forkhead box O4 level in human retinal vascular endothelial cells. Deficiency of forkhead box O4 could reverse high glucose-induced apoptotic and oxidative injury in human retinal vascular endothelial cells. Moreover, the inhibitory effects of Danggui Buxue decoction on high glucose-induced human retinal vascular endothelial cells were attenuated by forkhead box O4 overexpression. Danggui Buxue decoction may inhibit the apoptosis and oxidative damage of human retinal vascular endothelial cells induced by high glucose by down-regulating forkhead box O4.

Mesenchymal stem cell-derived exosomes inhibit the VEGF-A expression in human retinal vascular endothelial cells induced by high glucose.

To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor A (VEGF-A) in human retinal vascular endothelial cells (HRECs). Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis. HRECs were randomly divided into a normal control group (group A), a high glucose model group (group B), a high glucose group with 25 µg/mL (group C), 50 µg/mL (group D), and 100 µg/mL exosomes (group E). Twenty-four hours after coculture, the cell proliferation rate was detected using flow cytometry, and the VEGF-A level was detected using immunofluorescence. After coculture 8, 16, and 24h, the expression levels of VEGF-A in each group were detected using PCR and Western blots. The characteristic morphology (membrane structured vesicles) and size (diameter between 50 and 200 nm) were observed under transmission electron microscopy. The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis (NTA). The exosomal markers CD9, CD63, and HSP70 were strongly detected. The proliferation rate of the cells in group B increased after 24h of coculture. Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes (F=39.03, P<0.01). The upregulation of VEGF-A protein (group C: F=7.96; group D: F=17.29; group E: F=11.89; 8h: F=9.45; 16h: F=12.86; 24h: F=42.28, P<0.05) and mRNA (group C: F=4.137; group D: F=13.64; group E: F=22.19; 8h: F=7.253; 16h: F=16.98; 24h: F=22.62, P<0.05) in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes (P<0.05). hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.

Role and mechanism of necrostin-1 in promoting oxidative stress response of macrophages in high glucose condition.

To investigate the role and molecular mechanism of necrostatin-1 (Nec-1), a specific programmed cell necrosis inhibitor, in promoting the oxidative stress response of macrophages under high glucose (HG) environment. Macrophages were cultured in control (5.5 mmol·L-1 glucose) or HG (25 mmol·L-1 glucose) medium for 72 h. The HG+Nec-1 group was given HG and 5 μmol·L-1 Nec-1. Reactive oxygen species (ROS) level, malondialdehyde (MDA) activity, and superoxide dismutase (SOD) activity were measured by 2'-7'dichlorofluorescin diacetate, MDA, and SOD enzyme linked immunosorbent assay kits, respectively. Moreover, receptor interacting protein 1 (RIP1) expression was assessed through real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot (WB). Finally, after the expression of RIP1 in macrophages was silenced, the effect of HG environment on oxidative stress response was evaluated in the gene-deficient cells. The HG group had increased ROS level and MDA activity (P<0.000 1) and decreased SOD activity (P<0.000 1) compared with the control group. The HG+Nec-1 group had higher ROS level and MDA activity (P<0.000 1) and lower SOD activity (P<0.01) than the HG group. The qRT-PCR and WB results showed that RIP1 mRNA level (P<0.001) and protein expression level (P<0.000 1) in the HG group were significantly higher than those in the control group, and RIP1 mRNA and protein expression levels in the HG+Nec-1 group were significantly lower than those in the HG group (P<0.000 1). After RIP1 was silenced effectively (P<0.001) with si-RNA, the ROS level and MDA activity of the HG+si-RIP1 group decreased compared with those of the HG+si-negative control (si-NC) group (P<0.001), and SOD activity in the HG+si-RIP1 group increased than that in the HG+si-NC group (P<0.000 1). HG promotes oxidative stress on macrophages by upregulating RIP1 expression.

LncRNA MALAT1 induced by hyperglycemia promotes microvascular endothelial cell apoptosis through activation of the miR-7641/TPR axis to exacerbate neurologic damage caused by cerebral small vessel disease.

BackgroundThis study aimed to explore the effect of hyperglycemia-induced long noncoding RNA (lncRNA) metastasis-associated lung carcinoma transcript 1 (MALAT1) on microvascular endothelial cell activity. In addition, we investigated the possible downstream molecular regulatory mechanism in order to provide an adjunctive therapeutic target for the prognostic nerve recovery of cerebral small vessel disease (CSVD).MethodsA rat model of diabetes was induced by streptozotocin (STZ) injection in combination with a high-energy diet. The mixed model of CSVD and hyperglycemia was prepared by injection of homologous microemboli in vitro. Results of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and enzyme-linked immunosorbent assay (ELISA) showed that the inhibition of lncRNA MALAT1 by siRNA in a high-glucose environment effectively alleviated the cell damage caused by high glucose (HG) and reduced the rate of apoptosis. We found that the upregulation of downstream miR-7641 and TPR (translocated promoter region) reduced the occurrence of cell damage and apoptosis.ResultsThe results of neurological deficit score showed that the scores of ICH group, HG group and HG + ICH group were significantly higher than those of Sham group, and the differences were statistically significant. The qPCR results showed that the MALAT1 level of the model group was significantly different from that of the sham group, and the expression levels of damage markers vWF and ICAM-1 were detected by Western blot (WB), which were significantly higher in the model group than in the sham group. The MTT cell activity assay showed that the addition of miR-7641 inhibitor or TPR short hairpin RNA (shRNA) into normally cultured cells reduced cell activity. ELISA results showed that low expression of miR-7641 increased the apoptosis rate of microvascular endothelial cells. Western blot (WB) results showed that the protein expression levels of BAX and cleaved caspase-3 (c-caspase-3) were negatively correlated with miR-7641. The regulation of TPR expression showed similar results.ConclusionsHigh blood glucose level induced the increase of lncRNA MALAT1 and regulated the expression of TPR by activating miR-7641 to promote the initiation of apoptosis of microvascular endothelial cells, aggravating the neurological dysfunction caused by CSVD.

OR12. Augmentation of endothelial progenitor cell proliferation in stable coronary artery disease subjects using human umbilical cord blood-mesenchymal stem cell-derived secretome in hyperglycemia condition

Abstract Background and Aims The number of endothelial progenitor cells (EPC) was decreased in stable coronary artery disease (SCAD) and diabetic patients. Human umbilical cord blood-mesenchymal stem cell-derived secretome (secretome) has the potential to improve the proliferation of EPC. This study aims to figure out the effect of the secretome on EPC proliferation of SCAD patients and hyperglycemia conditions. Method and Results EPC’s were isolated from peripheral blood samples and cultured for three days in the Stemline II medium. Cultured EPC’s were incubated with various concentrations of secretome (2%, 10%, and 20%). The other groups were treated with high glucose (25 mmol/L), a combination of secretome and high glucose, vascular endothelial growth factors-only (positive group), and the basal medium only (negative group). MTT cell proliferation assay kit was used to evaluate the EPC’s proliferation at 72 hours after reagent addition. The absorbance of MTT was measured in optical density (OD). The 20% secretome group was significantly higher than the positive control group (1.59 vs. 1.43; p &amp;lt; 0.001). EPC proliferation in the secretome group is dose-dependent manner. EPC’s proliferation of the high glucose group was significantly lower than the negative control group (0.65 vs. 0.89; p &amp;lt; 0.001). EPCs proliferation OD was significantly higher between a combination of 20% secretome and high glucose than the secretome-only group (1.78 vs. 1.59; p &amp;lt; 0.001). Conclusion The secretome enhances the EPC proliferation of SCAD patients, even in a high glucose condition. The secretome is a new potential substance to modulate the EPC proliferation of SCAD patients.

Effects of Qijin granules on high glucose-induced proliferation, apoptosis and expression of nuclear factor- κB and MCP-1 in rat glomerular mesangial cells

Purpose: To investigate the effects of Qijin granules on high glucose-induced proliferation and apoptosis in rat glomerular mesangial cells (MC).Methods: MC cells from rats were passaged and cultured, and randomly divided into control group (CNG), high glucose group (HGG), Western medicine group (WMG, high glucose + Benazepril + Gliquidone), and Qijin granules 1/2/3 group (high glucose + different doses of Qijin granules). Mesangial cells proliferation was measured using MTT assay. The NF-κB, MCP-1 and inflammatory factors in supernatant were determined by ELISA. Apoptosis rate and cell cycle were assessed by flow cytometry. The apoptosis-related TGF-β1/Smad signaling pathway-related protein expressions were measured by Western blot.Results: The A-value and early apoptosis rate, apoptosis rate and S-phase percentage, and protein expressions of NF-κB, MCP-1, IL-6, IL-2, TNF-ɑ, Bax, Cyt-C, caspase-3, TGF-β1, and p-Smad3 of MC cells in the HGG at 12 h, 24 h and 48 h were higher than those in the CNG. The above indices were lower in the WMG, and Qijin granules 1/2/3 groups than in the HGG. The Bcl-2, Smad7 protein expression level and the percentage of G1 and G2/M phase were lower in the HGG than in the CNG, and the above indeices were higher in the WMG and Qijin granules 1/2/3 group than in HGG.Conclusion: Qijin granules can dose-dependently inhibit high glucose-induced proliferation and apoptosis in rat MC cells, block the cell cycle and reduce inflammatory responses. This may be related to the regulation of NF-κB, MCP-1 and TGF-β1/Smad signaling pathways. These findings provide theoretical and experimental basis for the clinical treatment of early diabetic nephropathy.

Autophagy is required for the promoting effect of angiogenic factor with G patch domain and forkhead-associated domain 1 (AGGF1) in retinal angiogenesis.

To investigate the effect of angiogenic factor with G patch domain and forkhead-associated domain 1 (AGGF1) on retinal angiogenesis in ischemic retinopathy and its association with autophagy. RF/6A cells were divided into the control group, hypoxia group and high-glucose group, and the expression of AGGF1 in cells was detected. C57BL/6J mice were divided into the control group, oxygen-induced retinopathy (OIR) group and diabetic retinopathy (DR) group, and AGGF1 expression in the retina was observed. RF/6A cells were then divided into the control group and different AGGF1 concentration groups, and the expression of autophagy marker, LC3 was detected. Then, RF/6A cells were divided into the control group, AGGF1 group, 3-methyladenine (3-MA, an early autophagy inhibitor)+AGGF1 group and chloroquine (CQ, a late autophagy inhibitor)+AGGF1 group, and the expression of autophagy markers, LC3 and p62, autophagic flux, as well as was key signaling pathway proteins in autophagy, PI3K, AKT, and mTOR was detected. Finally, the cell proliferation, migration and tube formation were detected in the four groups. AGGF1 expression in RF/6A cells and in the retinas of OIR and DR mouse model was found to be increased in the state of hypoxic and high glucose condition. AGGF1 treatment led to increased expressions of LC3 and decreased p62; therby induced autophagic flux, and the phosphorylation of PI3K, AKT and mTOR was down-regulated in RF/6A cells. When autophagy was inhibited by 3-MA or CQ, confirmed by corresponding changes of these indicators of autophagy, cellular proliferation, migration and tube formation of RF/6A cells were weakened by AGGF1 treatment when compared with that of AGGF1 treatment alone. This study experimentally revealed that AGGF1 activates autophagy to promote angiogenesis for ischemic retinopathy and inhibition of PI3K/AKT/mTOR pathway may be involved in the activation of autophagy by AGGF1.

The root extract of Scutellaria baicalensis Georgi promotes β cell function and protects from apoptosis by inducing autophagy

Scutellaria baicalensis Georgi (SBG) is a traditional Chinese medicine with a remarkable remedial effect on diabetes mellitus. However, the precise mechanism involved has not been fully elucidated yet. Here, we aimed to explore the anti-diabetes effects of its traditional decoction in vitro and elucidate the autophagy-related mechanism. This study was designed to investigate the effects of the water extract of SBG (WSB) on the β cell viability, insulin secretion and the mechanism related to autophagy. Detection of insulin secretion using an enzyme immunoassay method, and analysis of apoptosis rate in MIN-6cells by the flow cytometry with PI and Annexin V-FITC staining. In addition, the autophagy levels and pathways were evaluated from the number of autophagosomes and the expression of autophagy-related proteins. 3-Methyladenine (3-MA) was used as the autophagy inhibitor. Autophagosomes were observed using a confocal microscopy, and autophagy-related proteins (LC3-II/I, p62, S6k, p-AMPK/AMPK, p-mTOR/mTOR) were measured by Western blot. Here we detected a significant increase in insulin release from MIN-6cells after treated with WSB. It is about 1.6 times as much as that of the control group with 2.8mM glucose and 2.2 times more than the 16.8mM glucose group. At the same time, WSB increased the number of autophagosomes and the ratio of LC3 Ⅱ/LC3 Ⅰ, indicating that autophagy were activated in MIN-6cells. When inhibiting autophagy, there was no significant difference in insulin release between the two groups. The apoptotic rate of the high glucose group was as high as 33.23%. After pretreatment with WSB, the apoptotic rate decreased to 14.95%, and increased to 22.57% when treated with 3-MA and WSB. At the same time, WSB treatment enhanced the phosphorylation of AMPK, but had no significant effect on the expression of mTOR and S6K. Our data suggested that WSB increased insulin secretion and reduced apoptosis under high glucose by inducing autophagy through the AMPK pathway, which elucidated the mechanism of WSB in the treatment of diabetes.

The water extracts of Euonymus alatus (Thunb.) Siebold attenuate diabetic retinopathy by mediating angiogenesis

Ethnopharmacological relevanceEuonymus alatus (Thunb.) Siebold (family Celastraceae) is a deciduous woody shrub that is recorded in ShenNong BenCaoJing. It has been widely used for diabetes in traditional Chinese medicine.AIM OF THE STUDY: This study aimed to identify the most effective extract of Euonymus alatus (EA) against high glucose-induced endothelial cells in vitro, evaluate its pharmacological effect on retinopathy in diabetic mice and explore its underlying mechanism by RNA sequencing. MethodsRetinal vascular endothelial cells (RF/6A) were treated with normal glucose (5.5 mmol/L glucose), high glucose (25 mmol/L glucose) or high glucose plus methanol extracts of EA (MEA), ethyl acetate extracts of EA (EEA) or water extracts of EA (WEA). The cytotoxicity and cell viability were determined by Cell Counting Kit-8 (CCK-8) assay. Cell migration was examined using the Transwell assay, and tube formation ability was measured using the Matrigel assay. Then, the KK-Ay mice were administered WEA or water for 12 weeks. The velocities of ocular blood flow were determined by Doppler ultrasound. RNA sequencing and reverse transcription quantitative PCR (RT–qPCR) were performed on WEA-stimulated RF/6A cells to reveal the underlying mechanism. ResultsThe cytotoxicity assay found that 30 μg/mL MEA, 20 μg/mL EEA and 30 μg/mL WEA had no toxic effect on RF/6A cells. The cell viability results showed that MEA, EEA and WEA all decreased cell viability. Compared with the high-glucose group, both MEA and WEA decreased the number of migrated cells, while the inhibition rate of WEA was higher. The Matrigel results showed that 30 μg/mL WEA effectively reduced the total tube length. Moreover, WEA improved the haemodynamics of the central retinal artery. RNA sequencing coupled with RT–qPCR verified that WEA regulated angiogenesis-related factors in high glucose-stimulated RF/6A cells. ConclusionsWEA inhibits the migration and tube formation of RF/6A cells and improves diabetic retinopathy (DR) by mediating angiogenesis.

The Effects of Photobiomodulation on Bone Defect Repairing in a Diabetic Rat Model

The purpose of this study is to examine the prospective therapeutic effects of photobiomodulation on the healing of bone defects in diabetic mellitus (DM) using rat models to provide basic knowledge of photobiomodulation therapy (PBMT) during bone defect repair. For in vitro study, an Alizzarin red stain assay was used to evaluate the effect of PBMT on osteogenic differentiation. For in vivo study, micro-computed tomography (microCT) scan, H&E and IHC stain analysis were used to investigate the effect of PBMT on the healing of the experimental calvarial defect (3 mm in diameter) of a diabetic rat model. For in vitro study, the high glucose groups showed lower osteogenic differentiation in both irradiated and non-irradiated with PBMT when compared to the control groups. With the PBMT, all groups (control, osmotic control and high glucose) showed higher osteogenic differentiation when compared to the non-irradiated groups. For in vivo study, the hyperglycemic group showed significantly lower bone regeneration when compared to the control group. With the PBMT, the volume of bone regeneration was increasing and back to the similar level of the control group. The treatment of PBMT in 660 nm could improve the bone defect healing on a diabetic rat calvarial defect model.