Effects of metoprolol on phosphorylated connexin 43 expression in myocardial tissues and apoptosis of myocardial cells in heart failure rats

AIM：To explore the in vivo effects of metoprolol on the expression of phosphorylated connexin 43 (p-Cx43) in myocardial tissues and the apoptosis of myocardial cells in rats with heart failure (HF).METHODS：One hundred Sprague-Dawley rats were randomly divided into 5 groups (each n=20): sham group, HF group, low-dose (1.25 mg·kg-1·d-1) metoprolol treatment (MetoA) group, middle-dose (5 mg·kg-1·d-1) metoprolol treatment (MetoB) group and high-dose (20 mg·kg-1·d-1) metoprolol treatment (MetoC) group.The rats in HF group and metoprolol treatment groups were subject to abdominal aortic ligation, and different doses of metoprolol were given 4 weeks later till 8 weeks after operation.Echocardiography was conducted to monitor the hemodynamic parameters at the 4th and 8th weeks, and the rat hearts were taken at the 8th week after operation.The morphological changes and the proliferation of collagen fibers in myocardial tissues were observed by HE and Masson staining, respectively.The expression level of p-Cx43 was detected by Western blotting and the apoptosis of myocardial cells was assessed by TUNEL method.The relationship between p-Cx43 expression level and apoptotic index was analyzed by Pearson’s correlation.RESULTS：(1) Echocardiography showed that metoprolol could effectively improved cardiac hemodynamics in HF rats, and pathological findings suggested that metoprolol could effectively reverse HF-induced cardiac remodeling in a dose-dependent manner within the therapeutic dose range.(2) Western blotting showed that p-Cx43 expression in HF group was significantly higher than that in sham group (P<001), and that in all metoprolol treatment groups was significantly decreased compared with HF group (P<005 or P<001), among which pairwise comparisons also showed significant differences (P<001).(3) The myocardial apoptotic index in HF group ［(51.17±6.94)%］ was significantly increased compared with sham group ［(4.62±160)%, P<001］.Compared with HF group, myocardial apoptotic indexes in MetoA group ［(40.60±4.15)%］, MetoB group ［(30.66±4.00)%］ and MetoC group ［(22.24±5.69)%］ were significantly decreased (P<001), among which pairwise comparisons also showed significant differences (P<001).(4) The expression level of p-Cx43 was positively correlated with the apoptotic index (r=0.905, P<001).CONCLUSION: The mechanism of metoprolol against HF-induced myocardial apoptosis may be related to inhibition of p-Cx43 expression.     

Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition

AIM: To explore the effect of Pycnogenol on transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell activation. METHODS: Cultured LX-2 cells were treated with 5 μg/L TGF-β1 and different concentrations (0, 10, 25 and 50 mg/L) of Pycnogenol. The viability of the LX-2 cells under the conditions with or without autophagy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay. The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot. RESULTS: Compared with control group, 5 μg/L TGF-β1 treatment elevated the cell viability, and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05). However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most significant in the LX-2 cells (P<0.05). Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnogenol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells (P<0.05). CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition.     

Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in cardiomyocytes via inducing autophagy

AIM: This study aims to explore the effect of abietic acid (AA) on advanced glycosylation end products (AGEs)-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes. METHODS: H9c2 cells were divided into 5 groups. The cells in control group were treated with saline for 24 h. The cells in AGEs treatment group were treated with AGEs (100 mg/L) for 24 h. The cells in AGEs+AA (10, 25 and 50 μmol/L) groups were simulta-neously treated with AGEs (100 mg/L) and AA (10, 25 and 50 μmol/L) for 24 h. The cell viability was measured by MTT assay. The protein levels of myoglobin (Mb), creatine kinase MB isoenzyme (CK-MB), cardiac troponin I (cTnI), C/EBP homologous protein (CHOP), cleaved caspase-12, GADD34, BiP, LC3, P62 and beclin 1 were determined by Western blot. The levels of lactate dehydrogenase (LDH) were measured by ELASA. The apoptosis was analyzed by flow cytometry. RESULTS: The low concentration (<50 μmol/L) of abietic acid had no obvious effect on the viability of H9c2 cells. The high concentration (>50 μmol/L) of abietic acid decreased the viability of H9c2 cells. The levels of Mb, CK-MB, cTnI and LDH in AGEs group were higher than those in control group (P<0.05). Compared with AGEs group, the levels of Mb, CK-MB, cTnI and LDH in AGEs+AA (10, 25 and 50 μmol/L) groups were obviously reduced (P<0.05). Abietic acid at concentrations of 10, 25 and 50 μmol/L inhibited AGEs-induced apoptosis, elevated the protein levels of CHOP and cleaved caspase-12, and attenuated expression of GADD34 and BiP (P<0.05). Moreover, abietic acid at concentrations of 10, 25 and 50 μmol/L suppressed AGEs-induced decreased ratio of LC3-Ⅱ/LC3-Ⅰ and expression of beclin 1, and enhanced the expression of P62 (P<0.05). 3-Methyladenine, an inhibitor of autophagy, reversed the effect of abietic acid on the protein levels of LC3, Mb, cleaved caspase-12 and BiP (P<0.05). CONCLUSION: Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes via inducing autophagy.     

Urotensin Ⅱinduces pulmonary arterial smooth muscle cell proliferation and up-regulates Egr-1 expression through ERK1/2 pathway in rats

AIM: To investigate the effect of urotensinⅡ (UⅡ) on the proliferation of cultured rat pulmonary arterial smooth muscle cells (PASMCs), and to explore whether mitogen-activated protein kinase (MAPK) signaling pathways and early growth response factor-1 (Egr-1) involved in the regulation of the PASMCs proliferation stimulated by UⅡ. METHODS: The rat PASMCs were isolated and cultured in vitrowith explant culture technique. The proliferation of cultured PASMCs stimulated by different doses of UⅡwas detected by BrdU incorporation. The mRNA expression of extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase (SAPK), p38 MAPK and Egr-1 in cultured PASMCs treated with UⅡ, UⅡ-specific antagonist urantide, and ERK1/2 inhibitor PD98059 was detected by real-time PCR. The protein levels of phosphorylated ERK1/2 (p-ERK1/2), p-SAPK, p-p38 and Egr-1 in cultured PASMCs were determined by Western blotting. RESULTS: UⅡ at concentrations of 1 μmol/L, 0.1 μmol/L and 0.01 μmol/L increased the proliferation of cultured PASMCs in a dose-dependent manner (P<0.01 or P<0.05), with the maximal effect at a concentration of 1 μmol/L. However, urantide inhibited the promotion effect of UⅡ on PASMC proliferation (P<0.05). UⅡ up-regulated the mRNA expression of ERK1/2, SAPK and Egr-1 (P<0.01 or P<0.05), but not the p38 MAPK. However, the up-regulatory effect of UⅡ on ERK1/2 and Egr-1 expression was inhibited by PD98059 and/or urantide (P<0.01 or P<0.05). UⅡ also increased the protein levels of p-ERK1/2, p-SAPK and Egr-1 (P<0.01 or P<0.05), but the promotion effect was also inhibited by PD98059 and/or urantide (P<0.01 or P<0.05).CONCLUSION: UⅡ increases the proliferation of PASMCs, and U Ⅱand Egr-1 participates in UⅡ-mediated proliferation of cultured PASMCs through activation of ERK1/2 signal pathway.     

Nodosin induces apoptosis of HepG2 cells by increasing Apaf-1 expression and activating caspase-3

AIM:To investigate the effects of nodosin on apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS:HepG2 cells were treated with nodosin at different concentrations (1.25 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L and 20 μmol/L) for 24 h. The morphological changes of the HepG2 cells were observed by Hoechst 33258 staining and electron microscopy. The apoptotic rates were analyzed by flow cytometry. The mRNA expression of apoptotic protease-activating factor-1 (Apaf-1) was detected by RT-qPCR. The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:HepG2 cells showed obvious cell shrinkage and nucleus drift when treated with nodosin as the concentration was increased. Many apoptotic bodies were observed in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups. The mRNA expression of Apaf-1 was increased in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups as compared with control group (P<0.05). The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were increased with the increasing dose of nodosin (P<0.05). CONCLUSION:Nodosin induces the apoptosis of HepG2 cells. This effect was related to increasing Apaf-1 mRNA expression and subsequently promoting the activation of caspase-3.     

Effect of berberine on Helicobacter pylori-induced human gastric epithe-lial cells injury

AIM: To investigate the effect of berberine (Ber) on Helicobacter pylori (Hp)-induced human gastric epithelial cells (GES-1) injury and the underlying mechanism. METHODS: Berberine (5, 10 and 20 μmol/L) and PD98059 (20 μmol/L), a selective inhibitor of extracellular regulated protein kinases (ERK)1/2 signaling pathway, were added to Hp-infected GES-1 cells. The cell activity and apoptosis, the levels of interleukin (IL)-1β and IL-8, lactic dehydrogenase (LDH) activity and the protein levels of Bax, Bcl-2 and p-ERK1/2 in the GES-1 cells were determined by MTT assay, flow cytometry, ELISA, colorimetry and Western blot, respectively. RESULTS: Compared with control group, Hp significantly inhibited the cell activity, increased the apoptotic rate, LDH activity, IL-1β and IL-8 levels, the Bax and p-ERK1/2 protein levels but decreased the Bcl-2 protein level in GES-1 cells (P<0.05). However, these effects of Hp were reversed by berberine at medium-dose and high-dose, as compared with the Hp-infected GES-1 cells (P<0.05). Moreover, the protective effects of berberine were significantly enhanced by the co-incubation of berberine with PD98059, as compared with the berberine at higher dose (P<0.05). CONCLUSION: Berberine may attenuate Hp-induced human gastric epithelial GES-1 cells injury by anti-inflammation, promoting cell growth and anti-apoptosis via the inhibition of ERK1/2 signaling pathway.     

Effect of tetrandrine on radiosensitivity of nasopharyngeal carcinoma cells

AIM: To investigate the mechanism of the radiosensitizing effect of maximum non-cytotoxic doses of tetrandrine (Tet) on nasopharyngeal carcinoma cell lines CNE1 and CNE2.METHODS: The cells were treated with ma-ximum non-cytotoxic doses of Tet (for CNE1 cells at 1.5 μmol/L and for CNE2 cells at 1.8 μmol/L), irradiation at 4 Gy, or combination of irradiation and maximum non-cytotoxic doses of Tet. The cell cycle distribution was analyzed by flow cytometry. The protein levels of γ-H2AX, cleaved caspase-3, p-CDC25C, CDK1, p-CDK1, cyclin B1, ERK and p-ERK were determined by Western blot.RESULTS: The expression of γ-H2AX was increased in CNE1 cells and CNE2 cells after combined treatment with irradiation and maximum non-cytotoxic doses of Tet. The percentages of CNE1 cells and CNE2 cells at G₂/M phase in irradiation group were (18.09±0.42)% and (18.48±1.32)%, respectively, which were decreased to (15.88±1.04)% and (13.80±0.82)% in combined treatment group, respectively (P<0.05). Combined treatment enhanced the increase in the protein level of cleaved caspase-3 caused by irradiation. The protein levels of p-CDC25C and p-CDK1 were increased in a dose-dependent manner by Tet treatment (P<0.05), while the expression of CDK1 showed no difference among different doses of Tet treatments. The protein levels of p-CDC25C, p-CDK1 and CDK1 showed no difference after the treatment with maximum non-cytotoxic doses of Tet. The combined treatment with irradiation and the maximum non-cytotoxic doses of Tet decreased the protein levels of p-CDC25C and p-CDK1 (P<0.05), increased the expression of cyclin B1, and had no influence on the expression of CDK1 (P<0.05). The combined treatment resulted in an increase in the protein level of p-ERK1 (P<0.05).CONCLUSION: The maximum non-cytotoxic doses of Tet enhance the DNA damage and apoptosis in CNE1 cells and CNE2 cells caused by irradiation, and the mechanism might be associated with ending of G₂/M arrest via activation of ERK/CDC25C/CDK1/cyclin B1 pathways.     

Effect of simvastatin on function of mouse pancreatic beta cells and its mechanisms

AIM: To observe the influence of simvastatin on insulin secretion function of mouse pancreatic beta cell line MIN6 and to explore its possible mechanisms.
METHODS: MIN6 cells were randomly divided into normal control group and low-, middle-and high-concentration simvastatin treatment groups, which were cultured for 48 h with high-glucose DMEM containing 15% fetal bovine serum plus 0, 2, 5 and 10 μmol/L simvastatin, respectively. The insulin secretion of MIN6 cells was measured by radioimmunoassay. The content of ATP in MIN6 cells was measured by biochemiluminescence method. The mRNA and protein expression levels of inwardly rectifying potassium channel 6.2 (Kir6.2), voltage-dependent calcium channel 1.2 (CaV1.2) and glucose transporter 2 (GLUT2) were detected by real-time fluorescence quantitative PCR and Western blotting, respectively.
RESULTS: Compared with normal control group, middle-and high-concentration simvastatin treatment markedly decreased the synthesis and secretion of insulin in MIN6 cells (P<005). The content of ATP in MIN6 cells was markedly decreased in simvastatin treatment groups (P<005). The mRNA expression level of Kir6.2 in MIN6 cells was significantly up-regulated in simvastatin treatment groups (P<001), while the mRNA expression levels of CaV1.2 and GLUT2 were significantly down-regulated in middle-and high-concentration simvastatin treatment groups (P<001). The protein expression of Kir6.2 was significantly increased but that of CaV1.2 was significantly decreased in middle-and high-concentration simvastatin treatment groups (P<001), and the protein expression level of GLUT2 was markedly decreased in high-concentration simvastatin treatment group (P<001).
CONCLUSION: Simvastatin inhibits insulin synthesis and secretion in mouse pancreatic beta cell line MIN6 via suppressing ATP production, up-regulating the expression of Kir6.2 and down-regulating the expression of CaV1.2 and GLUT2.     

Hydrogen sulfide suppresses the increase in reactive oxygen species in neurons induced by angiotensin II via ERK1/2 signal pathway

AIM: To investigate the effect of hydrogen sulfide (H₂S) on the reactive oxygen species (ROS) level in medullary neurons induced by angiotensin II (Ang II). METHODS: Primarily cultured rat medullary neurons were divided into 5 groups as follows: control group, Ang II group, sodium hydrosulfide(NaHS) group, NaHS with Ang II group, and PD98059 (an inhibitor of p-ERK1/2) with Ang II group. ROS production was measured with dihydroethidium (DHE) staining. The expression of p-ERK1/2 and ERK1/2 was determined by Western blotting. The activity of neurons was detected by CCK-8 assay. RESULTS: Ang II at concentration of 100 nmol/L significantly increased ROS level in the neurons, but the effect was inhibited by NaHS at concentrations of 50~200 μmol/L, while NaHS alone had no influence on the ROS level in neurons. Additionally, PD98059 also depressed the ROS level in neurons induced by Ang II. Furthermore, the enhanced expression of p-ERK1/2 in the neurons induced by Ang II was significantly reduced by NaHS. CONCLUSION: H₂S remarkably inhibits the ROS level in the neurons induced by Ang II via activation of MAPK signal pathways, especially p-ERK1/2, indicating that H₂S rescues neurons from oxidative stress through declining the enhanced expression of p-ERK1/2.     

Role of thioredoxin nitration in doxorubicin-induced apoptosis of neonatal rat cardiomyocytes

AIM：To investigate the role of thioredoxin nitration in the apoptosis of neonatal rat cardiomyocytes (NRCMs) induced by doxorubicin (DOX). METHODS：Cardiomyocytes treated with DOX were isolated from newborn Sprague-Dawley rats and cultured in vitro. NRCMs were treated with DOX alone (DOX group), pretreated with Mn (III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP), a peroxynitrite (ONOO-) scavenger, and then treated with DOX (MnTMPyP+DOX group), or treated with MnTMPyP alone (MnTMPyP group). NRCMs without any treatment served as a normal control (control group). The viability of the cells was examined by MTT assay, and the apoptosis was measured by Hoechst 33258 nuclear staining kit. The activity of caspase-3 was detected by spectrophotometry. The expression of cleaved poly(ADP-ribose) polymerase 1 (PARP-1), apoptosis signal-regulating kinase 1 (ASK1), phosphorylated ASK1 (p-ASK1), p38 mitogen-activated protein kinase (p38 MAPK) and phosphorylated p38 MAPK (p-p38 MAPK) was measured by Western blotting. Immunoprecipitation and immunoblotting were performed to detect the formation of Trx-ASK1 and Trx-nitrotyrosine. RESULTS：DOX induced significant apoptosis of NRCMs. MnTMPyP could significantly attenuate the apoptosis induced by DOX. Compared with control group, Trx nitration in DOX group increased obviously. The increases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were also observed, besides the expression of Trx-ASK1 compound and p-ASK1 decreased significantly (P<005). MnTMPyP could decrease the nitration of Trx. The decreases in activity of caspase-3 and expression of cleaved PARP-1 and p-p38 MAPK were detected in MnTMPyP+DOX group, while the expression of Trx-ASK1 compound and p-ASK1 increased significantly (P<005). CONCLUSION: DOX could induce significant apoptosis of NRCMs and increase Trx nitration. The process was significantly attenuated by pretreatment with MnTMPyP. Therefore, Trx nitration may play an important role in doxorubicin-induced apoptosis of cardiomyocytes.     

CaMKII aggravates hypoxia/reoxygenation injury in H9c2 cells by activating apoptosis

AIM: To investigate the effect of Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) on hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: H9c2 cells were randomized into 4 groups:control group, KN-93 (an inhibitor of CaMKⅡ; 1 μmol/L) treatment group, H/R group and H/R+KN-93 (1 μmol/L) treatment group. The cells in KN-93 group and KN-93+H/R group were pretreated with KN-93 for 2 h before the other treatment was performed. The viability of H9c2 cells in each group was measured by CCK-8 assay. Lactate dehydrogenase (LDH) activity in the culture medium was detected. The protein levels of phosphorylated CaMKⅡ (p-CaMKⅡ), phosphorylated phospholamban (p-PLN) and cleaved caspase-3 were determined by Western blot. The apoptosis was analyzed by TUNEL staining and the flow cytometry. RESULTS: No significant difference of all indexes tested between control group and KN-93 group was observed. H/R treatment significantly reduced the cell viability, and increased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). KN-93 (1 μmol/L) significantly increased the cell viability, and decreased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). CONCLUSION: CaMKⅡ aggravates hypoxia/reoxygenation injury in the H9c2 cells by activating apoptosis.     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress. METHODS: H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H₂O₂ group. H9c2 cardiomyocytes in H₂O₂ group and high, middle and low doses of EDS groups were exposed to H₂O₂ for 6 h to establish the model of oxidative stress. The viability of the H9c2 cells was detected by CCK-8 assay. The apoptosis of H9c2 cells was analyzed by flow cytometry. The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorimetry. The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cytometry and confocal laser scanning microscopy. The protein levels of Bax, Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot. RESULTS: Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells. Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H₂O₂, but were decreased by EDS treatment in a dose-dependent manner. The levels of SOD and mitochondrial membrane potential of the H9c2 cells in H₂O₂ group were reduced significantly compared with control group, but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mitochondrial membrane potential in H₂O₂-treated H9c2 cells. The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H₂O₂ group showed significant elevation in comparison with control group, and the protein expression of Bcl-2 declined in H₂O₂ group compared with control group, but high, middle and low doses of ecdysterone treatments down-regulated the protein levels of Bax, cleaved caspase-3 and up-regulated the expression of Bcl-2 in H₂O₂-treated H9c2 cells. CONCLUSION: Ecdysterone attenuates the effect of H₂O₂-induced oxidative stress on H9c2 cardiomyocytes. The mechanism may be involved in scavenging oxidative stress products, increasing antioxidant enzyme activity and improving mitochondrial function.     

Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats

AIM: To evaluate the effects of antisense TGF-β1 oligodeoxynucleotide (AS TGF-β1) on the expression of TGF-β1, deposition of extracellular matrix (ECM) and the neointima formation in the arteries after balloon injury. METHODS: The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed. At the same time, sense TGF-β1 oligodeoxynucleotide (S TGF-β1) with the base sequence complement to AS TGF-β1 was synthesized as a control. The oligodeoxynucleotides were introduced into in vivo and in vitro experiments, respectively. RESULTS: The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner, and S TGF-β1 did not have the same effect. Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was observed. Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis. Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs. Fibronectin (FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (001~1 μmol/L)-dependent manner. AS TGF-β1 significantly increased the mRNA expression of contractile marker SM22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla, especially at the concentration of 001 μmol/L and 01 μmol/L. After treatment with AS TGF-β1 (90 μg·kg-1·d-1) for 28 d, the neointima formation was significantly inhibited, and the area ratio of intima/media was markedly decreased by 68% compared with untreated group, but S TGF-β1 had no effect on neointimal formation. CONCLUSION：The AS TGF-β1 specifically inhibits the protein expression of TGF-β1 in the VSMCs derived from injured arteries. Moreover, it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN. Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury. The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the alteration of VSMC phenotype after balloon injury.     

Quercitrin promotes apoptosis of gastric cancer cell line SGC7901 by inhibiting PI3K/AKT signaling pathway

AIM: To investigate whether quercitrin induces apoptosis of gastric cancer cell line SGC7901 by inhibition of PI3K/AKT signaling pathway. METHODS: The human gastric cancer SGC7901 cells were selected as the research object. The cytotoxicity of quercitrin was detected by MTT assay, and IC₅₀ value of quercitrin was calculated. The SGC7901 cells were divided into control group, quercitrin group (incubated with 200 μmol/L quercitrin), insulin-like growth factor-1 (IGF-1) group (incubated with 100 μg/L IGF-1) and quercitrin+IGF-1 group (incubated with 200 μmol/L quercitrin and 100 μg/L IGF-1). After 48 h, the apoptosis of SGC7901 cells was analyzed by flow cytometry, and the protein levels of cleaved caspase-3, p-AKT (Ser473), AKT, p-PI3K (Tyr508) and PI3K were determined by Western blot. RESULTS: The viability of SGC7901 cells was significantly decreased as the concentration of quercitrin increased, starting at 100 μmol/L (P<0.05). The IC₅₀ value of quercitrin for 48 h was 275.40 μmol/L. After treatment with 200 μmol/L quercitrin for 48 h, the apoptosis rate and the protein level of cleaved caspase-3 in quercitrin group were significantly increased (P<0.05), and the phosphorylated levels of AKT and PI3K were significantly decreased compared with control group (P<0.05). Treatment with quercitrin and IGF-1 inhibited the effect of quercitrin on SGC7901 cells compared with quercitrin group. CONCLUSION: Quercitrin may induce apoptosis of gastric cancer cell line SGC7901 by inhibiting the activation of PI3K/AKT signaling pathway.     

Anticancer function of Shp2 in lung adenocarcinoma A549 cells and rela-ted molecular mechanisms

AIM: To explore the anticancer function of Shp2 in lung adenocarcinoma A549 cells and the related molecular mechanisms. METHODS: The viability and proliferation of A549 cells treated with Shp2 specific inhibitor Phps-1 or cisplatin (DDP) were measured by CCK-8 assay and EdU assay. Annexin V-FITC/PI double staining was applied to detect apoptotic rate of A549 cells with different interventions. The protein levels of caspase-3-17p, Bcl-2, Bax, p-STAT3/STAT3 and p-ERK/ERK were determined by Western blot. RESULTS: Compared with control group, Phps-1 at the concentration of 20 μmol/L significantly increased the viability of A549 cells after 24 h of treatment (P<0.05). Meanwhile, the proliferation rate of A549 cells in Phps-1 20 μmol/L group was significant increased compared with control group (P<0.05). The apoptotic rate of A549 cells in DDP treatment group decreased from 13.01%±2.62% to 3.67%±0.93% after adding Phps-1 (P<0.05). Phps-1 down-regulated the protein levels of caspase-3-17p, Bax and p-ERK, but up-regulated p-STAT3.CONCLUSION: Shp2 is a tumor suppressor in A549 cells, which may be associated with the activation of STAT3 signal pathway.     

Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells via PI3K/Akt signaling pathway

AIM:To investigate the effect of apelin-13 on nicotine-induced apoptosis of cardiomyocytes and its potential molecular mechanism. METHODS:Rat H9c2 cells were treated with nicotine (10 μmol/L) to induced apoptosis. Flow cytometry was used to detect apoptotic rate. Western blot was used to determined the expression of related proteins. RESULTS:Compared with control group, nicotine treatment significantly increased the apoptotic rate of the H9c2 cells (P<0.01), and the protein levels of apoptosis-related proteins Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with nicotine group, apelin-13+nicotine significantly decreased the apoptotic rate of the H9c2 cells (P<0.01) and the the protein levels of Bax and cleaved caspase-3, but markedly increased the protein levels of Bcl-2, p-Akt, p-PI3K and APJ (P<0.05). Compared with apelin-13+nicotine group, apelin-13+nicotine+PI3K/Akt inhibitor LY294002 significantly increased the apoptotic rate of the H9c2 cells (P<0.01) and the protein levels of Bax and cleaved caspase-3, but markedly decreased the protein levels of Bcl-2, p-Akt and p-PI3K (P<0.05). CONCLUSION:Apelin-13 inhibits nicotine-induced apoptosis of H9c2 cells through PI3K/Akt signaling pathway.     

Role of endoplasmic reticulum stress in Bim-mediated cardiomyocyte apoptosis induced by hypoxia

AIM:To investigate the role of endoplasmic reticulum stress (ERS) in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia. METHODS:Cardiomyocytes were isolated from neonatal Sprague-Dawley rats aged 1~3 days, and primarily cultured in vitro. The antibody targeting α-striated muscle actin was used to identify the cardiomyocytes. The siRNAs targeting bim were transfected into cardiomyocytes with liposome, followed by detecting the expression of Bim by Western blotting. Cardiomyocytes were divided into five groups: blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+Bim-siRNA group. The cell viability was determined by MTT assay, and the cell apoptotic rate and the intracellular calcium concentration were measured by flow cytometry. The protein expression of caspase-12 and inositol 1,4,5-triphosphate (IP3) was detected by Western blotting. RESULTS:Immunohistochemical identification confirmed that rat cardiomyocytes were successfully cultured. Green fluorescence was observed in the cells transfected with negative control siRNA under fluorescence microscope. 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%). Compared with blank control group, the viability of cardiomyocytes in hypoxia group was significantly reduced (P<005). Compared with hypoxia+negative control siRNA group, the viability of cardiomyocytes in hypoxia+Bim-siRNA group was significantly increased (P<005). The apoptotic rate and the intracellular calcium concentration of cardiomyocytes were obviously increased in hypoxia group (P<0.01), and were both decreased after bim silencing (P<005 or P<0.01). The expression of caspase-12 and IP3 was up-regulated in hypoxia group (P<005), and was down-regulated after bim silencing (P<005 or P<0.01). CONCLUSION: Cardiomyocyte apoptosis induced by hypoxia can be inhibited by silencing the expression of bim gene. Caspase-12 and IP3, as markers of ERS, may participate in the process of Bim-mediated cardiomyocyte apoptosis induced by hypoxia.     

Angiotensin-(1-7)/Mas receptor axis protects cardiomyocytes against high glucose-induced injury by modulating nuclear factor-κB pathway

AIM: To study whether the angiotensin-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardiomyocytes against high glucose(HG)-induced injury by inhibiting nuclear factor-κB(NF-κB) pathway. METHODS: The cell viability was measured by CCK-8 assay. The intracellular levels of reactive oxygen species(ROS) were detected by DCFH-DA staining. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining. Mitochondrial membrane potential(MMP) was examined by JC-1 staining. The levels of NF-κB p65 subunit and cleaved caspase-3 protein were determined by Western blotting. RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose(HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated(p) NF-κB p65, peaking at 60 min. Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30 μmol/L and HG for 24 h inhibited HG-induced cytotoxicity, evidenced by an increase in cell viability. On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage, indicated by decreases in the number of apoptotic cells, cleaved caspase-3 level, ROS generation and MMP loss. However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor(Mas receptor). Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG. CONCLUSION: Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway.     

Pin1 inhibitor juglone induces apoptosis in human cervical cancer SiHa cells

AIM: To explore the effect of peptidyl-prolyl cis/trans isomerase (Pin1) inhibitor juglone on apoptosis of human cervical cancer SiHa cells. METHODS: Cultured SiHa cells were incubated with juglone at concentrations of 10, 20, 50, 80 and 100 μmol/L for 24 h. The SiHa cell activity was detected by methyl thiazolyl tetrazolium (MTT) assay. The cell apoptosis was analyzed by flow cytometry with Hoechst 33258 staining. The protein levels of cleaved caspase-3,8,9 and PTEN was determined by Western blotting. RESULTS: In different doses of juglone groups, the SiHa cell growth was greatly inhibited (P<0.05) in a dose-dependent manner as compared with control group. The IC₅₀ of juglone was 20.4 μmol/L. After treatment with juglone at concentration of 20 μmol/L for 12 h, the apoptosis of SiHa cells was induced, and the typical morphological changes of cell apoptosis such as karyopyknotic pyknic hyperfluorescence bolus, nuclear fragmentation and apoptotic body were observed by Hoechst 33258 staining. The early apoptotic rate was increased significantly as compared with the control. The protein levels of cleaved caspase-3, 8, 9 and PTEN were also increased significantly as compared with control group. CONCLUSION: Juglone significantly inhibits the cell activity and induces the apoptosis of SiHa cells in vitro by inhibiting the caspase pathway and increasing the expression of anti-oncogene.     

Effects of propofol on biological characteristics of colorectal cancer cells

AIM: To investigate the effect of propofol on the viability, invasion ability and apoptosis of colorectal cancer cells.METHODS: Propofol at 10, 25, 50 and 100 μmol/L was used to treat LoVo cells for 72 h, and propofol at 100 μmol/L was used to treat the LoVo cells for 12, 24, 48 and 72 h. The cell viability was measured by CCK-8 assay. The invasion ability of the LoVo cells treated with propofol at 100 μmol/L for 72 h was detected by Transwell assay. The cell cycle distribution and cell apoptotic rate were analyzed by flow cytometry. The protein levels of matrix metalloproteinase (MMP)-2, MMP-9, cleaved caspase-3, Notch1 and hairy and enhancer of split 1 (Hes1) were determined by Western blot.RESULTS: Propofol inhibited LoVo cell viability. The cell invasion ability, S stage cells, and the protein levels of MMP-2, MMP-9, Notch1 and Hes1 in propofol group were significantly lower than those in control group, and the apoptotic rate, G₀/G₁ cells and the protein level of cleaved caspase-3 were significantly higher than those in control group (P<0.01).CONCLUSION: Propofol inhibits the viability and invasion ability of colorectal cancer LoVo cells, blocks cell cycle and induces apoptosis. The mechanism is related to down-regulation of Notch1 signaling pathway.