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

Apoptosis ; Caspase 3/genetics* ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Pentacyclic Triterpenes ; Stomach Neoplasms/drug therapy*

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 investigate effects of the leaching liquids of 5 different kinds of dental alloys on L929 cells at cell level and molecular level.

METHODSThe fibroblast L929 cells of mouse were cultivated in vitro in leaching liquids of 5 different kinds of dental alloys, Au alloy (n = 8), Ag-Pt alloy (n = 8), Co-Cr alloy (n = 8), Ni-Cr alloy (n = 8), and Cu alloy (n = 8). The RPMI 1640 cell medium containing 10% fetal beef serum was used as control. The cytotoxicities of the 5 dental alloys were evaluated by means of methyl thiazolyl tetrazolium (MTT), and the effects of these alloys on the expression of caspase-3, caspase-8, and caspase-9 mRNA of L929 cells were examined using reverse transcription polymerase chain reaction (RT-PCR) method.

RESULTSAfter 48 hours culture the cytotoxicity of Cu alloy group was in Grade 4 and those of the other groups were all in Grade 0. The mRNA levels of caspase-8 had no change in all groups (P > 0.05). The mRNA levels of caspase-3 were as follows: Cu alloy (0.474 +/- 0.001), the negative control (0.527 +/- 0.003), Au alloy (0.528 +/- 0.013), Co-Cr alloy (0.615 +/- 0.007), Ag-Pd alloy (0.673 +/- 0.009), and Ni-Cr alloy (0.803 +/- 0.037). The mRNA levels of caspase-9 were as follows: Cu alloy (0.532 +/- 0.041), Au alloy (0.574 +/- 0.013), the negative control (0.578 +/- 0.010), Co-Cr alloy (0.617 +/- 0.009), Ag-Pd alloy (0.703 +/- 0.018), and Ni-Cr alloy (0.811 +/- 0.037). There were significant differences between the groups except the negative control group and Au alloy group.

CONCLUSIONSThe Cu alloy shows the highest cytotoxicity, and the leaching liquids of 5 different kinds of dental alloys may induce cell apoptosis through mitochondrion pathway.

Animals ; Apoptosis ; drug effects ; genetics ; Caspase 3 ; genetics ; metabolism ; Caspase 8 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Culture Media ; Dental Alloys ; pharmacology ; L Cells (Cell Line) ; Materials Testing ; Mice ; RNA, Messenger ; metabolism

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

OBJECTIVETo investigate the effects of caspase-3 siRNA on the neurobehavior of mice exposed to aluminum.

METHODSMale KunMing mice (3 months old) were randomly divided into 4 groups by weight:blank control group (4 microl normal saline), Al group (4 microl 0.5% AlCl3), Al plus empty vector group(3 microl 0.5% AlCl3 plus control siRNA expression vector)and Al plus RNAi group (3 microl 0.5% AlCl3 plus targeted siRNA expression vector). All groups were treated by lateral cerebral ventricle micro-injection for 5 days. The neurobehavior was tested by the Morris water maze test, Open-field and Step-down tests for all treated mice. Pathological changes in hippocampus was observed by electron microscopy, the caspase-3 gene expression levels were detected using RT-PCR.

RESULTSThe results of Step-down test indicated that as compared with control group, the latent time [LT, (44.67 +/- 10.60) s] in A1 group decreased significantly, the error number (3.63 +/- 0.52) in Al group increased significantly and the LT [(68.00 +/- 14.70) s] in Al plus empty vector group decreased significantly (P<0.05). the LT [(239.50 +/- 19.36) s] in Al plus RNAi group increased significantly and the error number in Al plus RNAi group decreased significantly, as compared with Al group (P<0.05). The results of Morris water maze test showed that as compared with control group, the LT in Al group increased significantly, and residence time in the former platform quadrant decreased significantly and the LT in Al plus empty vector group increased significantly (P<0.05). The LT in Al plus RNAi group was significantly longer than that in Al group (P<0.05). The results of open-field test demonstrated that as compared with control group, the time in the central grid in Al group and Al plus empty vector group increased significantly, the rearing number and the modification number in Al group and Al plus empty vector group decreased significantly (P< 0.05). As compared with Al group, the time in the central grid in Al plus RNAi group decreased, the inter-cell number, the rearing number and the modification number increased significantly (P<0.05). The results of electron microscopic examination exhibited that a slight change of hippocampal cells appeared in control group, the obvious pathological changes of hippocampal cells appeared in Al group and Al plus empty vector group, but the pathological changes of hippocampal cells in Al plus RNAi group significantly reduced as compared with Al group. The results of thionin staining indicated that the layers of neural cells of hippocampal CA3 were more clear and there was not obvious denatured injury of neural cells of hippocampal CA3 in control group. The number and Nissl body color of neural cells of hippocampal CA3 in Al group and Al plus empty vector group decreased significantly. After RNA interference, the number and Nissl body color of neural cells of hippocampal CA3 increased obviously. The expression levels of caspase-3 gene in Al group and Al plus empty vector group were 2.24 +/- 0.57 and 2.28 +/- 0.33, respectively, which were significantly higher than that (1.00 +/- 0.00) in control group (P<0.05). The expression level of caspase-3 gene in Al plus RNAi group was 0.44 +/- 0.08, which was significantly lower than those in Al group and control group (P<0.05).

CONCLUSIONAluminum can decrease the learning and memorizing ability, and inhibited the activity or exploration function of mice. It is suggested that Caspase-3 siRNA may reduce the neurotoxicity induced by aluminum to a certain extent.

Aluminum ; toxicity ; Animals ; Caspase 3 ; genetics ; Male ; Maze Learning ; Mice ; Mice, Inbred Strains ; Neurons ; RNA Interference ; RNA, Small Interfering ; genetics

To explore the effect of Huangqin Decoction on ulcerative colitis(UC) pyroptosis, and to explain the mechanism of pyroptosis based on NOD-like receptor thermoprotein domain 3(NLRP3)/cysteine proteinase 1(caspase-1) pathway. The animal model of UC induced with 3% dextran sodium sulfate(DSS) was established. The experimental animals were divided into control group, model group, low-dose(4.55 g·kg~(-1)), medium-dose(9.1 g·kg~(-1)) and high-dose(18.2 g·kg~(-1)) Huangqin Decoction groups and salazosulfapyridine group(0.45 g·kg~(-1)). While modeling, intragastric administration was given for 7 consecutive days. On the 8 th day, the mice were euthanized, the colon length was collected, and the histopathological changes were observed by HE staining. The content of interleukin-18(IL-18) was observed by ELISA. The content of lactatedehydrogenase(LDH)was determined by microplate method. TUNEL assay kit was used to detect the cell death. The immunohistochemical staining was used to detect the expressions of NLRP3 and apoptosis-associated speck-like protein containing a CARD(ASC). Western blot was used to detect the expressions of interleukin-1β(IL-1β), caspase-1 and gasdermin D(GSDMD).The experimental study showed that compared with normal group, the LDH content, TUNEL positive staining, inflammatory factors(IL-18, IL-1β), and proteins associated with pyroptosis were significantly increased(P<0.05). Compared with model control group, the LDH content, TUNEL positive staining, inflammatory factors(IL-18, IL-1β), and proteins associated with pyroptosis were decreased, and these results were more significant in high-dose groups(P<0.05). The results of HE staining showed that Huangqin Decoction could improve the pathological changes of colon. Huangqin Decoction could inhibit UC cell pyroptosis, and the mechanism may be closely related to NLRP3/caspase-1 signaling pathway.
Animals ; Caspase 1/genetics* ; Colitis, Ulcerative/drug therapy* ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Pyroptosis ; Scutellaria baicalensis

In this study, we investigated the mechanism of crude extract of Psammosilene tunicoides(CEPT) in the treatment of rheumatoid arthritis(RA) based on the Nod-like receptor protein 3(NLRP3) inflammasome. The collagen-induced arthritis(CIA) mouse model was established. On day 32 after the primary immunization, according to the arthritis score, the mice were randomly divided into model group, positive control(methotrexate) group, low-and high-dose CEPT groups, and normal group, with 10 mice in each group. According to the administration dose of each group, the mice were continuously administered for 21 days. Every four days during the administration, the paw edema degree, arthritis score, and spleen index of the mice were measured; histopathological examination was performed for the ankles of the mice; the contents of IL-1β and IL-18 in the serum were determined; the protein expression levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD(ASC), as well as the mRNA expression levels of NLRP3 and caspase-1 in the ankle joints of the mice were detected. The results showed that compared with those in the model group, the mice in the positive control group and CEPT groups had significantly decreased the contents of IL-1β and IL-18 in the serum and spleen index(P<0.01), significantly lowered arthritis score and degree of paw edema(P<0.01), alleviated arthritic infiltration of the knee, and down-regulated protein and mRNA levels of NLRP3, ASC, and caspase-1 in the ankle joint(P<0.01). These results suggest that P. tunicoides may reduce the paw edema and arthritis score and alleviate the inflammatory response in CIA mice by inhibiting the expression of NLRP3. This study provides a basis for the study of immune regulation of P. tunicoides in RA.
Animals ; Arthritis, Experimental/genetics* ; Arthritis, Rheumatoid/genetics* ; Caspase 1/genetics* ; Inflammasomes/genetics* ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics*

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