This study aims to investigate the effects and the molecular mechanism of Huangdi Anxiao Capsules(HDAX)-containing serum in protecting the rat adrenal pheochromocytoma(PC12) cells from diabetes-associated cognitive dysfunction induced by high glucose and whether the mechanism is related to the regulation of NOD-like receptor thermal protein domain associated protein 3(NLRP3)-mediated pyroptosis. The PC12 cell model of diabetes-associated cognitive dysfunction induced by high glucose was established and mcc950 was used to inhibit NLRP3. PC12 cells were randomized into control, model, HDAX-containing serum, mcc950, and HDAX-containing serum+mcc950 groups. Methyl thiazolyl tetrazolium(MTT) assay was employed to determine the viability, and Hoechst 33258/PI staining to detect pyroptosis of PC12 cells. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1 beta(IL-1β) and IL-18. Western blot was employed to determine the protein levels of postsynaptic density protein 95(PSD-95), NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), GSDMD-N, and cleaved cysteinyl aspartate specific proteinase-1(caspase-1), and RT-PCR to determine the mRNA levels of NLRP3, ASC, GSDMD, and caspase-1. The immunofluorescence assay was adopted to measure the levels and distribution of NLRP3 and GSDMD-N in PC12 cells. Compared with the control group, the model group showed decreased cell proliferation, increased PI positive rate, down-regulated protein level of PSD-95, up-regulated protein levels of NLRP3, ASC, GSDMD-N, GSDMD, and cleaved caspase-1, up-regulated mRNA levels of NLRP3, ASC, GSDMD, and caspase-1, and elevated levels of IL-1β and IL-18. Compared with the model group, HDAX-containing serum, mcc950, and the combination of them improved cell survival rate and morphology, decreased the PI positive rate, down-regulated the protein levels of NLRP3, ASC, GSDMD-N, GSDMD, and cleaved caspase-1 and the mRNA levels of NLRP3, ASC, GSDMD, and caspase-1, and promoted the secretion of IL-1β and IL-18. The findings demonstrated that HDAX-containing serum can inhibit the pyroptosis-mediated by NLRP3 and protect PC12 cells from the cognitive dysfunction induced by high glucose.
Rats ; Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Interleukin-18 ; Pyroptosis/physiology* ; Diabetes Mellitus ; Caspases ; Glucose ; RNA, Messenger

The clinical treatment of joint contracture due to immobilization remains difficult. The pathological changes of muscle tissue caused by immobilization-induced joint contracture include disuse skeletal muscle atrophy and skeletal muscle tissue fibrosis. The proteolytic pathways involved in disuse muscle atrophy include the ubiquitin-proteasome-dependent pathway, caspase system pathway, matrix metalloproteinase pathway, Ca-dependent pathway and autophagy-lysosomal pathway. The important biological processes involved in skeletal muscle fibrosis include intermuscular connective tissue thickening caused by transforming growth factor-β1 and an anaerobic environment within the skeletal muscle leading to the induction of hypoxia-inducible factor-1α. This article reviews the progress made in understanding the pathological processes involved in immobilization-induced muscle contracture and the currently available treatments. Understanding the mechanisms involved in immobilization-induced contracture of muscle tissue should facilitate the development of more effective treatment measures for the different mechanisms in the future.
Atrophy ; Autophagy ; Calcium ; metabolism ; Caspases ; metabolism ; Connective Tissue ; metabolism ; pathology ; Contracture ; etiology ; metabolism ; pathology ; therapy ; Fibrosis ; Humans ; Immobilization ; adverse effects ; Joints ; Lysosomes ; metabolism ; Matrix Metalloproteinases ; metabolism ; Muscle, Skeletal ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Proteolysis ; Signal Transduction ; physiology ; Transforming Growth Factor beta1 ; metabolism ; Ubiquitin ; metabolism

Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.
Adult ; Caspases/metabolism* ; Cell Death ; Enzyme Activation ; Humans ; Male ; Mitochondria/pathology* ; Necrosis ; Nitrosative Stress/physiology* ; Permeability ; Peroxynitrous Acid/pharmacology* ; Phosphatidylserines/metabolism* ; Spermatozoa/ultrastructure*

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.
*Apoptosis ; Caspases/metabolism ; Entamoeba histolytica/*enzymology/*growth & development ; Humans ; Hydrolysis ; Jurkat Cells ; Phosphatidylinositol 3-Kinases/*metabolism ; Protein Kinase C/*metabolism ; T-Lymphocytes/*parasitology/*physiology

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction

OBJECTIVETo evaluate whether Shenfu injection (SFI) protects against cardiac myocyte injury induced by Fupian injection (FPI) in vitro.

METHODSH9c2 cells were separately treated with FPI, Renshen injection (RSI) and SFI. Cell viability, lactate dehydrogenase (LDH) release, spontaneous beating rate of primative cardical cells, caspase-3/7 activity, cell apoptosis, and cytochrome P450 2J3 (CYP2J3) mRNA expression were analyzed.

RESULTSThe viability of H9c2 cells treated with SFI (37 and 75 mg/mL) was significantly higher than that of H9c2 cells treated with FPI (25 and 50 mg/mL) (P<0.05, P<0.01, respectively). LDH activity of H9c2 cells treated with SFI (75 mg/mL) was significantly decreased (P<0.01) compared with that of H9c2 cells treated with FPI (50 mg/mL). SFI (150 mg/mL) significantly attenuated FPI (100 mg/mL)-induced spontaneous beating rate decrease in primary myocardial cells after 4-hour treatment. Compared with FPI (12 and 25 mg/mL), SFI (18 and 37 mg/mL) treatment could effectively reverse the change of caspase-3/7 activity (P<0.01 and P<0.01, respectively). Compared with FPI (6 and 25 mg/mL), apoptotic cells decreased significantly (P<0.05, P<0.01, respectively) when H9c2 cells were incubated with SFI (9 and 37 mg/mL). The expression of CYP2J3 mRNA was down-regulated by FPI, while RSI and SFI could up-regulate the expression of CYP2J3 (P<0.01), which suggested the potential mechanism of protection of RSI against cardiac myocyte damage induced by FPI treatment.

CONCLUSIONThese observations indicate that SFI has the potential to exert cardioprotective effects against FPI toxicity. The effect was possibly correlated with the activation of CYP2J3.

Animals ; Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Cytochrome P-450 Enzyme System ; genetics ; physiology ; Drugs, Chinese Herbal ; pharmacology ; L-Lactate Dehydrogenase ; secretion ; Myocytes, Cardiac ; drug effects ; enzymology ; Rats

OBJECTIVEDeoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma (A549) cells.

METHODSThe cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 50% inhibitory concentration (IC50) value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase-contrast microscopy. The induction of apoptosis was evaluated using acridine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using fluorogenic substrate specific to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly (ADP-ribose) polymerase was also analyzed.

RESULTSDeoxyelephantopin exhibited cytotoxicity to A549 cells (IC50 = 12.287 μg/mL), however, there was no toxicity towards normal human lymphocytes. Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner. Acridine orange, ethidium bromide and Hoechst 33342 staining showed cell shrinkage, chromosomal condensation and nuclear fragmentation, indicating induction of apoptosis. Deoxyelephantopin increased apoptosis of A549 cells, as evidenced by more TUNEL-positive cells. DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population. Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through both extrinsic and intrinsic pathways.

CONCLUSIONThese results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.

Adenocarcinoma ; drug therapy ; pathology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Caspases ; physiology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Lactones ; pharmacology ; Lung Neoplasms ; drug therapy ; pathology ; Sesquiterpenes ; pharmacology

Acute and chronic neurodegenerative diseases are illnesses associated with high morbidity and mortality, and few or no effective options are available for their treatments. Many neurodegenerative diseases are included in them, for example, stroke, brain trauma, spinal cord injury, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, and Parkinson's disease. Given that central nervous system tissue has very limited, if any, regenerative capacity, it is of utmost importance to limit the damage caused by neuronal death. During the past decade, considerable progress has been made in understanding the process of cell death. In this article, we review the causes and mechanisms of neuronal-cell death, especially as it pertains to the caspases family of proteases associated with cell death. The results may be helpful to the experimental research and clinical application of neurodegenerative diseases.
Animals ; Apoptosis ; physiology ; Caspases ; metabolism ; Cell Death ; Humans ; Neurodegenerative Diseases ; enzymology ; pathology ; Neurons ; pathology ; Peptide Hydrolases ; metabolism

BACKGROUNDApoptosis is involved in the adaptive responses of bone to mechanical loading. The purpose of this study was to investigate the effects of tensile forces on osteoblast apoptosis and the related mechanism by analyzing the expression of caspases, Bcl-2, and Bax.

METHODSPrimary osteoblasts were harvested from neonatal rat calvaria and were subjected to cyclic tensile forces for 72 hours using Flexcell 4000 strain unit in Minimum Essential Medium (MEM) with 10% fetal calf serum (FCS) or with serum deprivation. Apoptosis was tested by flow cytometry using annexin V/PI staining. Caspase-3 activity was analyzed via Elisa. The gene expression of caspase-8, -9, Bcl-2, and Bax was quantified by reverse transcription (RT)-PCR.

RESULTSIn 10% FCS condition, no significant difference in cell apoptosis was found between the stretched and non-stretched osteoblast cultures. Serum withdrawal resulted in higher apoptosis rate in the osteoblasts with increased caspase-3 activity, and elevated expression of caspase-9 and Bax. Six-percent elongation of stretch attenuated the cell apoptosis induced by serum starvation, concurrent with a decrease in caspase-3 activity, a decline of caspase-8 expression, and an elevation of Bcl-2 level. On the contrary, 12% elongation of stretch increased caspase-3 activity and promoted the apoptosis with an elevated expression of caspase-8 and Bax. No significant change of caspase-9 expression was identified upon force application.

CONCLUSIONSThese results suggested that tensile forces regulate cell apoptosis of primary rat osteoblasts through caspase-3 and caspase-8 signaling cascade. Light forces rescue the cells from serum deprivation-induced apoptosis by elevating Bcl-2 expression, while heavy forces promote the apoptotic insult by inducing Bax expression.

Animals ; Apoptosis ; physiology ; Caspase 8 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Caspases ; genetics ; metabolism ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Osteoblasts ; cytology ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; bcl-2-Associated X Protein ; genetics ; metabolism

To observe the inhibitive effect of Baicalin against influenza A H1N1 virus infection in epithelial cell line A549, the cell proliferation and cytotoxicity were assayed by MTT, the cell cycle and the apoptosis were analyzed by flowcytometer using PI staining, the morphology of cellular nucleolus was observed by Hoechst 33258 staining and the effects of activation on caspase 3 and caspase 8/9 were also detected by immunofluorescent staining with a fluorescence microscope. The results showed that Baicalin exerted an inhibitive effect on CPE after influenza A H1N1 virus infection. The FACS with PI staining showed that the cell cycle of the infected cell was arrested at S phase, the Baicalin-treated group decreased S phase cell ratio and subG0 phase peak in comparison with the control (P < 0.05) and significantly promoted cell proliferation (# P < 0.05). Hoechst33258 staining suggested that Baicalin protected the cellular nucleolus against the influenza virus-induced apoptosis. Observation under the immunofluorescent microscope suggested that the activities of caspase-8 and caspase-3 were enhanced at 36 h post the influenza virus infection, but 100 microg/mL Baicalin suppressing the activation of caspase-8 and caspase-3 rather than that of caspase-9. In summary, this research confirmed that Baicalin inhibited the influenza A H1N1 virus strain infection in vitro, the drug obviously protected cells from apoptosis damages through regulating cell cycle and suppressed the activation of caspase-8 and caspase-3. The down-regulation was significant and showed a dose-dependent relationship.
Antiviral Agents ; pharmacology ; Apoptosis ; drug effects ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Flavonoids ; pharmacology ; Flow Cytometry ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology