Bcl-2 Protein Expression Research Articles

Glutathione's Role in Liver Metabolism and Hangover Symptom Relief: Dysregulation of Protein S-Glutathionylation and Antioxidant Enzymes.

Hangovers from alcohol consumption cause symptoms like headaches, nausea, and fatigue, disrupting daily activities and overall well-being. Over time, they can also lead to inflammation and oxidative stress. Effective hangover relief alleviates symptoms, prevents dehydration, and replenishes energy needed for daily tasks. Natural foods considered high in antioxidants and antiinflammatory properties may aid in the hepatic breakdown of alcohol. The study aims to investigate the impact of glutathione or its enriched yeast extract, which is recognized for its antioxidant characteristics, on alcohol metabolism and alleviating hangovers in a rat model exposed to binge drinking. In this study, glutathione and its enriched yeast extract controlled hangover behaviour patterns, including locomotor activity. Additionally, it enhanced the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) following ethanol ingestion (3 g/kg). Further, the incorporation of glutathione led to an increase in the expression of antioxidant enzymes, such as SOD and catalase, by activating the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway. This activation reduced the excessive production of reactive oxygen species (ROS) and malondialdehyde. Next, glutathione modulated the activity of cytochrome P450 2E1 (CYP2E1) and the protein expressions of Bax and Bcl2. Besides, in vitro and in vivo investigations with glutathione demonstrated a regulating effect on the pan-s-glutathionylation and its associated protein expression, glutaredoxin 1 (Grx1), glutathione-S-transferase Pi (GST-π), and glutathione reductase (GR). Together, these findings suggest that glutathione or its enriched yeast extract as a beneficial dietary supplement for alleviating hangover symptoms by enhancing alcohol metabolism and its associated Nrf2/Keap1 signalings.

Xuebijing Exerts Protective Effects on Myocardial Cells by Upregulating TRIM16 and Inhibiting Oxidative Stress and Apoptosis.

This study utilized transcriptomic sequencing combined with cellular and animal models to explore the potential mechanisms of Xuebijing in treating sepsis-induced myocardial dysfunction, also known as sepsis-induced myocardial injury. We investigated potential targets and regulatory mechanisms of XBJ injection using network pharmacology and RNA sequencing. The effects of XBJ on oxidative stress and apoptosis levels in human cardiac myocytes (AC16) and C57BL/6 mice exposed to lipopolysaccharide (LPS) were evaluated by Enzyme-Linked Immunosorbent Assay (ELISA), fluorescent probe, Fluorescent Quantitative Polymerase Chain Reaction (qPCR), Western Blot, Transmission Electron Microscopy, oxidative stress-related indicators detection kit, flow cytometry, and Immunohistochemistry (IHC). First, it was verified that XBJ can reduce the deformation of AC16 cardiomyocytes induced by LPS and the production and secretion of ROS (P <0.01). The transcriptome sequencing results showed that the TRIM16 gene was significantly increased after XBJ treatment, and the data of KEGG and GO analyses demonstrated that XBJ could inhibit the pathway expression of oxidative stress damage in AC16 cells, and PCR verified that XBJ could indeed increase the expression level of TRIM16 gene in AC16 cells (P <0.01). Basic animal and cell experiments showed that LPS could inhibit the expression of TRIM16 and NRF2 in cardiomyocytes (P <0.05) and promote the expression of Keap1 (P <0.01), while XBJ could significantly upregulate the expression levels of TRIM16 and NRF2 (P <0.01) and inhibit the expression of Keap1 (P <0.01), thereby affecting the expression levels of downstream proinflammatory cytokines and alleviating LPS-induced oxidative stress damage. In addition, XBJ also inhibited the expression of the pro-apoptotic proteins Bax and c-caspase3 (P <0.01), promoted the expression of the anti-apoptotic protein Bcl2 (P <0.01), and reduced LPS-induced apoptosis by upregulating TRIM16. Our comprehensive data demonstrated that TRIM16 is a key gene in the therapeutic action of Xuebijing in sepsis-induced myocardial dysfunction, protecting myocardial cells from injury through antioxidative stress and anti-apoptotic mechanisms.

Goose astrovirus induces apoptosis and endoplasmic reticulum stress in gosling hepatocytes

The ongoing Goose astrovirus (GoAstV) epidemic, which primarily infects goslings causing severe liver damage, has inflicted considerable damage on the poultry industry. Endoplasmic reticulum stress (ERS) is a significant modulator of several viral infections, while severe ERS may result in apoptosis. This study examined the roles and possible mechanisms of ERS and apoptosis in GoAstV-induced liver injury in goslings. Two hundred Xingguo gray geese were chosen and randomly separated into two groups (Con and Dis). The Dis group received a subcutaneous injection of GoAstV genotype 2 (GoAstV-2) JX01 (2 × 106 TCID50/0.2 mL), whereas the Con group received a subcutaneous injection of 0.2 mL physiological saline, both at 1 day of life. Subsequent analyses demonstrate that the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased following GoAstV infection. Hematoxylin and eosin (HE) staining revealed swollen and ruptured hepatocytes, with significant inflammatory cell infiltration. Electron microscopy revealed expansion of the endoplasmic reticulum (ER) and aggregation of chromatin at the periphery. TUNEL testing further demonstrated an increase in the quantity of positive cells. RT-qPCR and Western blot analyses indicated that GoAstV infection enhanced the expression of ER Ca2+ release channels (IP3R and RYR) and calmodulin-dependent protein kinase II (CaMKII), while decreasing the expression of ER Ca2+ uptake channels (SERCA). Further, GoAstV infection activated ERS-related factors, including GRP78, IRE1α, PERK, ATF6, eIF2α, ATF4, CHOP, TRAF2, and JNK, induced the expression of pro-apoptotic factors (Caspase-3, Caspase-9, and Bax), and inhibited the mRNA and protein expression of the anti-apoptotic factor Bcl-2. Correlation analysis further revealed a potential relationship among ERS gene expression, apoptotic gene expression, and liver injury. In summary, GoAstV infection can lead to liver injury by interfering with ER Ca2+ homeostasis, exacerbating ERS and inducing hepatocyte apoptosis.

Newly Synthesized Citral Derivatives Serve as Novel Inhibitor in HepG2 Cells.

2H-pyran compound 1 synthesized from 6-methylpyridine-2,4-diol and citral, has been used for Cu-catalyzed N-arylation with a range of arylboric acids to obtain arylated pyranopyridine core structure derivatives (yield up to 77 %). Among them, compound 3 h exhibited a much better inhibitory effect on HepG2 liver cancer cells compared to citral, and the IC50 value was 5.3 μM following exposure with the newly synthesized derivatives (herein named 3 h for short in this paper), which was lower than that of the cisplatin (6.5 μM). Meanwhile, the cell-cycle arrest of HepG2 cells occurred in the S phase, and the apoptosis of HepG2 cells was significantly increased with increasing drug concentration. In addition, real-time fluorescence quantification PCR and Western blotting experiments showed that the expression of apoptotic protein BAX was increased, while the expression of anti-apoptotic protein BCL2 was inhibited in a dose-dependent fashion. The results of these experiments indicated that apoptosis was promoted in HepG2 cells via apoptotic signaling pathway activated by 3 h. Furthermore, 3 h effectively decreased the phosphorylation levels of PI3 K, ATK and ERK, resulting in the inhibitions of MAPK/ERK and PI3 K/ATK signaling pathways. Briefly, 3 h has been found to show inhibitory effects on the survival of HepG2 liver cancer cells and may be used as anti-cancer drug in the future.

Estrogen hindrance escalates inflammation and neurodegeneration in the hippocampal regions of collagen-induced arthritis female Sprague-Dawley rats.

This study aims to investigate the effect of estrogen hindrance, i.e., menopause in women for instance with rheumatoid arthritis on the brain hippocampal region by using collagen-induced arthritis (CIA) female rat model (RA). CIA was induced in female rats by injecting bovine type II collagen and incomplete Freund's adjuvant. Estrogen receptor antagonist, fulvestrant (Ful), was given to RA rats to create estrogen hindrance. Control (C) and RA rats were injected with saline and DMSO, respectively, while RA + Ful rats received a 7-day fulvestrant injection. Following experiment completion, rats were sacrificed, and brains were harvested. Brains were stained with H&E and cresyl violet staining and morphological changes in the hippocampus were identified. Additionally, oxidative stress, inflammatory, and apoptosis markers' levels in the hippocampus were analyzed by qPCR, ELISA, and immunohistochemistry techniques. RA + Ful rats showed neuronal atrophy and reduced neurogenesis in the hippocampal regions. NOX4, NF-κB, IL-1β, IL-6, TNF-α, IKK-β, and Bax protein expression levels in the hippocampus were increased, whereas hippocampal Bcl-2, caspase-3, caspase-9, and IGF-1R protein expression levels were decreased. Furthermore, RA + Ful rats had lower levels of antioxidants PON-1 and catalase in the hippocampal regions. The changes in these molecular markers were statistically significant when compared to RA rats without Ful treatment (p < 0.05). Estrogen hindrance exaggerated oxidative stress, inflammation, and apoptosis which resulted in neuronal degeneration in the hippocampal regions in rheumatoid arthritis.

Gandou Bushen Decoction improves spermatogenesis and promotes spermatogenic cell proliferation in Wilson disease TX mice by activating testicular ERK signaling pathway

To investigate the therapeutic mechanism of Gandou Bushen Decoction (GDBSD) for improving reproductive disorders in male mouse models of Wilson disease (WD). Sixty male homozygous TX mice were randomized equally into 4 groups and treated with daily gavage of saline (WD model group), penicillamine (0.09 g/kg), or GDBSD (0.2 mL/10 g), or with intraperitoneal injection of U0126 (20 mg/kg) in addition to GDBSD gavage, with 15 male DL mice as control. After 4 weeks of treatment, copper content in testicular tissue of the mice was detected, and histopathology of the testes and epididymis was examined using HE staining and electron microscopy. TUNEL staining was used to identify apoptotic cells in the testes. The protein expressions of Bcl-2, Cytc, caspase-3, ERK, and p-ERK in the testicular tissue were evaluated with Western blotting, and BrdU-positive cells were detected with immunohistochemical labeling. Sperm density, viability, malformation rate and fertility levels of male mice were studied. Treatment with penicillamine and GDBSD obviously improved pathological changes of the testis, increased sperm density and motility, lowered sperm abnormality rate, fertility levels and increased testicular JOHNSEN score of TK mice, but the therapeutic effect of GDBSD was blocked by U0126. GDBSD treatment significantly lowered Cytc and caspase-3 expressions and increased Bcl-2 expression in the testicular tissue of TX mice (P < 0.05), while U0126 treatment significantly lowered testicular Bcl-2 expression level. No significant differences were found in total protein expression levels of ERK1/2 among the 5 groups, but p-ERK protein expression was significantly reduced in WD and U0126 groups and increased in penicillamine and GDBSD groups. GDBSD can improve spermatogenesis and enhance fertility of male TX mice with WD possibly by activating the ERK signaling pathway to enhance proliferation and reduce apoptosis of the spermatogenic cells.

Multifunctional approach with LHRH-mediated PLGA nanoconjugate for site-specific codelivery of curcumin and BCL2 siRNA in mice lung cancer

BackgroundLung cancer remains a leading cancer type, but current chemotherapy is limited by issues including poor drug delivery, toxicity, and resistance. To address these challenges, we developed a novel PLGA-PEG-LHRH (PPL) nanoconjugate system for improved drug delivery. Curcumin, known for its anticancer and P-gp inhibition properties, was co-loaded with bcl2siRNA (bclsR) to inhibit the bcl2 protein, thus overcoming both resistance mechanisms.ResultsThe PPL conjugate was successfully synthesized and characterized using FTIR, 1H NMR, XRD, XPS and BCA assay. Curcumin and bclsR-loaded PLGA nanoemulsions were prepared by double emulsion solvent evaporation method and characterized. The optimized nanoconjugate had size of 179 ± 16 nm, favorable zeta potential, high drug entrapment, and was confirmed via TEM. Controlled release studies indicated 83% drug release within 24 h. In vitro studies revealed significant cytotoxicity against A549 lung cancer cells, with the nanoconjugate showing IC50 of 8.24 µg/mL compared to 21.26 µg/mL for plain curcumin. Enhanced cellular uptake and effective targeting of A549 cells were observed. Molecular analyses demonstrated significant downregulation of MDR1 and Bcl2 RNA and protein expression, highlighting the nanoconjugates' ability to suppress resistance mechanisms. Pharmacokinetic studies in Wistar rats showed superior plasma drug concentrations, half-life, and AUC for the nanoconjugate versus pure drug suspension. Biodistribution studies showed increased drug accumulation in the lungs. In vivo efficacy studies in Balb/c mice demonstrated higher tumor inhibition ratios for CUR-siRNA PPL NPs (66.89%) and CUR-PPL NPs (59.84%) which was further confirmed with TNFα and p53 levels in blood. Histopathological studies showed good healing in the CUR-siRNA PPL NP- and CUR-PPL NP-treated mice compared to suspension.ConclusionFrom the study, it may be concluded that the PPL nanoconjugate system, loaded with curcumin and bcsR, can be potentially effective, multifunctional targeted approach for lung cancer therapy.Graphical

Design, synthesis, and antiproliferative activity evaluation of novel α-mangostin derivatives by ROS/MAPK signaling pathway

Novel hydroxamic acid and 3,6-amide modified α-mangostin derivatives were synthesized and evaluated their antiproliferative activities against KYSE 30 (esophageal cancer), HCT 116 (colon cancer), and HGC 27 (gastric cancer) cell lines. Most of the new derivatives displayed stronger anti-proliferative activities compared to α-mangostin. Among all the derivatives, compound 4a exhibited the most potent activity, with IC50 values of 0.57 ± 0.29 μM, 3.27 ± 0.16 μM, and 2.28 ± 1.02 μM against KYSE 30, HCT 116, and HGC 27 cells, respectively. Subsequent mechanism studies revealed that compound 4a inhibited cancer cells proliferation and colonies formation in a concentration-dependent manner. Additionally, compound 4a caused cell cycle arrest in a p53 dependent manner and induced apoptosis in p53 independent way. Meanwhile, 4a suppressed cell cycle related proteins (Cyclin D1 and cyclin B1) expression, increased pro-apoptotic proteins (cleaved PARP, cleaved caspase-7, and cleaved caspase-9) and decreased anti-apoptotic proteins (Bcl-2) expression. Moreover, 4a increased reactive oxygen species (ROS) levels in KYSE 30 cells and upregulated the expression of proteins related to the ROS related MAPK signaling pathway (p-ERK, p-p38, and p-JNK). These findings suggest that compound 4a holds promising potential as an antiproliferative agent by targeting MAPK signaling pathway to inhibit cell cycle progress, induce apoptosis and produce ROS in cancers.

1-Deoxynojirimycin affects high glucose-induced pancreatic beta-cell dysfunction through regulating CEBPA expression and AMPK pathway.

This study aims to explore the role of 1-Deoxynojirimycin (DNJ) in high glucose-induced β-cells and to further explore the molecular mechanism of DNJ effect on β-cells through network pharmacology. In the study, high glucose treatment of mouse INS-1 cells inhibited cell proliferation and insulin secretion, decreased the expression of Bcl-2 protein and Ins1 and Ins2 genes, promoted apoptosis, and increased cleaved caspase-3 and cleaved caspase-9 expression levels as well as intracellular reactive oxygen species (ROS) production. DNJ treatment significantly restored the dysfunction of INS-1 cells induced by high glucose, and DNJ showed no toxicity to normal INS-1 cells. Silencing CEBPA promoted, while overexpression of CEBPA relieved the dysfunction of pancreatic β-cells induced by high glucose. DNJ treatment partially restored the pancreatic β-cell dysfunction caused by silencing CEBPA. In conclusion, DNJ can inhibit high glucose-induced pancreatic β-cell dysfunction by promoting the expression of CEBPA.

Aristolochic acid I promotes renal tubulointerstitial fibrosis by up-regulating expression of indoleamine 2,3-dioxygenase-1 (IDO1)

Aristolochic acid I (AAI) is strongly nephrotoxic and can cause "Aristolochic acid nephropathy (AAN)". Aristolochic acid nephropathy is characterized by extensive renal interstitial fibrosis. However, the exact mechanism by which it occurs has not been fully elucidated. lt has been reported that indoleamine 2,3-dioxygenase-1 (IDO1) promotes renal fibrosis in renal disorders, but it is unclear how IDO1 functions in AAI-induced kidney fibrosis. In this work, we systematically examined the role of IDO1 in AAI-induced renal tubulointerstitial fibrosis. The results showed that AAI induced upregulation of IDO1 expression in renal tubular epithelial cells and mouse kidney. Inhibition of IDO1 expression reduced the levels of fibrosis-associated markers α-SMA, COL-I and FN and ameliorated renal tubular epithelial cell fibrosis. It also improved renal function, reduced collagen deposition, and ameliorated interstitial fibrosis in mice. Moreover, we discovered that inhibition of IDO1 decreased the expression of the apoptotic protein BAX, raised the expression of BCL-2 protein, and reduced apoptosis. The above studies suggest that IDO1 is a target of action in renal tubulointerstitial fibrosis caused by AAI, and inhibition of IDO1 may be a viable approach for the therapy of AAI-induced renal tubulointerstitial fibrosis.

Safety assessment and exploration of the mechanism of toxic effects of diallyl trisulfide: Based on acute and subacute toxicity and proteomics experiments

Ethnopharmacological relevanceGarlic (Allium sativum L.) is a widely consumed spice and condiment around the world, applied both as a food and as a traditional medicine, and is a natural strengthening agent for the body's circulatory and nervous systems. Diallyl trisulfide (DATS) is the major volatile organosulfur phytochemical found in garlic, with antithrombotic, anticoagulant, and antiplatelet activities as well as antioxidant, anti-infective, and other pharmacological effects. However, the safe dose and the underlying mechanisms of its toxic effects remain elusive. Aim of the studyDATS, an important pharmacologically active compound found in garlic, has garnered attention for its ability to fight cancer, antioxidant, anti-infective, and cardioprotective. The aim of this study was to evaluate the safety of DATS and to elucidate the potential mechanisms of its toxicity. Materials and methodsIn this study, ICR mice were selected for acute and subacute toxicity experiments according to OECD guidelines. The toxicity profile of DATS was analyzed by computer prediction software. Also, key differential proteins in spleen and serum were analyzed by proteomics. The binding stability of DAST to differential proteins was analyzed by molecular docking. Additionally, the regulatory relationship between DATS and differential proteins was verified by Western blot and ELISA experiments. ResultsThe results showed that the LD50 value of DATS in acute toxicity was 188.67 mg/kg. In subacute toxicity, water consumption and food intake were reduced in both male and female mice. In addition, the spleen and small intestinal organ coefficients were significantly elevated in male mice at the high dose of DATS; the blood biochemical indices ALB and TP were also significantly elevated. HE staining results showed significant damage to the spleen, liver, small intestine, and kidney of mice at high doses of DATS. Spleen and serum proteomics analyses showed that DATS significantly inhibited ZBP1 expression and upregulated TEC. ADMETlab 2.0 software predictions identified DATS as having potential genotoxicity, dermal sensitization, carcinogenicity, and respiratory and ocular toxicity. Docking results showed that the binding energies between DATS and TEC protein (PDB: 6F3F) and ZBP1 protein (PDB: 4KA4) were −3.7 kcal/mol, −2.4 kcal/mol, respectively. Western blot results showed that DATS-H significantly inhibited the expression of ZBP1 (only in male mice) and Bcl-2 proteins. ELISA results showed that DATS-H significantly increased the level of TEC protein both in male and female mice. ConclusionsLong-term administration of high-dose DATS may carry some risk of toxicity. Based on the amount of DATS in garlic, it is recommended that adults should not take more than 359 mg of DATS and 84.5 g of garlic per day. The mechanism of toxicity may be related to the fact that DATS significantly inhibits ZBP1 expression, upregulates TEC, and promotes apoptosis. This study provides valuable toxicological data for the effective evaluation of the long-term toxicity of DATS and offers an additional experimental basis for developing DATS as a healthy food or drug.

Synthesis and antitumor evaluation of glycyrrhetinic acid-dithiocarbamate hybrids.

Glycyrrhetinic acid (GA) is a naturally occurring triterpene compound. The aim of this study was to employ the pharmacophore hybrid strategy to merge GA with various dithiocarbamates and obtain novel compounds with better antitumor activities. We present a two-step synthetic protocol wherein the GA derivative underwent reaction with carbon disulfide and various secondary amines in a one-pot manner under mild conditions, facilitating the preparation of a series of structurally novel GA-dithiocarbamate derivatives. Bioassay screening revealed that the representative compound 3c demonstrated the capacity to reduce the mitochondrial membrane potential in Hep3B and Huh-7 cells, induce nuclear apoptosis, inhibit invasion and migration, and prompt both early and late apoptosis. Furthermore, our research findings indicated that this apoptotic phenomenon may be associated with the expression of Bcl-2, Bax, Bak, PARP, and cleaved-PARP proteins. Utilizing network pharmacology for predicting core targets and signaling pathways of compound 3c for hepatocellular carcinoma (HCC) treatment involved employing molecular docking models to demonstrate high affinity between compound and target protein. In conjunction with Western blot analysis, compound 3c may impact HCC through the PI3K-AKT-mTOR pathway.

Caffeic Acid Attenuates Neuronal Apoptosis, Oxidative Stress, and Memory Deficits via Antioxidant Properties in Aging Rats Induced by D-Galactose.

Aging is a main factor related to cognitive deficits. D-Galactose (D-gal), a monosaccharide, increases oxidative stress leading to cellular senescence, memory deficits, and neuronal apoptosis. Caffeic acid (CA) is an antioxidant that can interrupt free radicals and reduce oxidative stress. The present study purposely evaluated the benefits of CA in attenuating loss of neuronal apoptosis, oxidative stress, and memory in D-gal-activated rat brain aging. Male Sprague-Dawley rats were arbitrarily allocated into 6 groups (9 rats per group). The D-gal group was intraperitoneal (i.p.) injected with D-gal (50mg/kg). The CA groups were orally given 20 or 40mg/kg CA for 8weeks. During that time, the co-treatment groups were given 50mg/kg of D-gal and 20 or 40mg/kg of CA. The results reveal that animals receiving only D-gal showed memory deficit in both the novel object location (NOL) and novel object recognition (NOR) tests. Reduction in scavenging enzyme activities and levels of B-cell lymphoma 2 (Bcl-2) protein expression were detected in the D-gal group. Furthermore, D-gal treatment significantly enhanced in the number of p21 positive cells in the subgranular zone (SGZ) of the hippocampal dentate gyrus, Bcl-2 associated X protein (Bax) and caspase3 protein expression, and malondialdehyde (MDA) levels. By contrast, both 20 and 40mg/kg CA treatment alleviated these effects. These consequences confirmed that D-gal-activated brain aging led to enhancing apoptotic protein expression including Bcl-2, Bax, and caspase3 and memory impairments. Nevertheless, CA attenuated these effects in brain aging induced by D-gal via antioxidant properties.

Potential mechanism of perillaldehyde in the treatment of nonalcoholic fatty liver disease based on network pharmacology and molecular docking

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic metabolic liver diseases worldwide. Perillaldehyde (4-propyl-1-en-2-ylcyclohexene-1-aldehyde, PA) is a terpenoid compound extracted from Perilla, which has effective pharmacological activities such as anti-inflammatory, antidepressant, and anticancer. This study aimed to explore the pharmacological effects of PA in intervening with NAFLD and reveal its potential mechanisms. Firstly, we identified the core targets of PA intervention therapy for NAFLD through network pharmacology and molecular docking techniques. After that, in vitro animal experiments such as H&E and Masson staining, immunofluorescence, immunohistochemistry, and Western blot were conducted to validate the results network effectively pharmacology predicted. Network pharmacology analysis suggested that PPAR-α may be the core target of PA intervention in NAFLD. H&E and Masson staining showed that after low-dose (50 mg/kg) PA administration, there was a noticeable improvement in fat deposition in the livers of NAFLD mice, and liver tissue fibrosis was alleviated. Immunohistochemical and immunofluorescence analysis showed that low dose (50 mg/kg) PA could reduce hepatocyte apoptosis, decrease the content of pro-apoptosis protein Bax, and increase the expression of anti-apoptosis protein Bcl-2 in NAFLD mice. Western blot results confirmed that low-dose (50 mg/kg) PA could increase the expression of PPAR-α and inhibit the expression of NF-κB in NAFLD mice. Our study indicated that PA could enhance the activity of PPAR-α and reduce the level of NF-κB in NAFLD mice, which may positively affect the prevention of NAFLD.

Phenolics in soymilk manufactured from black and Proto soybeans by two continuous-ultrahigh-temperature-processing technologies inhibit DU145-prostate cancer cell proliferation through apoptosis.

Plant genotypes and processing technologies affect health properties of foods. How thermal processes with different sterilization values influence polyphenols in soymilk manufactured from different genotypes, particularly black soybean has not been well characterized. This study's aims were to investigate how one- and two-phase ultrahigh temperature (UHT) processing technologies, with wide differences of lethality (F0 158.5 and 6.35, respectively), affected anti-prostate cancer DU145-cell properties of black soymilk compared to light-yellow-Proto soymilk. Phenolics were extracted from soymilk and used for chemical, cell cycle and apoptosis analyses. Total isoflavones and genistein in black soymilk were significantly higher than Proto soymilk by either processing methods. Compared to one-phase processing, two-phase produced higher gallic acid in both soybeans, and higher oxygen radical absorbance capacity (ORAC) in black soymilk. Soymilk processed from both genotypes by both UHT methods inhibited DU145 cells. Two-phase-UHT processed black soymilk was more effective than one-phase UHT-processed soymilk. IC50 values (mg/mL) of black and yellow soy extracts against prostate cancer cells differed only by 11%-25%, which were lower than the differences of total isoflavone (29%-33%) or genistein (>50% between two beans). The mechanism by which soymilk inhibited DU145 cell proliferation was through apoptosis as evidenced by cell cycle analyses and expressions of caspase-3, Bcl-2, and PARP-1 proteins. Antioxidant properties, isoflavones, and phenolic acids were negatively correlated with prostate-cancer-cell inhibition IC50 (p<0.05) with ORAC having the highest coefficient (r=-0.98). Overall, two-phase-UHT processing of soybean would produce soymilk products with a higher health benefit than a one-phase UHT method. PRACTICAL APPLICATION: This study characterized the potential prostate cancer prevention effect of soymilk's phenolic extract in black soybean and compared with yellow soybean. The crude extract can be prepared much less costly than purified isoflavones and has potential to be developed into a dietary supplement. This study shows differences of soymilks made by continuous high-temperature processing of two soybean types and can serve as a scientific foundation for future clinical research and commercialization.

Early apoptosis detection in canine granulosa cells through the analysis of BCL-2 and BAX proteins during the follicular development associated with oocyte maturation

The objective of this study was to investigate the early follicular apoptosis in canine ovarian follicles by examining the expression of anti-apoptotic BCL-2 and pro-apoptotic BAX proteins throughout the estrous cycle associated with oocyte maturation. Follicular cells from preantral and antral follicles of varying sizes were isolated and grouped based on follicle type and estrous phase. Antral follicles underwent flow cytometry analysis, whereas preantral follicles were subjected to Western blotting. The meiotic capacity of oocytes from these different follicle types was evaluated through in vitro maturation. Statistical analysis included ANOVA and Duncan’s test. Results showed fluctuations in BCL-2 and BAX levels across different follicular stages and estrous phases. BCL-2 levels increased (P<0.05) with follicular development in antral follicles, particularly during estrus, while BAX exhibited variations peaking (P<0.05) during estrus. The BCL-2/BAX ratio in antral follicles was higher (P<0.05) in estrus and diestrus compared to anestrus and proestrus. Additionally, BCL-2 and BAX proteins were detected in preantral follicles, with varying expression levels (P<0.05) across estrous phases. The BCL-2/BAX ratio in preantral follicles was highest (P<0.05) during anestrus and estrus and decreased (P<0.05) in proestrus and diestrus. Oocytes from preantral follicles did not reach the MII stage, regardless of the levels of BCL-2/BAX. Conversely, oocytes obtained from large follicles during estrus showed the highest (P<0.05) maturation percentages, which were associated with the highest BCL-2/BAX ratio. These findings provide insights into the dynamic patterns of BCL-2 /BAX in canine follicles across the estrous cycle, shedding light on their potential roles in oocyte development.

Amitraz mechanisms of cytotoxicity in a characterized SH-SY5Y cells spheroid model

In recent years, spheroids (tridimensional cell cultures) have emerged as a more physiologically relevant replacement for monolayer models. Their distinctive advantage is the formation of an extracellular matrix that facilitates enhanced cellular interaction and communication, approximating the conditions observed in vivo. Therefore, the potential for conducting intricate cellular and molecular techniques in these models could offer a more precise assessment of pivotal proteins within various cellular pathways of interest. Amitraz (AMZ), an acaricide classified as a formamidine chemical, has been detected in honey at concentrations exceeding legal limits. The objective of this study was to characterize a spheroid model of SH-SY5Y cells and determine the cytotoxic effect of AMZ and its mechanisms of action on this spheroid. The formation of mature spheroids was observed on the seventh day following seeding. The results obtained with SH-SY5Y spheroids were an IC50 of 238.8 ± 17 µM and 224.3 ± 19 µM, respectively, after 24 and 48 h of exposure by the MTT assay. The findings revealed that AMZ did not exhibit any indications of inflammatory over-expression markers in the spheroids. Nevertheless, at 238.8 µM of AMZ, an increase incidence of late apoptosis within spheroid cells and Bcl-2 protein expression in peripheral spheroid cells were observed through annexin V and propidium iodide probe and immunofluorescence analysis. In conclusion, the results demonstrated that spheroids could be useful for an accurate assessment of toxicity, representing a viable alternative method for determining the mechanisms of action of AMZ and related compounds.

Liposomes-mediated enhanced antitumor effect of docetaxel with BRD4-PROTAC as synergist for breast cancer chemotherapy/immunotherapy.

It has been reported that proteolysis-targeting chimeras (PROTACs) can effectively degrade intracellular oncogenic proteins, providing an ideal strategy for cancer treatment. ARV825, a bromodomain-containing protein 4 (BRD4)-PROTAC, has demonstrated the capacity to enhance the antitumor effect of the classic chemotherapeutic agent docetaxel (DTX). However, there are three major challenges to the broader in vivo application of ARV825: poor solubility, poor permeability, and off-target effects. Additionally, the efficient co-delivery of ARV825 and DTX to tumor tissues for a synergistic therapeutic effect remains unresolved. In this study, liposomes were utilized as co-delivery vehicles for ARV825 and DTX to effectively address these issues. The well-established liposomes significantly improved the solubility of both ARV825 and DTX while maintaining a sustained release profile in blood-mimetic conditions. The co-loaded liposomes accumulated in tumor tissues via the enhanced permeability and retention (EPR) effect. After internalization, ARV825 effectively degraded intracellular BRD4 proteins and downregulated the expression of both Bcl-2 and PD-L1 proteins, thereby increasing tumor cell apoptosis and enhancing the tumor immune response. This, in turn, augmented the antitumor effect of DTX in vivo without undesired side effects. In conclusion, BRD4-PROTAC may serve as a promising synergistic agent alongside the conventional chemotherapeutic agent DTX, with liposomes functioning as effective co-delivery vehicles.

The Role of Serine Protease 8 in Mediating Gefitinib Resistance in Non-small Cell Lung Cancer.

This investigation aims to explore the expression levels of serine protease 8 (PRSS8) in gefitinib-resistant Non-Small Cell Lung Cancer (NSCLC) cell lines (PC9/GR) and elucidate its mechanism of action. We measured PRSS8 expression in gefitinib-resistant (PC9/GR) and sensitive (PC9) NSCLC cell lines using Western blot analysis. PRSS8-specific small interfering RNA (PRSS8-siRNA), a recombinant plasmid, and a corresponding blank control were transfected into PC9/GR cells. Subsequently, Western blot analyses were conducted to assess the expression levels of PRSS8, phosphorylated AKT (p-AKT), AKT, phosphorylated mTOR (p-mTOR), mTOR, and various apoptosis-related proteins within each group. Additionally, a cell proliferation assay utilizing Cell Counting Kit-8 (CCK8) was performed on each group treated with gefitinib. PRSS8 expression was markedly higher in PC9/GR cells compared to PC9 cells (p < 0.05). The group treated with PRSS8-siRNA exhibited significantly reduced protein expression levels of PRSS8, p-AKT, p-mTOR, β-catenin, and BCL-2 compared to the control siRNA (Con-siRNA) group, whereas expressions of Caspase9 and Bax were significantly increased. In the untransfected PC9/GR cells, protein expressions of PRSS8, p-AKT, pmTOR, and BCL-2 were significantly elevated when compared with the plasmid-transfected group, which also showed a significant reduction in Bax expression. The proliferative activity of the PRSS8-siRNA group postgefitinib treatment was significantly diminished at 24, 48, and 72 hours in comparison to the Con-siRNA group. The findings indicate that PRSS8 contributes to the acquisition of resistance to gefitinib in NSCLC, potentially through regulation of the AKT/mTOR signaling pathway.

Rational development of fingolimod nano-embedded microparticles as nose-to-brain neuroprotective therapy for ischemic stroke.

Ischemic stroke is one of the major diseases causing varying degrees of dysfunction and disability worldwide. The current management of ischemic stroke poses significant challenges due to short therapeutic windows and limited efficacy, highlighting the pressing need for novel neuroprotective treatment strategies. Previous studies have shown that fingolimod (FIN) is a promising neuroprotective drug. Here, we report the rational development of FIN nano-embedded nasal powders using full factorial design experiments, aiming to provide rapid neuroprotection after ischemic stroke. Flash nanoprecipitation was employed to produce FIN nanosuspensions with the aid of polyvinylpyrrolidone and cholesterol as stabilizers. The optimized nanosuspension (particle size = 134.0 ± 0.6nm, PDI = 0.179 ± 0.021, physical stability = 72 ± 0h, and encapsulation efficiency of FIN = 90.67 ± 0.08%) was subsequently spray-dried into a dry powder, which exhibited excellent redispersibility (RdI = 1.09 ± 0.04) and satisfactory drug deposition in the olfactory region using a customized 3D-printed nasal cast (45.4%) and an Alberta Idealized Nasal Inlet model (8.6%) at 15L/min. The safety of the optimized FIN nano-embedded dry powder was confirmed in cytotoxicity studies with nasal (RPMI 2650 and Calu-3 cells) and brain related cells (SH-SY5Y and PC 12 cells), while the neuroprotective effects were demonstrated by observed behavioral improvements and reduced cerebral infarct size in a middle cerebral artery occlusion mouse stroke model. The neuroprotective effect was further evidenced by increased expression of anti-apoptotic protein BCL-2 and decreased expression of pro-apoptotic proteins CC3 and BAX in brain peri-infarct tissues. Our findings highlight the potential of nasal delivery of FIN nano-embedded dry powder as a rapid neuroprotective treatment strategy for acute ischemic stroke.