Expression of CD40-CD40L during rat myocardial ischemia-reperfusion injury

AIM:To investigate the expression of CD40 and CD40 ligand(CD40L) during myocardial ischemia/reperfusion injury in rats.METHODS:In the rat modal with myocardial ischemia/reperfusion(MI/R),the changes of CD40 and CD40L expression in blood and myocardial tissues were detected by flow cytometry and immunohistochemistry, respectively. There were seven animal groups in the study, including the normal group (n=3), the ischemia 30 min group(I_{30 min},n=6),the ischemia/reperfusion 1 min,5 min,10 min,20 min and 30 min groups(each group, n=6).RESULTS:The indexes of the expression of CD40 and CD40L in I_{30 min} group were higher than those in control group,(P＜0.05); the indexes of R_{5 min},R_{10 min} groups were higher than those in the I_{30 min} group (P＜0.05);the indexes were the highest in R_{5 min} and then decreased in R_{10 min},R_{20 min},R_{30 min} group. The immunohistochemical results revealed that. CD40-CD40L expression increased on the cardiocyte membraneduring the ischemia/reperfusion injury.CONCLUSION:CD40 and CD40L may participate in the development of myocardial ischemia/reperfusion injury.     

Effect of PKC activition on cardiac myocyte apoptosis and Bcl- 2 expression during myocardial ischemia/reperfusion in rats

AIM: To explore the effect of PKC activition on cardiac myocyte apoptosis and expression of bcl- 2 during myocardial ischemia/reperfusion(I/R) in rats. METHODS: TUNEL,immunohistochemistry and in situ hybridization were used. RESULTS: The TUNEL data showed that the numbers of positive cardiac myocyte nucleus and the percentage of positive cardiac myocyte nucleus in PMA+IR_{3 h} group decreased significantly(P＜0.05,P＜0.01), compared to those in IR_3h group. The number of Bcl-2 protein positive cardiomyocytes and the percentage of Bcl-2 protein positive cardiomyocytes in PMA+IR_3h group were higher than those in IR_3h group (P＜0.01) bcl- 2 mRNA expression showed the same changes in PMA+IR_0h group compared to IR_1h group.CONCL USIONS:Activation of PKC decreased cardiomyocyte death during I/R.Upregulation of bcl-2 gene expression in cardiomyocytes during I/R may be one of the mechanisms of decreasing cardiomyocyte death by PCK activating during I/R.     

Effect of fucoxanthin on growth and apoptosis of HSC-T6 cells

AIM: To explore the effect of fucoxanthin (Fu) on the growth and apoptosis of HSC-T6 cells. METHODS: HSC-T6 cells were divided into blank control group, negative control group and drug groups (treated with different concentrations of Fu). The cell viability was detected by CCK-8 assay at 24 h, 48 h and 72 h after Fu treatment. The cell cycle distribution and apoptotic rate were analyzed by flow cytometry. The protein expression of Bcl-2 and Bax were detected by Western blot. RESULTS: Compared with blank control group, the viability of HSC-T6 cells was inhibited by Fu at concentrations of 15~75 μmol/L in a dose- and time-dependent manner (P < 0.01). The cell ratio of G₁ phase was significantly decreased (P < 0.01) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.01) in 60 μmol/L Fu group after 24 h. The cell ratio of G₁ phase was significantly decreased (P < 0.05) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.05) in 15 μmol/L and 30 μmol/L Fu groups in a dose-dependent manner after 48 h. The early cell apoptotic rates and total cell apoptotic rates were significantly increased in the Fu treatment groups in a dose-dependent manner (P < 0.05). The protein expression of Bax was significantly increased in the Fu treatment groups and the protein expression of Bcl-2 was significantly decreased in 30 μmol/L and 60 μmol/L Fu groups (P < 0.05).CONCLUSION: Fu inhibits the growth of HSC-T6 cells possiblely via arresting the cell cycle at S phase and G₂ phase. The apoptosis of HSC-T6 cells induced by Fu might be via down-regulating the protein expression of Bcl-2 and up-regulating the protein expression of Bax.     

Effect of epigallocatechin gallate on cardiomyocyte apoptosis induced by ischemia-reperfusion in rats

AIM: To observe the effects of epigallocatechin gallate (EGCG) on cardiomyocyte apoptosis induced by ischemia-reperfusion (IR) in rats. METHODS: The left anterior descending branch of coronary artery was ligated for 30 min and reperfused for 60 min to make a the myocardial ischemia-reperfusion model in rats. The experiment was divided into five groups: sham, ischemia/reperfusion (IR), EGCG (10 mg/kg and 20 mg/kg) and salvia miltiorrhizae (SM, 100 mg/kg) group. The apoptotic cardiomyocytes were detected by in situ end labeling method, and the expressions of Bcl-2 and Bax were shown through immunohistochemistry method. RESULTS: There was no apoptosis myocardial cell in sham operation group. The apoptosis index and expression of bax significantly increased, and bcl-2/bax reduced in IR group (P<0.01). In EGCG-treated group, however, the changes above were obviously alleviated (P<0.01). CONCLUSION: EGCG significantly inhibits cardiomyocyte apoptosis in ischemia-reperfusion rat hearts. The possible mechanism is to raise the ratio of Bcl-2/Bax proteins by increasing in the expression of bcl-2 gene and decreasing in the expression of bax gene.     

Effects of human thioredoxin on pneumocyte apoptosis and Bcl-2/Bax expression in rats with lung ischemia/reperfusion injury

AIM: To explore the relationship between apoptosis in the lung tissues and lung ischemia/reperfusion injury, and to observe the effects of human thioredoxin (hTrx) on apoptosis in lung ischemia/reperfusion injury. METHODS: The single lung in situ ischemia/reperfusion animal model was used. Eighty four Wistar rats were randomly divided into control group (control), groups of ischemia for 1 h and reperfusion for different times (IR1h, IR3h, IR5h), and groups of IR+human thioredoxin treatment (IR1h +hTrx, IR3h +hTrx and IR5h +hTrx). Transmission electron microscope (TEM), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunocytochemistry techniques were used to observe apoptosis, apoptosis signal-regulating kinase 1 (ASK1) and expression of Bcl-2 and Bax in various phases of lung ischemia/reperfusion. RESULTS: Cell apoptosis in lung tissues was significantly high, ASK1, Bcl-2 and Bax protein were up-regulated in lung tissues of lung ischemia/reperfusion injury as compared to control (all P<0.01). Compared to IR group, hTrx suppressed apoptosis as well as expression of ASK1 and Bax protein (P<0.01), Bcl-2 protein and the ratio of Bcl-2/Bax were up-regulated in lung tissues (all P<0.05 or P<0.01). There was a significant correlation between the expression of ASK1, Bax protein and cell apoptosis (r=0.775, r=0.814, respectively; all P<0.01). There was a negative correlation between cell apoptosis and Bcl-2/Bax protein (r=-0.275, P<0.05). CONCLUSION: Initiating cell apoptosis by the activation of Bcl-2/Bax system in lung tissues may contribute to the pathogenesis of lung ischemia/reperfusion injury. The protective effects of hTrx include suppressing the expression of ASK1, down-regulating the ratio of Bcl-2/Bax and blocking apoptosis in lung tissues in lung ischemia/ reperfusion injury.     

Effects of Tertram ethylpyrazine on expression of Fas/FasL mRNA in lung tissue with ischemia-reperfusion injury in rabbits

AIM: To study the expression of Fas/FasL mRNA in lung tissue with ischemia-reperfusion lung injury in rabbits and the relationship with the apoptosis,and to observe the effects of Tertram ethylpyrazine on them.METHODS: The pulmonary ischemia-reperfusion models in rabbits with occlusion of left pulmonary hilum for 1 h and then reperfusion 1,3,5 h respectively were used in this experiment.In TMP group,Tertram ethylpyrazine was intravenously dropped at dose of 60 mg/kg at 1 h before ischemia.The TUNEL technique was used to explore apoptotsis of pulmonary cells.In situ hybridization was performed on the rabbit lung tissue to assay the expression of Fas/FasL mRNA.RESULTS: Apoptosis of pulmonary cells was found in both IR group and TMP group.Compared with group IR,the apoptosis index (AI) was decreased obviously in group TMP (P<0.01).There was a significant positive correlation between the expression of Fas/FasL mRNA and the apoptosis of pulmonary cells (r₁=0.900,r₂=0.869,P<0.01).CONCLUSION: The activation of Fas/Fas-L system may contribute significantly to induce pneumocyte apoptosis in pulmonary ischemic injury.Tertram ethylpyrazine inhibits the activation of Fas/FasL system to decrease apoptosis in pulmonary tissue,which may protect the pulmonary tissues in ischemia injury.     

The mechanism of apoptosis induced by homoharringtonine in HL-60 cells

AIM: To study the signal transduction pathway of apoptosis initiation induced by homoharringtonine in HL-60 cells. METHODS: After establishing the model of apoptosis initiation induced by homoharringtonine in HL-60 cells, at the point of apoptosis initiation, molecular caspase-3, Bcl-2, Bax and Fas/FasL were measured with flow cytometry and transmission electron microscope. ERK2 and P38 expression in HL-60 cells were detected by using immunohistochemistry. RESULTS: The model of apoptosis initiation induced by homoharringtonine was established in HL-60 cells. At the point of apoptosis initiation, upregulation of caspase-3 and decrease in Bcl-2/Bax were observed. However, the expression of Fas/FasL did not significantly change. ERK2 expression decreased and P38 expression increased. CONCLUSIONS: Caspase-3, Bcl-2, Bax and mitogen activated protein kinase pathways were involved in signal transduction of apoptosis initiation induced by homoharringtonine in HL-60 cells.     

Effects of norepinephrine preconditioning and ischemic preconditioning on apoptosis and Bcl-2, Bax expression in rat myocardial cells during myocardial ischemic reperfusion

AIM: To study the effects of norepinephrine preconditioning(NE-P) and ischemic preconditioning (IP)on apoptosis and Bcl-2, Bax expression in rat myocardial cells in myocardial ischemic reperfusion (I/R). METHODS: The model of rat ischemic-reperfusion was used to conduct NE-preconditioning. Apoptotic myocytes were detected with TUNEL. Bcl-2, Bax expression were detected with immunohistochemistry. RESULTS: The rate of apoptosis cells in I/R group was higher, the rate of apoptosis cells in NE-P group and IP was lower significantly than that in I/R group(P＜0.01). The expression of Bcl-2 in I/R group was lower, but the expression of Bax was higher, the expression of Bcl-2 in NE-P group was higher significantly than that in I/R group(P＜0.01), the expression of Bax in NE-P group was lower than that in I/R group(P＜0.01). There was no significantly difference between NE-P and IP group in the above parameters (P＞0.05). CONCLUSION: NE-P reduced myocyte apoptosis by I/R in rats; The expression of Bcl-2 ,Bax genes played an important role in myocardial apoptosis.     

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.     

Effect of nitric oxide on apoptosis in lung injury following ischemia-reperfusion of hind limbs

AIM: To investigate the injury of lung and the role of cell apoptosis in the pathogenesis of acute lung injury following ischemia-reperfusion of hind limbs and the influence of nitric oxide (NO) to apoptosis.METHODS: Referring to our laboratory normal method,the model rats,which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made.L-arginine (L-Arg) and N-nitro-L-arginine methyl ester (L-NAME) was administrated respectively to these rats before the experiment.Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL),respectively.The radioimmunoassay (RIA) was used to detect level in the expression of TNF-α.The immunohistochemistry (IHC) method was used to detect the level in the expression of Bcl-2,Bax,caspase-3 and TNF-α.The morphologic changes were observed under microscope,respectively.The results of the RIA and the IHC were analyzed quantitatively by relative computer analytical system.RESULTS: After rats’s hind limbs suffered from ischemia-reperfusion,the apoptosis in alveolar epithelial cells and pulmonary vascular endothelial cells was found.The expression of TNF-α,caspase-3 and Bax increased.Compared with IR rats,the expressions of TNF-α,caspase-3 and Bax were not obvious in the L-Arg administrated group,but the expression of Bcl-2 was obvious in that group.Compared with IR rats,the expressions of TNF-α,caspase-3 and Bax were obvious in the L-NAME administrated group,but the expression of Bcl-2 was not obvious in that group even weaker than normal ones.CONCLUSION: Apoptosis participated in acute lung injury following ischemia-reperfusion of hind limbs.The excess expression of TNF-α related with apoptosis of alveolar epithelial cells and pulmonary vascular endothelial cells.NO may reduce the occurrence of apoptosis and other lung injury through down-regulating the level in the expression of TNF-α.     

Effects of ligustrazine ferulate on myocardial apoptosis and apoptosis-related protein expression in rats with myocardial ischemia-reperfusion injury

AIM:To observe the effects of ligustrazine ferulate on the apoptosis of myocardial cells in rats with myocardial ischemia-reperfusion injury, and to explore its possible mechanism. METHODS:Sixty male SD rats were randomly divided into five groups: sham-operation group, ischemia-reperfusion group, ligustrazine (4 mg/kg) group, low-dose (4 mg/kg) ligustrazine ferulate group and high-dose (8 mg/kg) ligustrazine ferulate group. The rat myocardial ischemia-reperfusion model was established by 30 min of myocardial ischemia followed by 120 min of reperfusion. Drugs were administered to the rats by jugular vein injection 10 min before reperfusion. After the reperfusion was finished, the biochemical indicators in serum and the histological indexes in myocardium were detected. RESULTS: Compared with ischemia-reperfusion group, ligustrazine ferulate lowered the serum levels of creatine kinase MB form, lactate dehydrogenase, cardiac troponin I and malondialdehyde, elevated the activity of total superoxide dismutase in serum and the expression of Bcl-2 protein in myocardium, decreased the expression of Bax protein and myocardial apoptotic index, and increased the Bcl-2/Bax ratio (all P<0.01). All the indicators in ligustrazine ferulate groups were dose-dependently superior to those in ligustrazine group (P<0.05 or P<0.01). CONCLUSION: Ligustrazine ferulate protects rats against myocardial ischemia-reperfusion injury. Its anti-apoptotic effect may be related to up-regulation of Bcl-2 and down-regulation of Bax.     

Mechanism underlying the antagonistic effect of folic acid on homocysteine-induced apoptosis in endothelial cell

AIM: To investigate the effects of homocysteine (Hcy) on apoptosis in SV40-transformed aortic rat endothelial cell line and the anti-apoptosis effects of folic acid. METHODS: Cells were treated with different concentrations of Hcy and folic acid, apoptosis was detected by TUNEL and annexin-V/ PI staining methods. Immunohistochemical assay was used to examine the expression of Bax and Bcl-2 in all groups. RESULTS: Both annexin-V/PI staining and TUNEL method showed that Hcy increased endothelial apoptosis in a dose-dependent manner, while folic acid reduced cell apoptosis. Hcy increased expression of Bax and Bcl-2 in endothelial cells, and folic acid decreased it. Bax/Bcl-2 ratio in 0.5 mmol/L Hcy and 5.0 mmol/L Hcy group were upregulated compared with control group (P<0.05 and P<0.01, respectively). Addition of 0.1 mmol/L folic acid decreased Bax/Bcl-2 ratio compared with the corresponding group without folic acid (P<0.05). Correlation analysis showed a strong relation between Bax/Bcl-2 ratio and apoptotic rate (P<0.01). CONCLUSION: Folic acid attenuates the apoptosis induced by Hcy in endothelial cells. Hcy may promote endothelial cell apoptosis via upregulation of Bax /Bcl-2 ratio, which can be partially antagonized by folic acid.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

Effects of ischemic preconditioning on cardiac myocyte apoptosis and expression of bcl-2 during myocardial ischemia/reperfusion in rats

AIM:To explore the effect of ischemic preconditioning on cardiac myocyte apoptosis and the expression of bcl-2 during myocardial ischemia/reperfusion in rats. METHODS:We use TUNEL,immunohistochemical and in situ hybridization(ISH) methods to detect the cardiac myocyte apoptosis and the expression of bcl-2 during myocardial ischemia/reperfusion in rats. RESULTS:①The numbers of positive cardiac myocyte nuclear and the percentage of positive cardiac myocyte nuclear in IP+I/R_3h group decreased significantly(P<0.05,P<0.01)compared with I/R_3h group,respectively.②The numbers of bcl-2 protein positive cardiomyocyte and the percentage of bcl-2 protein positive cardiomyocyte in IP+I/R_3h group were higher(P<0.01)than that of I/R_3h group,respectively.The numbers of positive bcl-2 mRNA cardiomyocyte and the percentage of positive bcl-2 mRNA cardiomyocyte in IP+I/R_1h group were higher(P<0.01)than that of I/R_1h group,respectively.CONCLUSION:① The first window of IP's protection could reduce cardiomyocyte apoptosis significantly.② Up-regulating the protein expression of bcl-2 in cardiomyocytes during I/R may be one of the mechanisms of first window of IP's protection.     

Ethyl acetate extract of Pleione bulbocodioides (Franch.) Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mitochondrial apoptosis pathway

AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC₅₀ of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G₂ phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.     

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 down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Effect of component II of broccoli polypeptide on glioma cell apoptosis

AIM：To explore the effect of component II of broccoli polypeptide on the apoptosis in glioma cells. METHODS：Human glioma SHG-44 cells were cultured and divided into control group and 3, 10, 30 and 100 mg/L component II of broccoli polypeptide groups. Cell viability was detected by MTT assay. The apoptotic rates were examined by Annexin V/PI staining. The morphological changes of the cells were observed under inverted microscope. The protein expression of Bax and Bcl-2 was detected by immunocytochemistry and Western blotting. The protein level of caspase-3 was also examined by Western blotting. RESULTS：Treatment with component II of broccoli polypeptide for 24 h, 48 h or 72 h induced significant inhibition of viability of SHG-44 cells in a time- and dose-dependent manner. The results of Annexin V/PI staining showed that the apoptotic rates were increased in treatment groups in a dose-dependent manner. The density of glioma cells was decreased after treated with increasing concentrations of the drug, and the apoptotic bodies were observed under inverted microscope at 72 h. The results of immunocytochemistry and Western blotting showed that the expression of Bax protein was increased but Bcl-2 protein expression was decreased, and the ratio of Bax/Bcl-2 was increased significantly compared with control group (P<0.05 or P<0.01). The level of caspase-3 protein was increased in 30 and 100 mg/L component II of broccoli polypeptide groups compared with control group (P<0.01). CONCLUSION：The component II of broccoli polypeptide increases the ratio of Bax/Bcl-2 and activates caspase-3 protein, thus inducing the apoptosis of glioma cells.     

Adenosine A2A receptor antagonist SCH58261 attenuates hypoxic-ischemic brain damage in a fetal rabbit model

AIM: To study the effect of adenosine A_2A receptor antagonist SCH58261 on hypoxic-ischemic brain damage (HIBD) in a mature fetal rabbit model.METHODS: Pregnant New Zealand white rabbits at gestational day 29 were selected and were randomly divided into sham-operated group, hypoxic-ischemic group, SCH58261 0.04 mg/kg group, SCH58261 0.12 mg/kg group and DMSO group. The intrauterine rabbit HIBD model was established. All pregnant rabbits were subjected to cesarean section 24 h after the sham operation or experimental procedure to induce hypoxic-ischemic injury in the fetus. The survival neonatal rabbits were kept in a neonatal incubator at 35℃. The general conditions of the newborn rabbits were recorded. The degree of neurobehavioral damage in the newborn rabbits was estimated by a neurobehavioral scoring protocol. The concentration of SCH58261 in the serum of pregnant rabbits, the serum of neonatal rabbits and the brain tissues of neonatal rabbits was measured by mass spectrometry. The mRNA expression of Bcl-2/Bax and protein levels of p-P38 mitogen-activated protein kinase (MAPK) in the cortex, hippocampus and striatum area in the brain of the neonatal rabbits were determined by real-time PCR and Western blot. RESULTS: SCH58261 was detected in the serum and brain tissues of the newborn rabbits. The SCH58261 concentration was approximately 40 μg/L in the brain tissue of the newborn rabbits. The mRNA expression of Bcl-2 in the cortex, hippocampus and striatum of brain tissues in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group was higher, and the mRNA expression of Bax was lower than those in HI group (P<0.05). The protein level of p-P38 MAPK in the cortex, hippocampus and striatum of brain tissues was reduced in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group compared with HI group (P<0.05). The protein level of p-P38 MAPK in SCH58261 0.12 mg/kg group was a little lower than that in SCH58261 0.04 mg/kg group (P<0.05). CONCLUSION: Adenosine A_2A receptor antagonist SCH58261 attenuates hypoxia-ischemia induced neonatal brain injury by blocking adenosine A_2A receptor, subsequently inhibiting p-P38 MAPK phosphorylation to reduce neuronal apoptosis.     

SPK1 interferes with LLC cell apoptosis by regulating Bcl-2/Bax pathway

AIM: To investigate whether sphingosine kinase 1 (SPK1) interferes with apoptosis of Lewis lung cancer (LLC) cells by regulating the Bcl-2/Bax pathway. METHODS: The SPK1 gene siRNA eukaryotic expression vector was constructed, and transfected into the LLC cells. The transfected LLC cells was observed under a fluorescence microscope. The apoptotic rate of LLC cells after transfection was analyzed by flow cytometry. The expression levels of SPK1, Bcl-2 and Bax in LLC cells after transfection were detected by Western blot. The protein levels of Bax and Bcl-2 were measured by ELISA. RESULTS: Transfected LLC cells emitted green fluorescence under a fluorescence microscope. Apoptosis in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group (P<0.01). Western blot analysis showed that the expression of Bax in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group, and the expression of Bcl-2 was lower than that in siRNA-SPK1-Neg group. The ELISA results showed that the protein level of Bax in siRNA-SPK1 group was significantly higher than that in siRNA-SPK1-Neg group (P<0.01), and the protein level of Bcl-2 in siRNA-SPK1 group was significantly lower than that in siRNA-SPK1-Neg group (P<0.01). CONCLUSION: The expression of SPK1 in LLC cells is related to the apoptotic rate. SPK1 may interfere with the apoptosis of LLC cells via Bcl-2/Bax pathway.