TUNEL Research Articles

Sunitinib alleviates hepatic ischemia reperfusion injury by inhibiting the JAK2/STAT pathway and promoting the M2 polarization of macrophages

Background Hepatic ischemia reperfusion injury (IRI) is a common liver surgery complication. This study aims to explore the effect and potential mechanism of Sunitinib – a multi-target tyrosine kinase inhibitor – on hepatic IRI. Methods We established a hepatic IRI model using C57BL/6 mice, and integrated 40 mg/kg of Sunitinib, solely or combined with 100 μg/kg of coumermycin A1 (C-A1), in the treatment strategy. H&E staining, TUNEL assay, and detection of serum ALT and AST activities were used to assess liver damage. Further, ELISA kits and Western Blots were utilized to determine IL-1β, TNF-α, IL-6, CXCL10, and CXCL2 levels. Primary macrophages, once isolated, were cultured in vitro with either 2 nM of Sunitinib, or Sunitinib in conjunction with 1 μM of C-A1, to gauge their influence on macrophage polarization. qPCR and Western blot were conducted to examine the level of p-STAT1/STAT1, p-STAT3/STAT3, p-JAK2/JAK2, and M1/M2 polarization markers. To quantify immune cell infiltration, we applied Immunofluorescence. Results Sunitinib pretreatment significantly alleviated liver injury and reduced p-STAT1/STAT1, p-STAT3/STAT3, p-JAK2/JAK2 levels. In vitro, Sunitinib treatment curbed M1 polarization induced by LPS + IFN-γ and bolstered M2 polarization triggered by IL-4. C-A1 application upregulated JAK2/STAT pathway phosphorylation and promoted LPS + IFN-γ-induced M1 polarization, which was reversed by Sunitinib treatment. In IL-4-stimulated macrophages, application of C-A1 activated the JAK2/STAT pathway and decreased M2-type macrophages, which was reversed by Sunitinib treatment either. Conclusion Sunitinib is capable of guiding the polarization of macrophages toward an M2-type phenotype via the inhibition of the JAK2/STAT pathway, thereby exerting a protective effect on hepatic IRI.

Pyrimidine compounds BY4003 and BY4008 inhibit glioblastoma cells growth via modulating JAK3/STAT3 signaling pathway

Glioblastoma (GBM) is a brain tumor characterized by its aggressive and invasive properties. It is found that STAT3 is abnormally activated in GBM, and inhibiting STAT3 signaling can effectively suppress tumor progression. In this study, novel pyrimidine compounds, BY4003 and BY4008, were synthesized to target the JAK3/STAT3 signaling pathway, and their therapeutic efficacy and mechanisms of action were evaluated and compared with Tofacitinib in U251, A172, LN428 and patient-derived glioblastoma cells. The ADP-Glo™ kinase assay was utilized to assessed the inhibitory effects of BY4003 and BY4008 on JAK3, a crucial member of the JAK family. The results showed that both compounds significantly inhibited JAK3 enzyme activity, with IC50 values in the nanomolar range. The antiproliferative effects of BY4003, BY4008, and Tofacitinib on GBM and patient-derived glioblastoma cells were evaluated by MTT and H&E assays. The impact of BY4003 and BY4008 on GBM cell migration and apoptosis induction was assessed through wound healing, transwell, and TUNEL assays. STAT3-regulated protein expression and relative mRNA levels were analyzed by western blotting, immunocytochemistry, immunofluorescence, and qRT-PCR. It was found that BY4003, BY4008 and Tofacitinib could inhibit U251, A172, LN428 and patient-derived glioblastoma cells growth and proliferation. Results showed decreased expression of STAT3-associated proteins, including p-STAT3, CyclinD1, and Bcl-2, and increased expression of Bax, a pro-apoptotic protein, as well as significant down-regulation of STAT3 and STAT3-related genes. These findings suggested that BY4003 and BY4008 could inhibit GBM growth by suppressing the JAK3/STAT3 signaling pathway, providing valuable insights into the therapeutic development of GBM.

Cross-talk between oxidative stress and lipid metabolism regulators reveals molecular clusters and immunological characterization in polycystic ovarian syndrome

BackgroundChanges in the oxidative stress and lipid metabolism (OSLM) pathways play important roles in polycystic ovarian syndrome (PCOS) pathogenesis and development. Consequently, a systematic analysis of genes related to OSLM was conducted to identify molecular clusters and explore new biomarkers that are helpful for the diagnostic of PCOS.MethodsGene expression and clinical data from 22 PCOS women and 14 normal women were obtained from the GEO database (GSE34526, GSE95728, and GSE106724). Consensus clustering identified OSLM-related molecular clusters, and WGCNA revealed co-expression patterns. The immune microenvironment was quantitatively assessed utilizing the CIBERSORT algorithm. Multiple machine learning models and connectivity map analyses were subsequently applied to explore potential biomarkers for PCOS, and nomograms were employed to develop a predictive multigene model of PCOS. Finally, the OSLM status of PCOS and the hub genes expression profiles were preliminarily verified using TUNEL, qRT‒PCR, western blot, and IHC assays in a PCOS mouse model.Results19 differential expression genes (DEGs) related to OSLM were identified. Based on 19 DEGs that were strongly influenced by OSLM, PCOS patients were stratified into two distinct clusters, designated Cluster 1 and Cluster 2. Distinct differences in the immune cell proportions existed in normal and two PCOS clusters. The random forest showed the best results, with the least cross-entropy and the utmost AUC (cross-entropy: 0.111 AUC: 0.960). Among the 19 OSLM-related genes, CXCR1, ACP5, CEACAM3, S1PR4, and TCF7 were identified by a Bayesian network and had a good fit with PCOS disease risk by the nomogram (AUC: 0.990 CI: 0.968–1.000). TUNEL assays revealed more severe DNA damage within the ovarian granule cells of PCOS mice than in those of normal mice (P < 0.001). The RNA and protein expression levels of the five hub genes were significantly elevated in PCOS mice, which was consistent with the results of the bioinformatics analyses.ConclusionA novel predictive model was constructed for PCOS patients and five hub genes were identified as potential biomarkers to offer novel insights into clinical diagnostic strategies for PCOS.

LncRNA NPTN-IT1-201 Ameliorates Depressive-like Behavior by Targeting miR-142-5p and Regulating Inflammation and Apoptosis via BDNF.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are widely expressed in the brain and are associated with the development of neurological and neurodegenerative diseases. However, their roles and molecular mechanisms in major depressive disorder (MDD) remain largely unknown. This study aimed to identify lncRNAs and miRNAs involved in the development of MDD and elucidate their molecular mechanisms. Transcriptome and bioinformatic analyses were performed to identify miRNAs and lncRNAs related to MDD. C57 mice were subjected to chronic unpredictable mild stress (CUMS) to establish a depression model. Lentiviruses containing either lncRNA NPTN-IT1-201 or miR-142-5p were microinjected into the hippocampal region of these mice. Behavioral tests including the sucrose preference test (SPT), tail suspension test (TST), and forced swim test (FST) were conducted to evaluate depressive-like behaviors. The results revealed that overexpression of lncRNA NPTN-IT1-201 or inhibition of miR-142-5p significantly ameliorated depressive-like behaviors in CUMS-treated mice. Dual-luciferase reporter assays confirmed interactions between miR-142-5p with both brain-derived neurotrophic factor (BDNF) and NPTN-IT1-201. ELISA analysis revealed significant alterations in relevant biomarkers in the blood samples of MDD patients compared to healthy controls. Histological analyses, including HE and Nissl staining, showed marked structural changes in brain tissues following CUMS treatment, which were partially reversed by lncRNA NPTN-IT1-201 overexpression or miR-142-5p inhibition. Immunofluorescence imaging demonstrated significant differences in the levels of BAX, Bcl2, p65, Iba1 among different treatment groups. TUNEL assays confirmed reduced apoptosis in brain tissues following these interventions. Western blotting showed the significant differences in BDNF, BAX, and Bcl2 protein levels among different treatment groups. NPTN-IT1-201 regulates inflammation and apoptosis in MDD by targeting BDNF via miR-142-5p, making it a potential therapeutic target for MDD.

GAB1 attenuates lipopolysaccharide‑mediated endothelial dysfunction via regulation of SOCS3.

Endothelial dysfunction is a crucial pathogenetic mechanism for sepsis. GRB2-associated binder 1 (GAB1) alleviates sepsis-induced multi-organ damage; however, to the best of our knowledge, its function in endothelial dysfunction in sepsis remains unclear. HUVECs were induced by lipopolysaccharide (LPS) to simulate endothelial cell injury under sepsis. Cell transfection was conducted to achieve GAB1 overexpression or suppressor of cytokine signaling 3 (SOCS3) knockdown. The expression levels of GAB1 and SOCS3 were detected by reverse transcription-quantitative PCR and western blotting. Cell viability, apoptosis and migration were assessed using Cell Counting Kit-8, TUNEL and wound healing assays, respectively. The production of cytokines and nitric oxide (NO) was detected using commercial kits. The interaction between GAB1 and SOCS3 was confirmed using a co-immunoprecipitation assay. GAB1 was downregulated in LPS-induced HUVECs. However, GAB1 overexpression significantly mitigated LPS-induced cell viability decrease and apoptosis in HUVECs, accompanied by upregulation of Bcl2 expression, and downregulation of Bax and cleaved caspase-3 expression. GAB1 also inhibited the production of pro-inflammatory cytokines and increased NO level, increased the levels of endothelial NO synthase (eNOS) and phosphorylated (p)-eNOS, and promoted migration in LPS-induced HUVECs. However, SOCS3 knockdown partially weakened the effects of GAB1 overexpression on cell viability, apoptosis, inflammation, p-eNOS, eNOS expression and NO levels in LPS-induced HUVECs. In addition, GAB1 and SOCS3 regulated Janus kinase 2 (JAK2)/STAT3 signaling in LPS-induced HUVECs. In conclusion, GAB1 exerted a protective effect against LPS-induced endothelial cell apoptosis, inflammation and dysfunction by modulating the SOCS3/JAK2/STAT3 signaling pathway.

Ruthenium Complex Suppresses Proliferation of Residual Hepatocellular Carcinoma after Incomplete Radiofrequency Ablation Therapy.

Radiofrequency ablation (RFA) is an effective therapy for hepatocellular carcinoma (HCC). However, incomplete radiofrequency ablation (IRFA) can promote the progression of residual cancer cells, which is a serious problem in the clinical application of RFA. Therefore, it is of great significance to explore the mechanism and countermeasures of the progression of residual tumors after IRFA. Our previous study confirmed that IRFA can activate the hypoxia/ autophagy pathway of residual tumors in mice and then induce the proliferation of residual tumor cells. Additionally, we found a metal ruthenium complex [Ru(bpy)2(ipad)](ClO4)2 (Ru, where bpy = 2,2'-bipyridine and ipad = 2-(anthracene-9,10-dione-2-yl)imidazo[4,5-f][1,10]phenanthroline) can effectively inhibit hypoxia-inducible factor (HIF-1α) and has good anti-tumor effect in a hypoxic environment; however, whether Ru could suppress the proliferation of residual tumor cells after IRFA is unknown. This study intends to evaluate the effect of Ru in suppressing the proliferation of residual hepatocellular carcinoma after IRFA in a mice model. The Hepa1-6 xenograft mouse model was established in C57BL/6 mice to simulate clinical IRFA. H&E staining was used to evaluate the biosafety of major organs in the treated mice. TUNEL assay was employed to assess the antitumor effect. Immunohistochemically and immunofluorescence staining was performed to detect the expression of HIF-1α and autophagy-related proteins. The ELISA assay was used to examine the cytokines of interferon-gamma (IFN-γ) and interleukin 10 (IL-10). Our findings revealed that the residual tumor relapsed via the HIF-1α/LC3B/P62 autophagy- related pathway after IRFA, while Ru could suppress this process. In addition, it was demonstrated that Ru could effectively activate the immune system of the mice and reverse the tumor immune suppression microenvironment after IRFA. The ruthenium complex Ru could suppress the proliferation of residual hepatocellular carcinoma cells after IRFA in the mice model. This study introduces a novel approach that combines the use of ruthenium complexes with IRFA, offering a potential solution to address the reoccurrence of residual liver cancer following IRFA in clinical settings.

Effect of lactose on the in vitro development of sheep secondary follicles

Considering that follicular development is an energy-dependent process, supplementation of the culture medium with energy substrates, such as lactose, would improve follicle viability and growth. Thus, the aim of this study was to evaluate the effect of lactose on morphology, development, glutathione (GSH) concentration, mitochondrial activity, DNA fragmentation, and meiotic resumption of oocytes from sheep secondary follicles cultured in vitro. Secondary follicles were isolated from the cortex of ovine ovaries and cultured individually for 18 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium and ascorbic acid (control medium: α-MEM+) or in α-MEM+ plus different concentrations of lactose (0.025, 0.05 and 0.1 M). After culture, some of the oocytes were subjected to TUNEL assay and in vitro maturation (IVM). Follicular morphology, glutathione (GSH) concentration and mitochondrial activity were evaluated at the end of the culture. At the day 18, the percentage of morphologically normal follicles was greater (P<0.05) in the treatment of 0.025 M lactose (92.5 %) compared to the control group (75.55 %). In addition, GSH concentrations increased (P<0.05) in treatment containing 0.025 M lactose compared to the other treatments. Furthermore, oocytes cultured in 0.025 M lactose had greater (P<0.05) mitochondrial activity levels than in α-MEM+ and 0.1 M lactose. The group α-MEM+ presented a increase of TUNEL-positive oocytes (35.09 %) compared to 0.025 lactose (9.09 %). The percentage of meiotic resumption was greater (P<0.05) in oocytes from secondary follicles cultured in 0.025 M lactose (54.5 %) than in α-MEM+ (45.5 %). In conclusion, 0.025 M lactose improved survival, GSH and active mitochondria levels and meiotic resumption of oocytes from in vitro cultured secondary follicles. Supplementation of the culture medium of preantral follicles with lactose can gradually provide energy to follicular cells, potentially enhancing the production of viable oocytes for biotechniques such as IVM and in vitro fertilization.

Morpho-Anatomical Degeneration of Dopaminergic Neurons in Adult Zebrafish Brain after Exposure to Nickel

Chronic exposure to heavy metals has been widely demonstrated to induce pathological features in different tissues and, in particular, in the central nervous system. Specific neurons, including dopaminergic neurons, were observed to be more susceptible to toxic agents. Several previous studies performed on zebrafish (Danio rerio) models observed that exposure to nickel (one of the most abundant heavy metals) induces impairment of memory and anxious-like behaviors. Nevertheless, this phenotypical evidence has not been associated with dopaminergic system damage, and no reports showing the effects of nickel on dopaminergic neurons are available. In this study, we aim to analyze the precise distribution and variation in dopaminergic neurons in adult zebrafish after chronic (96 h) exposure to nickel ions dissolved in water at different sub-lethal doses (0.4 mg L−1; 2 mg L−1 and 4 mg L−1). The effects of treatment on dopaminergic neurons were evaluated by measuring transcript and protein levels of tyrosine hydroxylase (TH), described as a dopaminergic neuron marker. As shown, the expression of the th1 and th2 genes was reduced in the entire brain of zebrafish treated with nickel. Immunostaining analysis allowed us to localize TH-expressing neurons mainly in the posterior tuberculum, where they were observed to be reduced after nickel treatment in a dose-dependent fashion. Consistently, the TUNEL assay revealed a significant increase in apoptosis of TH-expressing cells after treatment with 2 mg L−1 and 4 mg L−1 of nickel. Our findings represent the first evidence of the effect of nickel on the dopaminergic system.

Up-regulation of LPCAT1 is correlated with poor prognosis and promotes tumor progression in glioblastoma.

Glioblastoma (GBM) is a cancer with high malignancy because of its rapid proliferation and high metastatic ability. LPCAT1 is reported to play a tumor-promoting role in multiple cancers, but its precise molecular mechanism in GBM remains to be further explored. We aim to explore the biological role of LPCAT1 in GBM. In this study, the expression of LPCAT1 and its correlation with clinicopathological characteristics of GBM patients were analyzed based on The Cancer Genome Atlas (TCGA) dataset. Kaplan-Meier approach was adopted for plotting survival curves for patients showing different expression levels of LPCAT1. Meanwhile, LPCAT1 expression within 50 GBM tumor tissues and 30 non-tumor clinical samples was analyzed by qRT-PCR and western blot assays, respectively. Later, LPCAT1's effect on GBM tumorigenesis was analyzed in vivo and in vitro by CCK8, EdU proliferation, clone forming, scratch, TUNEL assays, and subcutaneous xenograft experiments. As a result, LPCAT1 expression elevated within GBM tumor tissues and cells. Overexpression of LPCAT1 enhanced GBM cell growth, invasion and migration, while accelerating cell cycle progression. LPCAT1 silencing significantly inhibited cell motility and proliferation in vivo and in vitro, and arrested U251 cells at G0/G1 phase. Moreover, LPCAT1 might play a role in GBM progression by activating the p-AKT-MYC signaling pathway. LPCAT1 activated AKT, which were synchronously up-regulated MYC to accelerate cancer progression. Knockdown of LPCAT1 induced the opposite changes to repress the viability and motility of GBM cells. LPCAT1 contributed to the progression of GBM by participating in the p-AKT-MYC axis.

Hepatoprotective potential of taxifolin in type 2 diabetic rats: modulation of oxidative stress and Bcl2/Bax/Caspase-3 signaling pathway.

Diabetes mellitus (DM) is a global metabolic problem. Several factors including hyperglycemia, oxidative stress, and inflammation play significant roles in the development of DM complications. Apoptosis is also an essential event in DM pathophysiology, -with B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X (Bax) determining apoptotic susceptibility. The present study aimed to elucidate the protective effects of two doses of taxifolin (TXF) on liver damage in diabetic rats and explore the possible mechanisms of action. DM was induced in eighteen rats through intraperitoneal injections of 50mg/kg streptozotocin and 110mg/kg nicotinamide. Diabetic rats received daily oral intubation of 25 and 50mg/kg TXF for 3 months. In the untreated diabetic group, there was a significant increase in fasting and postprandial glucose levels, glycosylated hemoglobin A1C (HbA1c), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), while insulin and adiponectin levels decreased significantly. Both TXF doses mitigated hyperglycemia, regulated cytokine production, and increased insulin level. Gene expressions and protein levels of Bax, caspase 3, and cytochrome c were significantly increased, while Bcl-2 was significantly decreased in the livers of diabetic rats, effects that were significantly ameliorated after TXF treatment. The results of the TUNEL assay supported the apoptotic pathway. Additionally, TXF significantly decreased lipid peroxidation and enhanced antioxidant enzyme activity in diabetic rats. Liver enzymes and histopathological changes also showed improvement. TXF mitigated diabetes-associated hepatic damage by reducing hyperglycemia, oxidative stress, inflammation, and modulating anti-/pro-apoptotic genes and proteins. A dose of 50mg/kg TXF was more effective than 25mg/kg and is recommended for consumption.

Therapeutic Potential of Silver Nitroprusside Nanoparticles for Melanoma.

Melanoma has gained considerable attention due to its high mortality and morbidity rate worldwide. The currently available treatment options are associated with several limitations such as nonspecificity, drug resistance, easy clearance, low efficacy, toxicity-related issues, etc. To this end, nanotechnology has garnered significant attention for the treatment of melanoma. In the present manuscript, we have demonstrated the in vitro and in vivo anticancer activity of silver nitroprusside nanoparticles (abbreviated as AgNNPs) against melanoma. The AgNNPs exhibit cytotoxicity against B16F10 cells, which has been investigated by several in vitro experiments including [methyl 3H]-thymidine incorporation assay, cell cycle and apoptosis analysis by flow cytometry, and ROS generation through DCFDA, DHE, and DAF2A reagents. Further, the internalization of nanoparticles was determined by ICPOES analysis, while their colocalization was analyzed by confocal microscopy. Additionally, JC-1 staining is performed to examine mitochondrial membrane potential (MMP). Cytoskeleton integrity was observed by phalloidin staining. Expression of different markers (Ki-67, cytochrome c, and E-cadherin) was checked using an immunofluorescence assay. The in vivo therapeutic efficacy of AgNNPs has been validated in the melanoma model established by inoculating B16F10 cells into the dorsal right abdomen of C57BL/6J mice. The intraperitoneal administration of AgNNPs reduced melanoma growth and increased the survivability of tumor-bearing mice. The in vivo immunofluorescence studies (Ki-67, CD31, and E-cadherin) and TUNEL assay support the inhibitory and apoptotic nature of AgNNPs toward melanoma, respectively. Furthermore, the various signaling pathways and molecular mechanisms involved in anticancer activity are evaluated by Western blot analysis. These findings altogether demonstrate the promising anticancer potential of AgNNPs toward melanoma.

β -estradiol alleviates Hypoxic-ischemic brain damage in neonatal rats through the GPER1 regulating AKT/NF-κB signal pathway

IntroductionResearch has established that estradiol (E2) offers neuroprotection against hypoxic-ischemic brain damage (HIBD) in neonatal rats, yet the underlying mechanisms are not fully understood. This study seeks to delineate whether E2's neuroprotective effects in neonatal HIBD are mediated through astrocytes by modulating the G Protein-Coupled Estrogen Receptor 1 (GPER1) receptor and the subsequent AKT Serine (AKT)/NF-κB signaling cascade.Material and methodsWe developed an in vivo HIBD model in neonatal rats and established primary cultures of astrocytes subjected to oxygen-glucose deprivation-reoxygenation (OGD-R) as an in vitro model. E2 and the GPER1 inhibitor (G15) were administered according to the experimental design. Protein expression levels of GPER1, phosphorylated AKT (p-AKT), NF-κB p65, and cleaved-caspase3 were examined using Western blot analysis. Apoptosis was assessed via the TUNEL assay, and the presence of TNF-α and IL-1β in the cell supernatant was quantified by ELISA. The localization of p-AKT and NF-κB p65 was determined through immunofluorescence.ResultsOur findings indicate that E2 treatment significantly reduced the volume of brain infarction and astrocyte apoptosis. E2 upregulated GPER1 and p-AKT expression while downregulating NF-κB p65 and cleaved-caspase3 levels in astrocytes and neonatal rats post-HIBD. Additionally, E2 diminished the secretion of TNF-α and IL-1β in the cell supernatant. The G15 inhibitor notably reversed the neuroprotective effects of E2 and the associated molecular changes.ConclusionsThese results suggest that E2 may exert neuroprotection in neonatal rats with HIBD by inhibiting astrocyte apoptosis and modulating the expression of GPER1, p-AKT, and NF-κB, thereby providing a potential therapeutic strategy for HIBD.

The effect of exclusive breastfeeding on synaptogenesis and cell survival in mouse offspring

The first thousand days of life are critical for determining a child's cognitive development in humans. Breastfeeding may provide the nutrients needed for brain development. Cognitive function has been widely associated with neuronal turnover, driven by synaptogenesis and apoptosis. To explore this hypothesis, this study aimed to examine the effect of exclusive breastfeeding (EBF) on cognitive function and neuronal turnover in the dentate gyrus of the mice hippocampus. We conducted cognitive function assessments using the Morris Water Maze (MWM) test. We also examined neuronal apoptosis with TUNEL assay and synaptogenesis using PSD-95 antibodies. The newborn mice (mixed gender) aged 0 days were randomly divided into two groups. The first group received exclusive breastfeeding (EBF, n = 14), while the second group (MF, n = 14) received mixed breast milk and formula milk. At postnatal day 21 (PND21), the MWM test was performed, followed by an assessment of neuronal apoptosis and synaptogenesis. In the MWM test, the escape latency of the EBF group was shorter than the MF group. There was a significant increase in PSD-95 expression and a decrease in TUNEL expression in the EBF group (p &lt; 0.001) than in the MF group. In conclusion, exclusive breastfeeding is associated with higher cognitive function. Exclusive breastfeeding affects neuronal turnover by increasing synaptogenesis and decreasing apoptosis in the dentate gyrus of the hippocampus. This has both scientific and clinical implications, pointing to nutritional practices early in life that could optimise the attainment of cognitive potential.

The use of matrine to inhibit osteosarcoma cell proliferation via the regulation of the MAPK/ERK signaling pathway.

Matrine is an alkaloid extracted from Sophorus beans of the legume family, and it has significant effects and a variety of pharmacological activities. Osteosarcoma(OS) is a common malignant bone tumor that is characterized by high incidence and rapid progression. There have been some preliminary studies on the therapeutic effect of matrine on OS, but the specific mechanism remains unclear. The aim of this study was to investigate the antitumor effect of matrine on HOS cells and the underlying molecular mechanism. The effects of matrine on the proliferation, apoptosis and cell cycle progression of HOS cells were determined by CCK-8 assay, TUNEL assay and flow cytometry in vitro. Wound healing and Transwell invasion assays were used to observe the effect of matrine on the migration and invasion of HOS cells. The mechanism underlying the antitumor effect of matrine on HOS cells was investigated by Western blotting. Matrine significantly inhibited HOS cell proliferation, promoted HOS cell apoptosis, and arrested HOS cells in the G1 phase of the cell cycle. Both wound healing and Transwell invasion assays showed that matrine inhibited HOS cell migration and invasion. Western blotting results showed that matrine inhibited the activation of the MAPK/ERK signaling pathway. We found that matrine also downregulated Bcl-2 expression, which may be related to protein synthesis inhibition. Matrine can inhibit the proliferation of HOS cells, arrest HOS cells in the G1 phase, and promote HOS cell apoptosis through the MAPK/ERK signaling pathway.

Obesity-associated non-oxidative genotoxic stress alters trophoblast turnover in human first-trimester placentas.

Placental growth is most rapid during the first trimester (FT) of pregnancy, making it vulnerable to metabolic and endocrine influences. Obesity, with its inflammatory and oxidative stress, can cause cellular damage. We hypothesized that maternal obesity increases DNA damage in the FT placenta, affecting DNA damage response and trophoblast turnover. Examining placental tissue from lean and obese non-smoking women (4-12 gestational weeks), we observed higher overall DNA damage in obesity (COMET assay). Specifically, DNA double-strand breaks were found in villous cytotrophoblasts (vCTB; semi-quantitative γH2AX immunostaining), while oxidative DNA modifications (8-hydroxydeoxyguanosine; FPG-COMET assay) were absent. Increased DNA damage in obese FT placentas did not correlate with enhanced DNA damage sensing and repair. Indeed, obesity led to reduced expression of multiple DNA repair genes (mRNA array), which were further shown to be influenced by inflammation through in vitro experiments using tumor necrosis factor-α treatment on FT chorionic villous explants. Tissue changes included elevated vCTB apoptosis (TUNEL assay; caspase-cleaved cytokeratin 18), but unchanged senescence (p16) and reduced proliferation (Ki67) of vCTB, the main driver of FT placental growth. Overall, obesity is linked to heightened non-oxidative DNA damage in FT placentas, negatively affecting trophoblast growth and potentially leading to temporary reduction in early fetal growth.

Mechanism of Action of Qingrekasen Granules in Alleviating Nephrotic Syndrome Evaluated by a Multi-Omics Approach

Objectives In this study, the efficacy of and mechanism of Qingrekasen Granules (QRKSG) is evaluated by metabolomics and transcriptomics using adriamycin (ADR)-induced nephrotic syndrome (NS) in rat model. Methods The model, benazepril, and QRKSG group received a single injection of 6.5 mg/kg ADR via the tail vein of the rats. The untreated group received an equal saline injection. The administration of drugs by gavage began after completing the modeling for one week. Benazepril was given at 0.9 mg/kg/d to the benazepril group and QRKSG was given at 1.62 g/kg/d to the QRKSG group. During gavage, 24 h urine was collected weekly. After four weeks of gavage, rats were anesthetized, and we collected the serum, feces, and kidney samples. The protective effect of QRKSG on NS was assessed by the detection of proteins in the urine at 24 h and serum biochemical indexes, as well as histopathological observation, TUNEL assay, and Western Blot of kidney samples. Moreover, gas chromatography-mass spectrometry (GC-MS) metabolomics sequencing of kidney metabolites helped investigate the significant differential metabolites produced by QRKSG that are implicated in ameliorating the pathological damage to the kidneys of NS rats. Subsequently, the significant targets of QRKSG that had an impact on the action of drugs were investigated by a transcriptome sequencing analysis. The correlation between both sets was also investigated. In addition, the effect of QRKSG on the gut microbiota of NS rats was investigated. Finally, the core targets were validated by molecular docking. Results The results indicated that QRKSG possess significant renoprotective effects in NS rats by reversing the abnormal urinary protein content and serum biochemical disorders, as well as improving renal pathological damage. Multiple genes and metabolites were shown to be back-regulated after QRKSG delivery, based on further multi-omics studies. The integrated metabolomics and transcriptomics analysis showed that QRKSG alleviated NS mainly by regulating amino acid metabolic pathways. Gut microbiota analysis demonstrated that QRKSG could alleviate NS by improving gut microbiota. The molecular docking results showed good binding ability of the QRKSG active ingredient to the core target, and among them, the ingredients with the best docking effect were mainly flavonoids and phenolic acid ingredients. The combined metabolomics and transcriptomics study findings were validated through a TUNEL assay and a Western Blot analysis. Conclusion We have concluded that ADR-induced NS in rats can be treated with QRKSG. The mechanisms used by QRKSG to reduce the symptoms of NS are through the multi-component, multi-target, and multi-pathway therapeutic modulation of inflammation, oxidative stress, energy homeostasis, and apoptosis. Additionally, QRKSG could alleviate NS by regulating gut microbiota.

The effects of olibanum on male reproductive system damage in a lipopolysaccharide induced systemic inflammation model in rat

BackgroundLipopolysaccharide (LPS) as a particle of Gram-negative bacteria is a main contributer in the pathogenesis of the male reproductive system infectious. Male infertility due to LPS is reported to be related to overproduction reactive oxygen species. This study aimed to investigate the effects of olibanum on oxidative stress and apoptosis in testes and sperm dysfunction induced by LPS. MethodsThe male (n = 28) rats were allocated in four groups: control, LPS (1 mg/kg, i.p., 14 days), LPS + Olibanum 100 (100 mg/kg, i.p., 14 days), and LPS + Olibanum 200 (200 mg/kg, i.p., 14 days). Germ cell apoptosis was determined by TUNEL assays and computed using the stereological method. Additionally, semen samples of the animals were analyzed for sperm count and morphology. Oxidative stress indicators were also determined. ResultsThe count of TUNEL-positive germ cells in LPS-treated rats was more than that in the controls. Treatment of the animals with olibanum significantly attenuated the number of apoptotic cells compared to the LPS group. The sperm count and those with a normal morphology in LPS-treated rats was lower than that in the controls. Administration of olibanum significantly improved the sperms with normal morphology and sperm count. Olibanum treatment also improved superoxide dismutase, catalase, and total thiol in testicular tissue and decreased malondialdehyde. ConclusionAdministering both doses of olibanum in LPS-treated rats had potentially a therapeutic value in reducing germ cell apoptosis, as well as improving sperm parameters.

Baicalein Enhances Radiosensitivity in Colorectal Cancer via JAK2/STAT3 Pathway Inhibition.

Radiation resistance is a crucial factor influencing therapeutic outcomes in colorectal cancer (CRC). Baicalein (BE), primarily derived from Scutellaria baicalensis, has demonstrated anti-CRC properties. However, the impact of BE on the radiosensitivity of CRC remains unclear. This study aimed to evaluate the radiosensitization effects of BE and elucidate its mechanism in CRC radiotherapy. We established an invitro radioresistant cell model (CT26-R) using parental CRC cells (CT26) subjected to ionizing radiation (IR). CT26-R cells were pretreated with or without BE, followed by transfection with pcDNA-NC and pcDNA-JAK2. The proliferation of CT26-R cells treated with BE and IR was assessed using a colony formation assay. A CRC animal model was developed in BALB/c mice via CT26-R cell transplantation. The radiosensitizing effect of BE on CRC was evaluated invivo. TUNEL assay was conducted to detect apoptosis in tumor tissue. The expression levels of p-STAT3, JAK2, PD-L1, and SOCS3 invitro and invivo were measured by western blotting. Our results demonstrated that BE significantly increased radiosensitivity invitro and invivo and enhanced apoptosis in tumor tissues. Additionally, BE significantly downregulated the expression of p-STAT3, JAK2, and PD-L1, and significantly upregulated SOCS3 expression. These invivo effects were reversed by pcDNA-JAK2. In summary, our data suggest that BE enhances CRC radiosensitivity by inhibiting the JAK2/STAT3 pathway.

Daphnetin protects neurons in an Alzheimer disease mouse model and normal rat neurons by inhibiting BACE1 activity and activating the Nrf2/HO-1 pathway.

The common neurodegenerative disorder Alzheimer disease (AD) is characterized by memory dysfunction and cognitive decline in the elderly. Neuropathological features include aggregated β-amyloid (Aβ) accumulation, neuroinflammation, and oxidative stress in the brain. Daphnetin (DAPH), a natural coumarin derivative, has the potential for inhibiting inflammatory and oxidative responses. We explored neuroprotective roles of DAPH treatment in the APP/PS1 transgenic mouse AD model. DAPH ameliorated spatial learning disabilities in Morris water maze tests and reduced Aβ deposition, assessed by immunohistochemistry. It also reduced the Aβ content in supernatants of neurons from fetal APP/PS1 mice, assessed by cell-based soluble ELISA. Molecular docking and fluorescence resonance energy transfer-based assay results suggested that DAPH could directly inhibit BACE1 activity. Furthermore, in vitro experiments utilizing isolated rat neurons assessing RNA expression profiling, immunofluorescence, TUNEL assay, and Western-blot analysis, suggested the potential of DAPH for regulating BDNF and GM-CSF expression and mitigating Aβ1-42-induced cortical injury, synaptic loss, and apoptosis. HO-1 and Nrf2 mRNA and protein expression were also increased in a dose-dependent manner. These results underscore the potential of DAPH as a neuroprotective agent in reversing memory deficits associated with AD and bolster its candidacy as a multitarget natural small-molecule drug for AD patients.

Modified Jiaoqi Powder enhances epithelial autophagy against TNF-triggered apoptosis in chronic ulcerative colitis

BackgroundA vicious cycle of dysregulated intestinal epithelial cell death, intestinal barrier defect, and subsequent inflammation response is core to chronic ulcerative colitis (UC). Modified Jiaoqi Powder (MJQP), a traditional Chinese medicine formula, has been clinically applied to treat chronic relapsing type and chronic persistent type of UC . Nevertheless, the underlying mechanism of MJPQ in chronic UC remains unknown. PurposeThe present study aimed to demonstrate the favorable effect and potential molecular mechanism of MJQP in chronic UC. MethodsThe chemical components of MJQP and MJQP drug serum were identified by LC-MS/MS. The curative effect of MJQP was evaluated in a well-established DSS-induced chronic UC mice model by measuring body weight, colon length, disease activity index (DAI) and histological scores . Serum cytokines, including interleukin (IL)-1β, IL-12, IL-13, IL-4, tumor necrosis factor-alpha (TNF-α) and INF-γ were measured using enzyme-linked immunosorbent assay. Western blotting, immunofluorescence, and MTT assay were used to analyze the effects of MJQP on colonic barrier function in chronic UC mice and human epithelial cell lines. TUNEL assay, western blotting, and flow cytometry were used to examine the related apoptosis indicators. An electron microscope was used to observe autophagosomes and autolysosomes, while western blotting and immunofluorescence were used to detect autophagy-associated proteins. Network pharmacology was used to predict potential targets and pathways of MJQP in UC. Finally, the TNF pathway-related proteins were detected by immunohistochemistry and western blotting. ResultsMJQP administration prevented the UC progression with faster weight gain, longer colon length, lower histological scores and DAI, and up/down- regulation of inflammatory factors. The expressions of tight junction proteins ki67 and E-cadherin increased dose-dependently after MJQP intervention. Moreover, MJQP treatment promoted the viability of NCM460 and Caco2 cells in a concentration-dependent manner. MJQP dose-dependently decreased the proportion of TUNEL-positive cells and attenuated the pro-apoptotic proteins cleaved-caspase 8 and cleaved-caspase 3 in colonic tissues. Flow cytometry also showed that MJQP dose-dependently decreased the apoptotic cell population of LPS-induced NCM460 and Caco2 cells. Electron microscopy revealed that autophagosomes and autolysosomes were significantly improved in the MJQP-treated groups. Additionally, autophagy-related proteins were significantly expressed after MJQP treatment. Network pharmacological analysis predicted that MJQP may alleviate chronic UC by affecting TNF-related signaling pathways and promoting intestinal epithelial cell proliferation. As anticipated, the TNF pathway-associated proteins were attenuated dose-dependently in colonic tissues after MJQP treatment. ConclusionThese results provide novel therapeutic strategies, indicating that MJQP may be a promising candidate treatment for chronic UC by promoting epithelial barrier restitution by enhancing epithelial autophagy against TNF–mediated apoptosis.