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.     

硒的营养研究进展

长期以来,微量元素硒是动物营养界所关注的重点和研究的热点。本文着重从硒的化学,自由基与抗氧化系统,硒蛋白,与凋亡、癌症以及生殖系统的关系等几个方面对硒的研究加以综述。     

氧化应激对Vero细胞的损伤及EGCG的保护作用

研究了氧化应激对肾细胞Vero细胞的损伤及EGCG的保护作用。用MTT、吖啶橙染色和DNA凝胶电泳等方法研究了H2O2和Cr6+应激对Vero细胞的损伤,及EGCG对凋亡细胞的保护作用及其机理。结果表明,H2O2和Cr6+剂量效应地抑制Vero细胞活性,IC50分别为175.6和9.8mgL-1;其中50 mgL-1 H2O2和400 mgL-1/2h Cr6+可诱导Vero细胞凋亡。0~60 mgL-1 EGCG有效抑制H2O2和Cr6+应激引起的细胞活性下降,且40 mgL-1 EGCG显著抑制细胞凋亡。对于Cr6+所诱导的细胞凋亡,EGCG的保护作用与EDTA和Vc的协同作用效果相当,表明清除活性氧和络合金属离子都有助于减轻Cr6+应激损伤。可见,EGCG通过清除活性氧和络合离子有效地保护了肾细胞免受应激损伤,这对易遭受活性氧损伤的肾脏无疑具有一定的临床价值。     

In vitro efficacy of doxorubicin and etoposide against a feline injection site sarcoma cell line

Feline injection site sarcoma (ISS) is a locally invasive tumor, in which surgical treatment is frequently combined with radiation or chemotherapy to improve tumor control. The focus of this study was to evaluate the cytotoxic effects of doxorubicin or etoposide on a feline injection site sarcoma cell line (JB) and to assess the impact of combining these drugs on cell death and cell cycle. Both single agent and combination drug administration increased cell death and significantly reduced the number of viable cells. Cells in G0/G1 were significantly reduced while the G2/M fraction was significantly increased following treatment. Collectively, combining doxorubicin and etoposide at the lower EC yielded comparable results to the EC50 of either drug alone in degree of cytotoxicity, level of apoptosis, and % of cells in G2/M. The results of this study indicate that doxorubicin and etoposide alone and in combination differentially alter ISS cell viability and cycle.     

Role of HIF-1α/VEGF pathway in treatment of cerebral ischemia-reperfusion injury by hyperbaric oxygen pretreatment

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway in hyperbaric oxygen (HO) pretreatment in rats with cerebral ischemia-reperfusion (IR) injury. METHODS: Healthy male SD rats (n=32) were randomly divided into control group IR group, HO-IR group and HO-IR-HIF-1α inhibitor group (HO-IR-I group). The IR model was established by middle cerebral artery occlusion. The corresponding blood vessels of the rats in control group were only exposed. The rats in HO-IR group and HO-IR-I group were treated with HO for 4 weeks before the animal modeling. The rats in HO-IR-I group received 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazol (YC-1) by intraperitoneal injection at 4 mg/kg before HO preconditioning every day. At 1 d and 7 d after modeling, the neurological assessment was evaluated.At the end of the 7 th day, after observation, the rats were sacrificed by anesthesia to measure the infarct volume of the brain tissue. The protein levels of HIF-1α, VEGF and apoptosis-related proteins Bax and Bcl-2 were determined by Western blot. The number of apoptotic cells was detected by TUNEL. RESULTS: Compared with control group, the neurological function score was decreased, while the cerebral infarction volume ratio, the protein levels of HIF-1α, VEGF, Bcl-2 and Bax, and the apoptotic cells were increased in IR group, HO-IR group and HO-IR-I group (P<0.05). Compared with IR group, the neurological function score, and the protein levels of HIF-1α,VEGF and Bcl-2 were increased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were decreased in HO-IR group and HO-IR-I group (P<0.05). Compared with HO-IR group, the neurological function score, and the protein levels of HIF-1α, VEGF and Bcl-2 were decreased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were increased in HO-IR-I group (P<0.05). CONCLUSION: The mechanism of HO preconditioning attenuating cerebral IR injury may be related to the regulation of apoptosis by inducing HIF-1α/VEGF signaling pathway activation.     

Effect of nicotine in vitro on expressions of apoptosis-related genes in human lung adenocarcinoma cells by cDNA microarray

AIM: To investigate effect of nicotine on growth of human lung adenocarnoma cells and expressions of apoptosis-related gene. METHODS: Lung adenocarcinoma cell line, SPC-A-1, was cultured in the presence of various concentrations (1-1 000 μg/L) of nicotine for 48 hours. MTT was applied to evaluate effect of nicotine in vitro on growth of SPC-A-1 cell line. After SPC-A-1 cells were treated with 100 μg/L for 48 hours, cDNA expression profile microarray was used to detect the expressions of 451 apoptosis-related genes in SPC-A-1 cell line. RESULTS: Significant proliferation in SPC-A-1 cells treated with nicotine (1-10 μg/L) was observed, but this effect decreased with increase in concentration of nicotine in culture. Growth inhibition rate of 1, 10, 100, 1 000 μg/L of nicotine was 27%, -40%, -40% and -93%. Microarray detection showed that significantly different expressions appeared in 80 of 451 apoptosis-related genes. 29 apoptosis-promoted genes and 26 apoptosis-inhibited genes were up-regulated significantly (CY3/CY5>2.0), and 25 genes were significantly down-regulated (CY3/CY5<0.5). CONCLUSION: Nicotine may promote growth of human lung adenocarcinoma cell through regulating many apoptosis-related gene expressions.     

Salviae miltiorrhizae injection antagonists renal-toxicity of streptomycin

AIM: To investigate the antagonistic and protective mechanism of salviae miltiorrhizae injection(SMI) on kidney toxicity damaged by streptomycin (SM). METHODS: 40 guined pigs were divided randomly into 4 groups: the control group, SM group, SMI+SM group and SMI group. Light microscope, transmission electron microscope (TEM), immunohistochemical staining, TdT mediated biotin dUTP nick-end cabelling (TUNEL) techniques and image quantitative analysis technique were used to observe the change of kidney after administration of SM and SMI. The expressions of iNOS and bFGF, apoptosis, and the activity of N-acetyl-D-glucosaminidase(NAG) were also observed. RESULTS: 10 d after administration of SM, the serious pathological change in SM group was observed under the light microscope and transmission electron microscope. Slight damage was found in SMI+SM group, and the number of apoptosis cells was fewer than that in SM group. The result of immunohistochemical implied that the iNOS expression in SM group was higher than that in SMI+SM group, while the bFGF expression was lower. The activity of NAG in SMI+SM group was lower than that in SM group (P<0.01). CONCLUSION: Salvia miltiorrhiza injection inhibits the increased expression of iNOS during kidney toxicity damaged by SM, reduces the activity of NAG, increases the expression of bFGF, and decreases the apoptosis, hence has the antagonism to the kidney toxicity damaged by SM.     

Effects of PDCD5 on hypoxia/reoxygenation-induced autophagy and apoptosis of cardiomyocytes

AIM: To investigate the influence of programmed cell death 5 (PDCD5) on apoptosis and autophagy in the cardiomyocytes exposed to hypoxia/reoxygenation (H/R) and its potential mechanism.METHODS: H9c2 cells were exposed to H/R. PDCD5 was downregulated by RNA interference. The cell viability was measured by MTT assay. TUNEL assay was used to detect cell apoptosis. The mRNA and protein levels were determined by RT-qPCR and Western blot.RESULTS: The expression of PDCD5 was upregulated in the cardiomyocytes after H/R injury. Furthermore, H/R injury obviously reduced the cell viability and enhanced the apoptosis and autophagy of the cardiomyocytes. However, knockdown of PDCD5 increased the cell viability, and attenuated H/R-induced apoptosis, accompany with reduction of Bax and augment of Bcl-2 expression. Additionally, silencing PDCD5 markedly inhibited H/R-induced autophagy by regulating the expression of LC3-II/LC3-I and beclin-1. Moreover, downregulation of PDCD5 suppressed NF-κB signaling by redu-cing the protein level of p-P65.CONCLUSION: Silencing PDCD5 suppresses H/R-induced H9c2 cells apoptosis and autophagy by blocking NF-κB signaling pathway. The result indicates a new strategy for the prevention and treatment of myocardial I/R injury.     

Regulatory effects of miR-195 on biological behaviors of lung cancer A549 cells

AIM: To investigate the regulatory effects of microRNA (miR)-195 on the biological behaviors, such as viability, apoptosis and migration, of lung cancer A549 cells, and to explore the related mechanisms. METHODS: After miR-195 mimics were transfected into the A549 cells, the cell viability, cell cycle distribution and apoptosis were measured by CCK-8 assay and flow cytometry. Transwell assay was used to detect cell migration ability. Furthermore, the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb/Rb were determined by Western blot. Dual-luciferase reporter assay was used to screen and identify the possible target genes of miR-195. RESULTS: Over-expression of miR-195 in the A549 cells inhibited the cell viability and induced cell cycle arrest, accompanied with the decrease in the cell migration ability and the increase in the apoptotic rate (P<0.05). Furthermore, the protein levels of cyclin D1, CDK2, Bcl-2 and p-Rb were significantly decreased (P<0.05). Dual-luciferase reporter assay demonstrated that MYB was a potential target gene of miR-195. Over-expression of MYB in the A549 cells partially reversed the effects of miR-195 on the cell viability, apoptosis and migration. CONCLUSION: miR-195 inhibits lung cancer A549 cell growth and migration, and promotes cell apoptosis by targeting MYB gene.     

Lutein suppresses t-BHP-induced apoptosis in retinal ganglion cells

AIM: To investigate the protective effects of lutein on retinal ganglion cells in vitro. METHODS: The effect of lutein on tert-butyl hydroperoxide (t-BHP)-treated retinal ganglion cells (RGC-5 cell line) was determined. The protein expression of Brn-3 and MAP-2 was examined by the method of immunocytochemistry to identify the RGC-5 cells. The RGC-5 cells were induced by a 24 h exposure of t-BHP, and the cell viability was examined by MTT assay. The apoptotic ratio of the RGC-5 cells after exposed to t-BHP or/ and lutein treatment was analyzed by flow cytometry assay with Annexin V-FITC /PI staining. The activation of caspase-3 was detected by immunocytochemistry and the protein levels of Bcl-2, Bax, cleaved caspase-3, JNK and c-Jun were determined by Western blot. RESULTS: The RGC-5 cells expressed Brn-3 and MAP-2 proteins. Lutein treatment prevented t-BHP-induced RGC-5 cell apoptosis and increased the cell activity. Compared with control group, exposure of the RGC-5 cells to t-BHP decreased the expression of anti-apoptotic protein Bcl-2, increased the Bax/Bcl-2 ratio, up-regulated the level of cleaved caspase-3, also promoted the phosphorylation of JNK and c-Jun. Lutein partly reversed the effects of t-BHP on the RGC-5 cells mentioned above. CONCLUSION: Lutein protects RGC-5 cells against t-BHP-induced apoptosis by up-regulating Bcl-2 expression and inhibiting caspase-3 activation through modulating the JNK signaling pathway.