Caspase-3 Protein Expression Research Articles

Piceatannol-Loaded Bilosome-Stabilized Zein Protein Exhibits Enhanced Cytostatic and Apoptotic Activities in Lung Cancer Cells.

Piceatannol (PIC) is a naturally occurring polyphenolic stilbene, and it has pleiotropic pharmacological properties. Moreover, PIC has cytotoxic actions among various cancer cells. In this work, preparations of PIC-loaded bilosome–zein (PIC-BZ) were designed, formulated, and characterized, and the optimized PIC-BZ cytotoxic activities, measured as half maximal inhibitory concentration (IC50), against lung cancer cell line was investigated. Box–Behnken design was utilized in order to examine the effect of preparation factors on drug entrapment and particle size. PIC-BZ showed a spherical shape after optimization, and its particle size was determined as 157.45 ± 1.62 nm. Moreover, the efficiency of drug entrapment was found as 93.14 ± 2.15%. The cytotoxic activity evaluation revealed that the adjusted formulation, which is PIC-BZ formula, showed a substantially smaller IC50 versus A549 cells. Cell cycle analysis showed accumulation of cells in the G2-M phase. Moreover, it showed in the sub-G1 phase, a rise of cell fraction suggestion apoptotic improving activity. Increased early and late phases of apoptosis were demonstrated by staining of cells with annexin V. Furthermore, the cellular caspase-3 protein expression was significantly raised by PIC-BZ. In addition, the wound healing experiment confirmed the results. To conclude, compared to pure PIC, PIC-BZ demonstrated a higher cell death-inducing activity against A549 cells.

Mitochondrial pathway of the lysine demethylase 5C inhibitor CPI-455 in the Eca-109 esophageal squamous cell carcinoma cell line.

BACKGROUNDEsophageal cancer is a malignant tumor of the digestive tract that is difficult to diagnose early. CPI-455 has been reported to inhibit various cancers, but its role in esophageal squamous cell carcinoma (ESCC) is unknown.AIMTo investigate the effects and mechanism of the lysine demethylase 5C inhibitor, CPI-455, on ESCC cells.METHODSA methyl tetrazolium assay was used to detect the inhibitory effect of CPI-455 on the proliferation of Eca-109 cells. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential were assessed by flow cytometry. Laser confocal scanning and transmission electron microscopy were used to observe changes in Eca-109 cell morphology. The protein expression of P53, Bax, lysine-specific demethylase 5C (KDM5C), cleaved Caspase-9, and cleaved Caspase-3 were assayed by western blotting.RESULTSCompared with the control group, CPI-455 significantly inhibited Eca-109 cell proliferation. Gemcitabine inhibited Eca-109 cell proliferation in a concentration- and time-dependent manner. CPI-455 caused extensive alteration of the mitochondria, which appeared to have become atrophied. The cell membrane was weakly stained and the cytoplasmic structures were indistinct and disorganized, with serious cavitation when viewed by transmission electron microscopy. The flow cytometry and western blot results showed that, compared with the control group, the mitochondrial membrane potential was decreased and depolarized in Eca-109 cells treated with CPI-455. CPI-455 significantly upregulated the ROS content, P53, Bax, Caspase-9, and Caspase-3 protein expression in Eca-109 cells, whereas KDM5C expression was downregulated.CONCLUSIONCPI-455 inhibited Eca-109 cell proliferation via mitochondrial apoptosis by regulating the expression of related genes.

Research on the effect of interfering with miRNA-155 on triple-negative breast cancer cells.

Triple negative breast cancer (TNBC) is a poor prognosis breast cancer with the highest mutation rate and limited treatment options. MiR-155 is highly expressed in TNBC, but its role and potential mechanism in TNBC remain to be elucidated. The aim of this study is to examine the effect of interfering with miRNA-155 on the inflammatory pathway of NLRP 3 in TNBC (MDA-MB-231). MiRNA-155-specific interference (Si-miR-155) on MDA-MB-231 cell was manifested by transfection of miRNA-155 inhibitor. Meanwhile, blank control (Blank) and negative control (NC) were set. Cell growth and proliferation rate were detected by MTT; apoptosis rate were detected by flow cytometry; colony forming test was used to detected cell viability; cell migration ability was detected by Wound healing assay; TNF-α, IL-18, IL-6 and IL-1β levels were detected by ELISA. The mRNA of miRNA-155, NLRP3, ASC, caspase-1 and Ki67 were detected by qRT-PCR. The expression levels of NLRP3, caspase-1, ASC and Ki67 were detected by Western blotting. The proliferation rate of Si-miRNA-155 group decreased, while the apoptosis rate increased significantly. After interfering with miRNA-155, the number of cancer cell colonies and the migration ability was decreased, and the secretion levels of IL-18, TNF-α, IL-6 and IL-1β were also inhibited. Moreover the mRNA and protein expression of NLRP3, caspase-1, ASC and Ki67 were significantly suppressed. Interference with miRNA-155 can inhibit the NLRP3 pathway of MDA-MB-231 cells, as well as the proliferation, migration and inflammatory factor secretion of MDA-MB-231 cell, and can accelerate its apoptosis.

Boswellia serrata suppress fipronil-induced neuronal necrosis and neurobehavioral alterations via promoted inhibition of oxidative/inflammatory/apoptotic pathways

Boswellic acid (BA) is a pentacyclic terpenoid derived from the gum-resin of Boswellia serrate. It is known for its strong antioxidant, anti-inflammatory, and anticancer properties. It has improved spatial learning and provides neuroprotection against trimethyltin-induced memory impairment. The aim of this study is to evaluate the possible neuroprotective activity of B. serrata extract (BSE) containing BA against fipronil (FPN)-induced neurobehavioral toxicity in Wister male albino rats. Sixty male rats were allocated equally into six groups. The first group served as control; the second and third groups received BSE at two different oral doses (250 or 500 mg/kg body weight [BW], respectively). The fourth group was orally intoxicated with FPN (20 mg/kg BW), whereas the fifth and sixth groups served as preventive groups and co-treated with FPN (20 mg/kg BW) and BSE (250 or 500 mg/kg BW, respectively). The experiment was conducted over 8 weeks period. Results revealed that co-treatment with BSE led to significant (p > 0.05) dose-dependent reduction in malondialdehyde (MDA), nitric oxide (NO), interleukin-6 (IL6), tumor necrosis factors-alpha (TNF-α), nuclear factor Kappa-B (NF-κB), Cyclooxegenase-2 (COX-2), prostaglandin E2 (PGE2), serotonin, and acetylcholine (ACh). Conversely, significant (p > 0.05) up regulation of catalase (CAT), glutathione peroxidase (GSH-Px), gamma-aminobutyric acid (GABA), and acetylcholine esterase (AChE) has reported in BSE-co-treated groups. In addition, significant (p > 0.05) promotion in neurobehaviours, histopathologic imaging of the cerebral, cerebellar, and hippocampal regions, and immunohistochemical expression of caspase-3 and glial fibrillary acidic protein (GFAP) were also reported in the BSE-treated groups in a dose-dependent manner. In conclusion, BSE (500 mg/kg BW) is a natural, promising neuroprotective agent that can mitigate FPN-induced neurobehavioral toxicity via the suppression of oxidative, inflammatory, and apoptotic pathways and relieve neuronal necrosis and astrogliosis.

Hydroxyurea affects in vitro porcine oocyte maturation through increased apoptosis and oxidative stress.

Hydroxyurea (HU) is an FDA-approved drug used to treat a variety of diseases, especially malignancies, but is harmful to fertility. We used porcine oocytes as an experimental model to study the effect of HU during oocyte maturation. Exposure of cumulus–oocyte complexes (COCs) to 20 µM (P<0.01) and 50 µM (P<0.001) HU reduced oocyte maturation. Exposure to 20 µM HU induced approximately 1.5- and 2-fold increases in Caspase-3 (P<0.001) and P53 (P<0.01) gene expression levels in cumulus cells, respectively, increased Caspase-3 (P<0.01) and P53 (P<0.001) protein expression levels in metaphase II (MII) oocytes and increased the percentage of apoptotic cumulus cells (P<0.001). In addition, HU decreased the mitochondrial membrane potential (Δφm) (P<0.01 and P<0.001) and glutathione (GSH) levels (P<0.01 and P<0.001) of both cumulus cells and MII oocytes, while increasing their reactive oxygen species (ROS) levels (P<0.001). Following parthenogenetic activation of embryos derived from MII oocytes, exposure to 20 µM HU significantly reduced total blastocyst cell numbers (P<0.001) and increased apoptosis of blastocyst cells (P<0.001). Moreover, HU exposure reduced the rate of development of two-celled, four- to eight-celled, blastocyst, and hatching stages after parthenogenetic activation (P<0.05). Our findings indicate that exposure to 20 µM HU caused significant oxidative stress and apoptosis of MII oocytes during maturation, which affected their developmental ability. These results provide valuable information for safety assessments of HU.

High Expression of Caspase-8 Associated with Improved Survival in Diffuse Large B-Cell Lymphoma: Machine Learning and Artificial Neural Networks Analyses

High expression of the anti-apoptotic TNFAIP8 is associated with poor survival of the patients with diffuse large B-cell lymphoma (DLBCL), and one of the functions of TNFAIP8 is to inhibit the pro-apoptosis Caspase-8. We aimed to analyze the immunohistochemical expression of Caspase-8 (active subunit p18; CASP8) in a series of 97 cases of DLBCL from Tokai University Hospital, and to correlate with other Caspase-8 pathway-related markers, including cleaved Caspase-3, cleaved PARP, BCL2, TP53, MDM2, MYC, Ki67, E2F1, CDK6, MYB and LMO2. After digital image quantification, the correlation with several clinicopathological characteristics of the patients showed that high protein expression of Caspase-8 was associated with a favorable overall and progression-free survival (Hazard Risks = 0.3; p = 0.005 and 0.03, respectively). Caspase-8 also positively correlated with cCASP3, MDM2, E2F1, TNFAIP8, BCL2 and Ki67. Next, the Caspase-8 protein expression was modeled using predictive analytics, and a high overall predictive accuracy (&gt;80%) was obtained with CHAID decision tree, Bayesian network, discriminant analysis, C5 tree, logistic regression, and Artificial Intelligence Neural Network methods (both Multilayer perceptron and Radial basis function); the most relevant markers were cCASP3, E2F1, TP53, cPARP, MDM2, BCL2 and TNFAIP8. Finally, the CASP8 gene expression was also successfully modeled in an independent DLBCL series of 414 cases from the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). In conclusion, high protein expression of Caspase-8 is associated with a favorable prognosis of DLBCL. Predictive modeling is a feasible analytic strategy that results in a solution that can be understood (i.e., explainable artificial intelligence, “white-box” algorithms).

Concise synthesis and biological evaluation of 2-Aryl-3-Anilinobenzo[b]thiophene derivatives as potent apoptosis-inducing agents

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure–activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.

Protective effect of melatonin entrapped PLGA nanoparticles on radiation-induced lung injury through the miR-21/TGF-β1/Smad3 pathway

Radiation-induced lung injury (RILI) is a complication commonly found in victims suffering from nuclear accidents and patients treated with chest tumor radiotherapy, and drugs are limited for effective prevention and treatment. Melatonin (MET) has an anti-radiation effect, but its metabolic period in the body is short. In order to prolong the metabolism period of MET, we prepared MET entrapped poly (lactic-co-glycolic acid) nanoparticles (MET/PLGANPS) for the treatment of RILI. As a result, the release rate of MET/PLGANPS in vitro was lower than MET, with stable physical properties, and it caused no changes in histopathology and biochemical indicators. After 2 weeks and 16 weeks of irradiation with the dose of 15 Gy, MET and MET/PLGANPS could reduce the expression of caspase-3 proteins, inflammatory factors, TGF-β1 and Smad3 to alleviate radiation-induced lung injury. MET/PLGANPS showed better therapeutic effect on RILI than MET. In addition, we also found that high expression of miR-21 could increase the expression levels of TGF-β1, and inhibit the protective effect of MET/PLGANPS. In conclusion, MET/PLGANPS may alleviate RILI by inhibiting the miR-21/TGF-β1/Smad3 pathway, which would provide a new target for the treatment of radiation-induced lung injury.

Effect of Andrographis paniculata polysaccharide on human retinoblastoma Y79 cell proliferation and apoptosis.

To explore the effect of the Andrographis paniculata (A. paniculata) polysaccharide on the proliferation and apoptosis of human retinoblastoma (RB) Y79 cells and its mechanism. The refined A. paniculata polysaccharide was obtained using techniques such as water extraction, ethanol precipitation, and decompression concentration. The inhibition effect of the A. paniculata polysaccharide on the proliferation of Y79 cells was detected by cell proliferation assay. Flow cytometry was used for the detection of cell apoptosis rate and cycle change. Real-time qunatitative polymerase chain reaction (RT qPCR)and Western blotting were used to detect the expression of cell apoptosis signal pathway-related factors (caspase-3, caspase-8, and caspase-9) and cell cycle signal pathway-related factors (CDK1 and cyclinB1) at the transcriptional and translational levels. Infrared and ultraviolet spectrum scanning showed that the extracted drug was a polysaccharide with high purity. After being treated with different concentrations of A. paniculata polysaccharide for different periods of time, the Y79 cells showed different degrees of proliferation inhibition. Flow cytometric observations showed that the cell apoptosis rate and the proportion of cells blocked in the G2/M phase were significantly increased after A. paniculata polysaccharide treatment. Further analysis revealed that the mRNA and protein expression of caspase-3, caspase-8, and caspase-9 in the A. paniculata polysaccharide treatment groups increased significantly compared with that in the control groups, while the expression of CDK1 and cyclinB1 decreased significantly. The A. paniculata polysaccharide could inhibit the proliferation and induce apoptosis of Y79 cells. Its possible mechanism is via the upregulation of caspase-3, caspase-8, and caspase-9 expression in the cell apoptotic signaling pathway and the downregulation of CDK1 and cyclinB1 expression in the cell cycle signaling pathway.

MIF inhibitor ISO-1 alleviates severe acute pancreatitis-associated acute kidney injury by suppressing the NLRP3 inflammasome signaling pathway

BackgroundAcute kidney injury (AKI) is an important complication of severe acute pancreatitis (SAP) with a poor prognosis. The methyl ester of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1), an inhibitor of macrophage migration inhibitory factor (MIF), has protective effects against many diseases. Our previous study confirmed MIF inhibition alleviated SAP. Here, we explored the effects of ISO-1 in an experimental mouse model of SAP-associated AKI induced by l-arginine. MethodsMice were randomly divided into four treatment groups (n = 6 each): control (CON), SAP, SAP + ISO-1, and ISO-1. Histopathologic examination was used to observe damage in pancreatic and renal tissues. Biochemical and enzyme-linked immunosorbent assays (ELISA) kits were used to measure the serologic indicators amylase, lipase, creatinine, uric acid, interleukin (IL)-6, and tumor necrosis factor (TNF)-α. Immunohistochemistry was used to detect protein expression of NLRP3, ASC and caspase-1, and the infiltration of myeloperoxidase (MPO)-positive neutrophils in kidney tissue. Western blotting was used to detect NLRP3, ASC and caspase-1 and IL-1β protein expression, and real-time PCR was used to measure MIF, IL-6, TNF-α, IL-1β and IL-18 mRNA levels in kidney tissue. ResultsISO-1 treatment alleviated pathological damage in pancreatic and renal tissues, and reduced the serum levels of amylase, lipase, creatinine, uric acid, IL-6 and TNF-α. ISO-1 also reduced protein expression of NLRP3, ASC, caspase-1 and IL-1β, mRNA expression of MIF, IL-6, TNF-α, IL-1β and IL-18, and the infiltration of MPO-positive neutrophils in kidney tissue. ConclusionISO-1 has a protective effect against experimental SAP-associated AKI. And the mechanism may be associated with ISO-1 inhibiting NLRP3 inflammasome signaling pathway.

Impact and mechanism of NEMO binding domain peptide on pulmonary inflammation and apoptosis of lung tissues in mice with acute respiratory distress syndrome

To investigate the effect of NEMO binding domain peptide (NBDP) on lung inflammation and apoptosis in mice with acute respiratory distress syndrome (ARDS) and its mechanism. Thirty-six male BALB/c mice were divided into normal saline (NS) control group, ARDS model group, NBDP negative control group and 6, 12 and 18 μg NBDP pretreatment group by random number table method, with 6 mice in each group. ARDS mouse model was reproduced by aerosol inhalation lipopolysaccharide (LPS) 50 μL. An equivalent among of NS was inhaled in NS control group. The mice in NBDP negative control group were inhaled the materials similar to the non-functional NBDP 30 minutes before the aerosol inhalation LPS; 6, 12 and 18 μg of NBDP 50 μL were respectively inhaled in NBDP pretreatment groups. After inhalation of LPS for 6 hours, mice were sacrificed to get lung tissue and observe the degree of pathological injury and edema. Western blotting was used to detect the phosphorylation of nuclear factor-κB (NF-κB) pathway related proteins [NF-κB inhibitor (IκB) kinaseα/β(IKKα/β), IκBα and NF-κB p65; p-IKKα/β, p-IκBα, p-p65] and the expression of caspase-3 in lung tissue. The bronchoalveolar lavage fluid (BALF) was collected and the levels of inflammatory markers such as myeloperoxidase (MPO), interleukins (IL-1β, IL-8), and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). ARDS model group had severe edema and hemorrhage, alveolar structure destruction, pulmonary hemorrhage and hyaline membrane formation etc. under light microscope, consistent with the pathological characteristics of ARDS lung tissue, suggesting that the ARDS model was successfully reproduced. ELISA showed that MPO, IL-1β, IL-8 and TNF-α levels of BALF in ARDS model group were obviously higher than those in NS control group. There were no significant differences in the above inflammatory indicators between NBDP negative control group and ARDS model group. The levels of MPO, IL-1β, IL-8 and TNF-α in NBDP pretreatment groups were significantly lower than those in ARDS model group in a dose-dependent manner, especially in 18 μg NBDP, the differences were statistically significant as compared with ARDS model group [MPO (ng/L): 393.32±19.35 vs. 985.87±101.50, IL-1β (ng/L): 43.05±5.11 vs. 97.68±10.88, IL-8 (ng/L): 84.64±2.32 vs. 204.00±17.37, TNF-α (ng/L): 229.13±17.03 vs. 546.73±62.72, all P < 0.05]. Western blotting showed that p-IKKα/β, p-IκBα, p-p65 and caspase-3 protein expressions in ARDS model group were significantly higher than those in NS control group. There was no significant difference in above NF-κB pathway and apoptosis-related protein expression between the NBDP negative control group and ARDS model group. The p-IKKα/β, p-IκBα, p-p65 and caspase-3 protein expression in NBDP pretreatment groups were significantly lower than those in ARDS model group in a dose-dependent manner, especially in 18 μg NBDP, the differences were statistically significant as compared with ARDS model group [p-IKKα/β protein (p-IKKα/β/β-actin): 0.15±0.02 vs. 0.42±0.04, p-IκBα protein (p-IκBα/β-actin): 0.10±0.01 vs. 0.93±0.30, p-p65 protein (p-p65/β-actin): 0.22±0.05 vs. 1.37±0.21, all P < 0.05]. NBDP can inhibit inflammatory response and apoptosis in ARDS lung tissue in a dose-dependent manner, and its mechanism is associated with interference NF-κB signaling pathway transduction.

Effect of astragaloside IV on the expression of NOD-like receptor protein 3 inflammasome in neonatal rats with hypoxic-ischemic brain damage

OBJECTIVE To study the effect of astragaloside IV (AS-IV) on NOD-like receptor protein 3 (NLRP3) inflammasome in neonatal rats with hypoxic-ischemic brain damage (HIBD). METHODS A total of 24 Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group, an HIBD group, and an AS-IV treatment group, with 8 rats in each group. After 24 hours of modeling, brain tissue was collected for hematoxylin-eosin staining, yo-PRO-1 staining, and EthD-2 immunofluorescent staining in order to observe the cerebral protection effect of AS-IV in vivo. HT22 cells were used to prepare a model of oxygen-glycogen deprivation (OGD), and a concentration gradient (50-400 μmol/L) was established for AS-IV. CCK-8 assay was used to measure the viability of HT22 cells. RT-PCR and Western blot were used to observe the effect of different concentrations of AS-IV on the mRNA and protein expression of NLRP3, gasdermin D (GSDMD), caspase-1, and interleukin-1β (IL-1β). RESULTS Yo-Pro-1 and EthD-2 staining showed that compared with the sham-operation group, the HIBD group had an increase in pyroptotic cells with a small number of necrotic cells, and the AS-IV group had reductions in both pyroptotic and necrotic cells. Compared with the sham-operation group, the HIBD group had significantly higher protein expression levels of NLRP3, IL-1β, caspase-1, and GSDMD (P < 0.05). Compared with the HIBD group, the AS-IV group had significant reductions in the protein expression levels of NLRP3, caspase-1, and GSDMD (P < 0.05). HT22 cell experiment showed that compared with the OGD group, the AS-IV group had inhibited mRNA and protein expression of NLRP3, GSDMD, caspase-1, and IL-1β, with the best therapeutic effect at the concentration of 200 μmol/L (P < 0.05). CONCLUSIONS AS-IV may alleviate HIBD in neonatal rats by inhibiting the expression of NLRP3, GSDMD, caspase-1, and IL-1β.

Up-Regulation of Bax/BCL-2 Ratio by 2-Methoxyestradiol Induces Apoptosis in Lymphoma Raji Cells

To investigate the effect of 2-methoxyestradiol (2-ME2) to lymphoma Raji cells and its mechanism. Different concentrations of 2-ME2 were used to treat lymphoma Raji cells. CCK8 method was used to detect the effect of 2-ME2 to proliferation of Raji cells. Flow cytometry FITC/PI double labeling method was used to detect early apoptosis of the cells. Western blotting was used to detect the effect of 2-ME2 to the expression of BCL-2, Bax, Caspase-3 and C-myc proteins in Raji cells. 2-ME2 significantly inhibited the proliferation of Raji cells. The inhibition rate increased with the increasing of drug concentration, and increased significantly with the prolongation of drug treatment time (r=0.9215). Flow cytometry FITC/PI double staining showed that the apoptotic rate of 2.5 μmol/L 2-ME2 treatment group was (33.79±1.63) %, while the apoptosis rate of the 48 h group was (51.90±2.72) %, and that of the control group was (7.08±0.36) %. After treated with 2.5 μmol/L 2-ME2 for 12 h, the expression of Bax protein was up-regulated, BCL-2 protein was down-regulated, caspase-3 protein expression was up-regulated, and C-myc protein expression was down-regulated, all of them showed a time-dependent relationship. 2-ME2 shows obvious inhibitory effect on lymphoma Raji cells in a dose- and time-dependent manner. Its mechanism of treatment on lymphoma Raji cells may be related to up-regulation of Bax/BCL-2 ratio and activation of Caspase-3 to induce apoptosis in cancer cells. Down-regulation of C-myc protein expression also participates in the apoptotic process.

Ginsenoside Re protects against chronic restraint stress-induced cognitive deficits through regulation of NLRP3 and Nrf2 pathways in mice.

Exposure to chronic stress negatively affects the development of cognition, characterized by learning and memory decline. Ginsenoside Re (GRe), an active compound derived from Panax ginseng, exhibited neuroprotective activity in various neurological diseases. In this study, the protective effect of GRe on chronic restraint stress (CRS)-induced memory deficit was investigated. The mice were experienced 35 days of the CRS induction. The GRe was administered daily orally (10, 20, or 40 mg/kg) during the next 3 weeks stress session and the behavior test period. The CRS-induced memory impairment mice were subjected to behavioral tasks, such as the Y-maze, novel objects recognition, and step-through passive avoidance tests. Nissl staining was used to examine the neuron numbers. The levels of antioxidant enzymes, malondialdehyde, and proinflammatory factor were determined by kits and ELISA assays. The expressions of brain-derived neurotrophic factor (BDNF), NOD-like receptor protein 3 (NLRP3), nuclear factor erythroid-2 related factor 2 (Nrf2) and synapse-associated proteins (synaptophysin, SYP, and postsynaptic density 95, PSD95) were measured by Western blotting. Behavioral assessments indicated that GRe could ameliorate the cognitive impairment of CRS-induced mice, as indicated by increased responses in Y-maze (p < .05), novel objects recognition (p < .01), and step-through passive avoidance tests (p < .01). In addition, GRe treatment significantly decreased the neuronal loss in CRS mice in histological examination. Moreover, chronic GRe treatment significantly ameliorated the down-regulated the expressions of BDNF, Nrf2, heme oxygenase (HO)-1, SYP, and PSD95, as well as up-regulated NLRP3, the adaptor protein ASC, and Caspase-1 protein expression in the hippocampus of CRS-treated mice. Taken together, these findings suggest that GRe has a potential therapeutic effect on memory impairment in C57BL/6J mice exposed to CRS paradigm.

Development of a protein signature to enable clinical positioning of IAP inhibitors in colorectal cancer

Resistance to chemotherapy-induced cell death is a major barrier to effective treatment of solid tumours such as colorectal cancer, CRC. Herein, we present a study aimed at developing a proteomics-based predictor of response to standard-of-care (SoC) chemotherapy in combination with antagonists of IAPs (inhibitors of apoptosis proteins), which have been implicated as mediators of drug resistance in CRC. We quantified the absolute expression of 19 key apoptotic proteins at baseline in a panel of 12 CRC cell lines representative of the genetic diversity seen in this disease to identify which proteins promote resistance or sensitivity to a model IAP antagonist [birinapant (Bir)] alone and in combination with SoC chemotherapy (5FU plus oxaliplatin). Quantitative western blotting demonstrated heterogeneous expression of IAP interactome proteins across the CRC cell line panel, and cell death analyses revealed a widely varied response to Bir/chemotherapy combinations. Baseline protein expression of cIAP1, caspase-8 and RIPK1 expression robustly correlated with response to Bir/chemotherapy combinations. Classifying cell lines into 'responsive', 'intermediate' and 'resistant' groups and using linear discriminant analysis (LDA) enabled the identification of a 12-protein signature that separated responders to Bir/chemotherapy combinations in the CRC cell line panel with 100% accuracy. Moreover, the LDA model was able to predict response accurately when cells were cocultured with Tumour necrosis factor-alpha to mimic a pro-inflammatory tumour microenvironment. Thus, our study provides the starting point for a proteomics-based companion diagnostic that predicts response to IAP antagonist/SoC chemotherapy combinations in CRC.

Effect of leonurine on peritoneal macrophages M1/M2 phenotypic differentiation via inhibiting overactivation of NLRP3 inflammasome

Objective To find the effect of leonurine on LPS-induced macrophages activation and its potential mechanism. Methods Mouse primary peritoneal macrophages were isolated and pretreated for 24 h with LPS and leonurine. MTT assay was used to detect the cell viability of macrophages. The production of IL-1β, IL-6, TNF-α and IL-18 in culture medium were tested by ELISA, and the production of NO was detected by Griess reagent. The mRNA expression of NLRP3, ASC, caspase-1, TNF-α, iNOS, Arg-1 and CD206 were detected by RT-PCR, and the protein expression of NLRP3, ASC and caspase-1 were detected by Western blotting. Results LPS can significantly increase the releases of NO、IL-1β、IL-6、TNF-α and IL-18 from macrophages. Leonurine can suppress the expression of pro-inflammatory factor levels, such as IL-1β (P Conclution Leonurine exerts beneficial effects through M1/M2 phenotypic differentiation of peritoneal macrophage via inhibiting overactivation of NLRP3 inflammasome. These findings suggest that leonurine might have a therapeutic potential for pelvic inflammatory disease.

The role and mechanism of human trophoblastic cell surface antigen 2 in the invasion and metastasis of ovarian cancer

Objective: To explore the role and molecular mechanism of trophoblastic cell surface antigen 2 (Trop2) in the invasion and migration of ovarian cancer. Methods: Through the data mining of Cancer Cell Line Encyclopedia and TCGA database, the clinical significance of Trop2 expression was analyzed. Western blot was used to detect Trop2 protein expression in ovarian cancer cell lines including A3O, A1780 and SKOV3. SKOV3 cells were used to construct Trop2-short hairpin RNA (shRNA) cell model. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect the SKOV3 mRNA expression in SKOV3-shRNA and SKOV3-NC cells. Cell counting kit-8 (CCK8) was used to detect the proliferation of SKOV3-shRNA cells and SKOV3-NC cells. Flow cytometry was used to detect cell cycle and apoptosis in two groups of cells. Transwell array was used to detecte the invasion and migration of SKOV3-shRNA cells and SKOV3-NC cells. Western blot was used to detect the protein expressions of AKT, p-AKT, β-catenin, caspase3, bcl-2, E-cadherin and vimentin. Results: Trop2 mRNA highly expressed in ovarian cancer, and was related to the tumor stage and patient survival. Compared with A3O cells, Trop2 overexpressed in A1780 and SKOV3 cells (P<0.05). The relative expression levels of Trop2 mRNA in SKOV3-NC group and SKOV3-shRNA group were 1.18±0.24 and 0.42±0.08, with statistically significant difference (P<0.05). The results of CCK-8 array showed that the cell viability of SKOV3-NC group was significantly higher than that of SKOV3-shRNA group (P<0.05). The proportion of G(0)/G(1) cells in SKOV3-NC and SKOV3-shRNA groups were (38.67±4.22)% and (60.24±8.17)%, respectively. G(0)/G(1) arrest was observed in SKOV3-shRNA cells (P<0.05). The apoptosis rate of SKOV3-shRNA group was (26.32±1.81)%, significantly higher than (6.54±1.32)% of SKOV3-NC group (P<0.05). The number of migrating SKOV3 cells in the SKOV3-shRNA and SkOV3-NC groups were 1 255.83±108.44 and 1 679.71±213.92, while the number of invading cells were 242.49±52.09 and 473.54±73.11, respectively. Compared with the SKOV3-NC group, the number of migrating and invading SKOV3-shRNA group was significantly reduced (all P<0.05). The expressions of p-AKT2, Bcl-2, vimentin and β-catenin were down-regulated, and the expressions of caspase 3 and E-cadherin were up-regulated in SKOV3-shRNA cells. There was no significant change in the total protein level of AKT. Conclusions: Trop2 expression is related to ovarian cancer stage and postoperative survival. Trop2 can promote ovarian cancer cell proliferation and metastasis by activating the AKT/β-catenin signaling pathway and knockdown of Trop2 inhibits the progression of ovarian cancer.

Selenium Attenuates S. aureus-Induced Inflammation by Regulation TLR2 Signaling Pathway and NLRP3 Inflammasome in RAW 264.7 Macrophages.

This study aimed to investigate the effects of selenium (Se) on the expression of Toll-like receptor (TLR) 2 and pyrin domain-containing protein (NLRP)3 inflammasome in macrophages infected by Staphylococcus aureus (S. aureus). RAW 264.7 macrophages were treated with 2 μmol/L Na2SeO3 for 12 h before infection with S. aureus for 2 h. Through Western blot, qRT-PCR, and ELISA analysis, the core molecules of TLR2 signaling pathway and NLRP3 inflammasome in RAW 264.7 macrophages were detected. Results showed that Se significantly reduced the elevated mRNA expression of TLR2, myeloid differentiation factor-88 (Myd88), NLRP3, Caspase-recruitment domain (ASC), and Caspase-1 induced by S. aureus. Furthermore, compared with I group, the protein expression of TLR2, Myd88, NLRP3, ASC, and Caspase-1 were suppressed in T group. In addition, the mRNA and protein expression of interleukin-1 beta (IL-1β) induced by S. aureus were also decreased after Se treatment. In conclusion, Se inhibits S. aureus-induced inflammation by suppressing the activation of the TLR2 signaling pathway and NLRP3 inflammasome in RAW 264.7 macrophages.

New organoselenides (NSAIDs-Se derivatives) as potential anticancer agents: Synthesis, biological evaluation and in silico calculations.

Herein we reported the synthesis of twenty new organoselenium compounds (2a-2j and 3a-3j) based on the hybridization of nonsteroidal antiinflammatory drugs (NSAIDs) skeleton and organoselenium motif (-SeCN and -SeCF3), the anticancer activity was evaluated against four types of cancer cell lines, Caco-2 (human colon adenocarcinoma cells), BGC-823 (human gastric cancer cells), MCF-7 (human breast adenocarcinoma cells), PC-3 (human prostatic cancer cells). Interestingly, the introduction of the -SeCN or -SeCF3 moiety in corresponding parent NSAIDs results in the significant effect on cancer cell lines. Moreover, the most active compound 3a showed IC50 values lower than 5μM against the four cancer cell lines, particularly to BGC-823 and MCF-7 with IC50 values of 2.5 and 2.7μM, respectively. Furthermore, three compounds 3a, 3g and 3i were selected to investigate their ability to induce apoptosis in BGC-823cells via modulating the expression of anti-apoptotic Bcl-2 protein, pro-inflammatory cytokines (IL-2) and proapoptotic caspase-8 protein. The redox properties of the NSAIDs-Se derivatives prepared herein were conducted by 2, 2-didiphenyl-1-picrylhydrazyl (DPPH), bleomycin dependent DNA damage and glutathione peroxidase (GPx)-like assays. Finally, molecular docking study revealed that an interaction with the active site of thioredoxin reductase 1 (TrxR1) and predicted the anticancer activity of the synthesized candidates. Overall, these results could serve a promising launch point for further design of NSAIDs-Se derivatives as potential anticancer agents.

Effect of Hedyotis diffusa Willd extract on gouty arthritis in rats

Purpose: To investigate the effect of Hedyotis diffusa Willd extract (HDWE) on gouty arthritis in rats.Method: Monosodium urate (MSU) crystal was injected into the ankle joint of rats to establish a rat model of gouty arthritis. HDWE (4.8, 9.6 and 19.2 g/kg) was administered to the rats treated with MSU crystals. The walking behavior of the rats was observed daily, and the gait score was calculated to evaluate the Oswestry disability index of rats. Levels of IL-1β and TNF-α in lavage fluid of articularcavities were measured using enzyme linked immunosorbent assay (ELISA) kits. The synovial tissues of joint of control, model and 19.2 g/kg HDWE group rats were obtained and NLRP3 inflammasome was analysed by Western blot.Results: The results showed that HDWE ameliorated the symptoms of gouty arthritis and gait score in rats significantly (p &lt; 0.05). Further pharmacological experiments showed that all doses of HDWE decreased the levels of inflammatory cytokines IL-1β and TNF-α (p &lt; 0.05), and inhibited NLRP3, caspase-1, ASC, IL-1β and IL-18 protein expressions of the lavage fluid of articular cavities in MSU crystal-treated rats (p &lt; 0.01).Conclusion: The results indicate that HDWE exhibits a significant effect in ameliorating gouty arthritis via inhibition NLRP3 inflammasome, and thus is a potential new drug choice for the treatment of gouty arthritis.&#x0D; Keywords: Hedyotis diffusa, Caspase, Gouty arthritis, Inflammatory cytokines, NLRP3 inflammasom