Importance of mitochondrial calcium uniporter in high glucose-induced cardiac myocyte apoptosis

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in high glucose(HG)-induced apoptosis of cardiac myocytes. METHODS: Cardiac myocytes were exposed to normal glucose (5.5 mmol/L glucose+ 19.5 mmol/L mannitol), HG (25 mmol/L glucose), or HG combined with 5 μmol/L spermine for 72 h. Mitochondrial free Ca²⁺ concentration ([Ca²⁺]_m), MCU at mRNA and protein levels, pyruvate dehydrogenase (PDH) activity, mitochondrial membrane potential (Δψ_m), the levels of ATP and reactive oxygen species (ROS), and apoptosis were determined. RESULTS: The [Ca²⁺]_m, the mRNA and protein levels of MCU, PDH activity, ATP levels, and Δψ_m were reduced (P<0.05), while ROS content and the protein levels of caspase-9 and caspase-3 were increased in HG group (P<0.05). Adding 5 μmol/L spermine returned these parameters toward control levels (P<0.05). Moreover, apoptosis was reduced by adding spermine and HG treatment (P<0.05). CONCLUSION: HG-induced cardiac myocyte apoptosis may be associated with the decreased MCU expression and activity, abnormal mitochondrial Ca²⁺ handling, deviant mitochon-drial respiratory chain, and mitochondrial dysfunction.     

Ligustilide inhibits angiogenesis and epithelial-mesenchymal transition in human hemangioendothelial cells by reducing VEGF levels

AIM To investigate the effect of ligustilide on human hemangioendothelial cells (HemECs) and to analyze its mechanism. METHODS The effect of ligustilide at different concentrations on the viability of HemECs was measured by CCK-8 assay. The HemECs were divided into control group and ligustilide （10, 25 and 50 μmol/L） treatment groups, and the proliferation of HemECs was detected by EdU staining. The effects of ligustilide on the angiogenesis of HemECs was tested by microtubule formation experiment. The protein expression of vascular endothelial growth factor (VEGF) and epithelial-mesenchymal transition (EMT)-related markers in HemECs cells was determined by Western blot. RESULTS There was no significant difference in the viability of the cells treated with ligustilide at the concentrations between 0.1~50 μmol/L compared with control cells. Compared with control group, ligustilide at 25 and 50 μmol/L significantly reduced the number of EdU-positive cells and microtubule-like structures (P<0.05), reduced the protein expression level of VEGF (P<0.05), increased the protein expression of E-cadherin, and decreased the protein expression of vimentin and β-catenin (P<0.05). Compared with control group, the expression of VEGF and vimentin was significantly up-regulated, and the protein expression of E-cadherin was significantly down-regulated in VEGF overexpression group (P<0.05). Compared with VEGF overexpression group, the expression of VEGF and vimentin in 50 μmol/L ligustilide-treated VEGF-overexpressing cells were significantly reduced (P<0.05), and the protein expression of E-cadherin was significantly increased (P<0.05). CONCLUSION Ligulide inhibits the proliferation of HemECs, and also inhibits the angiogenesis and EMT process of HemECs by reducing the level of VEGF.     

Niflumic acid inhibits the proliferation,migration and invasion of glioma U87 cells

AIM To investigate the effect of niflumic acid （NFA） on human glioma U87 cells and to clarify the potential mechanism. METHODS The U87 cells were cultured in vitro and divided into blank control group, and 50, 100 and 200 μmol/L NFA groups. MTT assay was performed to determine the viability of cells in various groups. Migration and invasion abilities were measured by real-time cell analysis （RTCA）. RESULTS The results of MTT assay showed that compared with blank control group, the viability of U87 cells was increased after treatment with NFA for 12 h （P<0.05 or P<0.01）, while the viability was significantly decreased after treatment with NFA for 24 and 48 h （P<0.05 or P<0.01） in a concentration-dependent manner. The results of RTCA showed that compared with control group, the cell migration and invasion abilities were inhibited in 100 and 200 μmol/L NFA groups （P<0.05 or P<0.01） and the inhibitory effects were more obvious in 200 μmol/L NFA group （P<0.01）. CONCLUSION NFA inhibits the viability, migration and invasion of human glioma U87 cells.     

Dasatinib inhibits the viability and migration of renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation in vitro

AIM To explore the effect of dasatinib on the viability, apoptosis and migration of human renal carcinoma cell lines 786-O and 769-P, as well as the molecular mechanism in vitro. METHODS 786-O cells and 769-P cells were treated with different concentrations （0~2 μmol/L） of dasatinib, and 0 μmol/L dasatinib was used as blank control group. MTT method was used to detect cell viability. Wound healing assay was used to detect the effect of dasatinib on migration. Hoechst 33258 staining was used to observe the effect of dasatinib on apoptosis. Flow cytometry was used to detect the effect of dasatinib on cell cycle. Western blot method was used to detected cell cycle- and apoptosis-related protein levels. RESULTS Dasatinib inhibited viability and migration of 786-O and 769-P cells, and the inhibitory effect of dasatinib increased with the concentration of dasatinib （P<0.05）. The IC₅₀ values of dasatinib against 786-O and 769-P cell lines were （0.958 7±0.028 8） μmol/L and （0.784 3±0.066 0） μmol/L, respectively. After treatment with dasatinib for 24 h, the expression of pro-apoptotic proteins cleaved caspase-3 and cleaved caspase-9 increased significantly （P<0.01）, while the expression of cyclin D1 decreased （P<0.05）. The cycle-related pathway proteins p53 and p21 increased （P<0.05）, while the level of p-AKT was decreased （P<0.05）. CONCLUSION Dasatinib impaired the viability and migration ability of human renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation.     

Effect of formononetin on viability,migration and invasion of ovarian cancer cells in vitro

AIM To observe the effect of formononetin on the viability, migration and invasion of ovarian cancer cells, and to explore its mechanism. METHODS Human ovarian serous cystadenocarcinoma SKOV-3 cells were cultured in vitro. The cells were treated with formononetin at 0, 25, 50 and 100 μmol/L for 48 h. The cell viability was measured by MTS assay. The migration and invasion abilities of the SKOV-3 cells were detected by scratch wound assay and Transwell assay. RT-qPCR and Western blot were used to detect the mRNA and protein levels of E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS The viability of SKOV-3 cells was decreased with the increase in the formononetin concentration compared with control group (P<0.01). The wound migration distance of the cells in 50 μmol/L formononetin group was less than that in control group (P<0.01). The number of invasive SKOV-3 cells across the Transwell sub-compartment was significantly decreased in 50 μmol/L formononetin group compared with control group (P<0.01). The mRNA and protein levels of E-cadherin in 50 μmol/L formononetin group were significantly higher than those in control group (P<0.01), while the mRNA and protein levels of MMP-9 in 50 μmol/L formononetin group were significantly lower than those in control group (P<0.01). CONCLUSION Formononetin inhibits the migration and invasion abilities of ovarian cancer SKOV-3 cells by increasing expression of E-cadherin and decreasing expression of MMP-9.     

Role of CRIF1 in cardiomyocyte apoptosis induced by palmitic acid

AIM: To investigate the effects of CR6-interacting factor 1 (CRIF1) on the apoptosis of mouse cardiomyocytes (MCMs) induced by palmitic acid. METHODS: The MCMs cultured with medium containing palmitic acid at 0 and 300 μmol/L for 24 h were divided into control group and palmitic acid group, respectively. In order to explore the effects of CRIF1 on MCMs injuries induced by high fat, MCM exposed to palmitic acid at 300 μmol/L for 24 h were divided into vehicle group, scrambled (Scra) siRNA group and CRIF1 siRNA group. The cells in vehicle group were only treated with transfection reagent, the cells in Scra siRNA group were given a treatment with transfection reagent and scrambled RNA, and the cells in CRIF1 siRNA group were given a treatment with transfection reagent and CRIF1 specific siRNA. In order to further confirm the specific mechanism of CRIF1 in high fat-induced MCM injuries, MCMs in CRIF1 siRNA group were divided into DMSO group and N-acetyl cysteine (NAC) group, and were given the same intervention of palmitic acid. RT-qPCR, Western blot and immunofluorescence staining were used to detect the mRNA and protein expression of CRIF1. The apoptotic rate was analyzed by flow cytometry, and the level of intracellular reactive oxygen species (ROS) was tested by DHE staining and ELISA. RESULTS: The expression of CRIF1 was significantly increased after exposure to palmitic acid (P<0.05). Compared with control group, the apoptotic rate was increased significantly in vehicle group and Scra siRNA group (P<0.05). The apoptotic rate was significantly increased in CRIF1 siRNA group (P<0.05). Compared with control group, the intracellular ROS content was significantly increased in vehicle group and Scra siRNA group (P<0.05). Compared with vehicle group and Scra siRNA group, the intracellular ROS content was significantly increased in CRIF1 siRNA group (P<0.05). Compared with DMSO group, the intracellular ROS content and the apoptotic rate were remarkably decreased in NAC group (P<0.05). CONCLUSION: With the inhibition of oxidative stress, CRIF1 may reduce the apoptosis of MCMs induced by high fat.     

Mechanism of quercetin improving rat coronary artery myogenic response under high glucose

AIM: To investigate the mechanism of quercetin improving rat coronary artery myogenic response under high glucose (HG) by measuring muscle tension of coronary arterial ring and recording voltage-gated K⁺ channel (Kv) current of coronary artery smooth muscle cells by whole cell patch clamp. METHODS: The coronary rings from the normal SD rats were acutely isolated, and then divided into 6 groups: (1) control group; (2) HG group; (3) HG+low dose (3 μmol/L) of quercetin group; (4) HG+moderate dose (10 μmol/L) of quercetin group; (5) HG+high dose (30 μmol/L) of quercetin group; (6) HG+C6303 (PKC inhibitor)+high dose of quercetin group. Determinations of coronary artery response to vasoconstrictor (60 mmol/L KCl or 0.1 mmol/L U46619) or vasodilator (ACh at 10^-9~10^-5 mol/L) were performed, and the percentage of coronary ring tension was calculated using the contraction as 100% caused by 60 mmol/L KCl. The rat coronary artery smooth muscle cells were acutely isolated for recording the Kv current using whole cell patch clamp. RESULTS: Compared with control group, the contraction amplitudes to 60 mmol/L KCl or 0.1 mmol/L U46619 were significantly increased under HG incubation. Quercetin intervention concentration-dependently reduced the coronary artery contraction amplitude. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. Compared with control group, the diastolic amplitude to ACh decreased significantly in HG group, and quercetin intervention concentration-dependently increased the coronary artery diastolic amplitude. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. Compared with control group, HG incubation inhibited Kv current of coronary artery vascular smooth muscle cells significantly, and quercetin intervention attenuated the inhibitory effect of HG on Kv current intensity. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. CONCLUSION: Quercetin has a protective effect on myogenic response of coronary artery under HG and the effects is related to the increase in Kv current and the activation of PKC in vascular smooth muscle cells.     

Effects of cardiac contractility modulation on ventricular electrical remodeling in rabbits with chronic heart failure

AIM: To investigate the effects of cardiac contractility modulation (CCM) on ventricular electrical remodeling in a rabbit model of chronic heart failure. METHODS: The rabbits were divided into sham group, heart failure(HF) group and HF+ CCM group. The rabbit model of chronic heart failure was established by ligating the ascending aortic root. Then electrical stimulations during the absolute refractory period were delivered lasting 6 h everyday for 4 weeks in rabbits of HF+ CCM group. The QTc and ventricular effective refrective period (VERP) were recorded. The protein and mRNA levels of Kv1.4, Kv4.3 and connexin 43 (Cx43) were determined by Western blot and RT-qPCR. RESULTS: Compared with sham group, QTc were significantly prolonged in HF rabbits at week 12 (P<0.05). CCM therapy shortened QTc of rabbits with heart failure at week 16 (P<0.05). Compared with sham group, VERP significantly increased in HF group and HF+ CCM group, while CCM therapy shortened VERP of rabbits with heart failure at week 16 (P<0.05). Compared with sham group, the mRNA and protein levels of Kv1.4, Kv4.3 and Cx43 were decreased in HF group and HF+ CCM group (P<0.05). However, CCM therapy restored the mRNA and protein levels of Kv1.4, Kv4.3 and Cx43 of rabbits with heart failure (P<0.05). CONCLUSION: CCM suppresses ventricular electrical remodeling in heart failure and the underlying mechanism may be associated with increasing Kv1.4, Kv4.3 and Cx43 expression.     

Curcumin inhibits Porphyromonas gingivalis LPS-induced inflammatory response in human gingival fibroblasts by up-regulating miR-124

AIM To investigate the effects of curcumin （Cur） on the inflammatory response of human gingival fibroblasts （HGFs） induced by Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS） and the role of microRNA-124 （miR-124） in this process. METHODS The HGFs were divided into control group, LPS group （10 mg/L LPS） and LPS+Cur （20, 40 and 80 μmol/L） groups （10 mg/L LPS+corresponding dose of Cur）. After treatment for 24 h, CCK-8 assay was used to measure the cell viability. ELISA was used to measure the levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the supernatant. The level of miR-124 in the cells was detected by RT-qPCR. The protein levels of nuclear factor kappa B （NF-κB） p-p65 in cytoplasm and nucleus were determined by Western blot, and the nuclear translocation of NF-κB p-p65 was evaluated by laser confocal microscopy. After transfection with mimic-NC or miR-124 mimic, the expression of miR-124 and NF-κB p-p65 protein in the cytoplasm and nucleus of the cells were also detected. RESULTS The cell viability, the level of miR-124 in the cells and NF-κB p-p65 protein level in cytoplasm of LPS group were lower than those in control group （P<0.05）, while the levels of IL-1β and TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were higher than those in control group （P<0.05）. The cell viability, the level of miR-124 in cells and NF-κB p-p65 protein level in the cytoplasm of LPS+Cur （40 and 80 μmol/L） groups were higher than those in LPS group （P<0.05）, while the level of TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were lower than those in LPS group （P<0.05）. The level of IL-1β in the supernatant of LPS+80 μmol/L Cur group was lower than that in LPS group （P<0.05）. The levels of miR-124 and NF-κB p-p65 protein level in the cytoplasm of miR-124 mimic group were higher than those in LPS group and mimic-NC group （P<0.05）, while the level of NF-κB p-p65 proteinlevel in the nucleus was lower than that in LPS group and mimic-NC group （P<0.05）. CONCLUSION Curcumin inhibits the inflammatory response of HGFs induced by Pg LPS, which may be achieved by up-regulating miR-124 and then inhibiting the nuclear translocation of NF-κB p-p65.     

NKT cells inhibit Ang II-induced phenotypic transformation of mouse vascular smooth muscle cells by down-regulating PDGFRβ

AIM To investigate the role of natural killer T (NKT) cells in the phenotypic transformation of vascular smooth muscle cells induced by hypertension in mice. METHODS The hypertension model in wild-type and CD1d gene knockout mice was established by continuous infusion of angiotensin II (Ang II) at a dose of 490 ng·kg^-1·min^-1 for 14 d, and the NKT cell specific agonist α-galactosylceramide (α-GC) was given at a dose of 100 μg/kg in wild-type mice. The blood pressure of the mice was monitored by non-invasive tail cuff method. The thickness of aortic wall was measured by HE staining, and the degree of aortic fibrosis was observed by Masson staining. The expression levels of α-smooth muscle actin (α-SMA), osteopontin (OPN) and platelet-derived growth factor receptor β (PDGFRβ) in aortic tissue were detected by Western blot. RESULTS Compared with control group, the blood pressure, the aortic wall thickness, the degree of aortic fibrosis, the secretory marker protein OPN expression and the PDGFRβ expression were all increased significantly after infusion of Ang II for 14 d (P<0.05), while the expression of contractile marker protein α-SMA was decreased (P<0.05). The above changes were aggravated after CD1d gene knockout (P<0.05), but were attenuated after administration of α-GC (P<0.05). CONCLUSION NKT cells reduces Ang II-induced phenotypic transformation of mouse vascular smooth muscle cells by reducing the expression of PDGFRβ, increasing the expression of contractile protein and decreasing the expression of secretory protein.     

Role of endoplasmic reticulum stress in trimethylamine N-oxide-induced oxidative stress in human umbilical vein endothelial cells

AIM: To investigate whether endoplasmic reticulum stress is involved in trimethylamine N-oxide (TMAO)-mediated oxidative stress in human umbilical vein endothelial cells (HUVECs). METHODS: The cell viability was examined by CCK-8 assay. The cells were stained by DCFH-DA, and the intracellular level of reactive oxygen species (ROS) was observed by phase-contrast microscopy and detected by flow cytometric analysis. The protein levels of phospho-IRE-1α, IRE-1α and GRP78/BiP were detected by Western blot. RESULTS: TMAO exerted no significant effect on the viability of HUVECs. For a long period (>24 h), even a low concentration (10 μmol/L) of TMAO increased the oxidative stress level in the HUVECs (P<0.05). TMAO increased the phosphorylation level of IRE-1α and significantly up-regulated the protein level of GRP78/BiP in HUVECs (P<0.01). Pretreatment with STF-083010, an inhibitor of IRE1α, for 1 h reduced TMAO-induced oxidative stress in HUVECs (P<0.05). CONCLUSION: Endoplasmic reticulum stress is involved in TMAO-induced oxidative stress in HUVECs.     

Expression relevance of GATA-1 and miR-451a in erythroid differentiation of K562 cells under hypoxia

AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O₂) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells.     

AT1-AA induces autophagy and apoptosis of H9c2 cardiomyocytes through oxidative stress

AIM: To investigate the effects of angiotensin Ⅱ type 1 receptor autoantibody (AT1-AA) on oxidative stress, autophagy and apoptosis in H9c2 cells, and to analyze the possible mechanism. METHODS: The rat H9c2 cells were cultured in vitro. The effect of AT1-AA at different concentrations for different time on the cell viability was measured by CCK-8 assay. Upon the optimum concentration (10^-5 mol/L) and time point (24 h) determined in this stu-dy, the experssion levels of autophagy- and apoptosis-related proteins were detected by Western blot, and the levels of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) were examined by oxidative stress kits. RESULTS: AT1-AA decreased cell viability in a concentration- and time-dependent manner, promoted apoptosis, and up-regulated the levels of autophagy and oxidative stress (P<0.05). The apoptosis of H9c2 cells induced by AT1-AA was decreased after pretreatment with autophagy inhibitor 3-methyladenine (P<0.05). The levels of autophagy and apoptosis in the H9c2 cells pretreated with α-lipoic acid were decreased (P<0.05). Pretreatment with angiotensin Ⅱtype 1 receptor inhibitor telmisartan inhibited oxidative stress, and significantly decreased the levels of autophagy and apoptosis induced by AT1-AA in the H9c2 cells (P<0.05). CONCLUSION: AT1-AA induces autophagy and apoptosis of H9c2 cells through oxidative stress.     

Effects of pterostilbene on cervical cancer cell autophagy and SIRT1-FoxO signaling pathway

AIM To study the effect of pterostilbene (PTE) on autophagy and SIRT1-FoxO signaling pathway in cervical cancer cells. METHODS Human cervical cancer HeLa cells were used as the study objects. CCK-8 assay and flow cytometry were used to determine the effects of PTE at different concentrations on the viability and apoptotic rate of HeLa cells. The mRNA expression of SIRT1 and FoxO in the cells was detected by qPCR. The number of autophagosomes in the cells was observed under transmission electron microscope.The protein levels of SIRT1, FoxO, LC3-Ⅰ,LC3-Ⅱ, p62, Bax and Bcl-2 in the cells were determined by Western blot. RESULTS After treatment with PTE for 24 and 48 h, the viability inhibitory rate of the HeLa cells was increased with increasing PTE concentration. Compared with 0 μmol/L PTE, after 15, 30 and 60 μmol/L PTE treatment, the apoptotic rate of the HeLa cells, the number of autophagosomes in the cells, and the protein levels of Bax, LC3-Ⅱ/LC3-Ⅰ, SIRT1 and FoxO were increased (P<0.05), while the protein levels of Bcl-2 and p62 were decreased (P<0.05) in a concentration-dependent manner. CONCLUSION PTE may induce autophagy and apoptosis of HeLa cells by activating SIRT1-FoxO signaling pathway, and inhibit HeLa cell viability.     

Procaine regulates CXCR7 and affects AKT/STAT3 signaling pathway to inhibit viability,migration and invasion of bladder cancer cells

AIM To investigate the effects of procaine (PCA) and CXC chemokine receptor 7 (CXCR7) on the viability, migration and invasion of bladder cancer cells and its potential mechanism. METHODS Human bladder cancer RT4 cells were treated with PCA at different concentrations, and were divided into PBS group (without PCA treatment), PCA group (treated with 4 mmol/L PCA), siRNA negative control (si-Con) group (transfected with si-Con), CX?CR7 siRNA (si-CXCR7) group (transfected with si-CXCR7), PCA+pcDNA group (treated with 4 mmol/L PCA and transfected with pcDNA) and PCA+pcDNA-CXCR7 group (treated with 4 mmol/L PCA and transfected with pcDNA-CX?CR7). The siRNA and pcDNA were transfected into the RT4 cells by liposome method. The mRNA expression of CX?CR7 in the RT4 cells was detected by RT-qPCR. The cell viability was measured by CCK-8 assay. The invasion and migration abilities of the cells were detected by Transwell assays. The protein levels of CXCR7, AKT, STAT3, p-AKT and p-STAT3 were determined by Western blot . RESULTS Compared with PBS group, the viability, migration ability and invasion ability of the RT4 cells treated with PCA at different concentrations were significantly decreased (P<0.05), and the expression of CXCR7 at mRNA and protein levels in PCA group was also significantly decreased (P<0.05). Compared with si-Con group, the expression of CXCR7 at mRNA and protein levels in si-CXCR7 group was significantly decreased, and the viability, migration ability and invasion ability of the cells were also significantly decreased (P<0.05). Compared with PCA+pcDNA group, the expression of CXCR7 at mRNA and protein levels in PCA+pcDNA-CXCR7 group was significantly increased, and the viability, migration ability and invasion ability of the cells were also significantly increased (P<0.05). Compared with PBS group, the protein levels of p-AKT and p-STAT3 in PCA group were significantly decreased(P<0.05). Compared with PCA+pcDNA group, the protein levels of p-AKT and p-STAT3 in PCA+pcDNA-CX?CR7 group were significantly increased (P<0.05). No significant difference in the protein levels of AKT and STAT3 among the groups was observed. CONCLUSION Treatment with PCA inhibits the viability, migration and invasion of bladder cancer cells by inhibiting the expression of CXCR7. Over-expression of CXCR7 reverses this effect of PCA. Its mechanism may be related to AKT/STAT3 signaling pathway.     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway, thus promoting the apoptosis of cardiomyocytes.     

Effects of simvastatin on proliferation of esophageal cancer cells through NF-κB p65 pathway

AIM To investigate the effects of simvastatin （SIM） on the proliferation of esophageal cancer Eca109 cells through NF-κB p65 pathway. METHODS Esophageal cancer Eca109 cells were cultured in vitro and treated with SIM at different concentrations （2 , 4, 8, 16 and 32 μmol/L）. The proliferation of Eca109 cells was measured by MTT assay and plate colony formation assay. The proliferation-related protein levels of cyclin D1, c-Myc, NF-κB p65 （p65） and IκB-α in the esophageal cancer Eca109 cells were determined by Western blot. ELISA was used to detect the levels of tumor necrosis factor α （TNF-α） and interleukin-6 （IL-6） in the culture supernatant of Eca109 cells. RESULTS Compared with control group, the inhibitory effects of SIM at 2 , 4 , 8 and 16 μmol/L on the viability of Eca109 cells were increased in a concentration-dependent and time-dependent manners （P<0.05）. Simultaneously, the inhibitory rates of SIM at 16 and 32 μmol/L on the viability of Eca109 cells showed no significant difference （P>0.05）. The inhibitory rates of SIM at 16 μmol/L on the viability of esophageal cancer cells was 50.61% at 48 h, which was closed to half of the inhibitory dose IC₅₀, and it was used as the optimum concentration and time for follow-up experiments. Compared with control group, the plate colony formation rate of Eca109 cells, the protein levels of cyclinD1, c-Myc, nuclear p65, TNF-α and IL-6 in 8 and 16 μmol/L groups were decreased, while the levels of cytosolic p65 and IκB-α proteins were increased （P<0.05）. No significant difference of plate colony formation rate in Eca109 cells, and the protein levels of cyclin D1, c-Myc, nuclear and cytoplasmic p65, IκB-α, TNF-α and IL-6 between 16 μmol/L SIM group and 32 μmol/L SIM group was observed （P>0.05）. CONCLUSION Simvastatin inhibits the proliferation of esophageal cancer Eca109 cells, and its mechanism may be related to up-regulation of IκB-α, down-regulation of cyclinD1 and c-myc, inhibition of p65 nuclear translocation, and the expression of TNF-α and IL-6 downstream of NF-κB p65 pathway.     

Inhibitory effect of Wendan decoction on formation of foam cells induced by ox-LDL

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on formation of foam cells. METHODS The optimal concentrations of Wendan decoction without cytotoxity to cells were selected by MTT assay. After Wendan decoction treatment, the formation of foam cells was examined by oil red O staining. The cholesterol efflux, cholesterol level, free cholesterol level and cholesterol esterification rate were analyzed using cholesterol efflux assay, total cholesterol assay and free cholesterol assay. The expression levels of macrophage membrane proteins, including CD36, scavenger receptor class A （SR-A）, ATP-binding cassette transporter A1 （ABCA1） and scavenger receptor class B type I （SR-BI）, were quantified by Western blot. RESULTS The optimal concentrations of Wendan decoction without cytotoxity to the cells were 0~6 g/L. Wendan decoction at the concentrations of 1.5, 3 and 6 g/L were selected for the experiments. Wendan decoction at these concentrations inhibited the formation of foam cells induced by oxidized low-density lipoprotein （ox-LDL）, and reduced the accumulation of intracellular lipids in a concentration-dependent manner （P<0.05 or P<0.01）. Wendan decoction also reduced intracellular total cholesterol level, cholesterol ester level and cholesterol esterification rate （P<0.05 or P<0.01）, promoted efflux of intracellular cholesterol （P<0.01）, and decreased the protein level of CD36 in THP-1 cell-derived macrophages （P<0.01） in a concentration-dependent manner. Wendan decoction at the concentration of 6 g/L significantly reduced the protein level of SR-A in THP-1 cell-derived macrophages （P<0.05）. At the concentrations of 3 and 6 g/L, Wendan decoction significantly increased the protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages （P<0.05 or P<0.01）. CONCLUSION Wendan decoction significantly inhibits ox-LDL-induced formation of foam cells by reducing cholesterol deposition and promoting cholesterol efflux, and its mechanism may be related to the down-regulation of CD36 and SR-A and the up-regulation of ABCA1 and SR-BI.