This paper aimed to investigate the effects of high mobility group box 1(HMGB1)-mediated pulmonary artery smooth muscle cell pyroptosis and immune imbalance on chronic obstructive pulmonary disease-associated pulmonary hypertension(COPD-PH) in rats and the intervening mechanism of Compound Tinglizi Decoction. Ninety rats were randomly divided into a normal group, a model group, low-dose, medium-dose, and high-dose Compound Tinglizi Decoction groups, and a simvastatin group. The rat model of COPD-PH was established by fumigation combined with lipopolysaccharide(LPS) intravascular infusion, which lasted 60 days. Rats in the low, medium, and high-dose Compound Tinglizi Decoction groups were given 4.93, 9.87, and 19.74 g·kg~(-1) Compound Tinglizi Decoction by gavage, respectively. Rats in the simvastatin group were given 1.50 mg·kg~(-1) simvastatin by gavage. After 14 days, the lung function, mean pulmonary artery pressure, and arterial blood gas of rats were analyzed. Lung tissues of rats were collected for hematoxylin-eosin(HE) staining to observe the pathological changes. Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR) was used to determine the expression of related mRNA in lung tissues, Western blot(WB) was used to determine the expression of related proteins in lung tissues, and enzyme linked immunosorbent assay(ELISA) was used to determine the levels of inflammatory factors in the lung tissues of rats. The ultrastructure of lung cells was observed by transmission electron microscope. The forced vital capacity(FVC), forced expiratory volume in 0.3 second(FEV_(0.3)), FEV_(0.3)/FVC, peek expiratory flow(PEF), respiratory dynamic compliance(Cdyn), arterial partial pressure of oxygen(PaO_2), and arterial oxygen saturation(SaO_2) were increased, and resistance of expiration(Re), mean pulmonary arterial pressure(mPAP), right ventricular hypertrophy index(RVHI), and arterial partial pressure of carbon dioxide(PaCO_2) were decreased by Compound Tinglizi Decoction in rats with COPD-PH. Compound Tinglizi Decoction inhibited the protein expression of HMGB1, receptor for advanced glycation end products(RAGE), pro caspase-8, cleaved caspase-8, and gasdermin D(GSDMD) in lung tissues of rats with COPD-PH, as well as the mRNA expression of HMGB1, RAGE, and caspase-8. Pulmonary artery smooth muscle cell pyroptosis was inhibited by Compound Tinglizi Decoction. Interferon-γ(IFN-γ) and interleukin-17(IL-17) were reduced, and interleukin-4(IL-4) and interleukin-10(IL-10) were incresead by Compound Tinglizi Decoction in lung tissues of rats with COPD-PH. In addition, the lesion degree of trachea, alveoli, and pulmonary artery in lung tissues of rats with COPD-PH was improved by Compound Tinglizi Decoction. Compound Tinglizi Decoction had dose-dependent effects. The lung function, pulmonary artery pressure, arterial blood gas, inflammation, trachea, alveoli, and pulmonary artery disease have been improved by Compound Tinglizi Decoction, and its mechanism is related to HMGB1-mediated pulmonary artery smooth muscle cell pyroptosis and helper T cell 1(Th1)/helper T cell 2(Th2), helper T cell 17(Th17)/regulatory T cell(Treg) imbalance.
Animals ; Rats ; Caspase 8 ; Pyroptosis ; HMGB1 Protein/genetics* ; Hypertension, Pulmonary/etiology* ; Pulmonary Disease, Chronic Obstructive/genetics*

OBJECTIVETo investigate the effects of trichloroethylene (TCE) toxicity on the normal human liver cells (L02 cells) and hepatocytes with CYP2E1 gene overexpression which was constructed through molecular cloning technology in our laboratory, then to explore the roles of CYP2E1 gene in TCE toxicity.

METHODSL02 cells and hepatocytes with CYP2E1 overexpression were treated with various doses of TCE (0,0.25, 0.5, 1.0, 2.0, 4.0 mmol/L) for 12h, the expression of apoptosis genes (Bcl-2, Caspase-3, Caspase-8, Caspase-9) and oncogenes (c-fos, c-myc, k-ras, p53) were determined by real-time fluorescent PCR.

RESULTSBcl-2 mRNA expression levels increased significantly in normal liver cells and CYP2E1-overexpressing cells after TCE treatment, Bcl-2 levels were 20%∼50%higher in CYP2E1-overexpressing cells than in L02 liver cells at doses of 0.25∼2.0 mmol/L TCE. Caspase-3, Caspase-8 and caspase-9 mRNA expression increased by 30%∼600% in CYP2E1-overexpressing cells at doses of 0.5∼4.0 mmol/L TCE when compared with L02 cells (P < 0.01). Additionally, c-fos, k-ras and c-myc mRNA expression levels were 25%∼120% higher in CYP2E1-overexpressing cells than in L02 cells (P < 0.01), p53 mRNA expression levels were lower 10%∼50% in CYP2E1-overexpressing cells than in L02 cells (P < 0.05 or P < 0.01).

CONCLUSIONSThere were significant differences for apoptosis gene and oncogene expression levels between normal liver cells and CYP2E1-overexpressing cells after they were treated with TCE, these findings indicated that CYP2E1 might play an important role in TCE metabolism in vivo.

Apoptosis Regulatory Proteins ; Caspase 3 ; Caspase 8 ; Caspase 9 ; Cytochrome P-450 CYP2E1 ; genetics ; Gene Expression ; Hepatocytes ; drug effects ; Humans ; Liver ; Proto-Oncogenes ; genetics ; RNA, Messenger ; Trichloroethylene ; toxicity

OBJECTIVETo investigate single nucleotide polymorphisms (SNPs) and the distribution of their haplotypes in caspase-8, -10 genes in Zhejiang Han nationality in China.

METHODSPCR, denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing were used to detect the SNPs in the 2nd-5th exons of caspase-10 gene, the 8th-10th exons of caspase-8 and their flanking sequences. Expectation Maximization (EM) algorithm was used for haplotype frequencies analysis and pairwise linkage disequilibrium (LD) test.

RESULTS(1) Two SNPs, A2823G and A12799G, were identified in caspase-10 gene, located in exon 2 and exon 5 respectively. A12799G was newly found with low informativeness. Three SNPs were identified in caspase-8 gene; A43466G, G51484A and G52951A were located in exon 8, exon 9 and intron 9, respectively. They do not change the primary structure of the encoded protein. (2) Linkage equilibrium was observed between A2823G in caspase-10 gene and the three sites in caspase-8 gene. A43466G and G52951A, and G51484A and G52951A in caspase-8 gene were also in linkage equilibrium. Their coefficients of disequilibrium were near 0. Whereas strong linkage disequilibrium was observed between A43466G and G51484A, because its coefficient of disequilibrium was near 1. (3) A total of 11 haplotypes were estimated within A2823G in caspase-10 gene and three sites in caspase-8 gene. A-2823/A-43466/G-51484/G-52951 was the main haplotype with a frequency of 0.3811. A-2823/A-43466/G-51484/A-52951 was the second haplotype with a frequency of 0.2536. The polymorphism information content of their haplotypes was 0.7106.

CONCLUSIONThe SNPs of caspase-8, -10 genes in Han Chinese of Zhejiang could be parsed into at least three different haplotype blocks. The polymorphism information content can be improved by using haplotype analysis of several SNPs.

Alleles ; Base Sequence ; Caspase 10 ; genetics ; Caspase 8 ; genetics ; China ; ethnology ; DNA ; analysis ; Ethnic Groups ; genetics ; Gene Frequency ; Haplotypes ; Humans ; Polymorphism, Single Nucleotide

OBJECTIVE: To investigate the relationships between caspase-8 (CASP8), fatty acid synthetase (Fas) gene polymorphisms and prognosis of non-Hodgkin's lymphoma patients in Han nationality. METHODS: The clinical data of 85 patients with non-Hodgkin's lymphoma were analyzed retrospectively. The polymorphisms of CASP8 and Fas gene were detected, and prognosis of the patients were analyzed. The polymorphisms of CASP8 and Fas gene in patients with different prognosis were compared, and the relationships between gene polymorphisms and the poor prognosis of the patients were investigated. RESULTS: The incidence rate of poor prognosis of the patients enrolled in the study was 65.88%. The polymorphisms of CASP8 and Fas genes in the patients with poor or good prognosis were in accordance with Hardy Weinberg's law of genetic balance. The frequencies of GG genotype and G allele at rs 1035142 of CASP8 gene, GA genotype and A allele at rs 1377 of Fas gene in patients with poor prognosis were lower than those of the patients with good prognosis (P<0.05). The frequencies of GT, TT and T alleles at rs 1035142 of CASP8 gene, GG and G alleles at rs 1377 of Fas gene in patients with poor prognosis were higher than those of the patients with good prognosis (P<0.05). The proportions of Ann Arbor stage III-IV and high malignancy in patients with poor prognosis were higher than those of the patients with good prognosis (P<0.05). Logistic multiple regression analysis showed that Ann Arbor stage III-IV, moderate malignant, high malignancy, CASP8 rs 1035142 GT genotype, CASP8 rs 1035142 TT genotype and Fas rs 1377 GG genotype were all the risk factors for the poor prognosis of the patients (P<0.05). CONCLUSION The poor prognosis rate of non-Hodgkin's lymphoma patients in Han nationality is relatively high, and the risk factors for the prognosis of the patients include Ann Arbor stage III-IV, moderate and high malignancy, CASP8 rs 1035142 GT genotype, CASP8 rs 1035142 TT genotype and Fas rs 1377 GG genotype.
Caspase 8/genetics* ; Ethnicity ; Fatty Acids ; Humans ; Ligases ; Lymphoma, Non-Hodgkin/genetics* ; Polymorphism, Genetic ; Prognosis ; Retrospective Studies ; fas Receptor

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

OBJECTIVEThe loss of caspase-8 expression correlates with unfavorable survival outcomes in neuroblastoma (NB). Caspase-8 gene inactivation is caused by methylation. This study aimed to explore the effect of the demethylation agent 5-azacytidine on caspase-8 expression and whether 5-azacytidine can increase the sensitivity of chemotherapy drug doxorubicin to NB cells.

METHODSCaspase-8 mRNA expression in NB cell lines (SH-SY5Y cells) was examined by RT-PCR before and after 5-azacytidine treatment. Survival rates of SH-SY5Y cells were detected using MTT analysis and compared among the doxorubicin alone treatment, 5-azacytidine along with doxorubicin treatment, and caspase-8 inhibitor+5-azacytidine+doxorubicin treatment groups.

RESULTSCaspase-8 mRNA was not expressed in untreated SH-SY5Y cell lines. Caspase-8 mRNA expression in SH-SY5Y cells was detectable 3 days after 5-azacytidine treatment, and increased significantly 5 days after 5-azacytidine treatment (P < 0.05). Survival rates of SH-SY5Y cells treated with 5-azacytidine along with different concentrations of doxorubicin (0.05, 0.1,0.25, 0.5 microg/mL) were (77.61 +/- 7.30)%, (57.35 +/- 6.64)%, (46.25 +/- 4.46)% and (35.59 +/- 5.12)%, respectively, which were significantly lower than those treated with doxorubicin alone (94.89 +/- 4.15%, 80.60 +/- 8.50%, 64.48 +/- 4.92% and 52.32 +/- 6.71%) (P < 0.01). Caspase-8 inhibitor pretreatment resulted in an increased survival rate of SH-SY5Y cells (92.95 +/- 3.48%, 78.39 +/- 4.28 %, 62.31 +/- 6.50% and 49.92 +/- 5.77%) compared with the 5-azacytidine+doxorubicin treatment group.

CONCLUSIONS5-azacytidine may enhance anti-tumor efficacy of doxorubicin to NB cell lines, possibly through an up-regulation of caspase-8 mRNA expression.

Antineoplastic Agents ; pharmacology ; Azacitidine ; pharmacology ; Caspase 8 ; genetics ; Caspase Inhibitors ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Synergism ; Humans ; RNA, Messenger ; analysis

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

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

OBJECTIVESTo investigate the mechanisms for human telomerase reverse transcriptase (hTERT) RNA interference (RNAi) in increasing hepatocellular carcinoma cell apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL).

METHODSCell apoptosis was identified by flow cytometry analysis after annexin V/PI double staining. Expression of apoptosis-related proteins, procaspase-8, -9, -3, Bax, Bcl-2 and hTERT, were identified by Western blotting analysis; telomerase activity and telomere length were detected by telomeric repeat amplification protocol (TRAP) and telomere amount and length assay (TALA) methods.

RESULTSHepatocellular carcinoma cell apoptosis induced by TRAIL were all significantly increased by hTERT RNAi (P less than 0.05). For example, apoptosis rates were enhanced from 5.53% (untransformed) to 10.35% (transformed) in HepG 2 cells and from 14.73% to 77.24% in SMMC 7721 cells after being treated by 100 ng/ml TRAIL for 24 h. Moreover, activation of procaspase-8, -9 and -3 in transformed cells after being treated by TRAIL were all significantly raised (P less than 0.05) in a dose-dependent manner. The expression of procaspase-8, -9 and Bcl-2 were effectively augmented (P less than 0.05), but expressions of Bax and hTERT were strikingly decreased (P less than 0.05). Meanwhile, telomerase activity was apparently suppressed and telomere length was markedly shortened (P less than 0.05). There were no remarkable differences in these effects between control cells and the untransformed cells (P more than 0.05).

CONCLUSIONEnhanced cell apoptosis induced by TRAIL through hTERT RNAi may be related to up-regulation of procaspase-8 and -9 expressions. However the down-regulation of hTERT expression, reduced telomerase activity and shortened telomere length may not be related to expressions of Bcl-2 and Bax.

Apoptosis ; Carcinoma, Hepatocellular ; pathology ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Humans ; Liver Neoplasms ; pathology ; RNA Interference ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Telomerase ; genetics ; metabolism

OBJECTIVETo investigate the effect of fragile histidine triad (FHIT) gene transfection on human colorectal cancer cell line SW480 through up-regulation of caspase-8 expression.

METHODSThe eukaryotic expression plasmid containing FHIT, pRc/CMV2-FHIT was prepared and purified, and then identified by restrictive enzyme digestion. pRc/CMV2-FHIT was transfected into SW480 cells, and positive cell clones (SW480-FHIT, study group) were selected and amplified. Empty plasmid-transfected SW480 cells(SW480-pRc/CMV2, negative control) and normal SW480 cells (bland control) were used as control. Methyl thiazolyl tetrazolium (MTT) assay was used to test the changes in the proliferation of SW480 cells. Cell-cycle kinetics and apoptosis were analyzed by flow cytometry (FCM). The changes of pro-caspase-8, caspase-8 mRNA and caspase-8 relative activity were analyzed by Western blot, semi-quantitative RT-PCR and colorimetric assay with pan labeled substrate, respectively.

RESULTSAt 96 hours after transfection, cell inhibition rates of the study group and the negative control group were 71.7% and 16.9%. G0/G1 ratio was (63.2±3.5)% and (50.6±2.1)%, optical density of caspase-8 mRNA band 107 and 41, and relative activity of caspase-8 0.43 and 0.25, respectively. All the differences above were statistically significant (P<0.05). When FHIT inhibitor was added, the relative activity of caspase-8 decreased to 0.22, comparable to that in the control group.

CONCLUSIONSFHIT gene transfection can significantly inhibit the proliferation and induce G0/G1 arrest in human colon cancer cell line SW480. The mechanism is related to the up-regulation of caspase-8 expression.

Acid Anhydride Hydrolases ; genetics ; Apoptosis ; Caspase 8 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; genetics ; pathology ; Humans ; Neoplasm Proteins ; genetics ; RNA, Messenger ; genetics ; Transfection