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 char...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.展开更多
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 apop...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.展开更多
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 ne...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.展开更多
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...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.展开更多
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...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.展开更多
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 diseas...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.展开更多
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...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.展开更多
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 ...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.展开更多
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...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.展开更多
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 transfo...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.展开更多
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 p...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.展开更多
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 dia...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.展开更多
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 p...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.展开更多
OBJECTIVE To investigate the neuroprotective effects of quercetin on central neurons against chronic high glucose in central neurons,in relation to Nrf2/ARE/Glo-1 activation.METHODS SH-SY5Y cells were cultured with hi...OBJECTIVE To investigate the neuroprotective effects of quercetin on central neurons against chronic high glucose in central neurons,in relation to Nrf2/ARE/Glo-1 activation.METHODS SH-SY5Y cells were cultured with high glucose(HG,70 mmol·L^(-1)),4-fold of the normal glucose(17.5 mmol·L^(-1)).Quercetin was set three concentrations(5,10,20μmol·L^(-1)),with Nrf2 activator sulforaphane(SFN)as a positive group(2.5μmol·L^(-1)).After 72 h,cells were collected for glyoxalase 1(Glo-1)activity and GSH level were by spectrophotometry;advanced glycation end-products(AGEs)as well as nuclear Nrf2 and p-Nrf2 levels by immunofluorescence;Glo-1,γ-glutamycysteine synthase(γ-GCS),Nrf2 and p-Nrf2 protein levels by Western blotting,and Glo-1 andγ-GCS m RNA levels by real-time qP CR.RESULTS Quercetin increased the cell viability of SH-SY5Y cells,and upregulated the levels of Glo-1 activity,protein,and m RNA in SH-SY5Y cells cultured with HG,accompanied by the elevated levels of glutathione,a cofactor of Glo-1 activity,and the reduced levels of AGEs.Meanwhile,quercetin could increase p-Nrf2 and Nrf2 levels in nucleus as well as p-Nrf2 levels in cytosol of SH-SY5Y cells exposed to chronic HG,accompanied by the elevated protein expression and m RNA levels ofγ-GCS,a known target gene of Nrf2/ARE signaling.Moreover,a PKC activator or a p38MAPK inhibitor pretreatment could significantly increase the protein expression ofγ-GCS in HG condition,but an alkylating agent for sulfydryl of cysteine in Keap 1,a negative regulator of Nrf2,pretreatment only showed an increased tendency ofγ-GCS protein,compared with without pretreatment;however,after pretreatment with those tool drugs,co-treatment with quercetin and HG had similar results to those of single tool drug pretreatment followed by HG exposure.CONCLUSION Firstly,quercetin can enhance Glo-1 function in central neurons,which is mediated by activation of Nrf2/ARE pathway,then exerts the neuroprotection against HG induced damage;moreover,PKC and p38 MAPK pathways may be involved in Nrf2 inactivation in chronic HG condition.展开更多
Previous reports have suggested that Ang-(1-7)may have a protective effect in endothelial cells against high glucose(HG)-induced cell injury thanks to a modulatory mechanism in the NF-κB signaling pathway.In this stu...Previous reports have suggested that Ang-(1-7)may have a protective effect in endothelial cells against high glucose(HG)-induced cell injury thanks to a modulatory mechanism in the NF-κB signaling pathway.In this study,we have examined whether NF-κB-IL-1βand Heme oxygenase-1(HO-1)pathways contribute to the protection of Ang-(1-7)against hyperglycemia-induced inflammation and oxidative stress in human umbilical vein endothelial cells(HUVECs).Our results indicate that time-varying exposures of HUVECs,from 1 h to 24 h,to high glucose concentrations result in an increased expression of phosphorylated(p)-p65 and HO-1 in a time-dependent manner.As an inhibitor of NF-κB,pyrrolidinedithiocarbamic acid(PDTC)suppressed IL-1βproduction induced by HG.Of note,HUVECs previously treated with Ang-(1-7)(2μM)for 30 min before being exposed to HG concentrations significantly ameliorated the HG-increased in p-p65 and IL-1βexpression;whereas obviously up-regulated the level of HO-1,along with inhibition of oxidative stress,inflammation,and the HG-induced cytotoxicity.Importantly,when HUVECs were previously treated either with PDTC or IL-1Ra for 30 min before being exposed to HG,it significantly prevented damages caused by high glucose concentrations mentioned above,while the treatment of HO-1 inhibitor Sn-protoporphyrin(SnPP)before exposure to both HG and Ang-(1-7)significantly blocked the protective effect exerted by Ang-(1-7)on endothelial cells against injuries induced by HG mentioned above.To conclude,the data of this study showed that activation and inhibition of the NF-κB-IL-1βpathway and HO-1 pathway may constitute an important defense mechanism against endothelial cell damage caused by HG concentrations.We additionally gave new evidence showing that exogenous Ang-(1-7)exerts a protective effect on HUVECs against the HG-induced cell injury via the inhibition and the activation of the NF-κB-IL-1βpathway and the HO-1 pathway,respectively.展开更多
Objective:To investigate the role of baicalein in phenotypic transformation of vascularsmooth muscle cells induced by high glucose.Methods:Rat vascular smooth muscle cellexperiments were divided into control group,bai...Objective:To investigate the role of baicalein in phenotypic transformation of vascularsmooth muscle cells induced by high glucose.Methods:Rat vascular smooth muscle cellexperiments were divided into control group,baicalein group,high glucose group,highglucose plus baicalein group,real time quantitative PCR were used for mRNA analysis of-SMA,SM22-αnd OPN,Western blot were used for protein analysis of α-SMA,SM22-αand OPN.Results:Comparing the high glucose group and the high glucose plus baicaleingroup,the level of α-SMA mRNA in high glucose group was 0.419±0.090,the level ofα-SMA mRNA in high glucose plus baicalein group was 0.699±0.079,the latter was 66.8%higher than the former.The level of α-SMA protein in high glucose group was 0.213±0.034,the level of α-SMA protein in high glucose plus baicalein group was 0.393±0.062,the latterwas 84.5%higher than the former.Baicalein could significantly inhibit the down-regulationof α-SMA gene expression induced by high glucose(P<0.05).Comparing the high glucosegroup and the high glucose plus baicalein group,the level of SM22-mRNA in high glucosegroup was 0.369±0.063,the level of SM22-α mRNA in high glucose plus baicalein groupwas 0.583±0.049,the latter was 58.0%higher than the former.The level of SM22-α proteinin high glucose group was 0.343±0.047,the level of SM22-protein in high glucose plusbaicalein group was 0.486±0.051,the latter was 41.7%higher than the former.Baicaleincould significantly inhibit the down-regulation of SM22-α gene expression induced by highglucose(P<0.05).Comparing the high glucose group and the high glucose plus baicaleingroup,the level of OPN mRNA in high glucose group was 2.023±0.281,the level of OPNmRNA in high glucose plus baicalein group was 1.511±0.091,the latter was 25.3%lowerthan the former.The level of OPN protein in high glucose group was 1.063±0.132,the levelof OPN protein in high glucose plus baicalein group was 0.761±0.089,the latter was 28.4%lower than the former.Baicalein could significantly inhibit the up-regulation of OPN geneexpression induced by high glucose(P<0.05).Conclusion:Baicalein can significantly inhibitthe high glucose-induced phenotypic transformation of vascular smooth muscle cells fromcontractile phenotype to synthetic phenotype.展开更多
Objective:To study the effect of mechanism of high glucose on apoptosis and cell cycle arrest of isletβcells via p27 pathway.Methods:Islet INS-1 cells were cultured and divided into groups.The control group was treat...Objective:To study the effect of mechanism of high glucose on apoptosis and cell cycle arrest of isletβcells via p27 pathway.Methods:Islet INS-1 cells were cultured and divided into groups.The control group was treated with ordinary medium,the high glucose group was treated with high glucose medium containing 25mmol/L glucose,the high glucose+si-NC group was treated with high glucose medium and transfected with NC siRNA,and the high glucose+si-P27 group was treated with high glucose medium and transfected with p27 siRNA.After 24 hours treatment,MTS assay was used to detect the cell viability A490,TUNEL assay was used to detect apoptosis rate,flow cytometry was used to detect the cell cycle distribution and western blot was used to detect the expression levels of P27,caspase-8 and cyclinD1.Results:Compared with those in the control group,the A490,the ratio of S phase and G2/M phase as well as the expression level of CyclinD1 decreased,while the apoptosis rate,the ratio of G0/G1 phase as well as the expression levels of P27 and caspase-8 increased in the high glucose group(P<0.05);compared with those in the high glucose group,the A490,cell cycle as well as the expression levels of P27,caspase-8 and cyclinD1 were not different from those in the high glucose+si-NC group(P>0.05);compared with those in the high glucose group and high glucose+si-NC group,the A490,the ratio of S phase and G2/M phase as well as the expression levels of cyclinD1 increased,while the apoptosis rate,the ratio of G0/G1 phase as well as the expression levels of p27 and caspase-8 decreased in the high glucose+si-P27 group(P<0.05).Conclusion:The apoptosis and cell cycle arrest induced by high glucose are related to P27 pathway activation.展开更多
OBJECTIVE:To explore the synergistic effect of deoxyribonucleaseⅠ(DNaseⅠ)knockdown combined with Schizandrin A(Sch A)in protecting islet beta-cells(β-cells)from apoptosis under high-glucose(HG)conditions.METHODS:Th...OBJECTIVE:To explore the synergistic effect of deoxyribonucleaseⅠ(DNaseⅠ)knockdown combined with Schizandrin A(Sch A)in protecting islet beta-cells(β-cells)from apoptosis under high-glucose(HG)conditions.METHODS:The concentration of Sch A was detected by Cell Counting Kit-8(CCK-8).High glucose-cultured rat insulinoma beta cell line(RIN-M5F)cells were treated with Sch A and transfected with DNaseⅠsmall interfering RNA(siRNA).Cell apoptosis rate and apoptosis-related protein level were examined by flow cytometry and Western blot method respectively.In addition,Na^(+)-K^(+)-adenosine triphosphatease(Na^(+)-K^(+)-ATPase)and Ca^(2+)-Mg^(2+)-ATPase activity,cell membrane potential,and intracellular Ca^(2+)concentration was also examined respectively.RESULTS:Our study revealed that HG stimulation can cause a significant increase in DNaseⅠlevel and cell apoptosis rate.However,Sch A combined with DNaseⅠknockdown can significantly decrease the cell apoptosis rate and apoptosis-related protein levels such as BAX(P<0.05)and Caspase-3(P<0.01).In addition,we also found that the combination of Sch A and DNaseⅠknockdown can dramatically increase cell membrane potential level,Na+-K+-ATPase,and Ca^(2+)-Mg^(2+)-ATPase activity.Meanwhile,intracellular Ca^(2+)concentration was also found to be significantly decreased by the synergistic effect of Sch A and DNaseⅠknockdown.CONCLUSION:Overall,our study reveals a synergistic effect of Sch A and DNaseⅠknockdown in protectingβ-cells from HG-induced apoptosis.展开更多
AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are r...AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are regulated,and whether this pathway could be specifically targeted.METHODS:hRPE cells were cultured in normal glucose or high glucose for 1,3,or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species(ROS)levels using a dihydroethidium kit.miR-27b-3p,Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunocytofluorescence(ICF),respectively.Western blot analyses were performed to determine nuclear and total Nrf2 protein levels.Nrf2,NQO1,and HO-1 expression levels by RT-qPCR,ICF,or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection.Finally,the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine.RESULTS:Persistent exposure to high glucose gradually suppressed hRPE Nrf2,NQO1,and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels.High glucose also promoted ROS release and inhibited cellular proliferation.Nrf2,NQO1,and HO-1 mRNA levels decreased after miR-27b-3p overexpression and,conversely,both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor.After treating hRPE cells exposed to high glucose with pyridoxamine,ROS levels tended to decreased,proliferation rate increased,Nrf2,NQO1,and HO-1 mRNA and protein levels were upregulated,and miR-27b-3p mRNA levels were suppressed.CONCLUSION:Nrf2 is a downstream target of miR-27b-3p.Furthermore,the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.展开更多
Objective:To study the effects of Lycium barbarum polysaccharide(LBP)on the proliferation,apoptosis,and autophagy of retinal pigment epithelial(RPE)cells cultured under high-glucose conditions.Methods:The ARPE-19 cell...Objective:To study the effects of Lycium barbarum polysaccharide(LBP)on the proliferation,apoptosis,and autophagy of retinal pigment epithelial(RPE)cells cultured under high-glucose conditions.Methods:The ARPE-19 cell line was randomly divided into a control group(normally cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12[DMEM/F-12]medium),a high-glucose group(HG;50 mmol/L glucose added to DMEM/F-12 medium),and a HG+LBP group(incubated in DMEM/F-12 medium containing 1 mg/mL LBP for 24 h,and then treated with 50 mmol/L glucose for 24 h).Following Ad-mCherry-GFP-LC3B infection,cell proliferation,apoptosis,mammalian target of rapamy-cin(mTOR)expression,and autophagic flux were determined by Cell Counting Kit-8(CCK-8),AnnexinV-APC/7-AAD Apoptosis Detection Kit,Western blot,and laser confocal microscopy,respectively.Results:The proliferation rate of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),while the proliferation rate of ARPE-19 cells in the HG+LBP group was significantly higher than that in the HG group(P<0.05).The apoptosis rate of ARPE-19 cells in the HG group was significantly higher than that in the control group(P<0.05),while the apoptosis rate of ARPE-19 cells in the HG+LBP group was significantly lower than that in the HG group(P<0.05).The relative expression of phosphorylated mTOR(p-mTOR)of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),with enhanced autophagic flux;when compared with the HG group,the HG+LBP group had significantly higher expression of p-mTOR(P<0.05),with diminished autophagic flux.Conclusion:LBP has a protective effect on RPE cells with high glucose-induced injury,and its mechanism may be related to LBP inhibition of high glucose-induced abnormal autophagy.展开更多
基金Supported by the Deanship of Scientific Research,Yarmouk University,Jordan,No.73/2022.
文摘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.
基金supported by the National-Natural Science Foundation of China(32072334),the General Project of the Education Department of Hunan Province(20C0959)the Changsha Natural Science Foundation(kq2007020).
文摘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.
文摘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.
基金Supported by Medical and Health Science and Technology Project of Zhejiang Province(No.2023KY1356).
文摘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.
基金Supported by the Shaanxi Province Traditional Chinese Medicine Project(No.SZY-KJCYC-2023-028)。
文摘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.
文摘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.
文摘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.
基金Supported by Shanghai Natural Science Foundation (No.19ZR1450500)National Foundation Cultivation Project of Tongji University (No.22120180285)the Good Physician Training Project of Yangpu District, Shanghai
文摘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.
基金supported by Research Projects of Wuhan Health Bureau(No.wx12c35)
文摘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.
基金supported by 2021 Nantong City Basic Research and People's Livelihood Science and Technology Plan Guiding Project (JCZ21133)。
文摘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.
文摘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.
基金the Natural Science Foundation of Hubei Province of China(No.2011CDC146).
文摘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.
文摘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.
基金supported by National Natural Science Foundation of China(81371210)Qing Lan Project of Jiangsu Province(2014)Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘OBJECTIVE To investigate the neuroprotective effects of quercetin on central neurons against chronic high glucose in central neurons,in relation to Nrf2/ARE/Glo-1 activation.METHODS SH-SY5Y cells were cultured with high glucose(HG,70 mmol·L^(-1)),4-fold of the normal glucose(17.5 mmol·L^(-1)).Quercetin was set three concentrations(5,10,20μmol·L^(-1)),with Nrf2 activator sulforaphane(SFN)as a positive group(2.5μmol·L^(-1)).After 72 h,cells were collected for glyoxalase 1(Glo-1)activity and GSH level were by spectrophotometry;advanced glycation end-products(AGEs)as well as nuclear Nrf2 and p-Nrf2 levels by immunofluorescence;Glo-1,γ-glutamycysteine synthase(γ-GCS),Nrf2 and p-Nrf2 protein levels by Western blotting,and Glo-1 andγ-GCS m RNA levels by real-time qP CR.RESULTS Quercetin increased the cell viability of SH-SY5Y cells,and upregulated the levels of Glo-1 activity,protein,and m RNA in SH-SY5Y cells cultured with HG,accompanied by the elevated levels of glutathione,a cofactor of Glo-1 activity,and the reduced levels of AGEs.Meanwhile,quercetin could increase p-Nrf2 and Nrf2 levels in nucleus as well as p-Nrf2 levels in cytosol of SH-SY5Y cells exposed to chronic HG,accompanied by the elevated protein expression and m RNA levels ofγ-GCS,a known target gene of Nrf2/ARE signaling.Moreover,a PKC activator or a p38MAPK inhibitor pretreatment could significantly increase the protein expression ofγ-GCS in HG condition,but an alkylating agent for sulfydryl of cysteine in Keap 1,a negative regulator of Nrf2,pretreatment only showed an increased tendency ofγ-GCS protein,compared with without pretreatment;however,after pretreatment with those tool drugs,co-treatment with quercetin and HG had similar results to those of single tool drug pretreatment followed by HG exposure.CONCLUSION Firstly,quercetin can enhance Glo-1 function in central neurons,which is mediated by activation of Nrf2/ARE pathway,then exerts the neuroprotection against HG induced damage;moreover,PKC and p38 MAPK pathways may be involved in Nrf2 inactivation in chronic HG condition.
基金supported by the Technology Planning Project of Huangpu District(201544-01)Medical Scientific Research Foundation of Guangdong Province(A2015287)+2 种基金Science and Technology Planning Project of Guangdong Province(2017ZC0474)Natural Science Foundation of Guangdong Province(2015A030313690)General Project of Dongguan City(Nos.201950715024922 and 2018507150241344).
文摘Previous reports have suggested that Ang-(1-7)may have a protective effect in endothelial cells against high glucose(HG)-induced cell injury thanks to a modulatory mechanism in the NF-κB signaling pathway.In this study,we have examined whether NF-κB-IL-1βand Heme oxygenase-1(HO-1)pathways contribute to the protection of Ang-(1-7)against hyperglycemia-induced inflammation and oxidative stress in human umbilical vein endothelial cells(HUVECs).Our results indicate that time-varying exposures of HUVECs,from 1 h to 24 h,to high glucose concentrations result in an increased expression of phosphorylated(p)-p65 and HO-1 in a time-dependent manner.As an inhibitor of NF-κB,pyrrolidinedithiocarbamic acid(PDTC)suppressed IL-1βproduction induced by HG.Of note,HUVECs previously treated with Ang-(1-7)(2μM)for 30 min before being exposed to HG concentrations significantly ameliorated the HG-increased in p-p65 and IL-1βexpression;whereas obviously up-regulated the level of HO-1,along with inhibition of oxidative stress,inflammation,and the HG-induced cytotoxicity.Importantly,when HUVECs were previously treated either with PDTC or IL-1Ra for 30 min before being exposed to HG,it significantly prevented damages caused by high glucose concentrations mentioned above,while the treatment of HO-1 inhibitor Sn-protoporphyrin(SnPP)before exposure to both HG and Ang-(1-7)significantly blocked the protective effect exerted by Ang-(1-7)on endothelial cells against injuries induced by HG mentioned above.To conclude,the data of this study showed that activation and inhibition of the NF-κB-IL-1βpathway and HO-1 pathway may constitute an important defense mechanism against endothelial cell damage caused by HG concentrations.We additionally gave new evidence showing that exogenous Ang-(1-7)exerts a protective effect on HUVECs against the HG-induced cell injury via the inhibition and the activation of the NF-κB-IL-1βpathway and the HO-1 pathway,respectively.
基金Hainan Provincial Natural Science Foundation of China(No.817141)National Natural Science Foundation of China(No.81460043).
文摘Objective:To investigate the role of baicalein in phenotypic transformation of vascularsmooth muscle cells induced by high glucose.Methods:Rat vascular smooth muscle cellexperiments were divided into control group,baicalein group,high glucose group,highglucose plus baicalein group,real time quantitative PCR were used for mRNA analysis of-SMA,SM22-αnd OPN,Western blot were used for protein analysis of α-SMA,SM22-αand OPN.Results:Comparing the high glucose group and the high glucose plus baicaleingroup,the level of α-SMA mRNA in high glucose group was 0.419±0.090,the level ofα-SMA mRNA in high glucose plus baicalein group was 0.699±0.079,the latter was 66.8%higher than the former.The level of α-SMA protein in high glucose group was 0.213±0.034,the level of α-SMA protein in high glucose plus baicalein group was 0.393±0.062,the latterwas 84.5%higher than the former.Baicalein could significantly inhibit the down-regulationof α-SMA gene expression induced by high glucose(P<0.05).Comparing the high glucosegroup and the high glucose plus baicalein group,the level of SM22-mRNA in high glucosegroup was 0.369±0.063,the level of SM22-α mRNA in high glucose plus baicalein groupwas 0.583±0.049,the latter was 58.0%higher than the former.The level of SM22-α proteinin high glucose group was 0.343±0.047,the level of SM22-protein in high glucose plusbaicalein group was 0.486±0.051,the latter was 41.7%higher than the former.Baicaleincould significantly inhibit the down-regulation of SM22-α gene expression induced by highglucose(P<0.05).Comparing the high glucose group and the high glucose plus baicaleingroup,the level of OPN mRNA in high glucose group was 2.023±0.281,the level of OPNmRNA in high glucose plus baicalein group was 1.511±0.091,the latter was 25.3%lowerthan the former.The level of OPN protein in high glucose group was 1.063±0.132,the levelof OPN protein in high glucose plus baicalein group was 0.761±0.089,the latter was 28.4%lower than the former.Baicalein could significantly inhibit the up-regulation of OPN geneexpression induced by high glucose(P<0.05).Conclusion:Baicalein can significantly inhibitthe high glucose-induced phenotypic transformation of vascular smooth muscle cells fromcontractile phenotype to synthetic phenotype.
基金National Natural Science Foundation of China(No.81970668)Shaanxi Science and Technology Planning Project(No.2019JM-116)。
文摘Objective:To study the effect of mechanism of high glucose on apoptosis and cell cycle arrest of isletβcells via p27 pathway.Methods:Islet INS-1 cells were cultured and divided into groups.The control group was treated with ordinary medium,the high glucose group was treated with high glucose medium containing 25mmol/L glucose,the high glucose+si-NC group was treated with high glucose medium and transfected with NC siRNA,and the high glucose+si-P27 group was treated with high glucose medium and transfected with p27 siRNA.After 24 hours treatment,MTS assay was used to detect the cell viability A490,TUNEL assay was used to detect apoptosis rate,flow cytometry was used to detect the cell cycle distribution and western blot was used to detect the expression levels of P27,caspase-8 and cyclinD1.Results:Compared with those in the control group,the A490,the ratio of S phase and G2/M phase as well as the expression level of CyclinD1 decreased,while the apoptosis rate,the ratio of G0/G1 phase as well as the expression levels of P27 and caspase-8 increased in the high glucose group(P<0.05);compared with those in the high glucose group,the A490,cell cycle as well as the expression levels of P27,caspase-8 and cyclinD1 were not different from those in the high glucose+si-NC group(P>0.05);compared with those in the high glucose group and high glucose+si-NC group,the A490,the ratio of S phase and G2/M phase as well as the expression levels of cyclinD1 increased,while the apoptosis rate,the ratio of G0/G1 phase as well as the expression levels of p27 and caspase-8 decreased in the high glucose+si-P27 group(P<0.05).Conclusion:The apoptosis and cell cycle arrest induced by high glucose are related to P27 pathway activation.
基金the Natural Science Foundation of China:the Protective Effect of Schizandrin A toβCell Apoptosis by DNaseⅠin Type 2 Diabetes(No.81803909)。
文摘OBJECTIVE:To explore the synergistic effect of deoxyribonucleaseⅠ(DNaseⅠ)knockdown combined with Schizandrin A(Sch A)in protecting islet beta-cells(β-cells)from apoptosis under high-glucose(HG)conditions.METHODS:The concentration of Sch A was detected by Cell Counting Kit-8(CCK-8).High glucose-cultured rat insulinoma beta cell line(RIN-M5F)cells were treated with Sch A and transfected with DNaseⅠsmall interfering RNA(siRNA).Cell apoptosis rate and apoptosis-related protein level were examined by flow cytometry and Western blot method respectively.In addition,Na^(+)-K^(+)-adenosine triphosphatease(Na^(+)-K^(+)-ATPase)and Ca^(2+)-Mg^(2+)-ATPase activity,cell membrane potential,and intracellular Ca^(2+)concentration was also examined respectively.RESULTS:Our study revealed that HG stimulation can cause a significant increase in DNaseⅠlevel and cell apoptosis rate.However,Sch A combined with DNaseⅠknockdown can significantly decrease the cell apoptosis rate and apoptosis-related protein levels such as BAX(P<0.05)and Caspase-3(P<0.01).In addition,we also found that the combination of Sch A and DNaseⅠknockdown can dramatically increase cell membrane potential level,Na+-K+-ATPase,and Ca^(2+)-Mg^(2+)-ATPase activity.Meanwhile,intracellular Ca^(2+)concentration was also found to be significantly decreased by the synergistic effect of Sch A and DNaseⅠknockdown.CONCLUSION:Overall,our study reveals a synergistic effect of Sch A and DNaseⅠknockdown in protectingβ-cells from HG-induced apoptosis.
基金Supported by National Natural Science Foundation of China(No.2020J01652)the Training Project for Young and Middleaged Core Talents in Health System of Fujian Province(No.2016-ZQN-62).
文摘AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are regulated,and whether this pathway could be specifically targeted.METHODS:hRPE cells were cultured in normal glucose or high glucose for 1,3,or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species(ROS)levels using a dihydroethidium kit.miR-27b-3p,Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunocytofluorescence(ICF),respectively.Western blot analyses were performed to determine nuclear and total Nrf2 protein levels.Nrf2,NQO1,and HO-1 expression levels by RT-qPCR,ICF,or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection.Finally,the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine.RESULTS:Persistent exposure to high glucose gradually suppressed hRPE Nrf2,NQO1,and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels.High glucose also promoted ROS release and inhibited cellular proliferation.Nrf2,NQO1,and HO-1 mRNA levels decreased after miR-27b-3p overexpression and,conversely,both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor.After treating hRPE cells exposed to high glucose with pyridoxamine,ROS levels tended to decreased,proliferation rate increased,Nrf2,NQO1,and HO-1 mRNA and protein levels were upregulated,and miR-27b-3p mRNA levels were suppressed.CONCLUSION:Nrf2 is a downstream target of miR-27b-3p.Furthermore,the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.
基金supported by the Supporting Fund of the First Affiliated Hospital of Xi'an Medical University(XYFYPT-2023-01).
文摘Objective:To study the effects of Lycium barbarum polysaccharide(LBP)on the proliferation,apoptosis,and autophagy of retinal pigment epithelial(RPE)cells cultured under high-glucose conditions.Methods:The ARPE-19 cell line was randomly divided into a control group(normally cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12[DMEM/F-12]medium),a high-glucose group(HG;50 mmol/L glucose added to DMEM/F-12 medium),and a HG+LBP group(incubated in DMEM/F-12 medium containing 1 mg/mL LBP for 24 h,and then treated with 50 mmol/L glucose for 24 h).Following Ad-mCherry-GFP-LC3B infection,cell proliferation,apoptosis,mammalian target of rapamy-cin(mTOR)expression,and autophagic flux were determined by Cell Counting Kit-8(CCK-8),AnnexinV-APC/7-AAD Apoptosis Detection Kit,Western blot,and laser confocal microscopy,respectively.Results:The proliferation rate of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),while the proliferation rate of ARPE-19 cells in the HG+LBP group was significantly higher than that in the HG group(P<0.05).The apoptosis rate of ARPE-19 cells in the HG group was significantly higher than that in the control group(P<0.05),while the apoptosis rate of ARPE-19 cells in the HG+LBP group was significantly lower than that in the HG group(P<0.05).The relative expression of phosphorylated mTOR(p-mTOR)of ARPE-19 cells in the HG group was significantly lower than that in the control group(P<0.05),with enhanced autophagic flux;when compared with the HG group,the HG+LBP group had significantly higher expression of p-mTOR(P<0.05),with diminished autophagic flux.Conclusion:LBP has a protective effect on RPE cells with high glucose-induced injury,and its mechanism may be related to LBP inhibition of high glucose-induced abnormal autophagy.
