Zinc Oxide Nanoparticles Ameliorate Histological Alterations Through Apoptotic Gene Regulation in Rat Model of Liver Ischemia-Reperfusion Injury.

Background: Organ ischemia-reperfusion (IR) is a common clinical condition associated with various situations such as trauma surgery, organ transplantation, and myocardial ischemia. Current therapeutic methods for IR injury have limitations, and nanotechnology, particularly zinc oxide nanoparticles (ZnO NPs), offers new approaches for disease diagnosis and treatment. In this study, we investigated the protective and anti-apoptotic effects of ZnO NPs in liver ischemia-reperfusion (IR) injury in rats. Methods: Forty-eight male rats were divided into six groups: sham, ZnO5, ZnO10, ischemia-reperfusion (IR), IR+ZnO5, and IR+ZnO10. The protective effect of ZnO NPs was evaluated by liver enzymes (AST, ALT, Bilirubin, ALP), biochemical (TAC, TNF-α, and MDA), molecular examinations (Bcl2, BAX), and histopathological evaluations (H&E, TUNEL). Results: Pre-treatment with ZnO5 and ZnO10 improved hepatic function in IR liver injury, attenuated the levels of oxidants (P = 0.03) and inflammatory mediators, and reduced apoptosis (P = 0). ZnO10 was found to have a greater effect on ischemic reperfusion injury than ZnO5 did. Histopathological examination also showed a dose-dependent decrease in alterations in the IR+ZnO5 and IR+ZnO10 groups. Conclusion: Administration of ZnO5 and ZnO10 improved liver function after IR. The findings of this study suggest that ZnO NPs have a protective effect against oxidative stress and apoptosis in liver ischemia-reperfusion injury in rats. These results may have important implications for developing advanced methods in ischemia-reperfusion treatment.

Directly targeting BAX for drug discovery: Therapeutic opportunities and challenges.

For over two decades, the development of B-cell lymphoma-2 (Bcl-2) family therapeutics has primarily focused on anti-apoptotic proteins, resulting in the first-in-class drugs called BH3 mimetics, especially for Bcl-2 inhibitor Venetoclax. The pro-apoptotic protein Bcl-2-associated X protein (BAX) plays a crucial role as the executioner protein of the mitochondrial regulated cell death, contributing to organismal development, tissue homeostasis, and immunity. The dysregulation of BAX is closely associated with the onset and progression of diseases characterized by pathologic cell survival or death, such as cancer, neurodegeneration, and heart failure. In addition to conducting thorough investigations into the physiological modulation of BAX, research on the regulatory mechanisms of small molecules identified through biochemical screening approaches has prompted the identification of functional and potentially druggable binding sites on BAX, as well as diverse all-molecule BAX modulators. This review presents recent advancements in elucidating the physiological and pharmacological modulation of BAX and in identifying potentially druggable binding sites on BAX. Furthermore, it highlights the structural and mechanistic insights into small-molecule modulators targeting diverse binding surfaces or conformations of BAX, offering a promising avenue for developing next-generation apoptosis modulators to treat a wide range of diseases associated with dysregulated cell death by directly targeting BAX.

Discovery and optimization of (2-naphthylthio)acetic acid derivative as selective Bfl-1 inhibitor.

Bcl-2 anti-apoptotic protein family suppresses cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Bfl-1 is a relatively understudied member of this family, though it has been implicated in the pathogenesis and chemoresistance of a variety of human cancers. Reported small molecular Bfl-1 inhibitors encountered the issue of either lack in potency or poor selectivity against its most homologous member Mcl-1. In order to tackle this issue, compound library was screened and a hit compound UMI-77 was identified. We modified its chemical structure to remove the characteristic of PAINS (pan-assay interference compounds), demonstrated the real binding affinity and achieved selectivity against Mcl-1 under the guidance of computational modeling. After optimization 15 was obtained as leading compound to block Bfl-1/BIM interaction in vitro with more than 10-fold selectivity over Mcl-1. We believe 15 is of great value for the exploration of Bfl-1 biological function and its potential as therapeutic target.

Inhibitory actions of oxyresveratrol on the PI3K/AKT signaling cascade in cervical cancer cells.

The phosphatidyl inositol 3-kinase (PI3K)/AKT signaling plays a critical role in cancer cell proliferation, migration, and invasion. This signal transduction axis in HPV-positive cervical cancer has been proved to be directly activated by E6/E7 proteins of the virus enhancing cervical cancer progression. Hence, the PI3K/AKT pathway is one of the key therapeutic targets for HPV-positive cervical cancer. Here we discovered that oxyresveratrol (Oxy) at noncytotoxic concentration specifically suppressed the phosphorylation of AKT but not ERK1/2. This potent inhibitory effect of Oxy was still observed even when cells were stimulated with fetal bovine serum. Inhibition of AKT phosphorylation at serine 473 by Oxy resulted in a significant decrease in serine 9 phosphorylation of GSK-3ß, a downstream target of AKT. Dephosphorylation of GSK-3ß at this serine residue activates its function in promoting the degradation of MCL-1, an anti-apoptotic protein. Results clearly demonstrated that in association with GSK-3ß activation, Oxy preferentially downregulated the expression of anti-apoptotic protein MCL-1. Furthermore, results from the functional analyses revealed that Oxy inhibited cervical cancer cell proliferation, at least in part through suppressing nuclear expression of Ki-67. Besides, the compound retarded cervical cancer cell migration even the cells were exposed to a potent enhancer of epithelial-mesenchymal transition, TGF-ß1. In consistent with these data, Oxy reduced the expression of ß-catenin, N-cadherin, and vimentin. In conclusion, the study disclosed that Oxy specifically inhibits the AKT/GSK-3ß/MCL-1 axis resulting in reduction in cervical cancer cell viability, proliferation, and migration.

Silibinin: an inhibitor for a high-expressed BCL-2A1/BFL1 protein, linked with poor prognosis in breast cancer.

Breast cancer (BC) accounts for 30% of all diagnosed cases of cancer in women and remains a leading cause of cancer-related deaths among women worldwide. The current study looks for a protein from the anti-apoptotic/pro-survival BCL-2 family whose overexpression reduces survivability in BC patients and a potential inhibitor for the protein. We found BCL-2A1/BFL1 protein with high expression linked to low survivability in BC. The protein shows prognosis in 8 out of 29 categories, whereas no other family member manifests this property. Out of 7379 compounds, three small molecules (CHEMBL9509, CHEMBL2104550 and CHEMBL3545011) form an H-bond with BCL-2A1/BFL1 protein's unique residue Cys55. Of the three small molecules, we found CHEMBL9509 (Silibinin) to be a potent inhibitor. The compound forms a stable H-bond with the residue Cys55 with the lowest binding energy compared to the other two compounds. It remains stable in the BH3 binding region for more than 100 ns, whereas the other two detach from the region. Additionally, the compound is found to be better than Venetoclax and Nematoclax. We firmly believe in the compound CHEMBL9509 potency to halt BC's progression by inhibiting the BCL-2A1/BFL1 protein, increasing patients' survivability.Communicated by Ramaswamy H. Sarma.

Design, Synthesis, and Potent Anticancer Activity of Novel Indole-Based Bcl-2 Inhibitors.

The Bcl-2 family plays a crucial role in regulating cell apoptosis, making it an attractive target for cancer therapy. In this study, a series of indole-based compounds, U1-6, were designed, synthesized, and evaluated for their anticancer activity against Bcl-2-expressing cancer cell lines. The binding affinity, safety profile, cell cycle arrest, and apoptosis effects of the compounds were tested. The designed compounds exhibited potent inhibitory activity at sub-micromolar IC50 concentrations against MCF-7, MDA-MB-231, and A549 cell lines. Notably, U2 and U3 demonstrated the highest activity, particularly against MCF-7 cells. Respectively, both U2 and U3 showed potential BCL-2 inhibition activity with IC50 values of 1.2 ± 0.02 and 11.10 ± 0.07 µM using an ELISA binding assay compared with 0.62 ± 0.01 µM for gossypol, employed as a positive control. Molecular docking analysis suggested stable interactions of compound U2 at the Bcl-2 binding site through hydrogen bonding, pi-pi stacking, and hydrophobic interactions. Furthermore, U2 demonstrated significant induction of apoptosis and cell cycle arrest at the G1/S phase. Importantly, U2 displayed a favourable safety profile on HDF human dermal normal fibroblast cells at 10-fold greater IC50 values compared with MDA-MB-231 cells. These findings underscore the therapeutic potential of compound U2 as a Bcl-2 inhibitor and provide insights into its molecular mechanisms of action.

Targeting apoptotic anticancer response with natural glucosinolates from cell suspension culture of Lepidium sativum.

BACKGROUND: Finding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential. METHODS: In this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography-mass spectrometric analysis (GC-MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines. RESULTS: The findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an IC50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells. CONCLUSIONS: The anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest.

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

Cytochrome C as a potential clinical marker for diagnosis and treatment of glioma.

Gliomas are the most prevalent kind of malignant and severe brain cancer. Apoptosis regulating mechanisms are disturbed in malignant gliomas, as they are in added forms of malignancy. Understanding apoptosis and other associated processes are thought to be critical for understanding the origins of malignant tumors and designing anti-cancerous drugs for the treatment. The purpose of this study was to evaluate the variation in the expression level of several apoptotic proteins that are responsible for apoptosis in low to high-grade glioma. This suggests a significant change in the expression of five apoptotic proteins: Clusterin, HSP27, Catalase, Cytochrome C, and SMAC. Cytochrome C, one of the five substantially altered proteins, is a crucial component of the apoptotic cascade. The complex enzyme Cytochrome C is involved in metabolic pathways such as respiration and cell death. The results demonstrated that Cytochrome C expression levels are lower in glioma tissues than in normal tissues. What's more intriguing is that the expression level decreases with an increase in glioma grades. As a result, the discovery shows that Cytochrome C may be a target for glioma prognostic biomarkers.

MPST deficiency promotes intestinal epithelial cell apoptosis and aggravates inflammatory bowel disease via AKT.

BACKGROUND & AIMS: Excessive inflammatory responses and oxidative stress are considered the main characteristics of inflammatory bowel disease (IBD). Endogenous hydrogen sulfide (H2S) has been reported to show anti-inflammatory activity in IBD. The main aim of this study was to explore the role of 3-mercaptopyruvate sulfurtransferase (MPST), a key enzyme that regulates endogenous H2S biosynthesis, in IBD. METHODS: Colonic MPST expression was evaluated in mice and patients with IBD. Various approaches were used to explore the concrete mechanism underlying MPST regulation of the progression of colitis through in vivo and in vitro models. RESULTS: MPST expression was markedly decreased in colonic samples from patients with ulcerative colitis (UC) or Crohn's disease (CD) and from mice treated with DSS. MPST deficiency significantly aggravated the symptoms of murine colitis, exacerbated inflammatory responses and apoptosis, and inhibited epithelium stem cell-derived organoid formation in an H2S-independent manner. Consistently, when HT29 cells were treated with TNF-α, inhibition of MPST significantly increased the expression of proinflammatory cytokines, the amount of ROS and the prevalence of apoptosis, whereas overexpression of MPST markedly improved these effects. RNA-seq analysis showed that MPST might play a role in regulating apoptosis through AKT signaling. Mechanistically, MPST directly interacted with AKT and reduced the phosphorylation of AKT. Additionally, MPST expression was positively correlated with AKT expression in human IBD samples. In addition, overexpression of AKT rescued IEC apoptosis caused by MPST deficiency, while inhibition of AKT significantly aggravated it. CONCLUSIONS: MPST protects the intestines from inflammation most likely by regulating the AKT/apoptosis axis in IECs. Our results may provide a novel therapeutic strategy for the treatment of colitis.

Ovarian tumor deubiquitinase 6A regulates cell proliferation via deubiquitination of nucleolin and caspase­7.

Most proteins maintain protein homeostasis via post­translational modifications, including the ubiquitin­proteasome system. Deubiquitinating enzymes (DUBs) have essential intercellular roles, such as responses to DNA damage, proteolysis and apoptosis. Therefore, it is important to understand DUB­related diseases to identify DUBs that target abnormally regulated proteins in cells. Ovarian tumor deubiquitinase 6A (OTUD6A) was previously reported as a downregulated DUB in HCT116 cells with p53 knockdown. Therefore, it was expected that the relationship between OTUD6A and p53 would affect cell proliferation. In the present study, putative substrates of OTUD6A related to the p53 signaling pathway were identified. Application of liquid chromatography­tandem mass spectrometry and proteomic analysis led to the identification of nucleolin (known to bind p53) as a binding protein. In addition, immunoprecipitation studies determined that caspase­7, an apoptotic protein, is associated with p53 signaling and is regulated by OTUD6A. It was further identified that OTUD6A regulates the protein stability of nucleolin, but not caspase­7. It was also demonstrated that OTUD6A acts as a respective DUB through the deubiquitination of K48­linked polyubiquitin chain of nucleolin and the K63­linked polyubiquitin chain of caspase­7. Furthermore, overexpression of OTUD6A induced cell proliferation via enhancing cell cycle progression of MCF7 cells. Taken together, OTUD6A may be proposed as a target for anticancer therapy.

Mechanism of periplakin on ovarian cancer cell phenotype and its influence on prognosis.

Background: The aim of this paper was to investigate the clinical significance of periplakin (PPL) expression in ovarian cancer (OV) tissues and to explore the influence and possible mechanism of PPL on OV apoptosis. Methods: PPL expression in OV tissues was detected by western blotting, and its correlation with the clinicopathological parameters and prognosis of OV patients was analyzed. The influence of PPL expression on the growth of OV cell lines was analyzed using the DepMap database. The biological function of PPL and related genes in tumors was studied using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis based on The Cancer Genome Atlas (TCGA) database. PPL expression in OV cell lines was detected by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression of apoptosis-related proteins in each group after PPL knockdown was detected by western blotting. Results: PPL expression in OV tissues was higher than that in normal ovarian tissues (P<0.05). PPL messenger RNA (mRNA) expression was highest in the OV cell line CAOV-4 and lowest in the OV cell line CoC1. PPL expression was decreased in the si-PPL-1, si-PPL-2, and si-PPL-3 groups, with significant inhibition in the si-PPL-1 and si-PPL-3 groups. Compared to that in the si-NC group, the cell proliferation rate in the si-PPL-1 and si-PPL-3 groups was decreased, and the apoptosis rate was increased. The expression of active caspase 3 and BCL-2-associated X (BAX) was increased, while that of B-cell lymphoma 2 (BCL-2) was decreased. Conclusions: PPL was highly expressed in OV tissues and cell lines, and this was related to the prognosis of OV patients. PPL might promote cancers by inhibiting OV apoptosis and could be a potential target of therapy for OV.

Brain-Targeted Codelivery of Bcl-2/Bcl-xl and Mcl-1 Inhibitors by Biomimetic Nanoparticles for Orthotopic Glioblastoma Therapy.

Glioblastoma (GBM) is among the most treatment-resistant solid tumors and often recurrs after resection. One of the mechanisms through which GBM escapes various treatment modalities is the overexpression of anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xl, and Mcl-1) in tumor cells. Small-molecule inhibitors such as ABT-263 (ABT), which can promote mitochondrial-mediated cell apoptosis by selectively inhibiting the function of Bcl-2 and Bcl-xl, have been proven to be promising anticancer agents in clinical trials. However, the therapeutic prospects of ABT for GBM treatment are hampered by its limited blood-brain barrier (BBB) penetration, dose-dependent thrombocytopenia, and the drug resistance driven by Mcl-1, which is overexpressed in GBM cells and further upregulated upon treatment with ABT. Herein, we reported that the Mcl-1-specific inhibitor A-1210477 (A12) can act synergistically with ABT to induce potent cell apoptosis in U87 MG cells, drug-resistant U251 cells, and patient-derived GBM cancer stem cells. We further designed a biomimetic nanomedicine, based on the apolipoprotein E (ApoE) peptide-decorated red blood cell membrane and pH-sensitive dextran nanoparticles, for the brain-targeted delivery of ABT and A12. The synergistic anti-GBM effect was retained after encapsulation in the nanomedicine. Additionally, the obtained nanomedicine possessed good biocompatibility, exhibited efficient BBB penetration, and could effectively suppress tumor growth and prolong the survival time of mice bearing orthotopic GBM xenografts without inducing detectable adverse effects.

Synthetic Analogs of Marine Alkaloid Aplysinopsin Suppress Anti-Apoptotic Protein BCL2 in Prostate Cancer.

Aplysinopsins are a class of indole alkaloids that possess various pharmacological activities. Although their action has been studied in regard to many diseases, their effect on prostate cancer has not yet been examined. Therefore, we synthesized a new series of aplysinopsin analogs and investigated their cytotoxic activity against prostate cancer. Five analogs showed high antitumor activity via suppressing the expression of the anti-apoptotic gene Bcl2, simulationously increasing the expression of the pro-apoptotic genes p53, Bax and Caspase 3. The inhibition of BCL2 led to the activation of BAX, which in turn activated Caspase 3, leading to apoptosis. This dual mechanism of action via apoptosis and cell cycle arrest induction is responsible for aplysinopsin analogs antitumor activity. Hence, our newly synthesized analogs are highly promising candidates for further preclinical studies against prostate cancer.

Early and late apoptosis protein expression (Bcl-2, BAX and p53) in traumatic brachial plexus injury.

OBJECTIVES: This research aims to analyze the expression of pro-apoptotic proteins (Bax, p53) and anti-apoptotic protein (Bcl-2) in the nerve roots of the brachial plexus following traumatic brachial plexus injury (TBPI) in the early and late stage. METHODS: A total of 30 biopsy samples were taken from the proximal stump of the postganglionic nerve roots of the TBPI patients' brachial plexus from January 2018 until September 2019. The samples were taken from patients within six months of trauma (early stage, group A) and more than six months following trauma (late stage, group B). Bcl-2, Bax, and p53 expressions in each group were measured and compared. RESULTS: We found significant differences in the Bcl-2 (p=0.04), Bax (p<0.0001), p53 (p<0.0001) expressions between group A and B. The Bcl-2/Bax expression ratio in group A and B was 2.26 and 0.22, respectively. Meanwhile, the Bcl-2/p53 expression ratio in group A and B was 1.64 and 0.23, respectively. CONCLUSION: Apoptosis is inhibited by Bcl-2 activities in the early stage following trauma. In the late stage, a significant decrease of Bcl-2 coupled with a substantial increase of Bax and p53 indicates a continuation of the apoptotic process.

The mechanism of miR-494 negatively regulating ROCK1 and PTEN in inhibiting apoptosis of acute pancreatitis cells / 中华急诊医学杂志

Objective:To explore the mechanism of miR-494 negatively regulating ROCK1 and PTEN in inhibiting apoptosis of pancreatic cells and participating in the occurrence and development of acute pancreatitis.Methods:Pancreatic acinar cells AR42J from rats were treated by caerulein, and then the levels of amylase, tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1) and IL-6 in the supernatant of cell culture were detected by ELISA to verify the cell model of acute pancreatitis. RT-PCR was used to detect the expression of miR-494 in normal AR42J cells (control group) and acute pancreatitis cell model (model group). Flow cytometry was used to detect the apoptosis of the control group, negative control miRNA transfected acute pancreatitis cell model (negative control group) and miR-494 transfected acute pancreatitis cell model (miR-494 transfection group). Western blot was used to detect the expression of ROCK1 and PTEN in the control group, negative control group and miR-494 transfection group.Results:The levels of amylase, TNF-α, IL-1 and IL-6 in the supernatant of AR42J cells treated with caerulein for 8 h and 12 h were significantly higher than those at 0 h and the control group ( P<0.05), indicating that the model was successfully constructed. The expression levels of miR-494 at 8 h, 12 h and 24 h after the establishment of acute pancreatitis cell model were significantly higher than those at 4 h and the control group ( P < 0.05). The apoptosis rate of the model group was significantly higher than that of the control group ( P<0.05), and the apoptosis rate of the miR-494 transfection group was significantly lower than that of the model group ( P<0.05). The expression levels of ROCK1 and PTEN in the miR-494 transfection group were significantly lower than those in the model group and negative control group ( P<0.05). Conclusions:When acute pancreatitis occurs, overexpression of miR-494 can inhibit the expression of pro-apoptotic protein, thus inhibiting the apoptosis of pancreatic acinar cells and promoting the development of acute pancreatitis.

[Menthol enhances interleukin-13-induced synthesis and secretion of mucin 5AC in human bronchial epithelial cells].

OBJECTIVE: To investigate the effect of interleukin (IL) -13 combined with cold stimulation on synthesis and secretion of mucin (MUC) 5AC in human bronchial epithelial cell line 16HBE and explore the role of transient receptor potential 8 (TRPM8) and anti-apoptotic factor B-cell lymphoblast-2 (Bcl-2) in this process. METHODS: 16HBE cells were stimulated with 10 ng/mL IL-13, 1 mmol/L menthol, or both (1 mmol/L menthol was added after 6 days of IL-13 stimulation), and the changes in the expression of MUC5AC, intracellular Ca2+ concentration and Bcl-2 expression were evaluated. The effects of ABT-263 (a Bcl-2 inhibitor) and BCTC (a TRPM8 ion channel inhibitor), alone or in combination, on MUC5AC expression in the cells were tested, and the changes in intracellular Ca2+ and Bcl-2 expression following BCTC treatment were observed. The cell viability was assessed using CCK-8 assay, the mRNA expressions of MUC5AC and Bcl-2 were detected with real-time quantitative PCR, the level of MUC5AC in the culture medium was measured with ELISA, and the intracellular Ca2+ fluorescence intensity was determined with flow cytometry. RESULTS: The mRNA and protein expressions of MUC5AC increased significantly in 16HBE cells following stimulation with IL-13, menthol, and both (P < 0.05), and were the highest in the combined treatment group with its peak level occurred at 24 h (P < 0.01). The intracellular Ca2+ fluorescence intensity and Bcl-2 mRNA expression were also increased in 16HBE cells after the stimulations (P < 0.05), and the increments were the most obvious in the combined treatment group (P < 0.01). Treatment with BCTC significantly lowered intracellular Ca2+ fluorescence intensity and the expressions of Bcl-2 and MUC5AC mRNA and protein in the cells stimulated with menthol or with both IL-13 and menthol (P < 0.05), but caused no significant changes in IL-13-stimulated cells (P > 0.05). Treatment with ABT-263 significantly lowered the mRNA and protein expressions of MUC5AC in the cells stimulated with IL-13 and menthol either alone or in combination (P < 0.05). CONCLUSIONS: Menthol combined with IL-13 produces a synergistic effect to promote the synthesis and secretion of MUC5AC in 16HBE cells possibly by activating TRPM8 receptor to upregulate the expression of Bcl-2.

Design and synthesis of quinoxaline-1,3,4-oxadiazole hybrid derivatives as potent inhibitors of the anti-apoptotic Bcl-2 protein.

Quinoxaline is one of the privileged heterocyclic fragments for drug molecules. Quinoxaline anticancer drug candidates XK469 and CQS exhibit antiproliferative and proapoptotic properties against various cancers. Based on their chemical structures, we therefore synthesized a series of quinoxaline-1,3,4-oxadiazole hybrids and assessed their anticancer potential on human leukemia HL-60 cells. Although these hybrids exerted significant inhibition of HL-60 cell proliferation, they showed high cytotoxicity on human normal cells (WI-38). Utilizing information from molecular modelling of the hybrids to the anti-apoptotic Bcl-2 protein, we added substructures including phenyl, piperazine, piperidine, and morpholine rings to their frameworks. The designed quinoxaline-1,3,4-oxadiazole hybrid derivatives successfully induced apoptotic response on HL-60 cells with low toxicity on WI-38 cells. Furthermore, RT-PCR analysis demonstrated that these derivatives predominantly inhibit Bcl-2 expression. Our findings highlight the great potential for the development of synthetic quinoxaline-1,3,4-oxadiazole hybrid derivatives as proapoptotic anticancer agents.

Structural characterization and cardioprotective activity of a novel polysaccharide from Fructus aurantii.

Fructus aurantii is a well-known plant commonly used to treat cardiovascular diseases in China. The Fructus aurantii polysaccharide CALB-3 has been shown to exhibit antioxidant activity in vitro and in vivo. We aimed to further characterize the structure of CALB-3 and investigate its protective effects against hypoxia/reoxygenation-mediated myocardial injury in vitro. To this end, here, the effect of CALB-3 was tested against hypoxia/reoxygenation-induced oxidative stress, antioxidant enzyme and reactive oxygen species levels, and apoptosis-related protein expression in H9c2 cells. Our results showed that CALB-3 had a molecular weight greater than 805.0 kDa and a â†’ 3)-α-Galp-(1→, →3, 4)-α-Galp-(1→, and →3)-ß-Arap-(1 â†’ backbone. Pretreatment with CALB-3 elevated survival rate, improved endogenous antioxidant enzyme activity, inhibited reactive oxygen species production, reduced mitochondrial membrane potential, downregulated the pro-apoptosis protein Bax, and upregulated the anti-apoptosis protein Bcl-2. The protective effects were correlated with Akt signaling, suggesting that CALB-3 exerts its cardioprotective effects via Akt signaling.

Effect of Yisui Jiedu Prescription on Apoptosis of Hippocampal Neurons Based onPI3K/Akt/Bad Signal Pathway in Rats with Vascular Dementia / 中国实验方剂学杂志

Objective:To investigate the effect of Yisui Jiedu prescription on hippocampal neuron damage in vascular dementia (VD) rats and to regulate phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/recombinant Bcl-2 associated death promoter (Bad) mechanisms of signaling pathways of neuronal apoptosis. Method:The 40 SD rats were divided into sham operation group， model group， donepezil hydrochloride group and Yisui Jiedu prescription group， with 10 rats in each group.VD animal model was prepared by bilateral carotid artery permanent ligation (2-VO) method.The sham operation group and the model group were intragastrically administered with normal saline， the donepezil hydrochloride group was intragastrically administered with donepezil hydrochloride 0.52 mg·kg-1. The Yisui Jiedu prescription group was administered with Yisui Jiedu prescription (11.11 g·kg-1)， 1 time/d . After 30 days， Morris water maze was used to test the learning and memory ability of rats， hematoxylin-eosin (HE) staining was used to observe the histomorphological structure of hippocampal CA1 region. Ultrasound of neuron in rat hippocampal CA1 region was observed by transmission electron microscopy (TEM).Real-time fluorescent quantitative（Real-time PCR） was used to detect the Akt， Bad mRNA expression.Western blot was used to detect the Akt， p-Akt and Bad protein expression in hippocampus. Result:Compared with sham operation group， the learning and memory ability of model group decreased significantly(P<0.05). The pathological structure and neuronal ultrastructure of the hippocampus were changed obviously. Hippocampal tissue Akt mRNA and the Akt，p-Akt protein expression level decreased significantly(P<0.05)， and the levels of Bad mRNA and protein were significantly increased (P<0.05). Compared with model group， Yisui Jiedu prescription group can significantly improve the learning and memory ability of rats， improve the neuronal cells and ultrastructural changes in hippocampal CA1 area，and increase the expression of Akt mRNA and Akt，p-Akt protein in hippocampus. Decreased Bad mRNA and Bad protein expression levels (P<0.05). Conclusion:Yisui Jiedu prescription can significantly improve the learning and memory ability of VD rats， improve the ultrastructural pathological changes of hippocampus and neurons， and repair damaged neurons， which may promote Akt phosphorylation and activate PI3K/Akt/Bad. The signaling pathway plays a role in the defense of neurons against apoptosis.