USP19 Stabilizes TAK1 to Regulate High Glucose/Free Fatty Acid-induced Dysfunction in HK-2 Cells

Objective Obesity-induced kidney injury contributes to the development of diabetic nephropathy(DN).Here,we identified the functions of ubiquitin-specific peptidase 19(USP19)in HK-2 cells exposed to a combination of high glucose(HG)and free fatty acid(FFA)and determined its association with TGF-beta-activated kinase 1(TAK1).Methods HK-2 cells were exposed to a combination of HG and FFA.USP19 mRNA expression was detected by quantitative RT-PCR(qRT-PCR),and protein analysis was performed by immunoblotting(IB).Cell growth was assessed by Cell Counting Kit-8(CCK-8)viability and 5-ethynyl-2′-deoxyuridine(EdU)proliferation assays.Cell cycle distribution and apoptosis were detected by flow cytometry.The USP19/TAK1 interaction and ubiquitinated TAK1 levels were assayed by coimmunoprecipitation(Co-IP)assays and IB.Results In HG+FFA-challenged HK-2 cells,USP19 was highly expressed.USP19 knockdown attenuated HG+FFA-triggered growth inhibition and apoptosis promotion in HK-2 cells.Moreover,USP19 knockdown alleviated HG+FFA-mediated PTEN-induced putative kinase 1(PINK1)/Parkin pathway inactivation and increased mitochondrial reactive oxygen species(ROS)generation in HK-2 cells.Mechanistically,USP19 stabilized the TAK1 protein through deubiquitination.Importantly,increased TAK1 expression reversed the USP19 knockdown-mediated phenotypic changes and PINK1/Parkin pathway activation in HG+FFA-challenged HK-2 cells.Conclusion The findings revealed that USP19 plays a crucial role in promoting HK-2 cell dysfunction induced by combined stimulation with HG and FFAs by stabilizing TAK1,providing a potential therapeutic strategy for combating DN.

Effects of high glucose and severe hypoxia on the biological behavior of mesenchymal stem cells at various passages

BACKGROUND Mesenchymal stem cells(MSCs)have been extensively studied for therapeutic potential,due to their regenerative and immunomodulatory properties.Serial passage and stress factors may affect the biological characteristics of MSCs,but the details of these effects have not been recognized yet.AIM To investigate the effects of stress factors(high glucose and severe hypoxia)on the biological characteristics of MSCs at different passages,in order to optimize the therapeutic applications of MSCs.METHODS In this study,we investigated the impact of two stress conditions;severe hypoxia and high glucose on human adipose-tissue derived MSCs(hAD-MSCs)at passages 6(P6),P8,and P10.Proliferation,senescence and apoptosis were evaluated measuring WST-1,senescence-associated beta-galactosidase,and annexin V,respectively.RESULTS Cells at P6 showed decreased proliferation and increased apoptosis under conditions of high glucose and hypoxia compared to control,while the extent of senescence did not change significantly under stress conditions.At P8 hAD-MSCs cultured in stress conditions had a significant decrease in proliferation and apoptosis and a significant increase in senescence compared to counterpart cells at P6.Cells cultured in high glucose at P10 had lower proliferation and higher senescence than their counterparts in the previous passage,while no change in apoptosis was observed.On the other hand,MSCs cultured under hypoxia showed decreased senescence,increased apoptosis and no significant change in proliferation when compared to the same conditions at P8.CONCLUSION These results indicate that stress factors had distinct effects on the biological processes of MSCs at different passages,and suggest that senescence may be a protective mechanism for MSCs to survive under stress conditions at higher passage numbers.

Human mesenchymal stem cells exhibit altered mitochondrial dynamics and poor survival in high glucose microenvironment

BACKGROUND Mesenchymal stem cells(MSCs)are a type of stem cells that possess relevant regenerative abilities and can be used to treat many chronic diseases.Diabetes mellitus(DM)is a frequently diagnosed chronic disease characterized by hyperglycemia which initiates many multisystem complications in the long-run.DM patients can benefit from MSCs transplantation to curb down the pathological consequences associated with hyperglycemia persistence and restore the function of damaged tissues.MSCs therapeutic outcomes are found to last for short period of time and ultimately these regenerative cells are eradicated and died in DM disease model.AIM To investigate the impact of high glucose or hyperglycemia on the cellular and molecular characteristics of MSCs.METHODS Human adipose tissue-derived MSCs(hAD-MSCs)were seeded in low(5.6 mmol/L of glucose)and high glucose(25 mmol/L of glucose)for 7 d.Cytotoxicity,viability,mitochondrial dynamics,and apoptosis were deplored using specific kits.Western blotting was performed to measure the protein expression of phosphatidylinositol 3-kinase(PI3K),TSC1,and mammalian target of rapamycin(mTOR)in these cells.RESULTS hAD-MSCs cultured in high glucose for 7 d demonstrated marked decrease in their viability,as shown by a significant increase in lactate dehydrogenase(P<0.01)and a significant decrease in Trypan blue(P<0.05)in these cells compared to low glucose control.Mitochondrial membrane potential,indicated by tetramethylrhodamine ethyl ester(TMRE)fluorescence intensity,and nicotinamide adenine dinucleotide(NAD+)/NADH ratio were significantly dropped(P<0.05 for TMRE and P<0.01 for NAD+/NADH)in high glucose exposed hAD-MSCs,indicating disturbed mitochondrial function.PI3K protein expression significantly decreased in high glucose culture MSCs(P<0.05 compared to low glucose)and it was coupled with significant upregulation in TSC1(P<0.05)and downregulation in mTOR protein expression(P<0.05).Mitochondrial complexes I,IV,and V were downregulated profoundly in high glucose(P<0.05 compared to low glucose).Apoptosis was induced as a result of mitochondrial impairment and explained the poor survival of MSCs in high glucose.CONCLUSION High glucose impaired the mitochondrial dynamics and regulatory proteins in hAD-MSCs ensuing their poor survival and high apoptosis rate in hyperglycemic microenvironment.

LIN28A attenuates high glucose-induced retinal pigmented epithelium injury through activating SIRT1-dependent autophagy

AIM:To evaluate the effects of LIN28A(human)on high glucose-induced retinal pigmented epithelium(RPE)cell injury and its possible mechanism.METHODS:Diabetic retinopathy model was generated following 48h of exposure to 30 mmol/L high glucose(HG)in ARPE-19 cells.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot tested the expression of the corresponding genes and proteins.Cell viability as well as apoptosis was determined through cell counting kit-8(CCK-8)and flow cytometry assays.Immunofluorescence assay was adopted to evaluate autophagy activity.Caspase 3 activity,oxidative stress markers,and cytokines were appraised adopting their commercial kits,respectively.Finally,ARPE-19 cells were preincubated with EX527,a Sirtuin 1(SIRT1)inhibitor,prior to HG stimulation to validate the regulatory mechanism.RESULTS:LIN28A was downregulated in HG-challenged ARPE-19 cells.LIN28A overexpression greatly inhibited HGinduced ARPE-19 cell viability loss,apoptosis,oxidative damage as well as inflammatory response.Meanwhile,the repressed autophagy and SIRT1 in ARPE-19 cells challenged with HG were elevated after LIN28A overexpression.In addition,treatment of EX527 greatly inhibited the activated autophagy following LIN28A overexpression and partly abolished the protective role of LIN28A against HG-elicited apoptosis,oxidative damage as well as inflammation in ARPE-19 cells.CONCLUSION:LIN28A exerts a protective role against HG-elicited RPE oxidative damage,inflammation,as well as apoptosis via regulating SIRT1/autophagy.

Vitamin B3 inhibits apoptosis and promotes autophagy of isletβcells under high glucose stress

Background:Hyperglycemia is a typical symptom of diabetes.High glucose induces apoptosis of isletβcells.While autophagy functions in cytoprotection and autophagic cell death.The interaction between autophagy and apoptosis is important in the modulation of the function of isletβcells.Vitamin B3 can induce autophagy and inhibit isletβapoptosis.Method:The mechanism of vitamin B3-mediated protective effect on the function of isletβcells was explored by the method of western blot,immunofluorescence and flow cytometry.Results:In the present study,high glucose stress increased the apoptosis rate,while vitamin B3 reduced the apoptosis rate.The effect of vitamin B3 on autophagy flux under normal and high glucose stress was also investigated.Vitamin B3 increased the number of autophagosomes and increased the light chain(LC)3-II/LC3-I ratio.In contrast,vitamin B3 decreased sequestosome 1(SQSTM1)/p62 protein expression and inhibited the phosphorylation of mammalian ribosomal protein S6 kinaseβ-1(p70S6K/S6K1),which was a substrate of mammalian target of rapamycin(mTOR)under normal and high glucose stress.To further verify the protective effect of vitamin B3 on apoptosis,we treated isletβcell RIN-m5F with autophagy inhibitor 3-methyladenine(3-MA).Vitamin B3 decreased the apoptosis rate under high glucose stress,while the inhibition of apoptosis by vitamin B3 was blocked after adding 3-MA.Conclusion:Our data suggested that vitamin B3 reduced the apoptosis rate ofβcells,possibly through inducing autophagy under high glucose stress.

Decreased TRPM7 alleviates high glucose-induced renal tubular epithelial cell injury by inhibiting the HMGB1/TLR4 signaling pathway

Objective:To explore the regulatory mechanism of transient receptor potential melastatin-7(TRPM7)in high glucose-induced renal tubular epithelial cell injury.Methods:The expression of TRPM7 in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells was detected by RT-qPCR.Then,the TRPM7 interference vector was constructed,and the downstream high mobility group box 1(HMGB1)/Toll-like receptor 4(TLR4)signaling pathway proteins were detected.Next,in addition to interference with TRPM7 expression,overexpression of HMGB1 in high glucose-induced HK-2 cells was performed.Cell activity,apoptosis,oxidative stress levels,and inflammation levels were determined by CCK8,TUNEL,Western blotting,immunofluorescence and related kits.Results:TRPM7 expression was upregulated in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells.Interference with TRPM7 reduced cell damage,epithelial-mesenchymal transition,oxidative stress,and inflammatory response in high glucose-induced HK-2 cells via inhibiting the HMGB1/TLR4 signaling pathway.However,the effects induced by TRPM7 silencing were abrogated by HMGB1 overexpression.Conclusions:Decreased TRPM7 alleviates high glucose-induced renal tubular epithelial cell injury by inhibiting the HMGB1/TLR4 signaling pathway.Further animal experiments and clinical trials are warranted to verify its effect.

Down-regulation of histone deacetylase 7 reduces biological activities of retinal microvascular endothelial cells under high glucose condition and related mechanism

AIM:To investigate the expression and effect of histone deacetylase 7(HDAC7)in human retinal microvascular endothelial cells(HRMECs)under high glucose condition and related mechanism,and the expression of HDAC7 in the retinal tissue in diabetic rats.METHODS:The expression of HDAC7 in HRMECs under high glucose and the retinal tissue from normal or diabetic rats were detected with immunohistochemistry and Western blot.LV-shHDAC7 HRMECs were used to study the effect of HDAC7 on cell activities.Cell count kit-8(CCK-8),5-ethynyl2’-deoxyuridine(EdU),flow cytometry,scratch test,Transwell test and tube formation assay were used to examine the ability of cell proliferation,migration,and angiogenesis.Finally,a preliminary exploration of its mechanism was performed by Western blot.RESULTS:The expression of HDAC7 was both upregulated in retinal tissues of diabetic rats and high glucosetreated HRMECs.Down-regulation of HDAC7 expression significantly reduced the ability of proliferation,migration,and tube formation,and reversed the high glucose-induced high expression of CDK1/Cyclin B1 and vascular endothelial growth factor in high glucose-treated HRMECs.CONCLUSION:High glucose can up-regulate the expression of HDAC7 in HRMECs.Down-regulation of HDAC7 can inhibit HRMECs activities.HDAC7 is proposed to be involved in pathogenesis of diabetic retinopathy and a therapeutic target.

Quercetin alleviates high glucose-induced Schwann cell damage by autophagy

Quercetin can reverse high glucose-induced inhibition of neural cell proliferation, and therefore may have a neuroprotective effect in diabetic peripheral neuropathy. It is difficult to obtain pri- mary Schwann cells and RSC96 cells could replace primary Schwann cells in studies of the role of autophagy in the mechanism underlying diabetic peripheral neuropathy. Here, we show that under high glucose conditions, there are fewer autophagosomes in immortalized rat RSC96 cells and primary rat Schwann ceils than under control conditions, the proliferative activity of both cell types is significantly impaired, and the expression of Berlin- 1 and LC3, the molecular mark- ers for autophagy, is significantly lower. After intervention with quercetin, the autophagic and proliferative activity of both cell types is rescued. These results suggest that quercetin can allevi- ate high glucose-induced damage to Schwann cells by autophagy.

Dexmedetomidine Attenuates High Glucose-induced HK-2 Epithelial-mesenchymal Transition by Inhibiting AKT and ERK

Objective To explore the protective effects of dexmedetomidine(Dex)against high glucose-induced epithelial-mesenchymal transition in HK-2 cells and relevant mechanisms.Methods HK-2 cells were exposed to either glucose or glucose+Dex for 6 h.The production of ROS,morphology of HK-2 cells,and cell cycle were detected.Moreover,the expression of AKT,p-AKT,ERK,pERK,PI3 K,E-Cadherin,Claudin-1,andα-SMA were determined and compared between HK-2 cells exposed to glucose and those exposed to both glucose and Dex with or without PI3 K/AKT pathway inhibitor LY294002 and ERK pathway inhibitor U0126.Results Compared with HK-2 cells exposed to high level of glucose,the HK-2 cells exposed to both high level of glucose and Dex showed:(1)lower level of ROS production;(2)cell morphology was complete;(3)more cells in G1 phase;(4)lower expression of p-AKT,p-ERK andα-SMA,higher expression of ECadherin and Claudin-1.PI3 K/AKT inhibitor LY294002 and ERK inhibitor U0126 decreased the expression of p-AKT,p-ERK andα-SMA,and increased the expression of E-Cadherin and Claudin-1.Conclusion Dex can attenuate high glucose-induced HK-2 epithelial-mesenchymal transition by inhibiting AKT and ERK.

MicroRNA-34a Regulates High Glucose-induced Apoptosis in H9c2 Cardiomyocytes

Hyperglycemia is an important initiator of cardiovascular disease, contributing to the de- velopment of cardiomyocyte death and diabetic complications. The purpose of the present study was to investigate whether high glucose state could induce apoptosis of rat cardiomyocyte cell line H9c2 through microRNA-mediated Bcl-2 signaling pathway. The expression of miR-34a and Bcl-2 mRNA was detected by using real-time PCR. Western blotting was used to examine the changes in apop- tosis-associated protein Bcl-2. Apoptosis of H9c2 cells was tested by using flow cytometry. The results showed that the expression of miR-34a was significantly elevated and that of Bcl-2 was strongly re- duced, and apoptosis of cardiomyocytes was apparently increased in the high-glucose-treated H9c2 cells as compared with normal-glucose-treated controls. In addition, we identified Bcl-2 gene was the target of miR-34a, miR-34a mimics reduced the expression of Bcl-2 and increased glucose-induced apoptosis, but miR-34a inhibitor acted as the opposite mediator. Our data demonstrate that miR-34a contributes to high glucose-induced decreases in Bcl-2 expression and subsequent cardiomyocyte apoptosis.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance the Osteoblastic Differentiation of Periodontal Ligament Stem Cells Under High Glucose Conditions Through the PI3K/AKT Signaling Pathway

Objective High glucose(HG)can influence the osteogenic differentiation ability of periodontal ligament stem cells(PDLSCs).Human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSC-exo)have broad application prospects in tissue healing.The current study aimed to explore whether hUCMSC-exo could promote the osteogenic differentiation of hPDLSCs under HG conditions and the underlying mechanism.Methods We used a 30 mmol/L glucose concentration to simulate HG conditions.CCK-8 assay was performed to evaluate the effect of hUCMSC-exo on the proliferation of hPDLSCs.Alkaline phosphatase(ALP)staining,ALP activity,and qRT-PCR were performed to evaluate the pro-osteogenic effect of hUCMSC-exo on hPDLSCs.Western blot analysis was conducted to evaluate the underlying mechanism.Results The results of the CCK-8 assay,ALP staining,ALP activity,and qRT-PCR assay showed that hUCMSC-exo significantly promoted cell proliferation and osteogenic differentiation in a dosedependent manner.The Western blot results revealed that hUCMSC-exo significantly increased the levels of p-PI3K and p-AKT in cells,and the effect was inhibited by LY294002(PI3K inhibitor)or MK2206(AKT inhibitor),respectively.Moreover,the increases in osteogenic indicators induced by hUCMSC-exo were significantly suppressed by LY294002 and MK2206.Conclusion hUCMSC-exo promote the osteogenic differentiation of hPDLSCs under HG conditions through the PI3K/AKT signaling pathway.

High glucose causes apoptosis of rabbit corneal epithelial cells involving activation of PERK-eIF2α-CHOP-caspase-12 signaling pathway

AIM: To investigate the effect of high concentration of glucose(HCG) on double stranded RNA-activated protein kinase-like ER kinase(PERK)-eukaryotic initiation factor-2α(eIF2α)-transcription factor C/EBP homologous protein(CHOP)-cysteine aspartate specific proteinase(caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells(RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48 h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 k Da glucose-regulated protein 78(GRP78), CHOP, B-cell lymphoma 2(Bcl-2), B-cell lymphoma-2-associated X protein(Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eI F2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs(P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12(P<0.05), and the alterations caused by glucose were in concentration-and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups(P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group(P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits(P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eI F2α in the HCG group(P<0.05), while still did not affect the expression of PERK and eIF2α among groups(P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose(P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOPcaspase-12 signaling pathway and promotes apoptosis of RCECs.

High glucose induces myocardial cell injury through increasing reactive oxygen species production

Objective: To study the injury effect and molecular mechanism of high glucose on myocardial cells. Methods: Myocardial cells H9 c2 were cultured and divided into the control group treated with DMEM containing 5.5 mmol/L glucose, the high glucose group treated with DMEM containing 35 mmol/L glucose, and the N-acetylcysteine(NAC) group pre-treated with 1000μmol/L NAC and treated with DMEM containing 1000 μmol/L NAC and 35 mmol/L glucose.The production of ROS and the expression of mitochondria pathway apoptosis molecules in cells as well as the contents of collagen and collagen metabolism molecules were measured.Results: After 8 h, 16 h and 24 h of treatment, ROS RFU as well as Bax, CytC, Caspase-3 and Caspase-9 protein expression in cells and Col-I, Col-Ⅲ, PINP and PⅢNP protein levels in culture medium of high glucose group were higher than those of control group, Bcl-2 protein expression were lower than those of control group, but CTX-Ⅰ protein levels in culture medium were not significantly different from those of control group; after 24 h of treatment, Bax, CytC,Caspase-3 and Caspase-9 protein expression in cells as well as Col-Ⅰ, Col-Ⅲ, PINP and PIIINP protein levels in culture medium of NAC group were lower than those of high glucose group whereas Bcl-2 protein expression was higher than that of high glucose group. Conclusions:High glucose can induce myocardial cell apoptosis, increase collagen synthesis and accelerate interstitial fibrosis by increasing the production of reactive oxygen species.

High Glucose Promotes the CTGF Expression in Human Mesangial Cells via Serum and Glucocorticoid-induced Kinase 1 Pathway

The role of serum and glucocorticoid-induced kinase 1 （SGK1） pathway in the connective tissue growth factor （CTGF） expression was investigated in cultured human mesangial cells （HMCs） under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 （SD） could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP （FP） under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 （KN） could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.

E26 transformation-specific 1 is implicated in the inhibition of osteogenic differentiation induced by chronic high glucose by directly regulating Runx2 expression

Chronic high glucose(HG) plays a crucial role in the pathogenesis of diabetes-induced osteoporosis by inhibiting the differentiation and proliferation of osteoblasts. This study aims to examine the role of E26 transformation-specific 1(ETS1) in the inhibition of osteoblast differentiation and proliferation caused by chronic HG, as well as the underlying mechanism. Chronic HG treatment downregulated ETS1 expression and inhibited differentiation and proliferation of MC3 T3-E1 cells. Downregulation of ETS1 expression inhibited the differentiation and proliferation of MC3 T3-E1 cells under normal glucose conditions, and ETS1 overexpression attenuated the damage to cells exposed to chronic HG. In addition, ETS1 overexpression reversed the decrease in runt-related transcription factor 2(Runx2) expression in MC3 T3-E1 cells treated with chronic HG. Using chromatin immunoprecipitation(ChIP) and luciferase reporter assays, we confirmed that ETS1 directly bound to and increased the activity of the Runx2 promoter. In summary, our study suggested that ETS1 was involved in the inhibitory effect of chronic HG on osteogenic differentiation and proliferation and may be a potential therapeutic target for diabetes-induced osteoporosis.

Green Tea Polyphenols Alleviate Autophagy Inhibition Induced by High Glucose in Endothelial Cells

Bovine aortic endothelial cells（BAECs）were cultured with high glucose（33 mmol/L）,4 mg/L green tea polyphenols（GTPs）or 4 mg/L GTPs co-treatment with high glucose for 24 h in the presence or absence of Bafilomycin-A1（BAF）.We observed that high glucose increased the accumulation of LC3-II.Treatment with BAF did not further increase the accumulation of LC3-II.

The effects of anti-vascular endothelial growth factor agents on human retinal pigment epithelial cells under high glucose conditions

AIM： To investigate the effects of high glucose levels and anti-vascular endothelial growth factor（VEGF） agents（bevacizumab,ranibizumab and aflibercept） on retinal pigment epithelium（RPE） cells.METHODS： ARPE-19 cells were cultured at different glucose levels（5.5 mmol/L,25 mmol/L,and 75 mmol/L）.Cell viability was evaluated by MTT assay at 3d after treatment with D-glucose.Cell migration ability was measured by wound healing assay at 3d.A cell death detection kit was used to assess apoptosis at 3 and 14 d.Cell proliferation was assessed by EdU assay at 3d.The culture medium was treated with anti-VEGF agents at clinically relevant concentrations.The experiment was then repeated at a different glucose level.RESULTS： The viability and migration of ARPE-19 cells were significantly decreased in the presence of 75 mmol/L as compared to 5.5 mmol/L glucose.The percentage of TUNEL-positive cells was significantly increased and the proliferative potential was decreased with 75 mmol/L compared to 5.5 mmol/L glucose.There were no significant differences in the results between 25 mmol/L and 5.5 mmol/L glucose.In the presence of 75 mmol/L glucose,the groups treated with anti-VEGF showed decreased cell viability and proliferation and increased apoptosis.However,there were no significant differences between the anti-VEGF groups.CONCLUSION： High glucose level decreases the viability,wound healing ability,and proliferation of RPE cells,while increasing apoptosis.Furthermore,anti-VEGF agents interfered with the physiological functions of RPE cells under high-glucose conditions,accompanied by decreases in cell viability and proliferation.

Gac fruit extracts ameliorate proliferation and modulate angiogenic markers of human retinal pigment epithelial cells under high glucose conditions

Objective: To investigate the impact of the extracts of Gac fruit parts(peel, pulp, seed, and aril) on the cell viability and angiogenesis markers of human retinal pigment epithelial(ARPE-19) cells under high glucose conditions. Methods: The effect of the extracts of Gac fruit peel, pulp, seed and aril on the ARPE-19 cells was determined using MTT viability assay, Trypan blue dye and morphological changes were observed using light microscopy. Enzyme-linked immunosorbent-based assay was performed to evaluate the effect of Gac fruit parts on the reactive oxygen species(ROS), vascular endothelial growth factor(VEGF) and pigmented epithelium-derived factor(PEDF) secretions. Results: High glucose(HG) at 30 mmol/L increased ARPE-19 cell viability and ROS and VEGF secretions. While, the exposure of ARPE-19 cells in high glucose condition to Gac fruit extracts led to inhibition of cell viability, induced morphological changes, decreased ROS and VEGF secretions, and increased PEDF level. Gac pulp, seed, and aril at 1 000 μg/mL showed significant inhibition activities [(7.5 ± 5.1)%,(2.7 ± 0.5)%,(3.2 ± 1.1)%, respectively] against HG-induced ARPE-19 cell viability. The findings also demonstrated that Gac aril at 250 μg/mL significantly decreased ROS and VEGF levels [(40.6 ± 3.3) pg/mL,(107.4 ± 48.3) pg/mL, respectively] compared to ROS [(71.7 ± 2.9) pg/mL ] and VEGF [(606.9 ± 81.1) pg/mL] in HG untreated cells. Moreover, 250 μg/mL of Gac peel dramatically increased PEDF level [(18.2 ± 0.3) ng/mL] compared to that in HG untreated cells [(0.48 ± 0.39) ng/mL]. Conclusions: This study indicates that the extracts of Gac peel, pulp, seed and aril reduced cell viability, minimized ROS generations and showed angiogenic activities. Therefore, our findings open new insights into the potentiality of Gac fruit against HG-related diabetic retinopathy disease.

β-elemene down-regulates HIF-lα, VEGF and iNOS in human retinal pigment epithelial cells under high glucose conditions

AIM:To investigate the effects and mechanism ofβ-elemene on the expressions of hypoxia-inducible factor-1α(HIF-lα),vascular endothelial growth factor(VEGF)and inducible nitric oxide synthase(iNOS)in human retinal pigment epithelial(RPE)cells under high glucose conditions.METHODS:ARPE-19 cell line was cultured under eight conditions:1)low glucose(LG;5.5 mmol/L);2)high glucose(HG;33 mmol/L);3)high glucose with 20μg/m Lβ-elemene(HG+20 E);4)high glucose with 40μg/m Lβ-elemene(HG+40 E);5)high glucose with SB203590[HG+SB203590,p38-mitogen-activated protein kinase(p38-MAPK)pathway inhibitor];6)high glucose with LY294002[HG+LY294002,phosphoinositide 3-kinase/protein kinase B(PI3 K/Akt)pathway inhibitor];7)high glucose with 40μg/m Lβ-elemene and SB203590(HG+40 E+SB203590);and 8)high glucose with 40μg/m Lβ-elemene and LY294002(HG+40 E+LY294002).Cells were treated in conditions 1-4 for 24 and 48 h,while for 48 h in conditions 5-8.Then m RNA and protein levels of HIF-1α,VEGF and iNOS in cells were measured by real-time polymerase chain reaction(q PCR),immunofluorescence and Western blotting,respectively.Furthermore,protein levels of total p38-MAPK,phosphorylated p38-MAPK(p38-MAPK-P),total Akt and phosphorylated Akt(Akt-P)in cells of conditions 2 and 4 which treated for 48 h were measured by Western blotting.RESULTS:The m RNA levels and protein levels of HIF-1α,VEGF and iNOS in cells were significantly reduced in conditions 3-8 when compared with those in condition 2(P<0.05).These reductions were more obvious in conditions treated for 48 h than in conditions treated for 24 h.The protein levels of p38-MAPK-P and Akt-P in cells of condition 4 were significantly lower than in condition 2(P<0.01).CONCLUSION:β-elemene down-regulates HIF-1α,VEGF and iNOS in ARPE-19 cells under a high glucose condition.The inhibitory effect ofβ-elemene is more significant when its concentration and treatment time are increased,as well as it is combined with SB203590 or LY294002 treatment.P38-MAPK and PI3 K/Akt signaling pathways may play a role in this inhibitory effect.

Hydrogen Sulfide Attenuates High Glucose-induced Myocardial Injury in Rat Cardiomyocytes by Suppressing Wnt/beta-catenin Pathway

Diabetic cardiomyopathy(DCM)is one of the major heart complications of diabetic patients.Hydrogen sulfide(H2S)is now recognized as an important signaling molecule and has been shown to attenuate the development of diabetic cardiomyopathy.However,the underlying mechanisms linking H2S and the development of DCM have not been fully elucidated.In the present study,we therefore sought to explore the role and mechanism of H2S in the pathogenesis of DCM by establishing high glucose-induced injury model in neonatal rat cardiomyocytes(NRCMs)and H9c2 cells.Using cystathionine gamma-lyase(CSE)overexpression and CSE interference vectors transfection,the cell viability,cell apoptosis,and oxidative stress were determined and compared between the treatment of high glucose induction and exgenous NaHS administration.Meanwhile,the relationship between the CSE/H2S system and Wnt/beta-catenin pathway was analyzed and discussed in the high glucose-induced cardiomyocytes.Our results indicated that H2S played an important protective role in high glucose-induced apoptosis and oxidative stress in cardiomyocytes,as shown by the decreased reactive oxygen species and malondialdehyde levels,and the increased activities of superoxide dismutase,catalase and glutathione peroxidase.Moreover,H:S could attenuate the Wnt/p-catenin signalling pathway and up-regulate the expression of haem oxygenase-1(HO-1)and NAD(P)H:quinone oxidoreductase 1(NQOl)in the diabetic myocardium cells.Together,these results demonstrated that H2S could attenuate high glucoseinduced myocardial injury in rat cardiomyocytes by suppressing Wnt/p-catenin pathway.