The aim of this study was to investigate the role of S100 calcium binding protein A16 (S100A16) in lipid metabolism in hepatocytes and its possible biological mechanism. HepG2 cells (human hepatoma cell line) were cultured with fatty acid to establish fatty acid culture model. The control model was cultured without fatty acid. Each model was divided into three groups and transfected with S100a16 over-expression, shRNA and vector plasmids, respectively. The concentration of triglyceride (TG) in the cells was measured by kit, and the lipid droplets was observed by oil red O staining. Immunoprecipitation and mass spectrometry were used to find the interesting proteins interacting with S100A16, and the interaction was verified by immunoprecipitation. The further mechanism was studied by Western blot and qRT-PCR. The results showed that the intracellular lipid droplet and TG concentrations in the fatty acid culture model were significantly higher than those in the control model. The accumulation of intracellular fat in the S100a16 over-expression group was significantly higher than that in the vector plasmid transfection group. There was an interaction between heat shock protein A5 (HSPA5) and S100A16. Over-expression of S100A16 up-regulated protein expression levels of HSPA5, inositol-requiring enzyme 1α (IRE1α) and pIREα1, which belong to endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway. Meanwhile, over-expression of S100A16 up-regulated the mRNA expression levels of adipose synthesis-related gene Srebp1c, Acc and Fas. In the S100a16 shRNA plasmid transfection group, the above-mentioned protein and mRNA levels were lower than those of vector plasmid transfection group. These results suggest that S100A16 may promote lipid synthesis in HepG2 cells through endoplasmic reticulum stress HSPA5/IRE1α-XBP1 pathway.
Endoplasmic Reticulum Stress ; Endoribonucleases ; physiology ; Heat-Shock Proteins ; physiology ; Hep G2 Cells ; Humans ; Lipid Metabolism ; Protein-Serine-Threonine Kinases ; physiology ; S100 Proteins ; physiology ; Triglycerides ; biosynthesis ; X-Box Binding Protein 1 ; physiology

BACKGROUND: Heat shock protein 70 (HSP70) exhibits protective effects against ultraviolet (UV)-induced premature skin aging. A standardized extract of Asparagus officinalis stem (EAS) is produced as a novel and unique functional food that induces HSP70 cellular expression. To elucidate the anti-photoaging potencies of EAS, we examined its effects on HSP70 expression levels in UV-B-irradiated normal human dermal fibroblasts (NHDFs). METHODS: NHDFs were treated with 1 mg/mL of EAS or dextrin (vehicle control) prior to UV-B irradiation (20 mJ/cm). After culturing NHDFs for different time periods, HSP70 mRNA and protein levels were analyzed using real-time polymerase chain reaction and western blotting, respectively. RESULTS: UV-B-irradiated NHDFs showed reduced HSP70 mRNA levels after 1-6 h of culture, which were recovered after 24 h of culture. Treatment with EAS alone for 24 h increased HSP70 mRNA levels in the NHDFs, but the increase was not reflected in its protein levels. On the other hand, pretreatment with EAS abolished the UV-B irradiation-induced reduction in HSP70 expression at both mRNA and protein levels. These results suggest that EAS is capable to preserve HSP70 quantity in UV-B-irradiated NHDFs. CONCLUSIONS EAS exhibits anti-photoaging potencies by preventing the reduction in HSP70 expression in UV-irradiated dermal fibroblasts.
Asparagus Plant ; Cells, Cultured ; Female ; Fibroblasts ; drug effects ; radiation effects ; HSP70 Heat-Shock Proteins ; biosynthesis ; Humans ; Middle Aged ; Plant Extracts ; pharmacology ; Polymerase Chain Reaction ; Skin ; drug effects ; radiation effects ; Skin Aging ; drug effects ; radiation effects ; Telomere ; metabolism ; Ultraviolet Rays ; adverse effects

OBJECTIVETo investigate the effect of heat shock factor 1 (HSF1) on airway hyperresponsiveness and airway inflammation in mice with asthma and possible mechanisms.

METHODSA total of 36 mice were randomly divided into four groups: control, asthma, HSF1 small interfering RNA negative control (siHSF1-NC), and siHSF1 intervention (n=9 each). Ovalbumin (OVA) sensitization and challenge were performed to induce asthma in the latter three groups. The mice in the siHSF1-NC and siHSF1 groups were treated with siHSF1-NC and siHSF1, respectively. A spirometer was used to measure airway responsiveness at 24 hours after the last challenge. The direct count method was used to calculate the number of eosinophils. ELISA was used to measure the serum level of OVA-specific IgE and levels of interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), and interferon-γ (IFN-γ) in lung tissues and bronchoalveolar lavage fluid (BALF). Quantitative real-time PCR was used to measure the mRNA expression of HSF1 in asthmatic mice. Western blot was used to measure the protein expression of HSF1, high-mobility group box 1 (HMGB1), and phosphorylated c-Jun N-terminal kinase (p-JNK).

RESULTSThe asthma group had significant increases in the mRNA and protein expression of HSF1 compared with the control group (P<0.05). The siHSF1 group had significantly reduced mRNA and protein expression of HSF1 compared with the siHSF1-NC group (P<0.05). The knockdown of HSF1 increased airway wall thickness, airway hyperresponsiveness, OVA-specific IgE content, and the number of eosinophils (P<0.05). Compared with the siHSF1-NC group, the siHSF1 group had significantly increased levels of IL-4, IL-5, and IL-13 and significantly reduced expression of IFN-γ in lung tissues and BALF (P<0.05), as well as significantly increased expression of HMGB1 and p-JNK (P<0.05).

CONCLUSIONSKnockdown of HSF1 aggravates airway hyperresponsiveness and airway inflammation in asthmatic mice, and its possible mechanism may involve the negative regulation of HMGB1 and JNK.

Animals ; Asthma ; etiology ; Bronchial Hyperreactivity ; etiology ; immunology ; Cytokines ; biosynthesis ; DNA-Binding Proteins ; analysis ; physiology ; Eosinophils ; physiology ; Female ; HMGB1 Protein ; analysis ; Heat Shock Transcription Factors ; Immunoglobulin E ; blood ; Mice ; Mice, Inbred BALB C ; Transcription Factors ; analysis ; physiology

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by abnormal proliferation of synoviocytes, leukocyte infiltration, and angiogenesis. The endoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded proteins in the ER leads to a condition known as ER stress. Failure of the ER's adaptive capacity results in abnormal activation of the unfolded protein response. Recently, we have demonstrated that ER stress-associated gene signatures are highly expressed in RA synovium and synovial cells. Mice with Grp78 haploinsufficiency exhibit the suppression of experimentally induced arthritis, suggesting that the ER chaperone GRP78 is crucial for RA pathogenesis. Moreover, increasing evidence has suggested that GRP78 participates in antibody generation, T cell proliferation, and pro-inflammatory cytokine production, and is therefore one of the potential therapeutic targets for RA. In this review, we discuss the putative, pathophysiological roles of ER stress and GRP78 in RA pathogenesis.
Animals ; Arthritis, Rheumatoid/genetics/*pathology ; Autoantibodies/immunology ; Cell Proliferation ; Cytokines/biosynthesis/immunology ; Endoplasmic Reticulum/immunology/pathology ; Endoplasmic Reticulum Stress/*immunology ; Haploinsufficiency/genetics ; Heat-Shock Proteins/*genetics/*immunology ; Humans ; Lymphocyte Activation ; Mice ; Neovascularization, Pathologic/genetics ; Protein Folding ; Synovial Membrane/cytology ; T-Lymphocytes/immunology ; Unfolded Protein Response/*immunology

The aim of this study was to examine the effects of endoplasmic reticulum (ER) stress on aldosterone (Aldo)-induced apoptosis of endothelial cells. Glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP, a hallmark of ER-associated apoptosis) were used to evaluate ER stress. Western blotting and real-time PCR were used to analyze indicators of ER molecule. Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry. Human umbilical vein endothelial cells (HUVECs) were stimulated with different concentrations of Aldo for different durations. Aldo promoted apoptosis of HUVECs and induced ER stress, as evidenced by increased expression of GRP78 and CHOP. siRNA knockdown of CHOP attenuated Aldo-mediated apoptosis. These results indicate that ER stress may be involved in Aldo-induced apoptosis of HUVECs.
Aldosterone ; pharmacology ; Apoptosis ; drug effects ; Endoplasmic Reticulum Stress ; drug effects ; Gene Expression Regulation ; drug effects ; Heat-Shock Proteins ; biosynthesis ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Transcription Factor CHOP ; biosynthesis

PURPOSE: To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. METHODS: Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. RESULTS: Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. CONCLUSIONS: Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease.
Animals ; Blotting, Western ; Cell Death/*genetics ; Cell Survival ; Cells, Cultured ; DNA/*genetics ; Disease Models, Animal ; Etoposide/toxicity ; *Gene Expression Regulation ; HSP72 Heat-Shock Proteins/biosynthesis/*genetics ; Immunohistochemistry ; Rats ; Retinal Degeneration/*genetics/metabolism/pathology ; Retinal Ganglion Cells/drug effects/*metabolism/pathology

OBJECTIVETo examine if the ultra-highly diluted homeopathic remedy Thuja 30C can ameliorate benzo(a)pyrene (BaP)-induced DNA damage, stress and viability of perfused lung cells of Swiss albino mice in vitro.

METHODSPerfused normal lung cells from mice were cultured in 5% Roswell Park Memorial Institute medium and exposed to BaP, a potent carcinogen, at the half maximal inhibitory concentration dose (2.2 μmol/L) for 24 h. Thereafter, the intoxicated cells were either treated with Thuja 30C (used against tumor or cancer) or its vehicle media, succussed alcohol 30C. Relevant parameters of study involving reactive oxygen species (ROS) accumulation, total glutathione (GSH) content, and generations of heat shock protein (hsp)-90 were measured; the cell viability and other test parameters were measured after treatment with either Thuja 30C or its vehicle media. Circular dichroism spectroscopy was performed to examine if Thuja 30C directly interacted with calf thymus DNA as target. For ascertaining if DNA damaged by BaP could be partially repaired and restituted by the remedy, 4',6-diamidino-2-phenylindole staining was performed.

RESULTSThuja 30C increased cell viability of BaP-intoxicated cells significantly, as compared to drug-untreated or drug-vehicle control. A minimal dose of Thuja 30C significantly inhibited BaP-induced stress level, by down-regulating ROS and hsp-90, and increasing GSH content. Thuja 30C itself had no DNA-damaging effect, and no direct drug-DNA interaction. However, it showed quite striking ability to repair DNA damage caused by BaP.

CONCLUSIONThuja 30C ameliorates BaP-induced toxicity, stress and DNA damage in perfused lung cells of mice and it apparently has no effect on normal lung cells.

Animals ; Benzo(a)pyrene ; toxicity ; Cell Survival ; drug effects ; DNA Damage ; drug effects ; Glutathione ; metabolism ; HSP90 Heat-Shock Proteins ; biosynthesis ; Homeopathy ; Mice ; Mice, Inbred Strains ; Reactive Oxygen Species ; metabolism ; Thuja

In response to viral infection, RIG-I-like RNA helicases detect viral RNA and signal through the mitochondrial adapter protein VISA. VISA activation leads to rapid activation of transcription factors IRF3 and NF-κB, which collaborate to induce transcription of type I interferon (IFN) genes and cellular antiviral response. It has been demonstrated that VISA is activated by forming prion-like aggregates. However, how this process is regulated remains unknown. Here we show that overexpression of HSC71 resulted in potent inhibition of virus-triggered transcription of IFNB1 gene and cellular antiviral response. Consistently, knockdown of HSC71 had opposite effects. HSC71 interacted with VISA, and negatively regulated virus-triggered VISA aggregation. These findings suggest that HSC71 functions as a check against VISA-mediated antiviral response.
Adaptor Proteins, Signal Transducing ; biosynthesis ; chemistry ; genetics ; metabolism ; Cell Aggregation ; genetics ; GPI-Linked Proteins ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; HSC70 Heat-Shock Proteins ; genetics ; metabolism ; Heat-Shock Response ; genetics ; Humans ; Interferon Regulatory Factor-3 ; genetics ; metabolism ; Interferon-beta ; genetics ; NF-kappa B ; genetics ; Prions ; metabolism ; Receptors, Retinoic Acid ; metabolism ; Viruses ; drug effects ; metabolism ; pathogenicity

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.
Animals ; Antigens, Protozoan/*biosynthesis ; Brain/parasitology ; Female ; *Gene Expression ; Heat-Shock Proteins/*biosynthesis ; Immunocompromised Host ; Life Cycle Stages ; Lung/parasitology ; Mice ; Protozoan Proteins/*biosynthesis ; Real-Time Polymerase Chain Reaction ; Toxoplasma/*genetics/physiology ; Toxoplasmosis, Animal

Heat shock protein 27 belongs to the heat shock protein family in the small molecular weight family. This review collected a number of literature to analyze the expression meaning and mechanism of HSP27,expounded HSP27 with inhibition of NO production, maintenance of cell protein stability and accelerated cell damage repair function. At the same time, HSP27 also has a resistance to apoptosis, protecting mitochondria, inhibiting activation of nuclear factor and other related functions. The heat shock protein 27 has protection in spinal cord ischemia-reperfusion.
Apoptosis ; HSP27 Heat-Shock Proteins ; physiology ; Humans ; Nitric Oxide ; biosynthesis ; Reperfusion Injury ; prevention & control ; Spinal Cord ; blood supply