FasL Gene Expression Research Articles

Measurement of gene expression of FAS and FASL genes and thyroid hormones in patients with thyroid dysfunction in Anbar Governorate

Thyroid hormones are known for their ability to control metabolism, growth and many other body functions. The aim of the study was to measure the gene expression of fas and fasl genes and thyroid hormones in patients with thyroiditis. Samples were collected from Ramadi Teaching Hospital and their ages ranged between 18-75 RNA was extracted from serum to measure gene expression using real-time PCR (qRT-PCR). The results of gene expression between FAS and FASL genes showed statistically significant differences at a probability level (P ≤ 0.05) with a decrease in FAS gene expression level by 0.350 in the patient group and an increase in FAS gene expression level by 28.07 in the patient group. while the results of hormone analysis showed statistically significant differences at the probability level (P ≤ 0.05). The results also showed that there were significant differences between the ages of patients compared to healthy people at a probability level, with high TSH and low T4, T3 in the serum of hypothyroid patients and high T4,T3 and low TSH in the serum of hyperthyroid patients, as well as significant differences between the ages of patients compared to healthy people (P ≤ 0.05). The increase in FASL gene expression in thyroid patients coincided with a decrease in FAS gene expression in thyroid patients and the study showed that FAS and FASL gene expression levels are closely correlated with thyroid hormones.

Dietary Supplementation with Rumen-Protected Arginine or N-Carbamylglutamate Enhances Fetal Liver Development in Nutrient-Restricted Pregnant Hu Ewes.

This study was conducted in nutrient-restricted pregnant Hu ewes to determine whether rumen-protected arginine (RP-Arg) or N-carbamylglutamate (NCG) supplementation affects fetal liver growth and development. From 35 d to 110 d of gestation, 32 Hu ewes were randomly divided into four groups: a control group (100% of the National Research Council (NRC) requirements), a nutrient-restricted group (50% of the NRC requirements), and two treatment groups (ARG and NCG, 50% of the NRC requirements, supplemented with 20 g/day RP-Arg or 5 g/day NCG, respectively). Fetal body weights, fetal liver growth performance, the capability of antioxidation, and the expression of the mRNA and proteins of apoptosis-related genes in the fetal liver were determined and analyzed at 110 d of gestation. The dry matter, water, fat, protein, and ash components of the fetal livers in the RG group were found to be lower than in the CG group, and these components were significantly higher in the NCG group than in the RG group (p < 0.05). A decrease in DNA, RNA, and protein concentrations and contents, as well as in protein/DNA ratios, was observed in the RG group in comparison to the CG group (p < 0.05). Compared with the RG group, the NCG group had higher concentrations of DNA, RNA, and protein, as well as higher protein/DNA ratios (p < 0.05). The RG group had lower concentrations of cholinesterase, nitric oxide, nitric oxide synthase, superoxide dismutase, alanine aminotransferase, and total protein than the CG group (p < 0.05). The RG group had higher levels of glutathione peroxidase, maleic dialdehyde, and aspartate aminotransferase than the CG group (p < 0.05). In the RG group, the mRNA and protein expression of p53 and Bax was significantly increased (p < 0.05) compared with the CG group, and the gene expression of FasL and Bcl-2, the ratio of Bcl-2 to Bax, and the protein expression of Bcl-2 in the RG group were lower (p < 0.05) than in the CG group. It appears that RP-Arg and NCG supplementation during pregnancy could influence fetal liver growth and development. A nutrition-based therapeutic intervention to alleviate reduced fetal growth can be developed based on this study, which has demonstrated that maternal undernutrition during pregnancy induces the maldevelopment of the fetal liver.

Cardiac Left Ventricular miRNA-26a Is Downregulated in Ovariectomized Mice, Upregulated upon 17-Beta Estradiol Replacement, and Inversely Correlated with Collagen Type 1 Gene Expression.

Heart failure with preserved ejection fraction (HFpEF) is more prevalent in post- compared to pre-menopausal women. The underlying mechanisms are not fully understood. Data in humans is confounded by age and co-morbidities. We investigated the effects of ovariectomy and estrogen replacement on the left ventricular (LV) gene expression of pro-inflammatory and pro-fibrotic factors involved in HFpEF and putative regulating miRNAs. Nine-week-old C57BL/6 female mice were subjected to ovariectomy (OVX) or SHAM operation. OVX and SHAM groups were sacrificed 1-, 6-, and 12-weeks post-surgery (T1/SHAM; T1/OVX; T6/SHAM; T6/OVX, T12/SHAM). 17β-estradiol (E2) or vehicle (VEH) was then administered to the OVX groups for 6 weeks (T12/OVX/E2; T12/OVX/VEH). Another SHAM group was sacrificed 12-weeks post-surgery. RNA and miRNAs were extracted from the LV apex. An early 3-fold increase in the gene expression of IL-1α, IL-6, Mmp9, Mmp12, Col1α1, and Col3α1 was observed one-week post-surgery in T1/OVX vs. T1/SHAM, but not at later time points. miRNA-26a was lower in T1/OVX vs. T1/SHAM and was inversely correlated with Col1α1 and Col3α1 expression 1-week post-surgery (r = -0.79 p < 0.001; r = -0.6 p = 0.007). miRNAs-26a, 29b, and 133a were significantly higher, while Col1α1, Col3α1, IL-1α, IL-6, Tnfα, Mmp12, and FasL gene expression was significantly lower in E2- compared to vehicle-treated OVX mice. miRNA-26a was inversely correlated with Col3α1 in T12/OVX/ E2 (r = -0.56 p = 0.02). OVX triggered an early increase in the gene expression of pro-inflammatory and pro-fibrotic factors, highlighting the importance of the early phase post-cessation of ovarian function. E2 replacement therapy, even if it was not immediately initiated after OVX, reversed these unfavorable changes and upregulated cardiac miRNA-26a, previously unknown to be affected by menopausal status.

Demonstrating the immunostimulatory and cytokine-augmentation effects of bacterial ghosts on natural killer cells and Caenorhabditis Elegans.

The potential of bacteria-based immunotherapy lies in its ability to inherently enhance immune responses. However, the "liveness" of bacteria poses risks of bacterial escape, nonspecific immuno-stimulation, and ethical concerns, limiting their acceptability in immunotherapy. In this scenario, nonliving empty bacterial-cell envelopes, named bacterial ghosts (BGs), have emerged as immuno-stimulants with the potential to side-step the limitations of live bacterial therapies. This study demonstrates the capability of BGs in modulating the functionality of NK-92 cells and Caenorhabditis elegans (C. elegans), as well as perform as cytokine-therapy adjuvants. BGs were obtained through a pH-driven culture method, and were validated for their structural and chemical integrity via electron microscopy and spectroscopy. In NK-92 cells, BGs have shown significant immuno-stimulation by boosting the gene-expression of perforin, granzyme-B, Fas-L, and interferon-gamma by factors of 3.5-, 1.5-, 12.5-, and 8.6-folds, respectively. Combined BG and IL-12 treatment yielded a notable 10.2-fold increase in interferon-gamma protein expression in 24 h. The BGs also significantly influenced the innate immune response in C. elegans through the upregulation of lysozyme genes viz., ilys-3 (8.8-fold) and lys-2 (3.1-fold). Our investigation into the impact of BGs on natural killer cells and C. elegans highlights its potential as a valid alternative approach for new-age immunotherapy and cytokine augmentation.

Plasma Levels of sFas-sFasL and FASL Gene Expression Are Associated with Tuberculosis.

Apoptosis of macrophages infected by Mycobacterium tuberculosis via Fas-FasL is an important immune mechanism against infection. This study investigated the association of tuberculosis (TB) with the presence of the polymorphisms FAS -670A/G and FASL -124A/G, the levels of sFas and sFasL, and the gene expression of FASL and cytokines. Samples of 200 individuals diagnosed with TB and 200 healthy controls were evaluated. Real-time PCR (genotyping and gene expression) and ELISA (dosages of sFas, sFasL, IFN-γ, and IL-10) tests were performed. There was no association of FAS -670A/G and FASL -124A/G polymorphisms with TB. The TB group exhibited high plasma levels of sFas and reduced plasma levels of sFasL (p < 0.05). The correlation analysis between these markers revealed a positive correlation between the levels of sFas and sFasL, sFasL and FASL expression, and between sFas and FASL expression (p < 0.05). In the TB group, there was a positive correlation between FASL expression and IFN-γ levels and higher levels of IL-10 compared to IFN-γ (p < 0.05). High levels of sFas and reduced levels of sFasL and FASL expression may contribute to the inhibition of apoptosis in infected cells and represent a possible bacterial resistance resource to maintain the infection.

Effect of valproic acid on extrinsic and intrinsic apoptotic pathways, cell viability and apoptosis in hepatocellular carcinoma PLC/PRF5 cell line.

Inhibitors of histone deacetylase enzymes induce various molecular and extracellular effects that lead to their anticancer role. In this study, the result of valproic acid on the expression of genes of extrinsic pathway and intrinsic pathway of apoptosis, cell viability, and apoptosis in liver cancer PLC/PRF5 cell line was designed. For this purpose, PLC/PRF5 liver cancer cells were cultured, and after the overlapping of the cells reached about 80%, the cells were collected with trypsin and after washing, they were cultured on a plate with a concentration of 3 x 105. After 24 hours, the culture medium was treated with a medium containing valproic acid (the control group received only DMSO). 24, 48, and 72 hours after treatment to determine the cell viability, apoptotic cells, gene expression, MTT, flow cytometry, and Real-time techniques. The results showed that valproic acid significantly inhibited cell growth, induced apoptosis, and decreased the expression of Bcl-2 and Bcl-xL genes. Also, it increased the expression of DR4, DR5, FAS, FAS-L, TRAIL, BAX, BAK, and APAF1 genes. In general, valproic acid seems to play its apoptotic role through intrinsic and extrinsic pathways in liver cancer.

The mechanism of immune related signal pathway Egr2-FasL-Fas in transcription regulation and methylated modification of Paralichthys olivaceus under acute hypoxia stress

Apoptosis genes Egr2, Fas and FasL are related to immune responses. However, the mechanism of these genes inducing apoptosis in fish are still not very clear. An acute hypoxia treatment (1.73 ± 0.06 mg/L) for 24 h was carried out on Japanese flounder (Paralichthys olivaceus). The increasingly dense apoptotic signals at 3 h, 6 h, 12 h by TUNEL in skeletal muscle indicated that hypoxia could quickly affect muscle growth and development. Furthermore, we concluded that the Egr2-FasL-Fas signal pathway, which was located at the upstream of apoptotic executor protein caspases, was related to the apoptosis by quantitative real-time PCR, protein concentration detection in ELISA and double gene in situ hybridization methods. The mechanism of the pathway was researched in transcription regulation and epigenetic modification by dual-luciferase reporter assay and bisulfite modified method, respectively. Egr2, as a transcription factor, could up-regulate the expression of FasL gene. And its binding site was mainly between −479 to −1 of FasL gene promoter. The 5th CpG dinucleotides (−514) methylation levels in FasL gene were significantly affected by hypoxia, and they were negatively correlated with its expressions. These suggested that the −514 site may be a very important site to regulate the FasL gene expression. Above results, we concluded that hypoxia activated the immune related signal pathway Egr2-FasL-Fas to induced skeletal muscle apoptosis to affect growth and development of Japanese flounder. The study revealed the mechanism of hypoxia induced apoptosis, which could provide a reference for fish immunity and aquaculture management.

Synergistic Anti-proliferative Effects of Lenalidomide and Dexamethasone on the HT-29 Cell Line Through Apoptotic Genes

Background: Colorectal cancer (CRC) is the third most common cancer among men and the second most common type of cancer among women worldwide. The resistance of tumor cells to apoptosis is caused by changes in the expression of anti-apoptotic or pro-apoptotic proteins. Histone deacetylase inhibitors (HDACi) are known to cause changes in gene expression. Objectives: The present study aimed at investigating the anti-proliferative effects of lenalidomide (LEN) as HDACi and dexamethasone (DEX) on the human colon cancer HT-29 cell line. Methods: The HT-29 cell line was treated with various concentrations of LEN and DEX individually and in combination for 24, 48, and 72 hours. Cytotoxicity was evaluated by MTT assay. The half-maximal inhibitory concentration (IC50) was measured, and quantitative real-time polymerase chain reaction (qRT-PCR) was also performed to examine the expression of Bcl2, Bax, Fas, and FasL genes. Results: The combination of LEN (1000 µM) with DEX (100 µM) showed potent synergistic anti-proliferative activities in a time- and dose-dependent manner. The combination of these drugs induced cell death by affecting the extrinsic and intrinsic apoptotic gene expression profiles. Conclusions: The combination of LEN with DEX can be proposed as a new therapeutic approach for CRC.

MiR-let-7i inhibits CD4+ T cell apoptosis in patients with acute coronary syndrome.

Abnormal CD4+ T cells appear in the peripheral blood of patients with acute coronary syndrome (ACS). Studies have confirmed that CD4+ T cells are resistant to apoptosis, but the specific mechanism has not been elucidated yet. The microRNA (miR)-let-7i plays an important regulatory role in the cardiovascular system and is widely involved in cell proliferation and apoptosis. In this study, we aimed to investigate its functional and regulatory roles in CD4+ T cell apoptosis. Apoptosis of CD4+ T cells was detected using TUNEL assay. Western blot analyses were used to detect the expression of Bcl-2 and Bax. Real-time polymerase chain reaction and western blot analyses were used to detect the expression of miR-let-7i, Fas and FasL. A miR-let-7i mimic or inhibitor was transfected into CD4+ T cells, and miR-let-7i activity was investigated using Cell Counting Kit-8 (CCK-8) and TUNEL assays. Apoptosis of CD4+ T cells in ACS patients was significantly decreased. Overexpression of miR-let-7i inhibited CD4+ T cell apoptosis and improved cell survival rates, while inhibition of miR-let-7i facilitated cell apoptosis. We also found that miR-let-7i negatively regulated Fas and FasL gene expression in CD4+ T cells. The present study identified that miR-let-7i significantly reduces Fas and FasL expression in ACS CD4+ T cells and inhibits apoptosis in these cells. Therefore, miR-let-7i may serve as a possible therapeutic target for the treatment of ACS.

Intrinsic and Extrinsic Cell Apoptotic Pathways in Patients with HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis: A Systematic Review.

We aimed to verify the influence of intrinsic and extrinsic cell apoptotic pathways on the inhibition of cellular apoptosis in patients with tropical spastic paralysis/myelopathy related to human T cell lymphotropic virus type 1. The databases accessed were PubMed, Scopus, Science Direct, and Web of Science. Neither the time of publishing nor the language of the articles was limited. The descriptors used for this systematic literature review were: Tropical Paraparesis, Proto-Oncogenic Protein C, Bcl-2, Bcl-X Protein, Bax protein, Fas ligand (FasL) protein, Fas receptor, TNF-related apoptosis-inducing ligand and Fas-associated protein with death domain (FADD)-like apoptosis regulating. The search resulted in 546 articles from which 9 articles were selected for analysis; ranging from serum levels of Bcl-2, Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) measured by enzyme-linked immunosorbent assay and the levels of cellular expression of Bcl-2 and Bcl-xL the TCD4+ lymphocytes accessed by western blot. Most studies accessed either gene expression or polymorphism of Fas, FasL, and TRAIL in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), whereas one study used flow cytometry and fluorescence to determine Fas expression. Increased Bcl-xL expression inhibited T lymphocyte apoptosis, whereas Bcl-2, serum levels, and cellular expression did not influence T lymphocyte apoptosis and serum levels of Fas were significantly higher and associated with markers of leukocyte activation in patients with HAM/TSP. In addition, Fas polymorphism (FAS-670AA) was associated with higher proviral load. There is a need for additional research on this issue since the number of patients was small and the studies presented higher heterogeneity.

In "Vitro" Lps-Stimulated Sertoli Cells Pre-Loaded With Microparticles: Intracellular Activation Pathways.

Sertoli cells (SC) are immune privileged cells with the capacity of modulating the immune response by expressing several immune-regulatory factors. SC have the capacity to respond to external stimuli through innate phagocytic and antibacterial activities. This evidence evoked a potential role of SC as drug carriers and therapeutic agents. Such stimuli drive SC towards a still unknown evolution, the clinical relevance of which as yet remains undisclosed. This study sought to investigate the effects of external stimuli in the form of polymeric microparticles (MP) and bacteria derived endotoxins, such as lipopolysaccharides (LPS), in order to identify the pathways potentially involved in cell phenotype modifications. Compared to single stimulation, when combined, MP and LPS provoked a significant increase in the gene expression of IDO, PD-L1, FAS-L, TLR-3, TLR-4, MHC-II, ICAM-1, TFGβ1, BDF123, BDF129, BDF3 and pEP2C. Western Blotting analysis demonstrated up-regulation of the ERK 1–2 and NF-kB p65 phosphorylation ratios. Our study, showing the exponential increase of these mediators upon combined MP and LPS stimulation, suggests a “switch” of SC function from typical cells of the blood-testicular barrier to nonprofessional tolerogenic antigen-presenting cells. Further studies should target the clinical and technological implications of such stimuli-induced SC transformation.

Epigenetic Mechanisms Underlying HIV-Infection Induced Susceptibility of CD4+ T Cells to Enhanced Activation-Induced FasL Expression and Cell Death.

Chronic immune activation and CD4 T cell depletion are significant pathogenic features of HIV infection. Expression of Fas ligand (FasL), a key mediator of activation-induced cell death in T cells, is elevated in people living with HIV-1 infection (PLWH). However, the epigenetic mechanisms underlying the enhanced induction of FasL expression in CD4 T lymphocytes in PLWH are not completely elucidated. Hence, the current work examined the effect of HIV infection on FasL promoter-associated histone modifications and transcriptional regulation in CD4 T lymphocytes in PLWH. Flow cytometric analysis was performed to examine the Fas-FasL expression on total CD4 T cells and naïve/memory CD4 T cell subsets. Epigenetic FasL promoter histone modifications were investigated by chromatin immunoprecipitation-quantitative real-time polymerase chain reaction analysis using freshly isolated total CD4 T lymphocytes from HIV-1 infected and noninfected individuals. All naïve/memory CD4 T cell subsets from PLWH showed markedly greater frequency of FasL expression. Notably, examination of functional outcome of FasL/Fas co-expression demonstrated the preferential susceptibility of Tcm and Tem subsets to activation-induced apoptosis. Importantly, these CD4 T cells collectively demonstrated a distinct FasL promoter histone profile involving a coordinated cross-talk between histone H3 modifications leading to enhanced FasL gene expression. Specifically, levels of transcriptionally permissive histone H3K4-trimethylation (H3K4Me3) and histone H3K9-acetylation (H3K9Ac) were increased, with a concomitant decrease in the repressive H3K9-trimethylation (H3K9Me3). The present work demonstrates that epigenetic mechanisms involving promoter-histone modifications regulate transcriptional competence and FasL expression in CD4 T cells from PLWH and render them susceptible to activation-induced cell death.

Effect of Eight Weeks of Resistance Training and Consumption of Tribulus Terrestris on Androgenic Receptor-1, Fas Ligand Gene Expression, and Lipid Profiles in Rats Exposed to Stanozolol

Background: Protective effect of medicinal plants on the heart has been reported, but the effect of resistance training (RT) and Tribulus terrestris (TT) on the heart exposed to anabolic-androgenic steroids (AAS) abuse is still unknown. Objectives: The present study aimed to investigate the effect of RT and TT on androgen receptor-1 (ar-1), Fas ligand (fasl) gene expression and lipid profiles in rats exposed to stanozolol (S). Methods: Thirty-five male rats were selected and divided into 7 groups as follows: (1) sham (normal saline/Sh), (2) stanozolol (S), (3) S+100 mg/kg TT (S+TT100), (4) S+ 50 mg/kg TT (S+TT50), (5) S+RT+TT, (6) S+RT+TT100, and (7) S+RT+TT50. Over a course of eight-week period, groups 3, 4, 6, and 7 received 50 and 100 mg/kg/d doses of TT peritoneally and groups 5-7 performed three sessions of increasing RT per week. Results: RT decreased plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) levels, as well as ar-1 and fasl gene expression in S-exposed rats (P&lt;0.05). TT50, TT100, SRTT100, and SRTT50 reduced ar-1 and fasl gene expressions (P&lt;0.05). TT50 reduced triglyceride (TG), cholesterol and increased high-density lipoprotein-cholesterol (HDL-C) (P≤0.01), and TT100 decreased LDL-C levels (P&lt;0.05). Additionally, SRTT100 reduced TG, cholesterol, and LDL-C levels and increased HDL-C level (P&lt;0.05), and SRTT50 decreased cholesterol level and increased HDL-C level in S-exposed rats (P&lt;0.05). Conclusion: RT and consumption of TT appear to have protective effects on the improvement of apoptosisdependent androgen receptor-1 and lipid profile in S-exposed rats.

In vitro effect of curcumin-mediated antimicrobial photodynamic therapy on fibroblasts: viability and cell signaling for apoptosis.

Although it was demonstrated that curcumin-mediated antimicrobial photodynamic therapy (aPDT) is effective for reducing the viability of microbial cells and the vitality of oral biofilms, the cytotoxicity of this therapeutic approach for host cells has not been yet elucidated. Hence, the aim of this study was to evaluate the cytotoxicity and apoptotic effects of curcumin-mediated aPDT on mouse fibroblasts. Cells were treated with 0.6 or 6 μmol.L-1 curcumin combined with 0.075 or 7.5 J.cm-2 LED at 455 nm. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while quantitative reverse transcriptase-PCR (qRT-PCR) was used to assess the expression of Bax, Bad, Bcl-2, VDAC-1, cytochrome C, and Fas-L genes for apoptosis. The differences between groups were detected by Kruskal-Wallis and post hoc Dunn's tests for MTT and CV assays and by ANOVA and post hoc Tukey test for qRT-PCR (P < 0.05). The effect of 0.6 μmol.L-1 curcumin plus 0.075 J.cm-2 LED (minimum parameter) did not differ statistically from control group; however, the combination of 0.6 μmol.L-1 curcumin plus 7.5 J.cm-2 LED reduced viable cells in 34%, while the combinations of 6 μmol.L-1 curcumin plus 0.075 and 7.5 J.cm-2 LED reduced viable cells in 47% and 99%, respectively. aPDT increased significantly the relative expression of Bax/Bcl-2, cytochrome C, VDAC-1, and Fas-L genes, without influence on the ratio Bad/Bcl-2. Therefore, curcumin-mediated aPDT activated Bcl-2 apoptosis signaling pathways in mouse fibroblasts regarding present conditions, reducing the viability of cells with the increase of curcumin concentrations and light energies.

Mechanism and significance of apoptosis of the immortalized human oral mucosal epithelial cells established by Lentivirus-mediated hTERT.

During the transition from human oral mucosal epithelial cells (HOMEC) to oral squamous cell carcinoma cells (Cal27), the cells must have undergone a precancerous state. To explore the malignant rule of HOMEC, plv-HOMEC was used as a model cell for the precancerous state to investigate plv-HOMEC's apoptosis by comparing human oral mucosal epithelial cells established by Lentivirus-mediated hTERT (plv-HOMEC) with HOMEC and human Cal27. The lentiviral particles overexpressing hTERT were packaged and transfected into primary HOMEC to obtain plv-HOMEC. Expression levels of NF-κB were detected in the cytoplasm and nucleus of Cal27, plv-HOMEC and HOMEC. The level of intracellular reactive oxygen species was measured to verify the endoplasmic reticulum pathway, cytochrome C expression was detected to verify the mitochondrial pathway, and FasL gene expression was detected to verify the death receptor apoptosis pathway. The total expression of NF-κB in plv-HOMEC increased, mainly due to the greater nuclear import of NF-κB, but it was still much lower than Cal27. The endoplasmic reticulum apoptosis pathway of plv-HOMEC was not significantly affected, and there were no significant differences between them and the HOMEC cells; the mitochondrial apoptosis pathway of plv-HOMEC was inhibited, and the expression of Cyt C was very close to that of Cal27, indicating that the characteristics of plv-HOMEC are so familiar with cancer cells; the death receptor apoptosis pathway of plv-HOMEC was also inhibited, and in this apoptotic pathway, plv-HOMEC were more similar to cancer cells than to HOMEC cells. The present data suggest that NF-κB nucleation may increase in the early stage of healthy cells' carcinogenesis, followed by inhibition of the mitochondrial pathway and the death receptor apoptotic pathway.

Hesperetin upregulates Fas/FasL expression and potentiates the antitumor effect of 5-fluorouracil in rat model of hepatocellular carcinoma

ABSTRACT This study aimed to investigate the antitumor efficacy of hesperetin (HST) either alone or combined with 5-fluorouracil (5-FU) in thioacetamide (TAA)-induced hepatocellular carcinoma (HCC) in rats. Moreover, possible HST molecular mechanism(s) of action were investigated. Sixty male Sprague-Dawley rats were randomized into six groups (n = 10/group); normal control (no treatment), HCC (TAA), HCC/carboxymethyl cellulose (CMC) (TAA+CMC), HST (TAA+HST in CMC), 5-FU (TAA+5-FU), HST/5-FU (TAA+HST+5-FU). Serum levels of liver enzymes (ALT and AST), bilirubin, albumin, total protein and alpha-Fetoprotein (AFP) along with hepatic contents of hepatic glutathione and mal0ndialdhyde were measured. Gene expression of Fas, FasL, caspase-8, caspase-3 and Bcl-2 were measured using real-time polymerase chain reaction. Histopathological examination was used to assess nodule size, fibrosis ratio and necroinflammatory score. HST showed improvements in hepatic functions, decreased levels of AFP and oxidative stress and restricted HCC progression. HST activated apoptosis through inducing Fas and FasL and inhibiting Bcl-2 gene expressions. Furthermore, HST/5-FU combination surpassed the results obtained from either HST or 5-FU groups. In conclusion, HST exerted its antitumor effect through induction of Fas/FasL apoptotic pathway and reduction of oxidative stress. HST and 5-FU combination provided prominent synergistic effects, thus it can be regarded as promising combination therapy for HCC.

Sheng Mai San protects H9C2 cells against hyperglycemia-induced apoptosis

BackgroundSheng Mai San (SMS) has been proven to exhibit cardio-protective effects. This study aimed to explore the molecular mechanisms of SMS on hyperglycaemia (HG)-induced apoptosis in H9C2 cells.MethodsHG-induced H9C2 cells were established as the experimental model, and then treated with SMS at 25, 50, and 100 μg/mL. H9C2 cell viability and apoptosis were quantified using MTT and Annexin V-FITC assays, respectively. Furthermore, Bcl-2/Bax signalling pathway protein expression and Fas and FasL gene expression levels were quantified using western blotting and RT-PCR, respectively.ResultsSMS treatments at 25, 50, 100 μg/mL significantly improved H9C2 cell viability and inhibited H9C2 cell apoptosis (p < 0.05). Compared to the HG group, SMS treatment at 25, 50, and 100 μg/mL significantly downregulated p53 and Bax expression and upregulated Bcl-2 expression (p < 0.05). Moreover, SMS treatment at 100 μg/mL significantly downregulated Fas and FasL expression level (p < 0.05) when compared to the HG group.ConclusionSMS protects H9C2 cells from HG-induced apoptosis probably by downregulating p53 expression and upregulating the Bcl-2/Bax ratio. It may also be associated with the inhibition of the Fas/FasL signalling pathway.

Synergistic effect of acupuncture and moxibustion on expression of renal Fas and FasL mRNAs and proteins in contrast-induced nephropathy diabetic rats

To observe the effect of acupuncture and moxibustion on renal Fas and Fas ligand (FasL) expression in contrast-induced nephropathy (CIN) diabetic rats, so as to explore its protective mechanisms. A total of 40 diabetic male SD rats were randomly divided into control, model, acupuncture, moxibustion and combined treatment (acupuncture combined with moxibustion) groups (n＝8 rats in each group). The diabetic model was established by i．p. injection of Streptozotocin (60 mg/kg), followed by regular raising for 9 weeks. Then, the manual acupuncture, moxibustion or acupuncture plus moxibustion at "Sanyinjiao"(SP6) "Shenshu"(BL23) and "Pishu"(BL20) was given to the diabetic rats, once daily for 7 days. On the 8th day, these diabetic rats received i．p. injection of compound Meglumine Diatrizoate Injection (10 mL/kg) for establishing CIN model. Then, the venous blood specimens were collected for detecting contents of serum urine nitrogen (BUN, with urease method) and creatinine (Scr, with picric acid method). The kindey tissues were exteriorized for detecting nitricoxide synthase (NOS, with oxidation-reduction method), malondialdehyde (MDA, with thiobarbituric acid method), superoxide dismutase (SOD, with xanthine oxidase technique) and total antioxidant capacity (T-AOC, with colorimetric technique). The expression levels of Fas and FasL in the kidney tissues were detected by quantitative real-time PCR and Western blot separately. The histopathological and ultrastructural changes of the kidney were observed under microscope (after H．E. staining) and transmission electron microscope, respectively. Following modeling and compared with the control group, the BUN, Scr and renal MDA contents, and Fas and FasL mRNA and protein expression levels were significantly increased (P<0.01, P<0.05), and renal NOS, SOD and T-AOC levels considerably decreased in the model group (P<0.05). After the interventions and compared with the model group, serum BUN and Scr contents and renal Fas mRNA and protein expression levels in both acupuncture group and combined treatment group, BUN in the moxibustion group, and renal MDA content and FasL mRNA and protein expression levels in the 3 treatment groups were obviously down-regulated (P<0.05，P<0.01), while renal NOS in the 3 treatment groups, and SOD and T-AOC levels in the combined treatment group were considerably up-regulated (P<0.01，P<0.05). The effects of the combined treatment were significantly superior to those of simple acupuncure and simple moxibustion in down-regulating serum BUN contents, and Fas and FasL mRNA and protein expression levels, and in up-regulating renal NOS, SOD and T-AOC activity (P<0.05). No significant differences were found between the acupuncture and moxibustion groups in down-regulating serum BUN, Scr and renal MDA contents, and Fas and FasL mRNA and protein levels, as well as in up-regulating NOS content (P>0.05). H．E. staining showed lobular changes of glomeruli, many local necrosis and vascular degeneration in the model group, and transmission electron microscope displayed thickened basement membrane of the glomerular blood loop, many vacuoles in the cytoplasm of renal tubular epithelial cells, severely swollen mitochondria with disordered, broken or disappeared cristae, and some apoptotic cells in the model group, which were relatively milder in the combined treatment group. Joint application of acupuncture and moxibustion can reduce the oxidative stress and renal injury in CIN diabetic rats, which may be associated with its function in down-regulating the expression of renal Fas and FasL genes and proteins. Acupuncture and moxibustion has a synergistic effect.

HTLV-1-infected asymptomatic carriers compared to HAM/TSP patients over-express the apoptosis- and cytotoxicity-related molecules.

HTLV-1 infection causes a chronic progressive debilitating neuroinflammatory disease which is called, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the host defense mechanisms against viral infection is apoptosis which may control HTLV-1 infection. Therefore, we aimed to investigate this process and its interaction with viral factors in HTLV-1-infected asymptomatic carriers (ACs) compared to HAM/TSP patients. Fas, FasL, TRAIL, perforin, granzyme A, granzyme B, and granulysin gene expression and serum levels of Fas, FasL, TRAIL, and granulysin in the peripheral blood of 21 sex- and age-matched healthy controls (HCs), ACs, and HAM/TSP patients were evaluated. Also, the level of granulysin secretion in the cell culture supernatant was measured. Finally, the correlation of the expression of these molecules with HTLV-1 proviral load (PVL), Tax, and HBZ mRNA expression was analyzed. ACs compared to HAM/TSP patients significantly over-expressed the Fas, FasL, TRAIL, perforin, and granzyme B molecules. Fas, FasL, TRAIL, and granulysin serum levels were not different among studied groups; whereas, the secretion of granulysin was significantly decreased in ACs and HAM/TSP patients compared to HCs. Also, HAM/TSP patients expressed higher levels of HTLV-1 PVL, Tax, and HBZ mRNA. In addition, in ACs, inverse correlations between the Fas, FasL, TRAIL, perforin, granzyme B, and granulysin levels with HBZ mRNA expression were seen. ACs compared to HAM/TSP patients over-expressed the apoptosis- and cytotoxicity-related molecules. It could be concluded that successful control of the HTLV-1 infection and suppression of HAM/TSP development stem from the strong apoptosis and cytotoxic activity in the peripheral blood of ACs.

Fas, FasL and Foxp3 gene expression in post-liver transplant autoimmune hepatitis patients with and without acute rejection.

Aim of the studyIn this study we investigated Fas, FasL and Foxp3 expression in relation to liver graft rejection and its severity in autoimmune hepatitis (AIH) patients.Material and methodsTwenty-three AIH patients including five post-transplant patients with acute rejection (AR) and 18 patients without AR (non-AR) were studied for Fas, FasL and Foxp3 gene expression in peripheral blood mononuclear cells on days 1, 3 and 7 after transplantation by real-time PCR. The relationships between gene expression and clinical features were determined.ResultsReal-time PCR showed various Fas gene expression levels with no significant difference between the days in AR patients (p = 0.52). In non-AR patients, Fas level increased from 0.98 ±0.24 fold on the first day to 1.89 ±0.42 fold on day 3 after transplantation (p < 0.01). In this group of patients, we also found a significant increase in FasL expression on day 7 (29.91 ±6.89 fold) compared to day 1 (13.50 ±7.44 fold, p < 0.05). Foxp3 gene expression in both groups showed decreased levels during the first week after transplantation. The decreased Foxp3 expression in AR patients was correlated with rejection activity index (r = 0.86, p < 0.0001).ConclusionsIncreased Fas and FasL gene expression levels in non-AR patients and decreased Foxp3 gene expression in both groups suggested the important role of these molecules in the alloreactive response after liver transplantation in AIH patients. Foxp3 expression might be useful for monitoring rejection severity.