Bcl-2 Expression Research Articles

MiR-484 as an "OncomiR" in Breast Cancer Promotes Tumorigenesis by Suppressing Apoptosis Genes.

Breast cancer (BC) is one of the most common causes of death among females. Cancer cells escape from apoptosis, causing the cells to proliferate uncontrollably. MicroRNAs (miRNAs) are known to regulate apoptosis in cancer cells. This study aimed to determine the change in miR-484 in different BC cells and its relationship with the apoptosis pathway. In the study, tumor and healthy tissue samples adjacent to the tumor were collected from 42 patients (6 benign, 36 malignant). Tissue samples were classified according to tumor type, tumor histological grade, proliferation index, and molecular subtypes. Gene expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR), and protein levels were determined using the Western Blot method. The results were analyzed using the delta-delta Ct method. Findings showed that miR-484 expression levels were higher in malignant tumors than in benign tumors, and higher in tumor tissues than healthy tissues. Additionally, it was determined that as Ki-67 levels and histological grade and aggressiveness increased, miR-484 expression levels also increased. In tumor tissue compared with healthy adjacent tissue, there was an increase in BCL2 expression and a decrease in Casp3 and Casp9 expression. Therefore, a positive correlation was found between miR-484 expression and BCL2, and a negative correlation was found between CASP3 and CASP9 expression. Our results show that miR-484 may play a roll as an onco-miR in BC. Increased miR-484 and BCL2, and decreased Casp3, in breast tumor tissues suggest that Casp9 expression may increase uncontrolled cell proliferation by suppressing apoptosis in BC cells and may contribute to tumor progression.

Differentiation ability of hematopoietic stem cells and mesenchymal stem cells isolated from human peripheral blood

Human hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the major stem cells of the bone marrow and are usually isolated from the peripheral blood. In the present study, we isolated these stem cells by an apheresis method from a donor who was administered granulocyte colony-stimulating factor (G-CSF). In vitro propagation of these stem cells showed a plastic-adherence property expressing CD73 and CD105 surface markers, which is a characteristic feature of MSCs. HSCs are non-adherent cells growing as a suspension culture, expressing CD150, CD133, CD34, and CD45 on their surface, which regulate the quiescence nature, and they derive energy from anaerobic glycolysis. The HSCs grow slowly compared to MSCs, are more viable, and survive for long periods under in vitro conditions, which are due to the expression of telomerase, BCL2, and Notch1 genes. The poor viability of MSCs in the culture due to the prominent expression of apoptotic genes BAX, caspase-3, and caspase-9 leads to rapid apoptosis. This was evident even in cells (astrocytes, osteocytes, and beta cells of the islets of Langerhans) differentiated from HSCs and MSCs, thus highlighting the importance of HSCs, the naive stem cells, in regeneration of tissues.

Immunoregulatory Effects of Codonopsis pilosula Polysaccharide Modified Selenium Nanoparticles on H22 Tumor-Bearing Mice

Codonopsis pilosula polysaccharide (CPP) and rare element selenium (Se) have been proved to exert various biological activities, and our previous study demonstrated that selenium nanoparticles modified with CPP (CPP-SeNPs) possessed significantly enhanced tumor cytotoxicity in vitro. This study aimed to investigated the inhibitory effects of CPP-SeNPs complex on H22 solid tumors via immune enhancement. In this study, the H22 tumor-bearing mice model was constructed, and the potential mechanisms of CPP-SeNPs antitumor effects were further explored by evaluating cytokines expression levels, immune cells activities and tumor cells apoptotic indicators in each group. The results demonstrated that CPP-SeNPs effectively exerted dose-dependent protective effects on the immune organs of tumor-bearing mice in vivo, leading to increase in peripheral white blood cell counts and inhibition of solid tumor growth with inhibitory rate of 47.18% in high-dose group (1.5 mg/kg). Furthermore, CPP-SeNPs treatment significantly elevated the levels of TNF-α, IFN-γ, and IL-2 in mice sera, enhanced NK cell cytotoxicity, augmented macrophage phagocytosis capacity, as well as increased both the amounts and proliferation activity of lymphocyte subsets. CPP-SeNPs improved the immune system’s ability to clear tumor cells by up-regulating Bax expression while down-regulating Bcl-2 expression within solid tumors, indicating the potential activation of mitochondrial apoptosis pathway. Therefore, CPP-SeNPs administration can effectively inhibit tumor growth by enhancing immune response in tumor-bearing mice, which might be relevant to the regulation of gut microbiota short-chain fatty acids metabolisms. These findings could provide theoretical support and data foundation for further development of CPP-SeNPs as functional food and drug adjuvants.

Matrine promotes colorectal cancer apoptosis by downregulating shank-associated RH domain interactor expression

BACKGROUND The 5-year survival rate of patients with colorectal cancer (CRC) in China is only 56.9%, highlighting the need for new therapeutic drugs. Previous studies have shown that matrine exhibits antitumor effects by inducing apoptosis. However, the mechanism by which matrine regulates antiapoptotic proteins in CRC remains unclear. AIM To identify apoptotic proteins from proteomics and investigate the role of matrine in impeding CRC apoptosis by regulating these proteins. METHODS Tumor and adjacent normal tissues were collected from 52 patients with CRC who underwent surgery between January and December 2021. Data-independent acquisition quantitative proteomic analysis was performed to identify differentially expressed apoptotic proteins. The selected apoptotic proteins were identified through their association with tumor-node-metastasis (TNM) stage and prognosis, then confirmed by immunohistochemical (IHC) staining in validation cohort. In vitro , the role of matrine or apoptotic proteins on cancer cells were analyzed. RESULTS Compared to normal tissues, 88 anti-apoptotic proteins from proteomic results were selected. Among them, Shank-associated RH domain interactor (SHARPIN) was identified because of its relationship with TNM stage and overall survival in TCGA database. In the IHC-confirmed cohort, SHARPIN was highly expressed in CRC tissues and localized in the cytoplasm. Higher SHARPIN expression was associated with TNM stage, carbohydrate antigen 153 levels, and gross type compared to low expression. SHARPIN knockdown promoted apoptosis, significantly upregulated the expression of Bcl-2 associated agonist of cell death, Bcl-2 associated X protein, caspase 3, and caspase 8, and downregulated B-cell lymphoma-2 (P < 0.05). Importantly, matrine treatment promoted apoptosis and reversed the proliferation, invasion, and migration of CRC cells by repressing SHARPIN. CONCLUSION SHARPIN was identified as an upregulated anti-apoptotic protein in CRC, and matrine exhibited anticancer effects by downregulating its expression. Thus, matrine appears to be a promising drug for CRC.

Piceatannol-3'-O-β-D-glucopyranoside inhibits neuroexcitotoxicity and ferroptosis through NMDAR/NRF2/BACH1/ACSL4 pathway in acute ischemic stroke.

Neuronal protection is a well-established method of acute ischemic stroke (AIS) treatment. The pharmacodynamic effect of Piceatannol-3'-O-β-D-glucopyranoside (Chinese name: Quzhazhigan, QZZG) on AIS has been reported, but the molecular mechanism of this effect remains unknown. The purpose of this study is to elucidate the pharmacodynamic effects and mechanisms of QZZG in the treatment of AIS. A combined network pharmacology and metabolomics approach was used to predict the key targets and pathways of QZZG in the treatment of AIS and to elucidate the mechanism of QZZG through experimental validation. In this study, QZZG improved histopathologic features and reduced infarct volume and neurologic deficit scores. Integrated network pharmacology and metabolomics revealed that QZZG may protect neurons by regulating glutamate and its receptors, and that glutamate is closely related to NMDAR1, NRF2, and Caspase-3. Pathway analysis results suggested that NMDAR-mediated Ca2+ inward flow is one of the critical pathways. In terms of neuroexcitotoxicity QZZG inhibited glutamate content, reduced Ca2+ inward flow, protected mitochondrial function, and reduced ROS, as well as being able to effectively inhibit the expression of NMDAR1, Caspase-3, Bax, and promote the expression of Bcl-2, NMDAR2A. In terms of ferroptosis QZZG promoted NRF2, HO-1, GPX4 and nuclear-NRF2, inhibited the expression of BACH1 and ACSL4, and suppressed Fe2+ accumulation and lipid peroxidation. Silencing of BACH1 resulted in elevated expression of NRF2 and decreased expression of ACSL4, which inhibited the sensitivity of neurons to ferroptosis. QZZG was able to further increase NRF2 expression under conditions of silencing BACH1. QZZG induced NRF2 and inhibited BACH1, ACSL4 was inhibited by ML385, and inhibition of NRF2 induced the expression of BACH1 and ACSL4, QZZG protects neurons in an NRF2-dependent manner. In summary, QZZG inhibited neuroexcitotoxicity and ferroptosis by regulating the NMDAR/NRF2/BACH1/ACSL4 pathway. The study provided a relatively novel perspective on the mechanism of traditional Chinese medicine (TCM) treatment of the disease.

Combined bisoprolol and trimetazidine ameliorate arsenic trioxide -induced acute myocardial injury in rats: targeting PI3K/GSK-3β/Nrf2/HO-1 and NF-κB/iNOS signaling pathways, inflammatory mediators and apoptosis

Background Arsenic-trioxide (ATO) is an effective therapy for acute promyelocytic leukemia. Unfortunately, its utility is hindered by the risk of myocardial injury. Both bisoprolol (BIS) and trimetazidine (TMZ) have various pharmacological features, including anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Aim The cardioprotective effects of BIS and TMZ were studied, and their mechanistic role in ameliorating ATO-induced myocardial injury. Materials and Methods Forty male Wistar rats were randomly allotted into five groups as follows: normal control group (received normal saline, orally), ATO group (7.5 mg/kg, orally), BIS (8 mg/kg, orally), TMZ (60 mg/kg, orally), and finally combination group (BIS+TMZ+ATO). Following 21 days, samples of serum and cardiac tissues were obtained to perform biochemical, molecular, and histopathological investigations. Results The present study showed that ATO caused myocardial injury evidenced by changes in serum biomarkers (Aspatate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase-MB, and cardiac troponin-1), electrolyte imbalance, and lipid profiles alongside histopathologic changes. In addition, ATO administration significantly elevated malondialdehyde, nicotinamide adenine dinucleotide phosphate hydrogen oxidase, myloperoxidase, total nitrite, inducible nitric oxide synthase, tumor necrosis factor-alpha, interleukin-1β, interleukin-6, 8-Hydroxy-2’-deoxyguanosine, nuclear factor NF-kappa-B p65 subunit, glycogen synthase kinase-3 beta, and caspase-3 expression contemporaneously with down-regulation of reduced glutathione, glutathione peroxidase, superoxide dismutase, catalase, heme oxygenase 1, nuclear factor erythroid 2–related factor 2, phosphatidylinositol-3 kinase, p-PI3K, and Bcl-2 expression. Interestingly, pretreatment with BIS and TMZ significantly reversed the detrimental effects of ATO-induced myocardial injury at both cellular and molecular levels. Otherwise, combining the two drugs displayed more enhancement than each drug alone. Conclusion The present research depicted that BIS and TMZ have the potential to protect the heart and provide therapeutic benefits by preventing acute heart injury induced by ATO. This is achieved by reversing the redox-sensitive pathway, reducing inflammation, and inhibiting apoptosis.

Effects of salinity stress on the growth performance, histological characteristics, and expression of genes related to apoptosis and immunity in channel catfish (Ictalurus punctatus).

Salinity is an important environmental factor affecting fish growth and development. To investigate the effects of salinity stress on the growth performance, tissue structures, and expression of genes related to apoptosis and immune response in large-sized channel catfish (Ictalurus punctatus) juveniles, an 8-week salinity stress experiment was conducted. Six salinity levels (3, 3.8, 4.7, 5.9, 7.3, and 9 psu) were set according to an equal logarithmic concentration gradient method, along with a control group (0), and growth performance and physicochemical indices were measured. The survival rate of channel catfish in all salinity groups was 100%. Body length, body weight, weight gain rate, and specific growth rate decreased with increasing salinity, with higher salinity levels resulting in more significant reductions in growth performance. At 9 psu, both the specific growth rate (SGR) and weight gain rate (WGR) decreased significantly to 0.61%/day and 40.86%, respectively (p < 0.05). Histological analysis results indicated that with the increase in salinity, the number of intestinal mucosal folds relatively increased, while the number of goblet cells gradually decreased. Hepatocytes became more densely packed, and the hepatic lobule gaps widened. Apoptosis detection results revealed that in the liver, the number of apoptotic cells at 4.7 psu salinity was more than that in the control group and at 9 psu salinity. In the intestine, the number of apoptotic cells at 9 psu salinity was significantly higher than in the other two groups (p < 0.05). Quantitative polymerase chain reaction (qPCR) analysis of the expression patterns of genes related to apoptosis and immunity showed that the expression levels of caspase 3, caspase 8, INF-I, IL-1β, and bax genes in the liver and intestinal tissues were higher in the experimental groups than in the control group, while the expression of bcl-2 decreased with increasing salinity in liver tissue but increased in intestinal tissue. These findings can provide theoretical guidance for the aquaculture of channel catfish in saline-alkali land.

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.

Efect of N-acetylcysteine on HepG2 cells which were induced into fatty liver cells.

Non-alcoholic fatty liver disease is a prevalent liver condition that can progress to fibrosis and cirrhosis. It also poses a risk for hepatocellular carcinoma, underscoring the importance of identifying effective treatments. N-acetylcysteine, an inhibitor of glutathione depletion, shows promise in modulating intracellular glutathione biosynthesis and combating oxidative stress, making it a potentially beneficial therapy for liver fibrosis in non-alcoholic fatty liver disease. This study assesses the impact of N-acetylcysteine on HepG2 cells which were induced into fatty liver cells was evaluated. HepG2 cells were cultured in DMEM and seeded onto six-well plates at a density of 5 × 105 cells. Following a 24-h incubation period, the cells were exposed to a medium inducing fat accumulation. Subsequently, the cells were treated with varying concentrations of N-acetylcysteine for 48 h. Some plates were utilized for Real-Time-PCR tests, while others underwent Oil Red staining. The findings indicated a significant increase in the expression of fatty acid β-oxidation genes in the group treated with 10mM N-acetylcysteine (p < 0.05), along with reduced expression of lipogenesis-related genes (p < 0.05) in N-acetylcysteine-treated groups. Analysis of apoptotic gene expression revealed decreased BAX expression but increased BCL2 expression in the N-acetylcysteine-treated groups. Oil Red staining demonstrated a dose-dependent reduction in lipid droplets compared to the control group. This study's results suggest that N-acetylcysteine has the potential to decrease lipid droplets and modulate lipid metabolism effectively.

Butorphanol Alleviates Hypoxia/Reoxygenation-Induced Myocardial Injury by Activating Wnt/β-Catenin Signal Pathway.

Ischemic heart disease remains a global health problem with high morbidity and mortality. Butorphanol, as a novel opioid, has been discovered with its cardioprotective properties. The purpose of this study was to explore that butorphanol can alleviate hypoxia/reoxygenation (H/R)-induced myocardial injury by activating the Wnt/β-catenin signal pathway. In this study, Cell Counting Kit-8 (CCK-8), lactate dehydrogenase (LDH) kit, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and Western blotting experiments were performed to observe butorphanol cardioprotective function and expression of Wnt signaling pathway proteins. For the main result, anti-apoptotic effect and higher expression of Wnt signaling pathway proteins were observed with the increasing concentrations of butorphanol. Compared with the H/R group, higher cellular viability, lower LDH release, and smaller apoptotic cell population were found in the butorphanol group. In addition, expression levels of apoptosis-related protein Bax and Cleaved Caspase-3 decreased, and increased expression of Bcl-2 were observed. Conversely, the protective effects of butorphanol were attenuated in the XAV939 group. In summary, butorphanol attenuates hypoxia/reoxygenation-induced myocardial injury by activating the Wnt/β-catenin signal pathway. Our work provides a theoretical basis for butorphanol's myocardial protective function.

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.

Comparison of biocompatibility of epoxy resin and bioceramic-based root canal sealers.

The purpose of this study is to compare the cytotoxicity of 2 bioceramic-based (Bioserra and AH Plus Bioceramic (Bio)) and 2 epoxy resin-based (Sealart and AH Plus) root canal sealers on the Saos-2 cell line. The cytotoxicity of the root canal sealers was determined using the MTT metabolic activity assay. The mRNA expression levels of Bcl-2, Beclin1, Cas-3, IL-6, LC3, MMP-9 and TNF-α genes were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and the expression of Beclin1 and LC3 proteins were assessed using western blot (WB) method. The canal sealers' total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were measured using photometric kits. AH Plus Bio significantly increased metabolic activity compared to the 2 resin-containing sealers, but there was no difference with Bioserra. Sealart was clearly the most cytotoxic group. QRT-PCR analysis revealed that the mRNA expressions of Bcl-2, Beclin1, Cas-3, IL-6, LC3, and MMP-9 in the Bioserra group, Bcl-2, Cas-3, and IL-6 in the Sealart group, Beclin1, Cas-3, IL-6, and MMP-9 in the AH Plus group, and Bcl2 in the AH Plus Bio group were stimulated. The OSI results showed no significant difference between the root canal sealer groups. In the study, the best biocompatibility results in MTT, qRT-PCR, WB and OSI were in the AH Plus Bio group. Although Bioserra, the study's other bioceramic paste, was found to be biocompatible according to MTT and OSI measurements, it increased the expression of all genes in qRT-PCR except for TNF-α, and also increased LC3 protein levels in WB. Since periradicular tissues can be directly affected by the materials used in root canal treatment, the sealers with high biocompatibility are being developed. In vitro studies examining the biocompatibility of newly produced root canal sealers are guiding for clinical success.

Dihydromyricetin Suppresses Lipopolysaccharide-Induced Intestinal Injury Through Reducing Reactive Oxygen Species Generation and NOD-Like Receptor Pyrin Domain Containing 3 Inflammasome Activation.

As an integral component of the gram-negative bacterial cellular envelope, excess production of lipopolysaccharide (LPS) regularly precipitates causing intestinal damage and barrier dysfunction in avian species. Dihydromyricetin (DHM), a naturally occurring constituent in rattan tea, exhibits protective characteristics against various tissue injuries. However, the intervention mechanism of DHM on intestinal injury induced by LPS in chickens has not been determined. Consequently, this study aimed to elucidate the mechanisms through which DHM mitigates LPS-induced intestinal damage in chickens through the reactive oxygen species (ROS)-NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Primary intestinal epithelial cells (IECs) were isolated and cultured from 14-day-old specific pathogen free (SPF) chicken embryos, and DHM ranging from 20 to 320 μmol/L increased cell survival rates. Additionally, DHM at 20 and 40 μmol/L demonstrated reduction in oxidative stress and ROS accumulation, mirroring the impact of ROS inhibitor (2.5 mmol/L NAC). DHM efficiently regulated ROS production, thereby augmenting ZO-1, occludin and claudin-1 expression to enhance barrier function; upregulating bcl-2 expression and downregulating bax and caspase-3 expression to regulate apoptosis and suppressing inflammation in IECs. Suppression of ROS subsequently attenuates NLRP3 inflammasome activation, leading to a remarkable downregulation of IL-1β, IL-18 and lactate dehydrogenase (LDH) secretion, consistent with direct inactivation of NLRP3 inflammasome (10 μmol/L MCC950). Notably, DHM diminished IL-1β and IL-18 levels and LDH activity via suppression of ROS-regulated NLRP3 and caspase-1 expression and activation. In summary, DHM prevents LPS-induced intestinal impairment by modulating ROS generation and NLRP3 inflammasome activation.

Pirfenidone promotes cell cycle arrest and apoptosis of triple‑negative breast cancer cells by suppressing Hedgehog/GLI1 signaling.

Breast cancer is a common malignant tumor in women and triple-negative breast cancer (TNBC) is the most challenging type of breast cancer with poor prognosis. We aimed to elucidate the effects of pirfenidone, a FDA-approved oral anti-fibrotic drug which has recently shown antitumor potential, in the progression of TNBC and the underlying mechanisms. After TNBC cells were treated with pirfenidone, cell viability was evaluated using CCK-8 assay. The EDU staining was applied for reflecting the ability of cell proliferation. Additionally, cell cycle distribution and apoptotic rate of TNBC cells exposed to pirfenidone were determined by flow cytometry. The levels of proteins associated with cell cycle, apoptosis and Hedgehog/gliomaassociated oncogene homolog (GLI)1 signaling was examined through western blot. Then, GLI1 was upregulated to analyze the proliferation, cell cycle and apoptosis of TNBC cells in the presence of pirfenidone to reveal the regulatory mechanism. Pirfenidone dose-dependently decreased the viability and proliferation of MDA-MB-231 and HCC-1937 cells. Besides, the distribution of TNBC cells in G0/G1 phase was significantly elevated by pirfenidone, accompanied by downregulated levels of CyclinD1, CDK4 and CDK6. Simultaneously, pirfenidone caused remarkably increased apoptotic MDA-MB-231 and HCC-1937 cells, coupled with downregulated BCL2 expression as well as upregulated Bax, cleaved caspase3 and cleaved PARP expression. Expression of proteins related to Hedgehog/gliomaassociated oncogene homolog (GLI)1 signaling was tested through western blot. Particularly, GLI1 and PTCH1 levels were dose-dependently inhibited after pirfenidone exposure. Interestingly, GLI1 overexpression attenuated the influences of pirfenidone on the proliferation, cell cycle and apoptosis of TNBC cells. Collectively, pirfenidone arrests the cell cycle and promotes apoptosis of TNBC cells by suppressing Hedgehog/GLI1 signaling.

Osteoking inhibits apoptosis of BMSCs in osteoporotic rats via PI3K/AKT signaling pathway.

This study aims to investigate whether Osteoking inhibits apoptosis of BMSCs in osteoporotic rats via the PI3K/AKT signaling pathway and to conduct a detailed exploration of this mechanism. The goal is to provide a theoretical basis for the clinical application of Osteoking in osteoporosis treatment. A rat model of osteoporosis was established through bilateral ovariectomy (OVX), followed by treatment with Osteoking. After ten weeks of therapy, BMD was evaluated. The biomechanics of the left tibia were measured, the left femur was sequenced, and the right tibia was stained using histomorphometric and Masson's staining methods. Peripheral serum was collected to measure bone-related markers, including E2, PINP, and CTX. RNA-Seq results were verified using the remaining bone samples. Comparative analysis demonstrated the efficacy of Osteoking in treating osteoporosis and provided preliminary insights into the underlying mechanisms. Primary BMSCs were cultured using bone marrow apposition. CCK8 assays were conducted to screen the intervention conditions of Osteoking and LY294002. Various concentrations of Osteoking-containing serum and LY294002 were tested separately to determine the optimal intervention concentration for drug delivery. The impact of Osteoking on lipid formation was also evaluated. Following treatment of BMSCs from OVX rats with Sham serum, OVX serum, OVX+LY294002 serum, and Osteoking+LY294002 serum, the expression of PI3K/AKT/mTOR, osteogenesis-related regulatory factors, and apoptosis-related regulatory factors was assessed. Flow cytometry was employed to evaluate apoptosis in BMSCs. Osteoking significantly improved whole-body BMD and bone biomechanical indices in OVX rats. It also significantly elevated the serum levels of E2 and PINP while reducing the level of CTX, which significantly improved bone microstructure and promoted new bone formation. RNA-seq analysis indicated that the therapeutic mechanism involved the PI3K/AKT signaling pathway. Osteoking increased the expression of RUNX2 and decreased the expression of PPAR-γ, a marker of lipogenesis, in OVX rats. Extraction of BMSCs for subsequent studies revealed a significant reduction in proliferation and osteogenic differentiation, along with an increase in lipogenic differentiation, in the OVX group. Osteoking treatment inhibited the expression of PPAR-γ and increased the expression of RUNX2 in BMSCs. Additionally, Osteoking reversed the LY294002-mediated inhibition of PI3K/AKT/mTOR signaling pathway activation, increased the expression of the apoptosis-protecting protein Bcl2, and decreased the expression of apoptosis-associated proteins Caspase3 and Bax. Osteoking markedly improved bone microstructure, biomechanics, and bone density in OVX rats. Osteoking-containing serum reversed the imbalance in lineage differentiation in OVX rats, characterized by reduced osteogenic differentiation and increased lipid differentiation of BMSCs. Furthermore, Osteoking-containing serum significantly increased BMSC proliferation and prevented apoptosis in OVX rats through the PI3K/AKT signaling pathway.

2-hydroxy-3-methyl anthraquinone promotes apoptosis and inhibits invasion of human hepatocellular carcinoma cells by targeting nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1/cellular tumor antigen p53 signaling pathway.

To investigate the anti-liver cancer effect of 2-hydroxy-3-methyl anthraquinone (HMA) and the specific mechanism based on nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1 (SIRT1)/cellular tumor antigen p53 (p53) pathway. Cell counting kit-8 method was used to observe the effect of HMA on the activity of human hepatocellular carcinoma cells (HepG2) cells. At 72 h and 80 μL HMA, the apoptosis rate of HepG2 cells in each group was measured by flow cytometry. Transwell was used to assay for cell invasion. The protein expression levels of SIRT1, p53, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase-9 (CASP9) and caspase-3 (CASP3) were detected by Western Blot. HMA significantly inhibited the proliferation of HepG2 cells, The half inhibiting concentration (IC50) of the HMA at 24, 48 and 72 h were examined and it were 126.3, 98.6, and 80.55 μM, respectively. Compared with the control group, the apoptosis rate of HMA, Selisistat (EX527), and HMA+ EX527 groups enhanced, while the apoptosis rate of SRT1720 diminished, demonstrating that inhibition of SIRT1 can lead to apoptosis of HepG2 cells. HMA+ EX527 group had the highest apoptosis rate, the lowest expression of SIRT1 and Bcl-2, and the highest expression of p53, Bax, CASP9 and CASP3. The number of invasions of HepG2 was significantly reduced after HMA and EX527 intervened. Western blot shows HMA could inhibit SIRT1, promote the expression of p53, and decrease the ratio of Bcl-2/Bax. HMA induced apoptosis in HepG2 cells, while inhibiting proliferation and invasion. The mechanism of HMA against HCC may be related to the SIRT1/p53 pathway.

Investigation of the Potential Mechanism of Compound Dragon's Blood Capsule against Myocardial Ischemia Based on Network Pharmacology.

Dragon's blood is widely consumed in China, Vietnam and Laos to promote blood circulation. A Compound Dragon's blood capsule (CDC) is a patented medicine composed of dragon's blood, notoginseng, and borneol. This combination is purported to stabilize coronary heart disease and myocardial ischemia. However, the possible mechanisms and the characterization of its drug targets' relevance at the systemic level remain unclear. The present study aims to reveal the potential mechanisms of CDC's anti-myocardial ischemia effect. The potential mechanisms were investigated by network pharmacology and qRT-PCR was used to verify the expression levels of key genes of PI3k-Akt pathway. S1PR2 and AGTR1 were the common targets, which involved 6 biological processes annotated by KEGG and GO analysis. The qRT-PCR results showed a remarkable increase in the expression of Pi3k, Pdk1, Akt, Mdm2, Bcl2, and mTOR. Results also showed a decline in the expression of P53 and Casp3 after CDC intervention. CDC has a significant anti-myocardial ischemia effect through the PI3k/Akt pathway, which demonstrates that CDC is a suitable adjuvant to treat CHD and provides a theoretical basis for its further clinical application.

Pink1/Parkin signaling mediates pineal mitochondrial autophagy dysfunction and its biological role in a comorbid rat model of depression and insomnia

Using a chronic unpredictable mild stress (CUMS) combined with multi-platform water environment sleep deprivation (SD) as an animal model, the occurrence and development of human depression combined with insomnia were simulated. The abnormal mitochondrial autophagy signaling caused by the putative kinase 1/Parkin E3 ubiquitin protein ligase (Pink1/Parkin) signaling pathway directly affects the normal secretion of melatonin by the pineal gland, which may explain the pathogenesis of depression combined with insomnia. This study aims to explore the depression-like behavior, sleep changes, central oxidative stress response, pineal mitochondrial autophagy damage, melatonin secretion, histopathological changes of the pineal gland, and the expression of Pink1/Parkin signaling-related factors in CUMS+SD rats. The results showed that the levels of reactive oxygen species (ROS) in cerebrospinal fluid of CUMS+SD rats significantly increased along with the inflammatory factors Interleukin-1β (IL-1β) and nuclear factor kappa-B (NF-κB) in cerebrospinal fluid. In addition, the number of pineal gland cells significantly decreased, cell boundaries became blurred, cell volume shrank, and apoptotic bodies appeared in the pineal gland tissue under HE staining, indicating pineal gland inflammation. Sleep deprivation further disrupted the levels of autophagy damage factors, including histamine (MDA), glutathione (GSH), and catalase (CAT), in the cerebrospinal fluid of CUMS + SD rats. Transmission electron microscopy of the pineal gland in CUMS+SD rats revealed damage to mitochondrial autophagy. The levels of 5-hydroxytryptamine (5-HT) and aromatic amine-N-acetyltransferase (AANAT) in the cerebrospinal fluid, as well as melatonin levels in the pineal gland, were significantly decreased. Additionally, the expression of IL-1β, NF-κB, Pink1, and Parkin in the pineal gland of CUMS+SD rats significantly increased. The expression of microtubule-associated protein 1 light chain 3-β (LC3), selective autophagy adaptor protein (P62), cytochrome c oxidase IV (COXIV), and mitochondrial outer membrane translocation enzyme 20 (TOM20) proteins downstream of the Pink1/Parkin signaling pathway was enhanced, while the expression of downstream brain-derived neurotrophic factor (BDNF), Beclin 1, and BCL2 interacting protein 3 (BNIP3) proteins was negatively regulated. Pink1/Parkin signaling may specifically respond to mitochondrial autophagy damage in the pineal gland, affecting the normal synthesis and secretion of melatonin in the pineal gland. In summary, mitochondrial autophagy damage in the pineal gland affects the normal secretion of melatonin in CUMS+SD rats, which is closely related to the specific autophagy signaling impairment of Pink1/Parkin pathway, which may mediate the occurrence of depression combined with insomnia.

Integrating Metabolomics, Histopathology, and Cardiac Marker Analysis to Assess Valsartan's Efficacy in Mitigating Dasatinib-Induced Cardiac Toxicity in Sprague-Dawley Rats.

Dasatinib (DASA) is associated with cardiotoxic effects, posing risks to patients. Valsartan (VAL) may offer protective benefits against these effects. This study evaluates the impact of DASA, VAL, and their combination on cardiac health. Wistar rats were treated with DASA, VAL, and a combination of VAL and DASA intraperitoneally every other day for 14 days. Body weight and survival rates were monitored. Serum levels of cardiac biomarkers (CPK, LDH, AST) were analyzed. Histopathological and immunohistochemical analyses assessed myocardial architecture and apoptosis-related protein expression. Metabolomic profiling was conducted using GC-MS to identify metabolic changes across treatment groups. The DASA group experienced significant weight loss and a 50% mortality rate, while the combination group had no mortality. Cardiac biomarkers like CPK, LDH, and AST were elevated in the DASA group but significantly reduced in the VAL + DASA group. Histopathological examination showed significant myocardial injury in the DASA group, with improved cardiac tissue morphology in the combination group. Immunohistochemical analysis revealed altered expression of apoptosis-related proteins, including caspase-3 and BCL-2, with improved levels in the combination group compared to DASA alone. Metabolomic profiling identified significant metabolic shifts, with 15 metabolites differentiating the treatment groups, and the VAL + DASA group mitigated the metabolic disturbances caused by DASA. The study suggesting VAL's potential therapeutic role in managing DASA-induced cardiac toxicity. The combination of VAL with DASA not only improved survival rates and reduced cardiac biomarker levels but also preserved myocardial architecture and normalized metabolic profiles. These findings highlight the importance of integrated approaches in evaluating drug efficacy and suggest VAL as a promising candidate for protecting cardiac function in preclinical models of DASA therapy.

Captopril inhibits the overproduction of proopiomelanocortin and adrenocorticotropic hormone in the pituitary gland of male diabetic mice in close relationship with an increase in glucocorticoid receptor expression.

Prior investigation shows that diabetic patients present hypothalamus-pituitary-adrenal (HPA) axis hyperactivity related to impaired negative feedback. This study investigates the effect of Captopril on the overproduction of adrenocorticotropic hormone (ACTH) and its precursor proopiomelanocortin (POMC) in the pituitary gland of male diabetic mice. Diabetes was induced by intravenous injection of alloxan into fasted Swiss-webster mice, and the animals were treated with Captopril for 14 consecutive days, starting 7 days post-diabetes induction. Plasma corticosterone levels were evaluated by ELISA, while pituitary gland expressions of angiotensin-II type 1 receptor (AT1), angiotensin-II type 2 receptor (AT2), ACTH, Bax, Bcl-2, KI-67, POMC, and glucocorticoid receptor (GR) were evaluated using immunohistochemistry or western blot. Diabetic mice showed pituitary gland overexpression of AT1, without altering AT2 levels, which were sensitive to Captopril treatment. Furthermore, diabetic mice presented hypercortisolism, along with an increase in the number of corticotroph cells, POMC and ACTH expression, and number of proliferative cells, and a decrease of GR expression in the pituitary gland. In addition, treatment with Captopril reduced systemic corticosterone levels, corticotroph and proliferative cell numbers, and Bcl-2, POMC, and ACTH expression in the pituitary gland of diabetic mice, besides increasing the expression of Bax and GR. In conclusion, these findings show that Captopril is a promising therapy for treating complications associated with HPA axis hyperactivity in diabetic patients, in a mechanism probably related to the downregulation of POMC production in the pituitary gland and subsequent reduction of systemic corticosterone levels.