CHIP participates in high glucose-induced vascular endothelial cell injury

AIM To investigate the effects of carboxy terminus of heat shock protein 70-interacting protein (CHIP) on high glucose (HG)-induced vascular endothelial cell injury. METHODS Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with 5.5 mmol/L glucose (normal glucose, NG) or 25.5 mmol/L glucose (HG) for 24 h. Down-regulation of CHIP expression by RNA interference was conducted. Before the experiment, mannitol was used to eliminate the interference of osmotic pressure. Subsequently, the cells was divided into 4 groups: NG+siRNA NC group, NG+siRNA CHIP group, HG+siRNA NC group, and HG+siRNA CHIP group. Additionally, MTT assay and TUNEL staining were used to detect the viability and apoptosis. The level of endothelin-1 (ET-1) was measured by ELISA, and the level of reactive oxygen species (ROS) was detected by fluorescence probe dihydroethidium. The level of nitric oxide (NO), and the activity of superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in the cells were detected by their respective kits. The mRNA expression of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) was detected by RT-qPCR. The protein levels of CHIP, NADPH oxidase (NOX) 2, NOX4, p38, p65, p-p38, p-p65, Bax and Bcl-2 were determined by Western blot. RESULTS Compared with NG+siRNA NC group, the cell viability was decreased, the apoptosis rate, the mRNA expression of IL-8 and MCP-1, and the level of ROS were increased (P<0.05), the activity of SOD was decreased (P<0.05), while the levels of ET-1, NO and iNOS and the protein levels of p-p38, p-p65 and Bax were increased in HG+siRNA NC group (P<0.05). Compared with HG+siRNA NC group, the inflammatory response, the oxidative stress, the apoptosis rate, and the protein levels of p-p38, p-p65 and Bax were significantly increased in HG+siRNA CHIP group (P<0.05). CONCLUSION Down-regulation of CHIP expression aggravates HG-induced vascular endothelial cell injury.     

Genipin inhibits oxidative stress and apoptotic damage in high glucose-induced H9c2 cardiomyocytes

AIM:To explore the effects of genipin (GEN) on high glucose (HG)-induced oxidative stress injury and apoptosis in H9c2 cardiomyocytes.METHODS:H9c2 cells were cultured in vitro and HG-induced injury model was established. H9c2 cells were divided into 4 groups:normal control (NC) group (glucose at 5.6 mmol/L), HG group (glucose at 50 mmol/L), NG+GEN group and HG+GEN group. The concentration of genipin was used at 10 μmol/L. The viability of the H9c2 cells was measured by CCK-8 assay. The intracellular malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by enzyme labeling and WST-1 methods, respectively. The activity of lactate dehydrogenase (LDH) in the cell culture supernatant was detected by microplate method. Fluorescent probe DCF was used to detect intracellular levels of reactive oxygen species (ROS). Nucleosome fragments was measured to evaluate cell apoptosis by ELISA. The intracellular mitochondrial membrane potential was detected by JC-1 method. The protein levels of Mn-SOD, cytochrome C (Cyt C), Bax and cleaved caspase-3 were determined by Western blot. RESULTS:Compared with HG group, the cell viability in HG+GEN group was increased significantly (P<0.05), the levels of MDA and LDH were decreased (P<0.05), SOD activity was increased (P<0.05), the levels of ROS and nucleosome fragments in HG+GEN group were decreased (P<0.05), and the mitochondrial membranes potential was notably increased (P<0.05). Compared with NG group, the activation of Mn-SOD was decreased, but the protein levels of Cyt C, Bax and cleaved caspase-3 were increased in HG group (P<0.05). Compared with HG group, the activation of Mn-SOD was increased, and the protein levels of Cyt C, Bax and cleaved caspase-3 were decreased in HG+GEN group (P<0.05).CONCLUSION:Genipin protects HG-induced H9c2 cardiomyocytes against oxidative stress injury and apoptosis.     

Effect of HMGA2 gene on high glucose-induced apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway

AIM:To investigate the effect of HMGA2 down-regulation on apoptosis and Notch signaling pathway in renal tubular epithelial cells exposed to high glucose (HG). METHODS:D-glucose at 5, 10, 20 and 30 mmol/L was used to stimulate human renal tubular epithelial HK-2 cells for 2 h, and D-glucose at 30 mmol/L was used to stimulate the HK-2 cells for 10 min, 60 min and 120 min. The protein expression of HMGA2 was determined by Western blot. The HK-2 cells were divided into normal glucose (NG) group, HG group, HG+si-HMGA2 group and HG+NC group, in which siRNA was transfected by Lipofectamine^TM 2000 for 48 h. Flow cytometry was used to analyze the apoptotic rate, reactive oxygen species (ROS) assay kit was used to detect ROS content, and Western blot was used to detect the protein levels of Notch1, Hes1 and Bcl-2. The HK-2 cells were treated with the Notch signaling pathway inhibitor DAPT, and then the cells were divided into HG group, HG+DAPT group and HG+si-HMGA2+DAPT group. The apoptotic rate was analyzed by flow cytometry. RESULTS:Exposure of the HK-2 cells to D-glucose at different concentrations for different time significantly increased the expression of HMGA2 (P<0.05). Compared with NG group, the protein expression of HMGA2, Notch1 and Hes1 in HG group was increased, the expression of Bcl-2/Bax was decreased, the apoptotic rate was increased, and the content of ROS was increased obviously (P<0.05). Compared with HG group, the protein expression of HMGA2, Notch1 and Hes1 of HG+si-HMGA2 group was decreased, the expression of Bcl-2/Bax was increased, the apoptotic rate was decreased, and the content of ROS was decreased significantly (P<0.05). The apoptotic rate in HG+DAPT group was significantly lower than that in HG group, while the apoptotic rate in HG+si-HMGA2+DAPT group was significantly lower than that in HG+DAPT group (P<0.05). CONCLUSION:Down-regulation of HMGA2 expression inhibits the apoptosis of renal tubular epithelial cells by regulating Notch signaling pathway and decreasing ROS production.     

Effect of bim silencing by siRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM：To investigate the effect of BH3-only protein Bim (Bcl-2 interacting mediator of cell death) on apoptosis of rat cardiomyocytes induced by hypoxia. METHODS：Rat cardiomyocytes were isolated from infant rats aged 1~3 days and then primarily cultured. The antibody targeting α-actin of striated muscle was used to identify the cardiomyocytes. The siRNAs of bim were transfected into the cardiomyocytes with liposome, and the expression of Bim was determined by Western blotting. The cardiomyocytes were divided into blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+bim-siRNA group.The frequency and rhythm of cardiomyocyte beating were observed and recorded under inverted microscope. The activity of lactate dehydrogenase (LDH) in the culture medium was assessed by automatic biochemical analyzer. The viability of the cells was analyzed by MTT assay. The cell apoptotic rate was measured by flow cytometry. The protein expression of Bim, Bax, Bcl-2, p-p38 MAPK and p38 MAPK was detected by Western blotting. RESULTS：Immunohistochemical identification confirmed that the rat cardiomyocytes were successfully cultured. The expression of Bim was obviously inhibited after transfected with bim-siRNAs and the silencing efficiency of bim-siRNA-2 was the highest (86.73%). The frequency of cardiomyocyte beating was slowed down after hypoxia and the rhythm was disordered, while the frequency of beating was obviously increased after silencting the expression of bim. Compared with control group, the LDH in the culture medium was increased (P<0.01), and the viability of the cardiomyocytes was reduced in hypoxia group (P<0.05). The apoptotic rate was increased (P<0.01). After transfection with bim-siRNA, the release of LDH was decreased, and the viability of the cardiomyocytes was increased. The apoptotic rate was decreased. The results of Western blotting showed that hypoxia increased the expression of Bax and p-p38 MAPK (P<0.05), and decreased the expression of Bcl-2 (P<0.01), while transfection with bim-siRNA reduced the effects caused by hypoxia (P<005). These were greatly related to the decrease of apoptosis. However, the expression of p38 MAPK was not changed. CONCLUSION：The apoptosis of cardiomyocytes induced by hypoxia can be inhibited by silencing the expression of bim gene by down-regulation of p-p38 MAPK and Bax expression and up-regulation of Bcl-2 expression.     

Effect of sulodexide on apoptosis of human dermal microvascular endothelial cells exposed to hypoxia

AIM: To investigate the effect of sulodexide (SDX) on the apoptosis of human dermal microvascular endothelial cells (HDMECs) exposed to hypoxia and its underlying mechanism. METHODS: The HDMECs were cultured and divided into normoxia control group cultured under normoxic condition; hypoxia control group cultured in a humid incubator maintained at 37℃ with 5% CO₂ and 1% O₂ for 24 h; treatment groups treated with SDX at 0.25, 0.5 and 1 LSU/mL for 24 h under hypoxic condition. The cell viability was measured by CCK-8 assay. The apoptotic rate of the HDMECs was analyzed by flow cytometry. The activity of caspase-3 in HDMECs was examined by caspase-3 activity assay kit. The expression of pro-apoptotic factor P53, caspase-3, Bax and anti-apoptotic factor Bcl-2 at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: SDX increased the viability of HDMECs exposed to hypoxia, but also decreased the apoptosis. Furthermore, SDX down-regulated the expression of pro-apoptotic factor P53, Bax and caspase-3 at mRNA and protein levels as well as the activity of caspase-3, while the expression of anti-apoptotic factor Bcl-2 was up-regulated. CONCLUSION: SDX significantly increases the viability and decreases the apoptosis of HDMECs exposed to hypoxia. Inhibition of the mitochondrial apoptosis pathway may be involved in the underlying mechanism.     

Effects of edaravone on high glucose-induced apoptosis in SH-SY5Y cells via regulating microRNA-25

AIM: To observe the effects of edaravone on high glucose-induced apoptosis of SH-SY5Y cells and its potential mechanism. METHODS: The SH-SY5Y cells were cultured in the DMEM medium with 100 mmol/L glucose and 100 μmol/L edaravone for 24 h. The viability of the SH-SY5Y cells was detected by MTT assay. The levels of ROS in the cells were determined by DCFH-DA fluorescent probing. The apoptotic rates of the cells were analyzed by flow cytometry. The protein expression of Bax and Bcl-2 in the cells were detected by Western blot. The expression levels of micro-RNA-25 (miR-25) were determined by real-time PCR. To further clarify the target sites of edaravone on inhibiting apoptosis induced by high glucose, miR-25 inhibitor was applied to the SH-SY5Y cells and the activity of caspase-3 was measured.RESULTS: Compared with control group, the cell viability was decreased significantly in model group, and the ROS level was increased significantly. The protein expression of Bax was up-regulated significantly, while the expression levels of Bcl-2 and miR-25 were significantly down-regulated. Compared with model group, the cell viability was increased significantly in edaravone group. The ROS level was decreased significantly. Meanwhile, the expression of Bax was down-regulated, while the expression of Bcl-2 and miR-25 was up-regulated with statistical significance. The caspase-3 activity of the cells incubated with 100 mmol/L glucose and miR-25 inhibitor was increased. However, no alteration of caspase-3 activity with edaravone added simultaneously was observed. CONCLUSION: Edaravone inhibits the apoptosis of SH-SY5Y cells induced by high glucose with the potential target site of miR-25.     

Effect of miR-24 on chemotherapy sensitivity in lung cancer A549 cells

AIM: To study the effect of microRNA (miR)-24 on chemotherapy sensitivity and its possible mechanisms in human lung adenocarcinoma A549 cells. METHODS: The expression of miR-24 in the A549 cells and A549/DDP cells was determined by real-time PCR. Transfection of miR-24 inhibitor was used to down-regulate the miR-24 level in the A549/DDP cells. The viability and apoptosis rate were measured by CCK-8 assay and flow cytometry, respectively. The protein levels of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, cytochrome C (Cyt C), phosphorylated extracellular signal regulated kinase (p-ERK) and P53 were detected by Western blot. Luciferase reporter assay was used to predict and identify the target genes of miR-24. RESULTS: The expression of miR-24 was significantly higher in the A549/DDP cells than that in the A549 cells (P<0.05). miR-24 inhibitor induced cell apoptosis and increased the sensitivity of the A549/DDP cells to cisplatin. Furthermore, miR-24 inhibitor down-regulated the ratio of Bcl-2/Bax, while up-regulated the protein levels of P53, p-ERK, cleaved caspase-9, cleaved caspase-3 and Cyt C. Incubation with U0126, a specific ERK inhibitor, partly reversed the viability of miR-24 inhibitor transfected A549/DDP cells. Bioinformatics analysis demonstrated that p53 was a potential target gene of miR-24. Co-teansfection of miR-24 inhibitor and P53 siRNA in A549/DDP cells partially reversed the effect of miR-24 inhibitor on cell viabiltiy. CONCLUSION: Down-regulation of miR-24 increases the sensitivity of A549/DDP cells to cisplatin. The mechanism may be related to directly targeting p53 gene and over-activation of ERK/P53 signaling pathway, thus promoting apoptosis via mitochondrial apoptosis pathway.     

Salvianolate attenuates oxidative stress injury of human endothelial EA.hy926 cells induced by hydrogen peroxide

AIM: To investigate the effect of salvianolate on oxidative damage induced by hydrogen peroxide in human endothelial EA.hy926 cells.METHODS: EA.hy926 cells were cultured in vitro and divided into the following groups:control group, damage group, and anti-damage groups (salvianolate+damage groups). The cell viability was measured by CCK-8 assay. The migration ability of the EA.hy926 cells was detected by Transwell assay. The content of nitric oxide (NO) in the culture supernatant of the EA.hy926 cells was examined. The levels of vascular endothelial growth factor (VEGF) were detected by ELISA. The apoptosis,mitochondrial membrane potential and intracellular superoxide anion content of the EA.hy926 cells were analyzed by flow cytometry. The protein levels of caspase-3, cleaved caspase-3, Bcl-2, Bax, NF-κB and p53 were determined by Western blot. RESULTS: Compared with damage group, the viability of EA.hy926 cells pretreated with salvianolate at different concentrations was significantly increased (P<0.05). The apoptotic rate was significantly decreased (P<0.05). Savianolate enhanced the migration ability of the cells. The levels of VEGF, NO and mitochondrial transmembrane potential were increased (P<0.05), and the intracellular ROS level was significantly decreased (P<0.05). The protein levels of NF-κB, p53, Bax and cleaved caspase-3 were significantly decreased, and the protein level of Bcl-2 was markedly increased(P<0.05). CONCLUSION: Savianolate reduces the damage of EA.hy926 cells by hydrogen peroxide exposure, and its mechanism may be related to the blocking of NF-κB signaling pathway.     

Protective effects of Zhouluotong extract Z-6 on Schwann cells damaged by high-glucose and PI3K/Akt/nNOS pathway

AIM:To explore the role of PI3K/Akt/nNOS in Zhouluotong extract resisting diabetic peripheral neuropathy. METHODS:The Schwann cells were divided into normal group (D-glucose 25 mmol/L), model group (D-glucose 100 mmol/L), Zhouluotong extract Z-6 + high glucose group, Zhouluotong + high glucose group, mecobalamine + high glucose group. The viability, nitric oxide content and the Ca2+-ATPase activity in Schwann cells were determined by Cell Counting Kit-8 , nitric oxide assay kit and Ca2+-ATPase assay kit, respectively. The apoptosis of Schwann cells were analyzed by flow cytometry. The expression of Bcl-2, Bcl-xL, Bax, Bak and caspase-3, and the phosphorylation levels of nNOS and Akt were determined by Western blotting. The signal pathway of PI3K/Akt was explored by dominant negative PI3K and Akt (δp85 and DN-Akt) transient transfection assay. RESULTS:Under high-glucose culture, the cell viability, nitric oxide content in culture supernatant, the expression of Bcl-2 and Bcl-xL, and the phosphorylation levels of Akt and nNOS in the Schwann cells were significantly increased. The cell apoptosis, the expression of Bax, Bak and caspase 3 in the Schwann cells were significantly decreased by Zhouluotong extract Z-6, compared with model group. Increased nitric oxide content and the up-regulation of nNOS were observed. However, the effects of blocking PI3K/Akt, the upstream pathway of nNOS , by transfection with DN-δp85 on Akt phosphorylation in the Schwann cells was still unclear. CONCLUSION: Zhouluotong extract Z-6 changes the phosphorylation of nNOS, and the expression of anti-apoptotic factors, caspase-3 and pro-apoptotic factors in Schwann cells under high-glucose culture, thus reducing apoptosis and elevating viability. The relationship to PI3K/Akt/nNOS pathway needs further investigation.     

Hypoxia promotes apoptosis of neural stem cells and down-regulates miR-26a

AIM: To investigate the effect of cobalt chloride (CoCl₂) on the apoptosis of neural stem cells (NSCs) and the expression of microRNA-26a (miR-26a) in vitro, and to explore the mechanisms of NSC apoptosis induced by CoCl₂. METHODS: NSCs were exposed to CoCl₂ at different doses (200~600 μmol/L) for 24 h. The cell viability and apoptosis were measured by CCK-8 assay and TUNEL method. The expression of miR-26a-3p, miR-26a-5p, GSK-3β, caspase-3, Bcl-2 and Bax was examined by real-time PCR. The protein levels of Bcl-2 and Bax were detected by Western blotting. RESULTS: The cell viability was inhibited and the apoptosis of NSCs was increased significantly by CoCl₂ in a dose-dependent manner (P<0.05). CoCl₂ at concentration of 400 μmol/L for 24 h was used to induce apoptosis and the expression of miR-26a was down-regulated compared with control (P<0.05). Exposure to CoCl₂ at concentration of 400 μmol/L up-regulated the expression of GSK-3β, caspase-3 and Bax, down-regulated the expression of Bcl-2 and Bcl-2/Bax (P<0.05). CONCLUSION: CoCl₂ at concentration of 400 μmol/L induces the apoptosis of NSCs obviously. CoCl₂ may induce the NSC apoptosis by mitochondrial apoptotic pathway. Declining miR-26a may be related to NSC apoptosis.     

TPX2 induces apoptosis of rectal cancer HR-8348 cells through p38 MAPK signaling pathway

AIM: To study the effect of targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression knockdown on the apoptosis of rectal cancer HR-8348 cells.METHODS: The HR-8348 cells transfected with TPX2 small interfering RNA (siRNA) served as TPX2 siRNA group. The non-transfected cells were used as control group. The cells transfected with siRNA negative control (siRNA-NC) were used as siRNA-NC group. The TPX2 siRNA-transfected cells exposed to p38 MAPK inhibitor SB203580 served as TPX2 siRNA+SB203580 group. The expression of TPX2 at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry. The protein levels of p38 MAPK, p-p38 MAPK, cleaved caspase-3 and Bcl-2 in the HR-8348 cells were determined by Western blot.RESULTS: After transfection, the expression of TPX2 at mRNA and protein levels was decreased in TPX2 siRNA-transfected cells (P<0.05). Transfection with siRNA-NC had no effect on TPX2 mRNA and protein levels in the cells. After knockdown of TPX2 expression, the viability of rectal cancer HR-8348 cells and the expression of Bcl-2 were decreased, while the apoptotic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK were increased significantly reduced (P<0.05). Compared with TPX2 siRNA group, the apopto-tic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK in TPX2 siRNA+SB203580 group were significantly decreased, while the viability was significantly increased (P<0.05).CONCLUSION: Knockdown of TPX2 expression promotes apoptosis of rectal cancer HR-8348 cells by activating p38 MAPK signaling pathway.     

Inhibition of autophgay enhances resveratrol-induced apoptosis of human chondrosarcoma cells

AIM: To investigate whether autophagy is up-regulated when resveratrol (Res) induces apoptosis in chondrosarcoma, and to study the effects of autophagy inhibitor combined with Res on chondrosarcoma. METHODS: SW1353 cells were divided into 4 groups: control group, Res group, 3-methyladenine (3MA) group, and Res+3MA group. Electron microscopy was used to observe the autophagyosomes in control group and Res group. At the same time, the viability of the cells in the 4 groups was detected by CCK-8 assay. TUNEL staining and Western blotting (for determining the levels of cleaved caspase-3, Bax and Bcl-2) were used to reflect levels of apoptosis in all groups. The expression of autophagy-related proteins Beclin 1, LC3-Ⅱ and p62 was detected by Western blotting. RESULTS: Exposure of the cells to Res resulted in a decrease in cell viability and an increase in the level of apoptosis (P<0.05). Compared with control group, the level of apoptosis was increased but the autophagy was decreased (P<0.05). Compared with Res group, the cell viability and the level of autophagy were decreased and the level of apoptosis was increased (P<0.05). CONCLUSION: Resveratrol induces apoptosis and autophagy, and inhibition of autophgay enhances resveratrol-induced apoptosis in chondrosarcoma.     

Effects of 6-gingerol on apoptosis of rat nucleus pulposus cells

AIM: To study the effect of 6-gingerol on the apoptosis of rat nucleus pulposus cells and its possible mechanism. METHODS: Rat nucleus pulposus cells were isolated and cultured. The effects of 6-gingerol and hydrogen peroxide (H₂O₂) at different concentrations on the viability of nucleus pulposus cells were measured by CCK-8 assay. After 6-gingerol treatment, the protein level of p-Akt was determined by Western blot. The cells were divided into 4 groups:control group, H₂O₂ group, 6-gingerol group (6-gingerol + H₂O₂) and LY294002 group (6-gingerol + H₂O₂ + LY294002). The apoptotic rate and the levels of reactive oxygen species (ROS) were analyzed by flow cytometry. TUNEL fluorescence staining was used to observe the number of apoptotic cells. The morphological changes of mitochondria were observed under transmission electron microscope, and Western blot was used to determine the protein levels of caspase-3, Bcl-2, Bax, p-Akt, Akt and p53. The mRNA expression of aggrecan and type II collagen was measured by RT-qPCR. RESULTS: The results of CCK-8 assay showed that the optimal concentration of 6-gingerol for promoting the viability of rat nucleus pulposus cells was 24 mg/L, and the exposure condition of H₂O₂ at 80 μmol/L for 6 h was appropriate for establi-shing the cell damage model. 6-Gingerol increased the protein level of p-Akt in a time-dependent manner. The apoptotic rate, ROS level and TUNEL positive cells in H₂O₂ group were significantly increased compared with control group. The mitochondrial edema was obvious in H₂O₂ group compared with control group. The protein levels of pro-apoptotic molecules caspase-3, Bax and p53 were significantly increased, while anti-apoptotic protein Bcl-2, and mRNA expression of aggrecan and type II collagen were significantly decreased compared with control group (P<0.05). 6-Gingerol exerted a protective effect against H₂O₂-induced apoptosis and promoted the expression of anti-apoptotic proteins. However, this effect was weakened after treatment with PI3K/Akt signaling pathway inhibitor LY294002. CONCLUSION: H₂O₂ induces damage and dysfunction of rat nucleus pulposus cells, and 6-gingerol may inhibit H₂O₂-induced apoptosis of nucleus pulposus cells by activation of PI3K/Akt signaling pathway.     

Berberine promotes anti-proliferative effects of doxorubicin in T24 bladder cancer cells by enhancing apoptosis

AIM: To observe the effect of berberine (Ber) on doxorubicin (DOX)-induced apoptosis in bladder cancer T24 cells. METHODS: The cells were exposed to DOX in the presence or absence of different concentrations of Ber. The viability of the cells was determined by CCK-8 assay. The apoptosis was measured by Hoechst 33258 staining and the protein levels of cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were detected by Western blotting.RESULTS: Ber enhanced the inhibitory effect of DOX on the viability of T24 cells and promoted DOX-induced apoptosis in T24 cells. DOX increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in DOX-treated T24 cells.CONCLUSION: Ber enhances the anti-proliferative effects of DOX through promoting apoptosis in bladder cancer cells.     

Baicalin inhibits high glucose-induced apoptosis of mouse glomerular mesangial cells via miR-141/Sirt1 signaling pathway

AIM:To investigate the therapeutic mechanism of baicalin for diabetic nephropathy involving microRNA-141 (miR-141)/silent information regulator 1 (Sirt1) signaling pathway. METHODS:Mouse glomerular mesan-gial cell line SV40-MES-13 was treated with high glucose (HG, 25 mmol/L glucose) to establish diabetic nephropathy cell model. Baicalin at 100 μmol/L was used to treat glomerular mesangial cells. qPCR and Western blot were performed to determine the expression levels of miR-141 and Sirt1. The regulatory relationship between miR-141 and Sirt1 was detected by dual-luciferase assay. The apoptosis of glomerular mesangial cells was analyzed by flow cytometry. RESULTS:Compared with control group, the cells treated with HG showed increased levels of miR-141 and apoptosis, and Sirt1 expression was decreased (P<0.01). Baicalin and miR-141 inhibitor suppressed the HG-induced effect on the levels of miR-141, Sirt1 and apoptosis. Knockdown of Sirt1 expression reversed the effect of miR-141 inhibitor on the levels of miR-141, Sirt1 and apoptosis. Over-expression of miR-141 reversed the effect of baicalin on the glomerular mesangial cells treated with HG. Up-regulation of Sirt1 abolished the effect of miR-141 over-expression on the glomerular mesangial cells. CONCLUSION:Baicalin inhibits the apoptosis of mouse glomerular mesangial cells via miR-141/Sirt1 signaling pathway, thus attenuating diabetic nephropathy.     

PQS attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibiting endoplasmic reticulum stress

AIM:To study the effect of Panax quinquefoliumsaponin (PQS) on cardiomyocyte apoptosis induced by thapsigargin (TG). METHODS:Primary cultured cardiomyocytes from neonatal SD rats were divided into control group, TG group, PQS (40 mg/L, 80 mg/L and 160 mg/L)+TG group, si-PERK+TG group, and mock+TG group. The cells were treated with 1 μmol/L TG for 24 h to induce apoptosis. The PERKgene in the cardiomyocytes was knocked down by RNAi. The cell viability was detected by CCK-8 assay. Apoptosis was analyzed by flow cytometry. Wes-tern blotting was used to determine the expression of ERS molecules GRP78, CRT, ATF4 and CHOP, anti-apoptosis protein Bcl-2 and pro-apoptosis protein Bax. RESULTS:Compared with control group, TG significantly and the apoptosis, reduced the cell viability (P<0.05), increased the phosphorylation of PERK and eIF2α, increased the expression of GRP78, CRT, ATF4, CHOP and pro-apoptosis protein Bax, and decreased the expression of anti-apoptosis protein Bcl-2 (P<0.05). Compared with TG group, PQS treatment (160 mg/L) significantly reduced the apoptosis and increased the cell viability (P<0.05). All the 3 different concentrations of PQS significantly increased the expression of anti-apoptosis protein Bcl-2 and reduced the expression of pro-apoptosis protein Bax (P<0.05) in a dose-dependent manner. PQS pretreatment and knockdown of PERK both reduced the protein levels of GRP78, CRT, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP and pro-apoptosis protein Bax, and increased the expression of anti-apoptosis protein Bcl-2 (P<0.05). CONCLUSION: PQS at concentration of 160 mg/L attenuated cardiomyocyte apoptosis induced by TG. PQS had the similar effect as PERKknockdown on cardiomyocyte apoptosis. The mechanism may be associated with inhibiting PERK-eIF2α-ATF4-CHOP pathway of ERS-related apoptosis.     

Tanshinone IIA prevents high glucose-induced human umbilical vein endothelial cell apoptosis

AIM: To investigate the effect of tanshinone IIA on the apoptosis of human umbilical vein endothelial cells(HUVECs) after high glucose treatment.METHODS: The cell viability was determined by MTT assay. The cell apoptotic rate was examined by flow cytometry with Annexin V-FITC/PI double staining. The expression of Bcl-2 and Bax, and the release of mitochondrial cytochrome C(Cyt C) were analyzed by Western blotting.RESULTS: Tanshinone IIA significantly inhibited high glucose-induced decrease in cell viability and increased the cell apoptosis. Additionally, after tanshinone IIA treatment, Bax expression and the release of mitochondrial Cyt C were significantly inhibited, while Bcl-2 expression was increased.CONCLUSION: Tanshinone IIA prevents high glucose-induced endothelial cell apoptosis via mitochondria-dependent pathway.     

Effects of CARHSP1 gene expression on viability,apoptosis and immune factors of vascular endothe-lial cells induced by hypoxia in atherosclerosis

AIM: To investigate the effect of calcium-regulated heat stable protein 1 (CARHSP1) gene expression on the viability, apoptosis and expression of interleukin-6 (IL-6) and C-reactive protein (CRP) in vascular endothe-lial cells induced by hypoxia.METHODS: The protein expression of CARHSP1 was detected by Western blot in atherosclerotic plaques. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia, and the cells were divided into normal culture group, hypoxia group, hypoxia+CARHSP1-siRNA group and hypoxia+pcDNA3.1-CARHSP1 group. The viability and apoptotic rate of the HUVECs were measured by CCK-8 assay and flow cytometry, respectively. The mRNA expression of IL-6 and CRP was detected by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS: The protein expression of CARHSP1 in atherosclerotic plaques was significantly higher than that in control group (P<0.05). Hypoxia significantly increased the expression of CARHSP1. The cell viability and the protein expression of Bcl-2 were significantly lower in hypoxia group than those in normal culture group (P<0.05). The apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were significantly higher than those in normal culture group (P<0.05). Compared with hypoxia group, the cell viability and protein expression of Bcl-2 were significantly increased in hypoxia+CARHSP1-siRNA group, while the apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were decreased significantly (P<0.05). The cell viability and protein expression of Bcl-2 were decreased significantly in hypoxia+pcDNA3.1-CARHSP1 group, while the apoptotic rate and the protein le-vels of IL-6, CRP, cleaved caspase-3 and Bax were increased significantly (P<0.05).CONCLUSION: The expression of CARHSP1 is increased in atherosclerotic plaques, and inhibition of CARHSP1 expression improves the viability, reduces the apoptosis, and down-regulates the expression of IL-6 and CRP in the HUVECs. Over-expression of CARHSP1 exerts the opposite effect.