Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

High-dose radiation activates caspases in tumor cells to produce abundant DNA fragments for DNA sensing in antigen-presenting cells, but the intrinsic DNA sensing in tumor cells after radiation is rather limited. Here we demonstrate that irradiated tumor cells hijack caspase 9 signaling to suppress intrinsic DNA sensing. Instead of apoptotic genomic DNA, tumor-derived mitochondrial DNA triggers intrinsic DNA sensing. Specifically, loss of mitochondrial DNA sensing in Casp9-/- tumors abolishes the enhanced therapeutic effect of radiation. We demonstrated that combining emricasan, a pan-caspase inhibitor, with radiation generates synergistic therapeutic effects. Moreover, loss of CASP9 signaling in tumor cells led to adaptive resistance by upregulating programmed death-ligand 1 (PD-L1) and resulted in tumor relapse. Additional anti-PD-L1 blockade can further overcome this acquired immune resistance. Therefore, combining radiation with a caspase inhibitor and anti-PD-L1 can effectively control tumors by sequentially blocking both intrinsic and extrinsic inhibitory signaling.

Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production.

Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-ß. In vivo, this precipitates an elevation in IFN-ß levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.

Apoptotic Caspases Suppress Type I Interferon Production via the Cleavage of cGAS, MAVS, and IRF3.

Viral infection triggers host defenses through pattern-recognition receptor-mediated cytokine production, inflammasome activation, and apoptosis of the infected cells. Inflammasome-activated caspases are known to cleave cyclic GMP-AMP synthase (cGAS). Here, we found that apoptotic caspases are critically involved in regulating both DNA and RNA virus-triggered host defenses, in which activated caspase-3 cleaved cGAS, MAVS, and IRF3 to prevent cytokine overproduction. Caspase-3 was exclusively required in human cells, whereas caspase-7 was involved only in murine cells to inactivate cGAS, reflecting distinct regulatory mechanisms in different species. Caspase-mediated cGAS cleavage was enhanced in the presence of dsDNA. Alternative MAVS cleavage sites were used to ensure the inactivation of this critical protein. Elevated type I IFNs were detected in caspase-3-deficient cells without any infection. Casp3-/- mice consistently showed increased resistance to viral infection and experimental autoimmune encephalomyelitis. Our results demonstrate that apoptotic caspases control innate immunity and maintain immune homeostasis against viral infection.

GD2-CART01 for Relapsed or Refractory High-Risk Neuroblastoma.

BACKGROUND: Immunotherapy with chimeric antigen receptor (CAR)-expressing T cells that target the disialoganglioside GD2 expressed on tumor cells may be a therapeutic option for patients with high-risk neuroblastoma. METHODS: In an academic, phase 1-2 clinical trial, we enrolled patients (1 to 25 years of age) with relapsed or refractory, high-risk neuroblastoma in order to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01). RESULTS: A total of 27 children with heavily pretreated neuroblastoma (12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line therapy) were enrolled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested (3-, 6-, and 10×106 CAR-positive T cells per kilogram of body weight) in the phase 1 portion of the trial, and no dose-limiting toxic effects were recorded; the recommended dose for the phase 2 portion of the trial was 10×106 CAR-positive T cells per kilogram. Cytokine release syndrome occurred in 20 of 27 patients (74%) and was mild in 19 of 20 (95%). In 1 patient, the suicide gene was activated, with rapid elimination of GD2-CART01. GD2-targeted CAR T cells expanded in vivo and were detectable in peripheral blood in 26 of 27 patients up to 30 months after infusion (median persistence, 3 months; range, 1 to 30). Seventeen children had a response to the treatment (overall response, 63%); 9 patients had a complete response, and 8 had a partial response. Among the patients who received the recommended dose, the 3-year overall survival and event-free survival were 60% and 36%, respectively. CONCLUSIONS: The use of GD2-CART01 was feasible and safe in treating high-risk neuroblastoma. Treatment-related toxic effects developed, and the activation of the suicide gene controlled side effects. GD2-CART01 may have a sustained antitumor effect. (Funded by the Italian Medicines Agency and others; ClinicalTrials.gov number, NCT03373097.).

Activation of caspase-9 on the apoptosome as studied by methyl-TROSY NMR.

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example.

The gut microbiota contributes to the infection of bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and causes significant economic losses. Gut microbiota is a large microbial community and has a variety of biological functions. However, whether there is a correlation between gut microbiota and BVDV infection and what kind of relation between them have not been reported. Here, we found that gut microbiota composition changed in normal mice after infecting with BVDV, but mainly the low abundance microbe was affected. Interestingly, BVDV infection significantly reduced the diversity of gut microbiota and changed its composition in gut microbiota-dysbiosis mice. Furthermore, compared with normal mice of BVDV infection, there were more viral loads in the duodenum, jejunum, spleen, and liver of the gut microbiota-dysbiosis mice. However, feces microbiota transplantation (FMT) reversed these effects. The data above indicated that the dysbiosis of gut microbiota was a key factor in the high infection rate of BVDV. It is found that the IFN-I signal was involved by investigating the underlying mechanisms. The inhibition of the proliferation and increase in the apoptosis of peripheral blood lymphocytes (PBL) were also observed. However, FMT treatment reversed these changes by regulating PI3K/Akt, ERK, and Caspase-9/Caspase-3 pathways. Furthermore, the involvement of butyrate in the pathogenesis of BVDV was also further confirmed. Our results showed for the first time that gut microbiota acts as a key endogenous defense mechanism against BVDV infection; moreover, targeting regulation of gut microbiota structure and abundance may serve as a new strategy to prevent and control the disease.IMPORTANCEWhether the high infection rate of BVDV is related to gut microbiota has not been reported. In addition, most studies on BVDV focus on in vitro experiments, which limits the study of its prevention and control strategy and its pathogenic mechanism. In this study, we successfully confirmed the causal relationship between gut microbiota and BVDV infection as well as the potential molecular mechanism based on a mouse model of BVDV infection and a mouse model of gut microbiota dysbiosis. Meanwhile, a mouse model which is more susceptible to BVDV provided in this study lays an important foundation for further research on prevention and control strategy of BVDV and its pathogenesis. In addition, the antiviral effect of butyrate, the metabolites of butyrate-producing bacteria, has been further revealed. Overall, our findings provide a promising prevention and control strategy to treat this infectious disease which is distributed worldwide.

Non-apoptotic caspase activation ensures the homeostasis of ovarian somatic stem cells.

Current evidence has associated caspase activation with the regulation of basic cellular functions without causing apoptosis. Malfunction of non-apoptotic caspase activities may contribute to specific neurological disorders, metabolic diseases, autoimmune conditions and cancers. However, our understanding of non-apoptotic caspase functions remains limited. Here, we show that non-apoptotic caspase activation prevents the intracellular accumulation of the Patched receptor in autophagosomes and the subsequent Patched-dependent induction of autophagy in Drosophila follicular stem cells. These events ultimately sustain Hedgehog signalling and the physiological properties of ovarian somatic stem cells and their progeny under moderate thermal stress. Importantly, our key findings are partially conserved in ovarian somatic cells of human origin. These observations attribute to caspases a pro-survival role under certain cellular conditions.

The pluripotent-to-totipotent state transition in mESCs activates the intrinsic apoptotic pathway through DUX-induced DNA replication stress.

The pluripotent mouse embryonic stem cell (mESCs) can transit into the totipotent-like state, and the transcription factor DUX is one of the master regulators of this transition. Intriguingly, this transition in mESCs is accompanied by massive cell death, which significantly impedes the establishment and maintenance of totipotent cells in vitro, yet the underlying mechanisms of this cell death remain largely elusive. In this study, we found that the totipotency transition in mESCs triggered cell death through the upregulation of DUX. Specifically, R-loops are accumulated upon DUX induction, which subsequently lead to DNA replication stress (RS) in mESCs. This RS further activates p53 and PMAIP1, ultimately leading to Caspase-9/7-dependent intrinsic apoptosis. Notably, inhibiting this intrinsic apoptosis not only mitigates cell death but also enhances the efficiency of the totipotency transition in mESCs. Our findings thus elucidate one of the mechanisms underlying cell apoptosis during the totipotency transition in mESCs and provide a strategy for optimizing the establishment and maintenance of totipotent cells in vitro.

Mixed lineage kinase 3 and CD70 cooperation sensitize trastuzumab-resistant HER2+ breast cancer by ceramide-loaded nanoparticles.

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our in vitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.

Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics.

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

Evaluating nephrotoxicity reduction in a novel polymyxin B formulation: insights from a 3D kidney-on-a-chip model.

This study aimed to assess the nephrotoxicity associated with VRP-034 (novel formulation of polymyxin B [PMB]) compared to marketed PMB in a three-dimensional (3D) kidney-on-a-chip model. To model the human kidney proximal tubule for analysis, tubular structures were established using 23 triple-channel chips seeded with RPTEC/hTERT1 cells. These cells were exposed to VRP-034 or PMB at seven concentrations (1-200 µM) over 12, 24, and 48 h. A suite of novel kidney injury biomarkers, cell health, and inflammatory markers were quantitatively assessed in the effluent. Additionally, caspase and cytochrome C levels were measured, and cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). Exposure to marketed PMB resulted in significantly elevated levels (P < 0.05) of four key biomarkers (KIM-1, cystatin C, clusterin, and OPN) compared to VRP-034, particularly at clinically relevant concentrations of ≥10 µM. At 25 µM, all biomarkers demonstrated a significant increase (P < 0.05) with marketed PMB exposure compared to VRP-034. Inflammatory markers (interleukin-6 and interleukin-8) increased significantly (P < 0.05) with marketed PMB at concentrations of ≥5 µM, relative to VRP-034. VRP-034 displayed superior cell health outcomes, exhibiting lower lactate dehydrogenase release, while ATP levels remained comparable. Morphological analysis revealed that marketed PMB induced more severe damage, disrupting tubular integrity. Both treatments activated cytochrome C, caspase-3, caspase-8, caspase-9, and caspase-12 in a concentration-dependent manner; however, caspase activation was significantly reduced (P < 0.05) with VRP-034. This study demonstrates that VRP-034 significantly reduces nephrotoxicity compared to marketed PMB within a 3D microphysiological system, suggesting its potential to enable the use of full therapeutic doses of PMB with an improved safety profile, addressing the need for less nephrotoxic polymyxin antibiotics.

NK-derived exosome miR-1249-3p inhibits Mycobacterium tuberculosis survival in macrophages by targeting SKOR1.

Murine Natural Killer cells were cultivated in vitro to isolate NK-derived exosomes. Subsequent quantification via qPCR confirmed enrichment of miR-1249-3p. Ana-1 murine macrophages were cultured in vitro and subsequently inoculated with Mycobacterium tuberculosis (MTB) strain H37Rv. NK-exo and NK-exo miR-1249-3p were separately applied to the infection model, followed by immunological assays conducted post-48-hour co-culture. Western blot analyses corroborated that NK-exo exhibited exosomal marker proteins Granzyme A (GzmA), Granzyme B (GzmB), and Perforin (PFN), alongside a notable enrichment of miR-1249-3p. Functionally, NK-exo augmented the expression levels of Caspase-9,-8, and -3, as well as PARP, while attenuating the expression of NLRP3, ASC, and Cleaved-Caspase-1. Furthermore, qPCR demonstrated an up-regulation of Caspase-9, -8, and -3, along with pro-apoptotic factors Bax and Bid, and a concomitant down-regulation of the anti-apoptotic factor Bcl-2. The expression levels of inflammatory markers ASC, NLRP3, Cleaved-Caspase-1, and IL-1ß were concomitantly decreased. ELISA findings indicated diminished levels of TNF-α and ROS secretion. NK-exo miR-1249-3p specifically targeted and attenuated the expression of SKOR-1, engendering up-regulation of apoptosis-associated proteins and down-regulation of inflammation-related proteins, consequently affecting cellular fate.Our empirical evidence substantiates that NK-exo induces macrophage apoptosis, thereby mitigating MTB survival. Furthermore, NK-exo miR-1249-3p directly targets and inhibits SKOR-1 expression, leading to macrophage apoptosis and consequently hampering the proliferation of MTB. The data implicate the potential therapeutic relevance of NK-exo and miR-1249-3p in managing drug-resistant tuberculosis.

Exploring the induction of endometrial epithelial cell apoptosis in clinical-type endometritis in yaks through the cyt-c/caspase-3 signaling axis.

Endometritis is a significant contributor to reduced productivity in yaks in Tibet, China. The Cyt-c/Caspase-3 signaling axis plays a crucial role in the mitochondrial pathway that triggers cell apoptosis due to endogenous factors. In this study, we examined the endometrial epithelial tissue of yaks with endometritis using pathological examination, immunohistochemical analysis, TUNEL staining, qRT-PCR, and Western blot. The results indicated significant changes in the apoptotic factors of the Cyt-c/Caspase-3 signaling axis. The expression levels of Bak1, Bax, Cyt-c, Apaf-1, Caspase-9, and Caspase-3 were significantly increased (P < 0.05), while the expression level of Bcl-2 was significantly decreased. Immunohistochemistry results revealed significant increase in Bak1, Bax, Cyt-c, Apaf-1, Caspase-9, and Caspase-3 expression in the cytoplasm compared to the healthy group, except for Bcl-2, which showed a significant decrease. Pathological section analysis demonstrated that clinical endometritis in yaks led to structural damage, bleeding, congestion, and inflammatory cell infiltration in the endometrial epithelium. Our study findings indicated that clinical endometritis in yaks can modulate apoptosis of endometrial epithelial cells via the Cyt-c/Caspase-3 signaling pathway, resulting in different levels of damage. This research is pioneering in exploring cell apoptosis induced by clinical endometritis in yaks, offering novel insights and potential strategies for the future prevention and treatment of endometritis in yaks.

Therapeutic effects of genistein in experimentally induced ulcerative colitis in rats via affecting mitochondrial biogenesis.

Ulcerative colitis (UC) is an inflammatory bowel disease that affects the mucosa of the colon, resulting in severe inflammation and ulcers. Genistein is a polyphenolic isoflavone present in several vegetables, such as soybeans and fava beans. Therefore, we conducted the following study to determine the therapeutic effects of genistein on UC in rats by influencing antioxidant activity and mitochondrial biogenesis and the subsequent effects on the apoptotic pathway. UC was induced in rats by single intracolonic administration of 2 ml of 4% acetic acid. Then, UC rats were treated with 25-mg/kg genistein. Colon samples were obtained to assess the gene and protein expression of nuclear factor erythroid 2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1), peroxisome proliferator-activated receptor-gamma coactivator (PGC-1), mitochondrial transcription factor A (TFAM), B-cell lymphoma 2 (BCL2), BCL2-associated X (BAX), caspase-3, caspase-8, and caspase-9. In addition, colon sections were stained with hematoxylin/eosin to investigate the cell structure. The microimages of UC rats revealed inflammatory cell infiltration, hemorrhage, and the destruction of intestinal glands, and these effects were improved by treatment with genistein. Finally, treatment with genistein significantly increased the expression of PGC-1, TFAM, Nrf2, HO-1, and BCL2 and reduced the expression of BAX, caspase-3, caspase-8, and caspase-9. In conclusion, genistein exerted therapeutic effects against UC in rats. This therapeutic activity involved enhancing antioxidant activity and increasing mitochondrial biogenesis, which reduced cell apoptosis.

Cytomorphological Disparities in Invasive Breast Cancer Cells following Neoadjuvant Endocrine Therapy and Chemotherapy.

INTRODUCTION: Neoadjuvant endocrine therapy (NAE) offers a breast-conserving surgery rate and clinical response rate similar to those of neoadjuvant chemotherapy (NAC), while presenting fewer adverse events and lower pathological complete response rates. The assessment of pathological response determines degenerative changes and predicts the prognosis of breast cancer treated with NAC. This study clarified the degenerative changes occurring in breast cancer following NAE. METHODS: Our study encompassed two groups: NAE, consisting of 15 patients, and NAC, comprising 18 patients. Tissue samples were obtained from core needle biopsies and surgeries. Nuclear and cell areas were calculated using Autocell analysis. Furthermore, we assessed markers associated with microtubule depolymerization (KIF2A) and initiators of apoptosis (caspase-9). RESULTS: In the NAC group, we observed significant increases in both cytoplasmic and cell areas. These changes in cytoplasm and cells were notably more pronounced in the NAC group compared to the NAE group. After treatment, KIF2A exhibited a decrease, with the magnitude of change being greater in the NET group than in the NAC group. However, no discernible differences were found in caspase-9 expression between the two groups. CONCLUSION: Our findings indicate that NAE induces condensation in cancer cells via cell cycle arrest or apoptosis. Conversely, NAC leads to cell enlargement due to the absence of microtubule depolymerization. These discrepancies underscore the importance of accounting for these distinctions when establishing criteria for evaluating pathological responses.

The selective proapoptotic impact of the myricetin-loaded alginate-cellulose hybrid nanocrystals (MAC-NCs) on the human AGS gastric cancer cells.

BACKGROUND: Myricetin, a flavanol present in fruits, tea, and vegetables, has the potential to reduce chronic diseases like gastric cancer by promoting cell death and stopping cell growth. However, its limited bioactivity due to its short lifespan and poor solubility in water has been a challenge. The current research focuses on incorporating myricetin into alginate-cellulose hybrid nanocrystals to enhance its selective proapoptotic effects on human AGS gastric cancer cells. METHODS: MAC-NCs, myricetin-loaded alginate-cellulose hybrid nanocrystals, were synthesized using a combined co-precipitation/ultrasonic homogenization method and characterized through Dynamic Light Scattering (DLS), Fourier Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscope (FESEM), and Zeta-potential analyses. Their cytotoxic activity was tested on cancerous (AGS) and normal (Huvec) cells, revealing selective toxicity. Apoptotic markers, Caspase 8 and Caspase 9, gene expression was measured, and cell death type was confirmed using DAPI staining and flow cytometry on AGS cells. RESULTS: Synthesized MAC-NCs, measuring 40 nm, showed significant selective toxicity on human gastric cells (IC50 of 31.05 µg/mL) compared to normal endothelial cells (IC50 of 214.26 µg/mL). DAPI and annexin flow cytometry revealed increased apoptotic bodies in gastric cells, indicating apoptosis. However, the apoptosis was found to be independent of Caspase-8 and Caspase-9. CONCLUSION: The current study provides critical insights into the therapeutic potential of MAC-NCs for gastric cancer treatment. Based on the notable induction of apoptosis in the AGS cancer cell line, the synthesized MAC-NCs exhibit promising potential as a selective anti-gastric cancer agent. However, further in-vivo studies are necessary to confirm and quantify the nanoparticle's selective toxicity and pharmaceutical properties in future investigations.

Exercise-activated hepatic autophagy combined with silymarin is associated with suppression of apoptosis in rats subjected to dexamethasone induced- fatty liver damage.

AIM: There is a need for effective treatments for non-alcoholic fatty liver disease (NAFLD) that are economically inexpensive, and have few side effects. The present study aimed to investigate exercise training and silymarin on hepatocyte death factors in rats with liver damage. METHODS: Forty-nine male Wistar rats were assigned to seven groups: sedentary control, fatty liver control (DEX), fatty liver + high-intensity interval training (HIIT), fatty liver + HIIT + silymarin (HIIT + SILY), fatty liver + continuous training (CT), fatty liver + CT + silymarin (CT + SILY), and fatty liver + silymarin (SILY). A subcutaneous injection of dexamethasone for 7 days was used to induce fatty liver in rats. Masson's trichrome and hematoxylin-eosin staining were done to evaluate hepatic injury. The hepatocyte apoptosis was determined by TUNEL assay. Real-Time PCR was conducted to evaluate the gene expressions of caspase-9, adenosine monophosphate-activated protein kinase (AMPKα1), mitofusin 2 (Mfn2), and damage-regulated autophagy modulator (DRAM). Liver tissue changes and serum levels of liver enzymes were also evaluated. RESULTS: Liver apoptosis was decreased in the CT, HIIT, HIIT + SILY and CT + SILY groups compared to the DEX group. Both continuous and high-intensity training models produced beneficial alterations in liver morphology and hepatic injuries that were significant in exercise training + silymarin group. This impact was accompanied by increased AMPKα1 and DRAM gene expression and decreased caspase-9 and Mfn2 gene expression. Liver enzyme levels were high in the DEX group and treatment with silymarin significantly reduced it. CONCLUSION: Silymarin supplementation combined with interval or continuous training substantially improves DEX-induced hepatic steatosis and hepatocyte injury mostly through suppressing liver apoptosis and upregulating autophagy, which may provide a novel perspective for NAFLD treatment.

Dexpanthenol exhibits antiapoptotic and anti-inflammatory effects against nicotine-induced liver damage by modulating Bax/Bcl-xL, Caspase-3/9, and Akt/NF-κB pathways.

Chronic tobacco use can lead to liver damage and inflammation due to the accumulation of various toxins in the body. This study aimed to investigate the correlation between the molecular mechanisms of nicotine-induced liver injury, the caspase cascade, and the Akt/NF-κB signaling pathway, as well as the protective effects of dexpanthenol (DEX). Male rats were subjected to intraperitoneal injections of nicotine at a concentration of 0.5 mg/kg/day and/or DEX at a concentration of 500 mg/kg/day for 8 weeks. After the treatment period, liver function tests were conducted on serum samples, and tissue samples were analyzed for protein levels of Akt, NF-κB, Bax, Bcl-xL, Caspase-3, and Caspase-9, along with histopathological changes. Additionally, assessments of oxidative stress markers and proinflammatory cytokines were carried out. Nicotine administration led to elevated levels of IL-6, IL-1ß, MDA, TOS, and oxidative stress index, accompanied by decreased TAS levels. Moreover, nicotine exposure reduced the p-Akt/Akt ratio, increased NF-κB, Bax, Caspase-3, and Caspase-9 protein levels, and decreased the antiapoptotic protein Bcl-xL levels. DEX treatment significantly mitigated these effects, restoring the parameters to levels comparable to those of the control group. Nicotine-induced liver injury resulted in oxidative stress, inflammation, and apoptosis, mediated by Bax/Bcl-xL, Caspase-3, Caspase-9, and Akt/NF-κB pathways. Conversely, DEX effectively attenuated nicotine-induced liver injury by modulating apoptosis through NF-κB, Caspase-3, Caspase-9, Bax inhibition, and Bcl-xL activation.

Granulosa cells undergo BPA-induced apoptosis in a miR-21-independent manner.

Bisphenol A (BPA) is a harmful endocrine disrupting compound that alters not only classical cellular mechanisms but also epigenetic mechanisms. Evidence suggests that BPA-induced changes in microRNA expression can explain, in part, the changes observed at both the molecular and cellular levels. BPA is toxic to granulosa cells (GCs) as it can activate apoptosis, which is known to contribute to increased follicular atresia. miR-21 is a crucial antiapoptotic regulator in GCs, yet the exact function in a BPA toxicity model remains unclear. BPA was found to induce bovine GC apoptosis through the activation of several intrinsic factors. BPA reduced live cells counts, increased late apoptosis/necrosis, increased apoptotic transcripts (BAX, BAD, BCL-2, CASP-9, HSP70), increased the BAX/Bcl-2 ratio and HSP70 at the protein level, and induced caspase-9 activity at 12 h post-exposure. miR-21 inhibition increased early apoptosis and, while it did not influence transcript levels or caspase-9 activity, it did elevate the BAX/Bcl-2 protein ratio and HSP70 in the same manner as BPA. Overall, this study shows that miR-21 plays a molecular role in regulating intrinsic mitochondrial apoptosis; however, miR-21 inhibition did not make the cells more sensitive to BPA. Therefore, apoptosis induced by BPA in bovine GCs is miR-21 independent.

5(S)-5-Carboxystrictosidine from the Root of Mappianthus iodoides Ameliorates H2O2-induced Apoptosis in H9c2 Cardiomyocytes via PI3K/AKT and ERK Pathways.

5(S)-5-carboxystrictosidine (5-CS) is a compound found in the root of Mappianthus iodoides, a traditional Chinese medicine used for the treatment of coronary artery disease. The aim of the present study was to investigate the protective effect of 5-CS against oxidative stress-induced apoptosis in H9c2 cardiomyocytes and the underlying mechanisms. 5-CS pretreatment significantly protected against H2O2-induced cell death, LDH leakage, and malondialdehyde (MDA) production, which are indicators for oxidative stress injury. 5-CS also enhanced the activity of SOD and CAT. In addition, 5-CS pretreatment significantly inhibited H2O2-induced apoptosis, as determined by flow cytometer, suppressed the activity of caspase-3 and caspase-9, and attenuated the activation of cleaved caspase-3 and caspase-9. 5-CS also increased Akt and ERK activation altered by H2O2 using Western blot analysis. The PI3K-specific inhibitor LY294002 abolished 5-CS-induced Akt activation. The ERK-specific inhibitor PD98059 abolished 5-CS-induced ERK activation. Both LY294002 and PD98059 attenuated the protective effect of 5-CS on H9c2 cardiomyocytes against H2O2-induced apoptosis and cell death. Taken together, these results demonstrate that 5-CS prevents H2O2-induced oxidative stress injury in H9c2 cells by enhancing the activity of the endogenous antioxidant enzymes, inhibiting apoptosis, and modulating PI3K/Akt and ERK signaling pathways.