OBJECTIVETo investigate the effects of microRNA-144 (miR-144) expression on H9C2 (2-1) myocytes.

METHODSMiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with Lipofectamine(TM) 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry.

RESULTSReal-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84 ± 838.52) compared with negative (2.06 ± 0.73) and blank (1.00 ± 0.00) control group (all P < 0.01). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group, while no significant difference was found between the latter 2 groups.

CONCLUSIONMiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.

Animals ; Apoptosis ; genetics ; Caspase 3 ; metabolism ; Cell Line ; MicroRNAs ; genetics ; metabolism ; Muscle Cells ; metabolism ; Rats ; Sincalide ; metabolism ; Transfection

The objective of this study was to investigate the apoptosis-inducing effect and underlying mechanism of naringenin (NGEN) on K562 cells in vitro. The inhibition of NGEN on K562 cells was evaluated by means of MTT assay so as to observe the cytotoxicity of NGEN; The morphological changes of the cells treated by NGEN were observed by transmission electron microscope; cell apoptosis rate influenced by NGEN was assessed by flow cytometry; the enzyme activity changes of caspase-3 and caspase-8 in the process of NGEN-induced K562 apoptosis were detected by Caspase Colorimetric Assay Kit; immunohistochemistry technique was used to detect FAS, FASL protein expression in K562 cells. The results showed that the growth of cells was inhibited by NGEN in dose-and time-dependent manners (p<0.05); NGEN-induced K562 cells apoptosis and sub-G1 peak was observed; some typically early and final phase changes of cell apoptosis were revealed under transmission electron microscope; the enzyme activity of caspase-3 and caspase-8 and the expression of FAS remarkably increased, meanwhile the expression of FASL was down-regulated (p<0.05). It is concluded that NGEN exerts anti-cancer effect by inducing K562 cell apoptosis, and the regulation of the expression of FAS and FASL. The caspase-3 and caspase-8 co-pathway brings about one of the mechanisms.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Fas Ligand Protein ; genetics ; metabolism ; Flavanones ; pharmacology ; Humans ; K562 Cells ; fas Receptor ; genetics ; metabolism

OBJECTIVETo study the role of apoptosis and the expression of apoptosis-related genes Fas ligand (FasL), Fas, caspase-3 and caspase-8 in an animal model of non-alcoholic steatohepatitis (NASH).

METHODSAn experimental progressive NASH model was established by feeding male C57BL6/J mice with a high fat, methionine-choline deficient (MCD-) diet for two days, five days, ten days, three weeks and eight weeks. Control mice were fed methionine-choline supplemented (MCD+) diet. Hepatic steatosis, inflammation and fibrosis were graded by examining their H and E stained liver sections. Hepatocyte apoptosis was detected by TUNEL assay. Expressions of mRNA and protein of FasL, Fas and caspase-8 were performed by quantitative real time RT-PCR and Western blot. Caspase-3 activity assay was conducted using ApoAlert caspase-3 assay kit.

RESULTSIn MCD- mice, minimal hepatic steatosis was observed at day 5, and by day 10, mild steatosis with inflammatory infiltration was found. Severe steatohepatitis was noted at week 3, and fibrosis at week 8. TUNEL assay showed that apoptotic index in MCD- group was higher than that in MCD+ group at week 3 (15.59%+/-4.87% vs 5.17%+/-3.19%, P less than 0.05) and at week 8 (11.29%+/-3.22% vs 5.41%+/-1.54%, P less than 0.05). Compared to MCD+ group, the expression of FasL was dramatically increased on day 10 and in week 3 in MCD- mice both at the mRNA and protein levels (P less than 0.05 and P less than 0.01). Expression of Fas mRNA was up-regulated in weeks 3 and 8 (P less than 0.01), and expression of Fas in protein level was higher at week 8 (P less than 0.01) in MCD- group. Expression of caspase-8 significantly increased at the mRNA level at week 3 and week 8 (P less than 0.01 and P less than 0.05 respectively) and at the protein level at week 8 (P less than 0.05) in MCD- group. In all of the time points except for day 5, caspase-3 activities were significantly more enhanced in MCD- group than that in MCD+ group (P less than 0.05).

CONCLUSIONSIn our experimental NASH model, hepatic apoptosis was frequently detected. Increased apoptosis was probably attributable to up-regulation of apoptosis-related genes, such as FasL/Fas system, and activation of the caspase pathway. These changes may provoke hepatic apoptosis and the development of inflammation and fibrosis.

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Fas Ligand Protein ; metabolism ; Fatty Liver ; genetics ; metabolism ; pathology ; Hepatocytes ; metabolism ; Liver ; pathology ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVETo observe the expression of caspase-3 in the kidney of a rat model of renal tubular damage induced by endotoxin and hypoxia and explore the mechanism of renal tubular damage.

METHODSTen rats were anesthetized with artificial ventilation and received 2 mg/kg lipopolysaccharide (LPS) injection through the penile vein. The FiO2 was reduced 90 min later from 21% to 5%, and the ventilation was withdrawn after another 90 min. Immediately after ventilation withdrawal, the kidney of the rats were obtained for immunocytochemistry and HE staining.

RESULTSHE staining showed obvious hyperemia in most of the glomeruli, mild swelling of the endothelial and mesangial cells, severe swelling and turbidity in the proximal tubular epithelial cells without obvious changes in most of the distal proximal tubules. A small portion of the interstitial epithelial cells showed swelling and turbidity, and the entire renal interstitium appeared hyperemic but without inflammatory cell infiltration. Immunocytochemistry detected the presence of caspase-3 in the cytoplasm, and most of the distal renal tubule cells were positive for caspase-3, while only occasional cells showed caspase-3 positivity in the proximal tubular epithelial cells. Most of the proximal tubular epithelial and glomerulus cells were negative for caspase-3.

CONCLUSIONSEndotoxin and hypoxia can induce renal damage, particularly in the proximal renal tubule cells, and the distal tubular epithelial cells sustain relatively light damage. Caspase-3 is strongly expressed in the distal renal tubular cells, suggesting that in renal tubular damage induced by endotoxin and hypoxia, cell degeneration, necrosis and apoptosis coexist in the tubular epithelial cells; degeneration and necrosis occur primarily in the proximal tubular epithelial cells, while apoptosis is obvious in the distal renal cells.

Animals ; Caspase 3 ; genetics ; metabolism ; Hypoxia ; chemically induced ; Kidney ; metabolism ; Kidney Tubules ; metabolism ; pathology ; Lipopolysaccharides ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To explore the mechanism of formaldehyde inducing ovarian toxicity in female rats by observing the effect of formaldehyde on the expression of Fas and caspase-8 mRNA, and the activity of caspase-3 and caspase-8 of ovary tissues in female rats. METHODS: Forty female Sprague-Dawley(SD) rats were randomly divided into a control group and 3 formaldehyde groups at different concentrations. The rats in the formaldehyde groups were intraperitoneally injected different doses of formaldehyde (20.0,2.0 and 0.2 mg/kg) continuously for 14 days.After 14 days, all rats were sacrificed and their ovaries were collected for detecting the expression of Fas and caspase-8 mRNA with RT-PCR, the protein expression of Fas with Western blot, and the activities of caspase-8 and caspase-3 with spectrophotometric method. RESULTS: Compared with the control group, the expression of Fas mRNA and its protein and caspase-8 mRNA and the activity of caspase-8 and caspase-3 of ovary tissues in the rats treated with formaldehyde significantly increased with dose (P<0.05). CONCLUSION The increase of Fas gene expression and the activity of caspase-8 and caspase-3 may be the important mechanism of ovarian toxicity induced by formaldehyde in female rats.
Animals ; Apoptosis ; drug effects ; genetics ; Caspase 3 ; genetics ; metabolism ; Caspase 8 ; genetics ; metabolism ; Environmental Pollutants ; toxicity ; Female ; Formaldehyde ; toxicity ; Ovary ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; fas Receptor ; genetics ; metabolism

Objective: To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of DRAIC in lung cancer tissues and corresponding adjacent normal tissues of 40 patients with lung adenocarcinoma who underwent surgery in Tangshan People's Hospital from 2019 to 2020. Lung adenocarcinoma cells A549 and H1299 were cultured in vitro and divided into si-NC group, si-DRAIC group, miR-NC group, let-7i-5p mimics group, si-DRAIC+ inhibitor-NC group, and si-DRAIC+ let-7i-5p inhibitor group. CCK-8 method and clone formation experiment were used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Transwell array was used to detect the cell migration and invasion. Western blot was used to detect the protein expressions of Caspase-3, Caspase-9, Bcl-2 and Bax. The double luciferase reporter gene experiment was used to verify the regulatory relationship between DRAIC and let-7i-5p. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and Pearson correlation analysis was used for correlation analysis. Results: Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (P<0.05), but the expression level of let-7i-5p decreased (P<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (r=-0.737, P<0.05). The absorbance value of A549 and H1299 cells in the si-DRAIC group at 48, 72 and 96 hours were lower than those in the si-NC group (P<0.05), the number of clones formed [(91.00±6.08 vs. 136.67±6.51); (50.67±1.53 vs. 76.67±4.51)], the number of migration [(606.67±31.34 vs. 960.00±33.06); (483.33±45.96 vs. 741.67±29.67)], the number of invasion [(185.00±8.19 vs. 447.33±22.05); (365.00±33.87 vs. 688.00±32.97)] were lower than those in the si-NC group (P<0.05). However, the apoptosis rates of cells [(13.43±2.79)% vs. (4.53±0.42)%; (23.77±1.04)% vs. (6.60±1.42)%] were higher than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC group were higher than those in si-NC group, and the protein expression of Bcl-2 was lower than that in si-NC group (P<0.05). DRAIC is located in the cytoplasm. DRAIC targeted and negatively regulated the expression of let-7i-5p. The absorbance values of A549 and H1299 cells in the let-7i-5p mimics group at 48, 72 and 96 hours were lower than those in the miR-NC group (P<0.05), the number of clones formed [(131.33±14.47 vs. 171.33±6.11); (59.33±4.93 vs. 80.33±7.09)], the number of migration [(137.67±3.06 vs. 579.33±82.03); (425.00±11.14 vs. 669.33±21.13)], the number of invasion [(54.00±4.36 vs. 112.67±11.59); (80.00±4.58 vs. 333.33±16.80)] were lower than those in the miR-NC group (P<0.05). However, the apoptosis rates of cells [(14.57±1.10)% vs. (6.97±1.11)%; (23.97±0.42)% vs. (7.07±1.21)%] were higher than those in the miR-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in let-7i-5p mimics group were higher than those in miR-NC group, and the protein expression of Bcl-2 was lower than that in miR-NC group (P<0.05). The absorbance values of A549 and H1299 cells in the si-DRAIC+ let-7i-5p inhibitor group at 48, 72 and 96 hours were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05), the number of clones formed [(82.00±5.29 vs. 59.00±5.57); (77.67±4.93 vs. 41.33±7.57)], the number of migration [(774.33±35.81 vs. 455.67±19.04); (569.67±18.72 vs. 433.67±16.77)], the number of invasion [(670.33±17.21 vs. 451.00±17.52); (263.67±3.06 vs. 182.33±11.93)] were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05). However, the apoptosis rates of cells [(7.73±0.45)% vs. (19.13±1.50)%; (8.00±0.53)% vs. (28.40±0.53)%] were lower than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC+ let-7i-5p inhibitor group were higher than those in si-DRAIC+ inhibitor-NC group, and the protein expression of Bcl-2 was lower than that in si-DRAIC+ inhibitor-NC group (P<0.05). Conclusion: DRAIC is highly expressed in lung adenocarcinoma, and DRAIC promotes the proliferation, migration and invasion of lung adenocarcinoma cells and inhibits apoptosis by targeting let-7i-5p.
Humans ; Adenocarcinoma/genetics* ; Apoptosis/genetics* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Caspase 9/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Lung/metabolism* ; MicroRNAs/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Long Noncoding/genetics*

OBJECTIVETo identify the effect of NEP on Abeta -induced apoptosis in PC12 cells.

METHODSPC12 cells that stably express NEP is generated and the effect of NEP on the process of apoptosis induced by Abeta is analyzed, including the viability of the cells, the production of LDH, ROS and ATP, the activity of Caspase-3.

RESULTSNEP could improve the viability of cells and the production of ATP, inhibit the release of LDH and ROS. In the same time, the activity of caspase-3 descended (P < 0.05). But iNEP had not significant effect on cells apoptosis (P > 0.05).

CONCLUSIONNEP has the protective effect on Abeta-induced apoptosis in PC12 cells.

Amyloid beta-Peptides ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Endopeptidases ; genetics ; metabolism ; PC12 Cells ; Rats ; Transfection

OBJECTIVETo investigate the effect of p38 mitogen-activated protein kinase (p38MAPK) on the expression of COX-2 and caspase-3 in the substania nigra (SN) of mice with MPTP-induced Parkinson disease (PD).

METHODSC57BL/CN mice were treated with MPTP to prepare a subacute PD model, and their behavioral changes following the treatment were observed. Immunohistochemistry and Western blotting were performed to detect the expression of tyrosine hydroxylase (TH), COX-2 and phosphorylation of P38MAPK in the SN and their changes following treatment with SB203580, a specific inhibitor of P38MAPK.

RESULTSThe 7-day model group showed typical symptoms of PD with decrements of TH-positive neurons and TH protein level in the SN of the midbrain by about 65% and 75%, respectively (P<0.01). In the 3-day model group, the COX-2-, caspase-3- and phosphorylated P38MAPK-immunoreactive cells and their protein levels in the SN increased markedly with obvious loss of TH-positive neurons. Administration of SB203580 obviously lessened the above changes (P<0.01).

CONCLUSIONP38MAPK regulates the inflammation and apoptosis in the SN of the mouse model of subacute PD, and SB203580 may provide some neuroprotective effect.

Animals ; Caspase 3 ; genetics ; metabolism ; Cyclooxygenase 2 ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Parkinson Disease ; metabolism ; Signal Transduction ; Substantia Nigra ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUNDA nonsteroidal anti-inflammatory drug, indomethacin, has been shown to have anti-leukemic activity and induce leukemic cell apoptosis. This study was to elucidate the mechanism of indomethacin-induced K562 cell apoptosis.

METHODSK562 cells were grown in RPMI 1640 medium and treated with different doses of indomethacin (0 micromol/L, 100 micromol/L, 200 micromol/L, 400 micromol/L, 800 micromol/L) for 72 hours. The cells were harvested, and cell viability or apoptosis was analyzed using MTT assay and AO/EB stain, combining laser scanning confocal microscopy (LSCM) technique separately. For the localization and distribution of intracellular caspase-3 or caspase-8 protein, immunofluorescence assay was carried out. To reveal the activation of caspase-3 or caspase-8 in indomethacin-treated cells, Western blot detection was used. The change in intracellular free calcium was determined by Fluo-3/Am probe labeling combined with LSCM.

RESULTSIndomethacin could lead to K562 cell apoptosis and inhibit cell viability in a concentration-dependent manner. An increased expression of intracellular caspase-3 or caspase-8 was observed at higher doses of indomethacin (400 - 800 micromol/L). Western blot results showed upregulation and activation in both caspase-3 and caspase-8 protein. Under indomethacin intervention, the levels of intracellular free calcium showed a significant increase. Blocking the activity of cyclooxygenase did not abolish the effects of indomethacin on K562 cell apoptosis.

CONCLUSIONSActivation and upregulation of caspase-3 or caspase-8 protein were responsible for Indomethacin-induced K562 cell apoptosis. Variation of intracellular free calcium might switch on the apoptotic pathway and the proapoptotic effect of indomethacin might be cyclooxygenase-independent.

Apoptosis ; drug effects ; Calcium ; metabolism ; Caspase 3 ; Caspase 8 ; Caspases ; genetics ; metabolism ; Cyclooxygenase Inhibitors ; pharmacology ; Enzyme Activation ; Gene Expression Regulation, Enzymologic ; drug effects ; Humans ; Indomethacin ; pharmacology ; K562 Cells

This study was aimed to investigate the effects of SUV39H1 siRNA on proliferation and apoptosis of acute myelogenous leukemia KG-1 cell line. The small interfering RNA (siRNA) targeting SUV39H1 gene was designed and transfected into KG-1 cells by Lipofectamine(TM) 2000. Cell growth affected by SUV39H1 siRNA was determined by MTS method. Cell apoptosis was measured by flow cytometry. The expressions of P15 and anti-apoptosis protein such as BCL-2, procaspase-9, procaspase-3 and C-MYC were detected by Western blot. The results indicated that siRNA targeting SUV39H1 inhibited proliferation of KG-1 cells. Proliferated rates were (76.43 ± 1.98)%, (51.31 ± 1.84)%, (37.31 ± 1.61)%, (18.94 ± 3.22)% respectively after transfection with SUV39H1 siRNA at 30, 60, 120, 240 nmol/L for 48 h, while P15 expression was upregulated. Apoptotic cells significantly increased, apoptotic rates were (40.2 ± 5.1)%, (56.8 ± 4.8)%, (71.6 ± 5.6)% respectively after transfection with siRNA targeting SUV39H1 at 30, 60, 120 nmol/L (P < 0.05). The protein expression of BCL-2, procaspase-9, procaspase-3, C-MYC was downregulated after transfection. It is concluded that the siRNA targeting SUV39H1 inhibits cell growth and induces cell apoptosis of KG-1 cell line, which may be a new therapeutic target in human leukemia.
Apoptosis ; genetics ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Methyltransferases ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Repressor Proteins ; genetics