Objective To investigate the expressions of tumor necrosis factor related apoptosis inducing ligand （TRAIL） and its receptors (DR4, DcR1) in human rectal cancer tissues and normal rectal tissues. MethodsThe expressions of TRAIL and its receptors (DR4, DcR1) in 31 cases of human rectal cancer tissues and 20 cases of normal rectal tissues were detected by immunohistochemical staining. ResultsThe positive expression rates of TRAIL, DR4 and DcR1 （32.26%， 29.03%， 0） were lower than those of normal rectal tissues （55.00%， 70.00%， 65.00%）, the difference was statistically significant(P=0.015， P=0.000， P=0.000). There were no relation between the expressions of TRAIL, DR4 and DcR1 and clinicopathologic characteristics (Pgt;0.05). ConclusionThe expressions of TRAIL and its receptors (DR4, DcR1) in human rectal cancer tissues were lower than those of normal rectal tissues, which may suggest that the apoptotic effect induced by the interaction between TRAIL and its receptors has attenuated in human rectal cancer.
This study aims to investigate the effects of small interference RNA (siRNA) targeting PML-RARα mRNA on the activity of the acute promyelocytic leukemia cell line NB4. The proliferation inhibition was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry after siRNA treatment. The results showed that the cell growth of siRNA treated group was inhibited, and the apoptosis of NB4 could be induced. The siRNA targeting PML-RARα mRNA might be a valid therapy of acute promyelocytic leukemia.
Pulmonary arterial hypertension(PAH) is a kind of pulmonary hypertension disease. Recently, the researches of its pathogenesis have reached more and more deeply. The treatment of pulmonary arterial hypertension is individual and systematic, not only relying on medicine treatment. The treatment of PAH is as follows: common treatment, non-specific medicine treatment, targeted medicine treatment, NO breath-in treatment, gene treatment, intervention and surgery treatment.The article reviews the main treatment of pulmanory arteral hypertesion to provide new thought and evidence in clinic.
ObjectiveTo investigate the in vitro effect of pseudolaric acid B (PAB) on apoptosis of protoscolece cells and its regulatory effects on angiogenesis and cell apoptosis in the the lesion-host microenvironment tissue in vivo, as well as its possible mechanisms, in order to provide a basis for the clinical development of new alternative drugs for Echinococcus multilocularis. MethodsIn vitro experiments: the protoscoleces, vesicles, germinal cells, human foreskin fibroblasts (HFFs) and normal human liver cells were treated with different concentrations of PAB (0, 2.5, 5, 10, 20, 40, 80, 160 and 320 μmol/L) for 7, 5, 5, 5 and 5 days, then evaluated the survival rate of the protoscoleces, the release level of phosphoglucose isomerase (PGI) from the vesicles, the viability of the germinal cells, as well as the viability of HFFs and normal human liver cells. The protoscoleces and vesicles were fixed with 2.5% glutaraldehyde and used for scanning electron microscopy and transmission electron microscopy observation. Animal experiments: the protoscoleces were isolated from the abdominal lesions of the protected gerbils, and then infected 18 C57BL/6J mice by intraperitoneal injection to establish models, dividing into 3 groups with 6 mice in each group. The model group was given 0.3 mL of PBS by gavage daily, the albendazole (ABZ) group was given 0.3 mL ABZ (100 mg/kg) daily by gavage, the PAB group was given 0.3 mL of PAB (40 mg/kg) by gavage daily. After continuous gavage for 6 weeks, the lesion host microenvironment tissue was taken and ELISA was used to detect the expression levels of vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS) and cysteinyl aspartate specific proteinase3 (caspase3), the expression levels of nitric oxide (NO) was detected using a biochemical detection kit, Western blot was used to detect the expression levels of BCL2-associated X protein (Bax), B-cell lymphoma-2 (Bcl2), caspase3, cleaved-caspase3, VEGF, vascular endothelial growth factor receptor 2 (VEGFR2), phosphatidylinositol 3 kinase (PI3K), phosphorylated PI3K (p-PI3K), protein kinase B (AKT) and phosphorylated AKI (p-AKT) protein. ResultsIn vitro experiments: the protoscoleces of Echinococcus multilocularis were cultured with different concentrations of PAB for 7 days in vitro, the protoscoleces of 40, 80, 160 and 320 μmol/L group all died after 6, 4, 2 and 1 day, respectively; PAB exhibited a certain time and concentration dependence on the protoscoleces of Echinococcus multilocularis. After PAB treatment, the release of PGI in culture supernatant of Echinococcus multilocularis gradually increased with the increase of PAB concentration [concentration for 50% of maximal effect value was (24.40±1.42) μmol/L], the vitality of germinal cells was significantly inhibited [half maximal inhibitory concentration value was (15.94±2.55) μmol/L]. PAB had no significant toxicity to mammalian cells. When 20 μmol/L PAB intervention in the protoscoleces for 3 days, the expression levels of Bax and caspase3 proteins were upregulated, while the expression level of Bcl2 protein was downregulated. Animal experiments: compared with the model group, the wet weight of lesions in the PAB and ABZ groups decreased (P<0.01), and the inhibition rates of lesion growth in the PAB and ABZ groups were 91.03% and 74.44%, respectively. The expression of proliferation and angiogenesis indicators (Ki67, CD34, VEGF, VEGFR2, eNOS, NO) were downregulated in the lesion host microenvironment tissues of mice in the ABZ and PAB groups (P<0.05), while the expression of apoptosis related proteins (caspase3, cleaved-caspase3 and Bax) were upregulated and the expression of PI3K/AKT signaling pathway related proteins (p-PI3K and p-AKT) were downregulated (P<0.05). ConclusionPAB has a strong in vitro and in vivo effect against Echinococcus multilocularis, and its mechanism may be related to the inhibition of PI3K/AKT signaling pathway, leading to increased apoptosis and decreased angiogenesis.
【 Abstract 】 Objective To probe into the role of inositol 1, 4, 5-trisphosphate (IP3) and bax gene expression in apoptosis of HepG2 cells induced by genistein (Gen). Methods HepG2 cells were treated with different concentrations including 20, 40, 60 and 80 μ mol/L Gen as HepG2 cells cultured with 0 μmol/L Gen for 72 h was control; HepG2 cells were treated with 60 μmol/L Gen for 6, 12, 24, 48 and 72 h as HepG2 cells treated with 60 μmol/L Gen for 0 h was control. IP3 content, bax mRNA expression and apoptosis rate were assayed by IP3- ［ 3H ］ Birtrak assay, RT-PCR and flow cytometry, respectively. ResultsHepG2 cells incubated with each concentration of Gen for 72 h ， IP3 content was lower than that of control 〔 (17.7 ± 1.3), (11.2 ± 0.9), (4.9 ± 0.5), (4.8 ± 0.3) pmol/106 cells vs (29.4 ± 0.5) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression (RI which was the gray degree multiply area of bax/the gray degree multiply area of β -actin) was higher than that of control (0.26 ± 0.02, 0.33 ± 0.05, 0.35 ± 0.06, 0.38 ± 0.05 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate was higher than that of control 〔 (10.1 ± 0.9)%, (18.7 ± 1.6)%, (28.7 ± 2.5)%, (27.9 ± 2.0)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. HepG2 cells were incubated with 60 μ mol/L Gen for 6, 12, 24, 48 and 72 h ， IP3 content was lower than that of control 〔 (22.6 ± 0.9), (12.0 ± 1.4), (7.5 ± 0.8), (5.6 ± 0.5), (4.3 ± 0.6) pmol/106 cells vs (29.2 ± 0.6) pmol/106 cells 〕 , P ＜ 0.01 ； bax mRNA expression was higher than that of control incubated with 60 μ mol/L Gen for above 12 h (0.25 ± 0.06, 0.29 ± 0.02, 0.30 ± 0.02, 0.35 ± 0.04 vs 0.09 ± 0.01), P ＜ 0.01 ； The apoptosis rate in groups incubated with 60 μ mol/L Gen for 24, 48 and 72 h was significantly higher than that in control 〔 (7.4 ± 0.5)%, (20.5 ± 2.0)%, (30.7 ± 1.6)% vs (2.6 ± 0.1)% 〕 , P ＜ 0.01. ConclusionGen induces apoptosis of HepG2 cells by reducing IP3 production and increasing bax gene expression.
ObjectiveTo investigate the effects of phenethyl isothiocyanate (PEITC) on apoptosis and proliferation of breast cancer SK-BR-3 cells. MethodsSK-BR-3 cells were treated with different concentrations (0, 10, 30, 50 μmol/L) of PEITC respectively. The proliferation capacity of SK-BR-3 cells was detected by MTT and BrdU staining methods. The cell apoptosis was detected by TUNEL and flow cytometry methods. The protein and mRNA expressions levels of indexes related apoptosis such as Bcl-2, Bax, and MCL-1 and indexes related endoplasmic reticulum stress (ERS) such as PERK, eIF2α, CHOP, IRE1α, ATF6α were detected by Western blot and quantitative real-time PCR (qRT-PCR), respectively. ResultsCompared with the control group (0 μmol/L PEITC treatment group), the results of MTT and BrdU staining methods showed that the proliferations of SK-BR-3 cells in the 10, 30 and 50 μmol/L PEITC treatment group were decreased in turn with the increase of concentration. The results of TUNEL and flow cytometry methods showed that the apoptosis rates of SK-BR-3 cells in the 10, 30 and 50 μmol/L PEITC treatment group were increased in turn with the increase of concentration. The results of Western blot and qRT-PCR methods showed that the protein and mRNA expression levels of anti-apoptotic indexes (Bcl-2, MCL-1) were decreased with the increase of concentration, while the expression levels of protein and mRNA of the pro-apoptotic index (Bax) and ERS-related indexes (PERK, eIF2α, CHOP, IRE1α, ATF6α) increased with the increase of concentration. ConclusionFrom the preliminary results of this study, PEITC can promote the apoptosis of breast cancer SK-BR-3 cells and inhibit cell proliferation, which might be achieved by regulating the expression levels of indexes related apoptosis and ERS.
Objective To investigate the pathogenesis of acute lung injury in rats induced by intra-peritoneally injection of perforative peritonitis ascitic fluids（PPAF） and the role of L-arginine （L-Arg） in acute lung injury in this model. Methods Perforative peritonitis （PP） models were established in 60 rats and PPAF were collected. Forty-eight rats were randomly divided equally into NS group，PPAF group, and L-Arg group. Rats were randomly subjected to death at 7 h and 12 h. Peripheral blood WBC were counted，levels of NO and malondialdehyde （MDA） in serum were examined. Lung injury score and wet/dry ratio were evaluated， and level of myeloperoxidase （MPO） in lung tissues and lung cell apoptosis were tested. Results WBC count of peripheral blood, levels of NO and MDA in serum， level of MPO in lung tissue, lung injury score， wet/dry ratio， and lung cell apoptosis rate in PPAF group were significantly higher than that in NS group at each time point（P＜0.01）. Level of NO in serum in L-Arg group was higher than that in PPAF group （P＜0.01）， but lower level of MDA in serum， lower level of MPO in lung tissue and lung injury score，lower wet/dry ratio， and lung cell apoptosis rate were observed in L-Arg group（P＜0.05）. In PPAF group and L-Arg group， level of NO in serum， wet/dry ratio, and lung cell apoptosis rate were higher at 12 h than that at 7 h（P=0.000）. Serum NO level was in negative correlation with serum MDA level （r=-0.257，P=0.021）， MPO level in lung tissue（r=-0.444， P=0.011），and lung cell apoptosis（r=-0.351， P =0.010） in PPAF group and L-Arg group， but serum MDA level was in positive correlation with cell apoptosis（r=0.969， P＜0.001） in each group. Conclusions Acute lung injury rats model can be established by intra-peritoneally injection of PPAF. Enhanced oxidizing reaction and cell apoptosis take part in the occurrence of acute lung injury. L-Arg plays a protective role in acute lung injury.
ObjectiveTo investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia.MethodsThe skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment.ResultsAfter identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased (P<0.05), and the level of ROS also significantly increased (P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased (P<0.05), the relative expression of Bcl-2 protein significantly increased (P<0.05), the cell apoptosis rate and level of ROS also significantly decreased (P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak.ConclusionInhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.
Objective To investigate the inhibition effect of silence of a disintegrin and metalloproteinase 17 (ADAM17) gene on proliferation and apoptosis of HT29 colon cancer cells and its possible mechanism. Methods HT29cells were divided into 3 groups: cells of interference group were transfected with recombinant lentivirus vector, cells of negative control group were transfected with negative recombinant lentivirus vector, and cells of blank control group were treated with PBS. The expression of ADAM17 mRNA was detected by real-time PCR, the expressions of ADAM17 protein, caspase3, protein kinase B (Akt), glycogen synthase kinase-3β (GSK3β), phospho-protein kinase B (P-Akt), phospho-glycogen synthase kinase-3β (P-GSK3β) protein were detected by Western blot method, the cell proliferation was detected by MTT assay, and the apoptosis rate was detected by Annexin V-FITC/PI cell death detection kit. Results Compared with the control group and the negative control group, the interference group was related to low expressions of ADAM17 mRNA and its protein, low optical density value at the same time point (24, 48, and 72 h), high apoptosis rate, high expression level of caspase3 protein, but low expression levels of P-Akt and P-GSK3β protein (P<0.05). Conclusion Silent ADAM17 gene could significantly induces apoptosis and inhibits the proliferation of HT29 cells, which maybe via inhibiting Akt/GSK3β signaling pathway.
Objective To investigate the effect of heme oxygenase 1 (HO-1) on the apoptosis of human degenerated nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α), and explore its possible molecular mechanism. Methods The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8 (CCK-8) method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium (control group), TNF-α (TNF-α group), TNF-α and CoPP 10 μmol/L (CoPP group), and TNF-α and ZnPP 15 μmol/L (ZnPP group), respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1 (EMP-1), HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α (TNF-α stimulated group), TNF-α and pyrrolidine dithiocarbamate (PDTC) 5 μmol/L (TNF-α+PDTC stimulated group), respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured. Results The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group. Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group (P<0.05). Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased (P<0.05), while in ZnPP group it increased (P<0.05). Western blot showed that the expression of HO-1 protein in TNF-α group was decreased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were increased when compared with the control group (P<0.05). Compared with TNF-α group, the expression of HO-1 protein in CoPP group increased, and the expressions of cleaved Caspase-3, EMP-1, and p-P65 proteins were reduced (P<0.05); the expression of HO-1 protein in ZnPP group decreased (P<0.05), the expressions of cleaved Caspase-3 and EMP-1 proteins increased (P<0.05), and the expression of p-P65 protein was not significantly changed (P>0.05). Compared with TNF-α stimulated group, the cell apoptosis rate in TNF-α+PDTC stimulated group was significantly reduced (t=3.076, P=0.031). Conclusion HO-1 can inhibit the apoptosis of degerated NP cells induced by TNF-α, and its mechanism effect is by inhibiting the nuclear factor кB signaling pathway.