Abstract： Objective To investigate the effects of calcium preconditioning (CP) on immature myocardial cell apoptosis and apoptosisregulated proteins. Methods The experiment was carried out from June 2000 to December 2001 in the Renmin Hospital of Wuhan University. Twelve rabbits with the age of 1421 d and the weight of 230300 g were divided into 2 groups with 6 in each group by random digital table. For rabbits in the ischemia/reperfusion group (I/R group), after Langendorff models were routinely set up, KrebsHenseleit (KH) solution was perfused for 20 minutes and reperfused for 120 minutes after 45 minutes of ischemia. For rabbits in the CP group, after Langendorff models were established, KH solution was perfused for20 minutes, and 45 seconds’ noncalcium KH solution perfusion and 5 minutes’ KH solution perfusion were repeated 3 times before 45 minutes of ischemia and 120 minutes of reperfusion of KH solution. In situ apoptosis identification and semiquantitative analysis were used to detect the myocardial cell apoptosis; agarose gel electrophoresis was used to detect the nucleosomal ladder of DNA fragments; and the expression of bcl-2, bax and fas were detected with Western blot method. Results The apoptosis rate for the CP group was lower than that of the I/R group (4.53%±1.22% vs. 12.30%±2.12%,t=7.780, P=0.000). Nucleosomal ladder of DNA fragments of the CP group was lower than that of the I/R group (OD value: 56 460±1 640 vs. 135 212±3 370,t=51.460,P=0.000). The expression of bcl-2 in the I/R group was lower than that of the CP group (OD value: 13 217±1 770 vs. 31 790±1 018,t=22.280, P=0.000). The expression of bax (OD value: 30 176±1 025 vs. 7 954±730, t=43.260, P=0.000) and fas (OD value: 29 197±1 233 vs. 8 140±867, t=34.220, P=0.000) in the I/R group was higher than that of the CP group. Conclusion CP can affect the expression of myocardial bcl-2, bax, and fas, and decrease immature myocardial cell apoptosis.
ObjectiveTo explore the effect of DDX46 silencing on growth and apoptosis in esophageal squamous cell carcinoma cell TE-1 by the shRNA. MethodsThe relative expression of DDX46 mRNA in TE-1 cells was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and compared with immortalized human esophageal squamous cell Het-1A. DDX46 shRNA-expressing lentivirus was applied to silence DDX46 (experimental group), and non-silencing control lentivirus was added (control group) with a multiplicity of infection of 5 in TE-1 cells. In both groups, cell growth was monitored using high content screening, cell colony-forming capacity was measured by colony formation assay, cell apoptosis were determined by flow cytometry. Further, the Stress and Apoptosis Signaling Antibody Array Kit was used to detect the changes of signaling molecules in TE-1 cells after DDX46 knockdown. ResultsCompared with the control group, cell counting after DDX46 silencing showed that TE-1 cell growth was significantly inhibited (P<0.001). Colony formation assay showed that cell colony-forming capacity was significantly inhibited (P<0.01). Annexin V-APC flow cytometry showed a significant increase in apoptosis (P<0.001). In PathScan® Antibody Array, the expression levels of Akt (Ser473, phosphorylation) and IκBα (Total, N/A) significantly decreased (P<0.01), and the expression of Caspase-3 (Asp175, cleaved) increased (P<0.05). ConclusionDDX46 is overexpressed in TE-1 cells. Targeted gene silencing of DDX46 inhibits cell growth, and induces cell apoptosis. DDX46 silencing probably by negative regulation of Akt/NF-κB signaling pathway, to play a role in inhibiting TE-1 cells growth and inducing apoptosis.
Objective To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Methods Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2’-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. Results After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G2 phase decreased significantly, and the proportion of cells at G1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes. ConclusionmiR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.
ObjectiveTo investigate the effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells.MethodsThe MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis.ResultsAfter 48 hours of culture, the cells of 2 groups had grown into slender fusiform at the bottom of the bottle, and there was no significant difference in cell morphology between 2 groups. Immunofluorescence staining showed that the expression of collagen type Ⅰ was positive in the experimental group and weakly positive in the control group; the integrated absorbance (IA) value of the experimental group was 187 600±7 944, which was significantly higher than that of the control group (14 230±1 070) (t=43.680, P=0.001). The expression of collagen type Ⅲ was weakly positive in the experimental group and the control group, and there was no significant difference in IA value between 2 groups (t=1.976, P=0.119). ALP staining and alizarin red staining were positive in the experimental group and negative in the control group. TUNEL staining was positive in the experimental group and negative in the control group; the rate of TUNEL staining positive cells in the experimental group was 35.82%±2.99%, which was significantly higher than that in the control group (2.28%±0.51%) (t=23.420, P=0.002).ConclusionFTY720-P can promote the osteogenic differentiation of MC3T3-E1 cells with speeding up maturation and mineralization of extracellular matrix and affect the apoptosis of the cells.
HtrA serine peptidase 2 (HTRA2) is a serine protease existing in the mitochondrial gap. Among the four members of the human HtrA serine peptidase family, HTRA2 is the only protease with clear localization in the cell. It plays a dual role in the maintenance of mitochondrial homeostasis and the promotion of cell apoptosis. HTRA2 has been found to be associated with a variety of tumors. Meanwhile, the expression of HTRA2 can enhance the sensitivity of chemotherapy and radiotherapy, and can be used as a diagnostic and prognostic marker for malignant tumors and a target for combined therapy. This article reviews the structure, biological function and role of HTRA2 in malignant tumors, in order to provide clues and basis for early diagnosis and individualized treatment of tumor patients.
Objective To determine the anti-apoptosis effects of heme oxygenase-1 (HO-1) on lung injury after cardiopulmonary bypass (CPB), and to investigate its probable mechanisms. Methods A total of 144 male Wistar rats with wight of 250-350 g were divided into 3 groups: group A (control group), group B (cobalt protoporphyrin, CoPP), and group C [CoPP and zinc protoporphyrin (ZnPP)] randomly. A modified rat model of CPB-induced lung injury was established. And then the lung tissues were taken at different times for the relevant indicators test: before CPB (T0), immediately after CPB (T1), 2 h after CPB (T2), 6 h after CPB (T3), 12 h after CPB (T4), and 24 h after CPB (T5). The expression of HO-1 and Bcl-2 protein in each group was tested by immunohistochemistry, and cell apoptosis by TUNEL. Results The HO-1 protein expression in group B was significantly higher than that in groups A and C at any given time point, so was the HO-1 activity (P<0.05). There was no significant difference in Bcl-2 expression of lung tissue before CPB among each group (P>0.05). The Bcl-2 protein reduced gradually after CPB. The expressions of Bcl-2 protein in group B at all time points after bypass were significantly higher than that in groups A and C (P<0.05). The apoptosis index (AI) showed no significant difference before CPB in each group (P>0.05), and increased gradually after CPB. AI in group B at any time point after bypass was lower than that in groups A and C (P<0.05). The HE staining results showed that the damage of lung tissue in group B obviously reduced compared with groups A and C. Conclusion CoPP can induce a large amount of HO-1 expression in the lung tissue, and it is still highly expressed after CPB. So it plays an important role in anti-apoptosis through the up-regulation of Bcl-2 protein expression.
Objective To investigate the effect of ginkgolide B (GB) on cysteinyl aspartate specific proteinase-3 (Caspase-3)/chromosome 10 deletion phosphatase-tension protein homologue (PTEN)/protein kinase B (Akt) pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes. Methods H9C2 cells were cultured in vitro. A control group was cultured in serum-free DMEM high glucose medium at 37°C and 5% CO2 for 28 hours. The remaining groups were prepared with hypoxia/reoxygenation models. A GB low-dose group and a GB high-dose group were treated with GB pretreatment with final concentration of 50 μmol/L and 200 μmol/L respectively at 1 h before hypoxia/reoxygenation. A carvedilol group was treated with carvedilol of a final concentration of 10 μmol/L at 1 h before hypoxia/reoxygenation. The proliferation and apoptosis of H9C2 cells were detected, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), reactive oxygen species (ROS), PTEN, Akt, phosphorylated Akt (p-Akt) and Caspase-3 in H9C2 cells were also detected. Results Compared with the control group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in other groups decreased, and the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 increased (P<0.05). Compared with the hypoxia/reoxygenation group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in all GB dose groups and the carvedilol group increased; the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 decreased, and the effect of GB was in a dose dependent manner; however, the effect of GB was not as strong as carvedilol (P<0.05). Conclusion GB can inhibit H9C2 cell apoptosis and promote H9C2 cell proliferation by activating Caspase-3/PTEN/Akt pathway.
Objective To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 μmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8 (CCK-8). At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results CCK-8 tests showed that the absorbance (A) value of each group was not significant at 0 and 24 hours (P>0.05); but the differences between groups were significant at 48 and 72 hours (P<0.05). Flow cytometry results showed that, with the ursolic acid concentration increasing, the G1 phase of U2-OS cells increased, the S phase and G2/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups (P<0.05). Compared with the 0 μmol/L group, the relative expressions of cyclin D1 mRNA and protein in 10, 20, and 40 μmol/L groups significantly decreased (P<0.05); whereas, there was no significant difference in relative expression of Caspase-3 mRNA between groups (P>0.05). However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences between groups (P<0.05). Conclusion Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS, induce the down-regulation of cyclin D1 expression leading to G0/G1 phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.
This paper aims to study the effects of traditional Chinese medicine Euphorbia esula on multidrug resistant human gastric cancer cells in the cell proliferation, migration, invasion and apoptosis, and to study the apoptosis-inducing pathway. Different dilutions of Euphorbia esula extract were used to process human multidrug resistant gastric cancer SGC7901/ADR cells. Cell proliferation inhibition phenomenon was determined by MTT experiment. Nuclear morphological changes of apoptotic cells and apoptotic indexes were observed and determined by Hochest33528 staining followed with fluorescence microscope observing. Flow cytometry was used to detect cell apoptosis rate. Cell migration and invasion ability were observed and determined by Transwell method. Spectrophotometry was used to detect caspase-3 and caspase-9 enzyme activity. Western blotting was used to detect subcellular distribution of cytochrome c. The results showed that Euphorbia esula extract had obvious inhibition effect on proliferation of gastric cancer multidrug resistant SGC7901/ADR cells, which was time- and concentration-dependent. After processing multidrug resistant gastric cancer SGC7901/ADR cells with Euphorbia esula extract, the apoptotic index and apoptosis rate were significantly increased than those in the control group, which showed a time- and dose-dependent mode; but if a caspase inhibitor was added, apoptosis index was not obviously increased. Transwell method showed that migration and invasion ability of the Euphorbia esula extract-processed SGC7901/ADR cells dropped significantly. Spectrophotometry showed that in Euphorbia esula extract-processed SGC7901/ADR cells, caspase-3 and caspase-9 expression were increased, which had significant differences with the control group. Western blotting test showed that the distribution of cytochrome c decreased in mitochondria, while increased in the cytoplasm (i.e., cytochrome c escaped from mitochondria to the cytoplasm). In conclusion, Euphorbia esula extract could inhibit the proliferation, migration and invasion, and induce apoptosis in human gastric cancer multidrug resistant SGC7901/ADR cells; and cytochrome c, caspase-9 and caspase-3 might be involved in cell apoptosis induced by Euphorbia esula extract, suggesting endogenous or mitochondrial apoptotic pathway.
ObjectiveThis study is aimed to determine the expression of ubiquitin-specific peptidase 39 (USP39) protein in the colorectal cancer (CRC) tissues, and the effect of silencing USP39 gene on the cell growth and cell cycle distribution of CRC cells.Methods① The expressions of USP39 protein in CRC tissues and its paracancerous tissues were determined by immunohistochemical staining method. ② By lentiviral infection, Lv-shUSP39 (KD-1 and KD-2 group) and Lv-shCon (shCon group) were transferred into SW1116 and HCT116 cells, and cells of blank control group did not received any treatment (Con group). To determine the role of USP39 gene in cell growth, MTT assay was performed to draw growth curve, and cell cycle distribution of CRC cells in the 4 groups were determined by flow cytometer.Results① The expression of USP39 protein was higher in CRC tissues compared to adjacent tissues (P=0.007). ② For SW1116 and HCT116 cells, the cell proliferation ability of KD-1 and KD-2 groups were remarkably decreased than those in corresponding shCon and Con groups on 3, 4, and 5-day (P<0.05). ③ Flow cytometry assay showed that, the percentage of G0/G1 phase cells were decreased obviously (P<0.05), while increased significantly in percentage of G2/M phase and number of sub-G1 phase cells in KD-1 group compared with that in the Con group and shCon group of SW1116 and HCT116 cells (P<0.05).ConclusionsThe expression of USP39 protein is highly expressed in CRC tissues. Knockdowning of USP39 gene can inhibit cell proliferation and promote cell apoptosis.