Expression Of Apoptotic Markers Research Articles

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids

BackgroundInduced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin.MethodsDifferentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy.ResultsWe observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles.ConclusionOur data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Impact of combined alpha-lipoic acid and mitoquinone supplementation on myocardial infarction in aged rats: Heart performance and molecular mechanisms

BackgroundThis study aimed to investigate the effects of combined alpha-lipoic acid (ALA) and mitoquinone (Mito Q) supplementation on cardiac function and the underlying mechanisms in aged rats with myocardial infarction (MI). MethodsThe aged rats underwent left anterior descending artery (LADA) occlusion for 30 min, followed by reperfusion for 24 h. ALA (100 mg/kg, gavage) and Mito Q (10 mg/kg, IP) were administered daily for two weeks before ischemia. Cardiac function, inflammatory, and apoptotic markers were evaluated 24 h after ischemia. ResultsThe results of this study indicated that the administration of the combination of ALA and Mito Q significantly improved cardiac function. This improvement was linked to a reduction in the expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (P < 0.001) and apoptotic markers (Bax, caspase-3, and Cyt-c), as well as a decrease in the percentage of TUNEL-positive cells (P < 0.001). ConclusionThe study revealed that combined intervention synergistically mitigated cardiac dysfunction by suppressing inflammatory and apoptotic pathways in aged rats with MI. Further research is needed to validate the potential of ALA and Mito Q as therapeutic options for elderly people at risk of heart attacks.

Renoprotective effect of Nesfatin-1 in Adenine-Induced Chronic kidney Disease: An in vitro and in vivo study

Chronic Kidney Disease (CKD) presents a significant global health challenge with limited treatment options. Nesfatin-1, an anorexigenic peptide, has demonstrated antioxidant, anti-inflammatory, and anti-apoptotic properties in various diseases. However, the role of nesfatin-1 in CKD remains unclear. This study investigates the potential renoprotective effects of nesfatin-1 in adenine-induced CKD mice and in NRK-52E renal epithelial cells.Male C57BL/6J mice and NRK-52E renal epithelial cells were administered adenine to induce CKD. Various aspects of renal function, histopathology, oxidative stress, inflammation, apoptosis, and renal interstitial fibrosis were assessed and downstream pathways were investigated.Adenine-fed mice exhibited reduced nesfatin-1 expression and increased markers of kidney damage, including elevated blood urea nitrogen (BUN), serum creatinine, and histological abnormalities, reactive oxygen species (ROS), inflammation, apoptosis, and fibrosis. Treatment with nesfatin-1 in adenine induced mice significantly reversed these changes. Nesfatin-1 also lowered calcium levels and the expression of inflammatory markers, including IL-1β, IL-6, TNF-α, and Nf-kB. Furthermore, nesfatin-1 reduced the expression of apoptotic markers (Caspase-3, Caspase-1, Bax/Bcl2 ratio) and restored the balance of Bcl2 and MMP. Lastly, nesfatin-1 attenuated fibrotic markers (Tgf-β, Smad2/3,4, type IV collagen, α-SMA) in both adenine-induced CKD mice and NRK-52E cells.In conclusion, our results suggest that nesfatin-1 may enhance kidney function in adenine-induced CKD mice and NRK-52E cells. The renoprotective effects of nesfatin-1 are likely associated with its antioxidant, anti-inflammatory, anti-apoptotic, and anti-fibrotic properties.

Mycobacterium tuberculosis PE_PGRS38 Enhances Intracellular Survival of Mycobacteria by Inhibiting TLR4/NF-κB-Dependent Inflammation and Apoptosis of the Host.

Mycobacterium tuberculosis (Mtb) ranks as the most lethal human pathogen, able to fend off repeated attacks by the immune system or medications. PE_PGRS proteins are hallmarks of the pathogenicity of Mtb and contribute to its antigenic diversity, virulence, and persistence during infection. M. smegmatis is a nonpathogenic mycobacterium that naturally lacks PE_PGRS and is used as a model to express Mtb proteins. PE_PGRS has the capability to evade host immune responses and enhance the intracellular survival of M. smegmatis. Despite the intense investigations into PE_PGRS proteins, their role in tuberculosis remains elusive. We engineered the recombinant M. smegmatis strain Ms-PE_PGRS38. The result shows that PE_PGRS38 is expressed in the cell wall of M. smegmatis. PE_PGRS38 contributes to biofilm formation, confers permeability to the cell wall, and shows variable responses to exogenous stresses. PE_PGRS38 downregulated TLR4/NF-κB signaling in RAW264.7 macrophages and lung tissues of infected mice. In addition, PE_PGRS38 decreased NLRP3-dependent IL-1β release and limited pathogen-mediated inflammasome activity during infection. Moreover, PE_PGRS38 inhibited the apoptosis of RAW264.7 cells by downregulating the expression of apoptotic markers including Bax, cytochrome c, caspase-3, and caspase-9. In a nutshell, our findings demonstrate that PE_PGRS38 is a virulence factor for Mtb that enables recombinant M. smegmatis to survive by resisting and evading the host's immune responses during infection.

Dantrolene alleviates mitochondrial dysfunction and neuroinflammation in traumatic brain injury by modulating the NF-ĸβ/Akt pathway

Traumatic brain injury (TBI) triggers a bevy of changes including mitochondrial dysfunction, apoptosis, oxidative stress, neurobehavioural impairment, and neuroinflammation, among others. Dantrolene (DNT), a muscle relaxant which inhibits intracellular Ca2+ signaling from the ER, has been repurposed as a potential neuroprotective agent in various neurological diseases. However, there have been limited studies on whether it can mitigate TBI-induced deficits and restore impaired mitochondrial dynamics. This study sought to evaluate whether Dantrolene can potentially provide neuroprotection in an in vivo model of TBI. Male wistar rats subjected to TBI were treated with DNT (10 mg/kg) 1 h and 12 h post surgery. Animals were assessed 24 h post-TBI to evaluate neurobehavioural deficits and cerebral edema. We evaluated the protein expressions of apoptotic, autophagic, and neuroinflammatory markers by immunoblotting, as well as Mitochondrial Membrane Potential (MMP) and Reactive Oxygen Species (ROS) via Flow Cytometry to ascertain the effects of DNT on TBI. We further analysed immunofluorescence staining with Glial Fibrillary Acidic Protein (GFAP) and immunohistochemistry with NF-κβ to investigate neuroinflammation. H&E staining was also performed post-TBI. Our findings revealed DNT administration inhibits mitochondria-mediated apoptotis and reduces heightened oxidative stress. DNT treatment was also found to reverse neurobehavioural impairments and offer neuroprotection by preserving neuronal architechture. We also demonstrated that DNT inhibits neuronal autophagy and alleviates neuroinflammation following TBI by modulating the NF-κβ/Akt signaling pathway. Thus, our results suggest a novel application of DNT in ameliorating the multitude of deficits induced by TBI, thereby conferring neuroprotection.

Up-regulation of inflammatory, oxidative stress, and apoptotic mediators via inflammatory, oxidative stress, and apoptosis-associated pathways in bovine endometritis

Endometritis is the inflammation of the endothelial lining of the uterine lumen and is multifactorial in etiology. Escherichia (E.) coli is a Gram-negative bacteria, generally considered as a primary causative agent for bovine endometritis. Bovine endometritis is characterized by the activation of Toll-like receptors (TLRs) by E. coli, which in turn triggers inflammation, oxidative stress, and apoptosis. The objective of this study was to investigate the gene expression of inflammatory, oxidative stress, and apoptotic markers related to endometritis in the uteri of cows. Twenty uterine tissues were collected from the abattoir. Histologically, congestion, edema, hyperemia, and hemorrhagic lesions with massive infiltration of neutrophil and cell necrosis were detected markedly (P < 0.05) in infected uterine samples. Additionally, we identify E. coli using the ybbW gene (177 base pairs; E. coli-specific gene) from infected uterine samples. Moreover, qPCR and western blot results indicated that TLR2, TLR4, proinflammatory mediators, and apoptosis-mediated genes upregulated except Bcl-2, which is antiapoptotic, and there were downregulations of oxidative stress-related genes in the infected uterine tissue. The results of our study suggested that different gene expression regimes related to the immune system reflex were activated in infected uteri. This research gives a novel understanding of active immunological response in bovine endometritis.

Betterment of testicular dysfunction: Obesity induced testicular damage regulated by Syzygium paniculatum

BackgroundDiet is the most important environmental factor but consumption of high calorie diet leads to obesity which contributes to the development of various diseases, mainly leads to testicular dysfunction and infertility in males. It was reported that traditional medicinal plant extracts showed preventive and ameliorative effect on the testis, spermatogenesis and also fertility in rats fed on high fat diet. With this context, we set out to see whether an aqueous extract of Syzygium paniculatum fruit may help restore normal function to male rat testes that had become dysfunctional. PurposeThe goal of our research was to determine preventive functions of Syzygium paniculatum against testicular dysfunction caused by a high fat diet induced obesity. Study designAll rats were grouped into three of six rats each (n = 6), Group 1-Control rats (C); Group 2-HFD obese rats (Ob C); Group 3-HFD fed obese rats treated with 100 mg/kg bw/day of AESPF. MethodsA high fat diet rat model was established to examine the effects of Syzygium paniculatum. Eighteen Albino Wister rats (n = 6) divided into three groups, which was control group, HFD group and HFD+ AESPF treated group. All rat groups had a 17 weeks treatment period, and their physical, biochemical, testicular, and cytotoxic effects were assessed along with spermatogenic marker for cellular apoptosis (Bax, Cleaved caspase 3, Caspase-3), cell proliferation (PCNA), cell survival (Bcl2), testicular inflammatory pathway regulators (TNF-α, NF-B, IL-6) and peroxisome proliferator activated receptor (PPAR) were evaluated by western blotting. The protective effects of S. Paniculatum on testes were evaluated by using histopathological examination. ResultsThe obese rats which given an HFD showed a substantial rise in their obesity index, aberrant hormone levels, and sperm counts along with the elevated expressions of inflammatory and apoptotic markers in the testis. Treatment with AESPF for 17 weeks improved testicular function, as measured by increased spermatogenesis and semen quality, lower expression of apoptotic and inflammatory proteins in the testes, and an increase in testosterone level and antioxidant capacity. Histopathological results corroborate the enhanced testicular relative weight and histological picture seen after AESPF treatment for 17 weeks. According to our findings, the metabolic and reproductive systems were dysfunctional after HFD intake, however AESPF restored normalcy. ConclusionThe administration of the aqueous extract of Syzygium paniculatum fruit (AESPF) reduced HFD induced spermatogenesis dysfunction and apoptosis. Obesity-related testicular dysfunction in men might be treated with Syzygium paniculatum fruit as a new therapeutic medication.

The relationship between oxidative stress and apoptosis of histopathological changes in the ovary made by mad honey containing grayanotoxin

The purpose of this study is to determine the effects of grayanotoxin in mad honey on ovarian tissue folliculogenesis in terms of cell death and nitric oxide expression. Three groups of 18 female Sprague-Dawley rats were formed. The first group received mad honey (80 mg/kg), the second group received normal honey (80 mg/kg), and the third group was the control. The first and second groups received normal and mad honey by oral gavage for 30 days before being sacrificed under anesthesia. Caspase 3 immunostaining showed a moderate to strong response, particularly in the mad honey group. In the mad honey group, immunostaining for caspase 8 and caspase 9 revealed a moderate immunoreaction in the granulosa cells of the Graaf follicles. The majority of Graaf follicles exhibited TUNEL positive in the mad honey group. The iNOS immunoreaction revealed a high level of expression in the mad honey group. In all three groups, eNOS immunostaining showed weak reactivity. According to the findings of apoptotic and nitric oxide marker expression, it was determined that mad honey may result in an increase in follicular atresia in ovarian follicles when compared to normal honey and control groups.

Cyclovirobuxine D inhibits hepatocellular carcinoma growth by inducing ferroptosis of hepatocellular carcinoma cells

ObjectiveHepatocellular carcinoma (HCC) is one cancer with high death rates. Nowadays, there are no effective drugs to treat it. Cyclovirobuxine D (CVB-D) is the primary ingredient of the traditional Chinese medicine (TCM) Buxus microphylla. Here, we try to explore the impacts of CVB-D on human HCC cells and explain the potential mechanisms.MethodsHepG2 and Huh-7 cells were used for our experiments. The cell viability and half inhibitory concentration (IC50) were detected by MTT assays. The apoptosis ratio was examined by Annexin V-FITC/7AAD staining and flow cytometry (FCM). The Fe2+ content was examined by ferrous ion content assays. The malondialdehyde (MDA) content was evaluated by lipid peroxidation MDA assays. The reactive oxygen species (ROS) level was examined by the DCFH-DA probe. The expression of apoptotic markers (Bax and Bcl-2) and ferroptosis-related proteins (GPX4 and FSP1) was detected by western blotting. The in vivo curative effect of CVB was explored using xenograft models established in C-NKG mice.ResultsThe cell viability could be inhibited by CVB-D in HepG2 and Huh-7 cells. The IC50 value of CVB-D on HepG2 and Huh-7 cells are 91.19 and 96.29 µM at 48 h, and 65.60 and 72.80 µM at 72 h. FCM showed that the apoptosis rate was increased by CVB-D in HepG2 and Huh-7 cells. Next, ferrous ion content assays showed that the level of Fe2+ was increased by CVB-D in HepG2 and Huh-7 cells. Then, we found the level of MDA and ROS was increased by CVB-D. And the Fe2+ promotion by CVB-D could be reversed by Fer-1. Additionally, western blotting assays showed that the expression of GPX4 and FSP1 was inhibited by CVB-D in HepG2 and Huh-7 cells. Moreover, in vivo, CVB-D displayed excellent anticancer effects in HCC tumor-bearing C-NKG mice.ConclusionCVB-D suppresses the growth in HCC cells through ferroptosis.

Molecular mechanisms of resveratrol and its silver nanoparticle conjugate in addressing sepsis-induced lung injury.

Sepsis is a life-threatening condition characterized by a systemic inflammatory response to infection. Despite extensive research on its pathophysiology, effective therapeutic approaches remain a challenge. This study investigated the potential of resveratrol (RV) and silver nanoparticle-enhanced resveratrol (AgNP-RV) as treatments for sepsis-induced lung injury using a rat model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). The study focused on evaluating changes in oxidative status (TAS, TOS, and OSI) and the expression of inflammatory and apoptotic markers (IL-1β, TNF-α, P2X7R, TLR4, Caspase-3, and Bcl-2) in lung tissue. Both RV and AgNP-RV demonstrated potential in mitigating oxidative stress, inflammation, and apoptosis, with AgNP-RV exhibiting greater efficacy than RV alone(p < 0.05). These findings were corroborated by histopathological analyses, which revealed reduced tissue damage in the RV- and AgNP-RV-treated groups. Our study highlights the therapeutic potential of RV and, particularly, AgNP-RV in combating sepsis-induced oxidative stress, inflammation, and apoptosis. It also underscores the promise of nanoparticle technology in enhancing therapeutic outcomes. However, further investigations are warranted to fully understand the mechanisms of action, especially concerning the role of the P2X7 receptor in the observed effects. Nonetheless, our research suggests that RV and AgNP-RV hold promise as novel strategies for sepsis management.

High fructose Corn Syrup recast glucose transporter-5, Wnt, NF-κB signalling and mitochondrial apoptosis in an animal model of oral oncogenesis

BackgroundWnt signalling pathway, is mediated by members of T-cell factor (TCF) transcription factors family, is essential for the control of epithelial cell proliferation and death. Glucose transporter-5 (GLUT5), fructose-specific transporter, is also important in allowing transcellular fructose uptake. The goal of this work to determine how the High fructose Corn Syrup (HFCS) affected Wingless-related integration site (Wnt) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in the 7,12 –dimethylbenzaanthracene (DMBA)-induced hamster buccal pouch carcinogenesis (HBPCs) model. MethodsFour groups of hamsters were created. For 12 weeks, 0.5 % DMBA was applied 3 times/week to the left side buccal pouches of the hamsters in groups (2 & 4). Additionally, the animals in groups (3 & 4) were given through drinking water of HFCS 25 %. The control animals were from group 1. By using western blot analysis, signalling network markers of the GLUT-5, Wnt, TCF-4, GSK-3β and NF-κB as well as mitochondrial apoptotic pathway marker expression B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cyclooxygenase – 2 (COX-2) was assessed. ResultsDrinking water uptake of 25% HFCS encouraged progress of HBP carcinomas by constitutive stimulating of the Wnt pathway via GSK-3β overexpression. HFCS suppressed Wnt signalling which contributed the NF-κB attenuation and changes the signalling markers in apoptotic network. ConclusionsOur hypothesis suggests a mechanically crosstalk between Wnt and NF-κB signalling pathways in HBP carcinomas that is developed by HFCS. HFCS that targets the Wnt pathway and its downstream signalling mediators could be additive reason for cancer development.

Abstract 5262: MBO enhances anti-tumor immunity in pancreatic ductal adenocarcinoma by inducing immunogenic apoptosis

Abstract Pancreatic ductal adenocarcinoma (PDAC) poses significant therapeutic challenges due to its aggressive nature and limited treatment options. In this study, we explored the anti-cancer potential of MBO, a compound selected through drug repurposing strategy based on its cytotoxic ability and modulation of key survival pathways in PDAC. Our preliminary in vitro experiments demonstrated MBO's potent cytotoxicity against various PDAC cell lines, inhibiting cellular growth and clonogenicity. MBO induced G1 phase cell cycle arrest in a concentration-dependent manner. In the next step, in vivo, studies using xenograft and orthotopic mouse models confirmed MBO's ability to suppress tumor growth and increase CD8+ T cell infiltration. MBO demonstrated a favorable tumor suppressive activity wherein it suppressed tumor growth by approximately 49% and 56% in xenograft and orthotopic models, respectively. A CD8+ T cell depletion experiment revealed partial dependence on CD8+ T cells for MBO's overall anti-tumor efficacy. Further, combining MBO with anti-PD1 therapy exhibited a synergistic effect, enhancing overall T cell infiltration, particularly CD8+ T cells. Our mechanistic studies revealed that MBO induced immunogenic apoptosis, as evidenced by increased expression of immunogenic cell death markers (HSP90 and calreticulin) and apoptotic markers. To further delineate the mechanism, we performed RPPA profiling. RPPA analysis indicated MBO's impact on mitochondrial gene expression and stress response pathways, suggesting its involvement in disrupting cellular survival mechanisms along with stress-induced immunogenic marker expression. Overall, this study demonstrates MBO as a promising anti-cancer agent with the potential to serve as an immune adjuvant. Particularly, in combination with immunotherapies, MBO can serve as a potential option to improve immunotherapeutic response in PDAC tumors providing valuable insights for developing novel therapeutic strategies in PDAC. Citation Format: Shreyas Ramchandra Gaikwad, Sanjay K. Srivastava. MBO enhances anti-tumor immunity in pancreatic ductal adenocarcinoma by inducing immunogenic apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5262.

Development of a Novel Peptide with RGD Tumor Homing Motif: Evaluation of its Anticancer Potential in Hepatocellular Carcinoma and Colon Cancer Cells

Background: Peptide-based therapy has emerged as a promising avenue for treating various disorders, and recent research has highlighted the potential of anti-cancer peptides (ACPs) in cancer treatment. In this context, this study aimed to design a novel peptide incorporating a tumor-homing peptide (RGD) and C-amidation to enhance its anticancer activity, particularly against liver (HepG2) and colon (HCT-116) cancer cell lines. Objectives: The primary objective was to design a peptide with improved anticancer properties by leveraging the tumor-homing capabilities of RGD and enhancing its activity through C-amidation. The study sought to evaluate the cytotoxicity of the designed peptide against red blood cells (RBCs) and normal Vero cells. Furthermore, the anticancer efficacy of the peptide was assessed in hepatocellular carcinoma (HepG2) and colon cancer (HCT-116) cell lines. The specific objectives included examining the apoptotic induction and morphological changes in treated cells compared to untreated cells. Methods: The peptide was designed using the ACPred-FL bioinformatics tool, and its cytotoxicity was assessed through hemolysis assays against RBCs and normal Vero cells. Anticancer activity was evaluated against HepG2 and HCT-116 cell lines. The analysis of apoptotic induction involved measuring the relative gene expression of oncogenic marker BCL2 and apoptotic markers (BAX, BID, CAS-8). Additionally, Cytopathological examination and Western Blot analysis were employed to study morphological changes and confirm the quantification of relevant markers. Results: The designed peptide, consisting of twelve amino acids with a molecular mass of 1230.6233 Da and an isoelectric point of 9.81, exhibited low erythrocyte lysis and minimal toxicity to normal cells. The IC50 values demonstrated significant anticancer activity against both HepG2 (36.49±2.6 μg/mL) and HCT-116 (11.03±2.5 μg/mL) cell lines. Treated cells exhibited a significant decrease in the oncogenic marker BCL2 and an upregulation of apoptotic markers (BAX, BID, CAS-8). Western Blot analysis confirmed these findings, and Cytopathological examination revealed scattered apoptotic and degenerative changes. Conclusion: The designed peptide displayed remarkable anticancer activity against hepatocellular carcinoma and colon cancer cell lines, effectively modulating apoptotic and oncogenic markers. These findings highlight the potential of the peptide as a therapeutic agent for cancer treatment, emphasizing its clinical significance in combating liver and colon cancers. Nonetheless, further research and development are warranted to explore the translational potential of this peptide in clinical settings.

Inhibition of both NOX and TNF-α exerts substantial renoprotective effects in renal ischemia reperfusion injury rat model

Background and aimsAcute kidney injury (AKI) due to renal ischemia-reperfusion injury (RIRI) is associated with high morbidity and mortality, with no renoprotective drug available. Previous research focused on single drug targets, yet this approach has not reached translational success. Given the complexity of this condition, we aimed to identify a disease module and apply a multitarget network pharmacology approach. MethodsIdentification of a disease module with potential drug targets was performed utilizing Disease Module Detection algorithm using NADPH oxidases (NOXs) as seeds. We then assessed the protective effect of a multitarget network pharmacology targeting the identified module in a rat model of RIRI. Rats were divided into five groups; sham, RIRI, and RIRI treated with setanaxib (NOX inhibitor, 10 mg/kg), etanercept (TNF-α inhibitor, 10 mg/kg), and setanaxib and etanercept (5 mg/kg each). Kidney functions, histopathological changes and oxidative stress markers (MDA and reduced GSH) were assessed. Immunohistochemistry of inflammatory (TNF-α, NF-κB) apoptotic (cCasp-3, Bax/Bcl 2), fibrotic (α-SMA) and proteolysis (MMP-9) markers was performed. ResultsOur in-silico analysis yielded a disease module with TNF receptor 1 (TNFR1A) as the closest target to both NOX1 and NOX2. Targeting this module by a low-dose combination of setanaxib, and etanercept, resulted in a synergistic effect and ameliorated ischemic AKI in rats. This was evidenced by improved kidney function and reduced expression of inflammatory, apoptotic, proteolytic and fibrotic markers. ConclusionsOur findings show that applying a multitarget network pharmacology approach allows synergistic renoprotective effect in ischemic AKI and might pave the way towards translational success.

FOXN2, identified as a novel biomarker in serum, modulates the transforming growth factor-beta signaling pathway through its interaction with partitioning defective 6 homolog alpha, contributing to the pathogenesis of gastric cancer

Background and Objective Dysregulated expression of Forkhead Box N2 (FOXN2) has been detected in various cancer types. However, the underlying mechanisms by which FOXN2 contributes to the onset and progression of gastric cancer (GC) remain largely unexplored. This study aimed to elucidate the potential role of FOXN2 within GC, its downstream molecular mechanisms, and its feasibility as a novel serum biomarker for GC. Methods Tissue samples from GC patients and corresponding non-cancerous tissues were collected. Peripheral blood samples were obtained from GC patients and healthy controls. The expression of FOXN2 was determined using quantitative real-time PCR, western blotting, and immunohistochemistry. The expression of FOXN2 in GC cells was modulated by transfection with small interfering RNA (siRNA) or the pcDNA 3.1 expression vector. Cell proliferation was assessed using the Cell Counting Kit-8 and 5-ethynyl-2’-deoxyuridine incorporation assays. The migratory and invasive capacities of cells were evaluated by Transwell assays, apoptosis rates were measured by flow cytometry, and the expression of proliferative, apoptotic, and epithelial-mesenchymal transition (EMT) markers were assessed by western blot analysis. Results FOXN2 was found to be overexpressed in the serum, tissues, and cells of GC, correlating with distant metastasis and TNM staging. FOXN2 demonstrated diagnostic value in differentiating GC patients from healthy individuals, with higher levels of FOXN2 being indicative of poorer survival rates. Silencing FOXN2 in vitro inhibited the proliferation, invasion, migration, and EMT of GC cells, while promoting apoptosis. FOXN2 was shown to regulate the transforming growth factor-beta (TGFβ) receptor signaling pathway in GC cells via its interaction with Partitioning Defective 6 Homolog Alpha (PARD6A). Conclusion In summary, our data suggest that FOXN2 acts as an oncogenic factor in GC, modulating the TGFβ pathway by binding to PARD6A, thereby influencing gastric carcinogenesis. This study underscores the functional significance of FOXN2 as a potential serum biomarker and therapeutic target in GC.

Investigating the Role of 17-Beta Estradiol in the Regulation of the Unfolded Protein Response (UPR) in Pancreatic Beta Cells.

Diabetes mellitus is clinically defined by chronic hyperglycemia. Sex differences in the presentation and outcome of diabetes exist with premenopausal women having a reduced risk of developing diabetes, relative to men, or women after menopause. Accumulating evidence shows a protective role of estrogens, specifically 17-beta estradiol, in the maintenance of pancreatic beta cell health; however, the mechanisms underlying this protection are still unknown. To elucidate these potential mechanisms, we used a pancreatic beta cell line (BTC6) and a mouse model of hyperglycemia-induced atherosclerosis, the ApoE-/-:Ins2+/Akita mouse, exhibiting sexual dimorphism in glucose regulation. In this study we hypothesize that 17-beta estradiol protects pancreatic beta cells by modulating the unfolded protein response (UPR) in response to endoplasmic reticulum (ER) stress. We observed that ovariectomized female and male ApoE-/-:Ins2+/Akita mice show significantly increased expression of apoptotic UPR markers. Sham operated female and ovariectomized female ApoE-/-:Ins2+/Akita mice supplemented with exogenous 17-beta estradiol increased the expression of adaptive UPR markers compared to non-supplemented ovariectomized female ApoE-/-:Ins2+/Akita mice. These findings were consistent to what was observed in cultured BTC6 cells, suggesting that 17-beta estradiol may protect pancreatic beta cells by repressing the apoptotic UPR and enhancing the adaptive UPR activation in response to pancreatic ER stress.

Effects of Herpud1 in Methamphetamine-Induced Neuronal Apoptosis.

Methamphetamine (METH) is an illicit psychoactive substance that can damage various organs in the body, especially the nervous system. We hypothesized that expression of homocysteine-inducible endoplasmic reticulum-resident with ubiquitin-like domain member 1 (Herpud1) protein would alleviate the induction of apoptosis following METH administration. To test this hypothesis, we analysed the changes in Herpud1 expression and apoptosis in PC12 cells under different concentrations and exposure times of METH. Moreover, we examined the effects of Herpud1 knockdown on METH-induced neuronal apoptosis. Flow cytometry and Western blot analyses were used to evaluate apoptosis levels and the expression of apoptotic markers (cleaved caspase-3) in PC12 cells following Herpud1 knockdown by synthetic small interfering RNA (siRNA). Our results showed that Herpud1 expression was upregulated in PC12 cells following METH treatment, while endoplasmic reticulum stress (ERS) and apoptosis were also increased. Conversely, Herpud1 knockdown reduced METH-induced ERS and apoptosis levels in vitro. These results suggest that Herpud1 plays an essential role in METH-induced neuronal ERS and apoptosis and may represent a potential therapeutic gene target in METH-induced neurotoxicity.

Evaluation of telomere length, reactive oxygen species, and apoptosis in spermatozoa of patients with oligospermia.

50% of cases of infertility are caused by male factor, which acquired or congenital problems may bring on. Male infertility can be caused by oligospermia and asthenozoospermia, which are common. Since the same mutations that cause azoospermia in some people also cause oligozoospermia in others, oligozoospermia may be thought of as a less severe form of azoospermia. Studies have demonstrated telomere length, catalase activity, super oxide dismutase (SOD), and DNA fragmentation can be influential factors for male infertility. The amount of apoptosis, oxidative stress factors, telomere length, and DNA fragmentation were some aspects of healthy sperm that we chose to look into in this study and compare to oligospermia individuals. Oligospermia patients (n = 24) and fertile men (n = 27) semen samples were collected, and the apoptosis rate of sperms in both groups was analyzed (Flow cytometry). Also, gene expression of apoptotic and antiapoptotic markers and telomere length were examined (real-time polymerase chain reaction). The sperm DNA fragmentation kit was used to determine DNA fragmentation and to evaluate catalase and SOD activity; the specific kits and methods were utilized. Higher expression levels of caspase3 (p = .0042), caspase8 (p = .0145), caspase9 (p = .0275), and BAX (p = .0202) mRNA were observed in patients who had oligospermia. In contrast, lower mRNA expression of BCL-2 (p = .0009) was detected in this group. In addition, telomere length was decreased in the oligospermia group (p < .0001) compared to the health group. Moreover, the frequency of apoptosis is induced in patients (p = .0026). The catalase activity is low (p = .0008), but the SOD activity is high (p = .0015) in the patient group. As a result of our findings, we may list the sperm cell apoptosis rate, telomere length, the degree of sperm DNA fragmentation, and lastly, the measurement of significant and efficient oxidative stress markers like SOD and catalase in semen plasma among the principal diagnostic characteristics for oligospermia. Future studies will be better able to treat oligospermia by showing whether these indicators are rising or falling.

Sex-related differences in delayed doxorubicin-induced cardiac dysfunction in C57BL/6 mice.

Cancer survivors may experience long-term cardiovascular complications due to chemotherapeutic drugs such as doxorubicin (DOX). The exact mechanism of delayed DOX-induced cardiotoxicity has not been fully elucidated. Sex is an important risk factor for DOX-induced cardiotoxicity. In the current study, we identified sex differences in delayed DOX-induced cardiotoxicity and determined the underlying molecular determinants of the observed sexual dimorphism. Five-week-old male and female mice were administered intraperitoneal injections of DOX (4mg/kg/week) or saline for 6weeks. Echocardiography was performed 5weeks after the last dose of DOX to evaluate cardiac function. Thereafter, mice were sacrificed and gene expression of markers of apoptosis, senescence, and inflammation was measured by PCR in hearts and livers. Proteomic profiling of the heart from both sexes was conducted to determine differentially expressed proteins (DEPs). Only DOX-treated male, but not female, mice demonstrated cardiac dysfunction, cardiac atrophy, and upregulated cardiac expression of Nppb and Myh7. No sex-related differences were observed in DOX-induced expression of most apoptotic, senescence, and pro-inflammatory markers. However, the gene expression of Trp53 was significantly reduced in hearts of DOX-treated female mice only. The anti-inflammatory marker Il-10 was significantly reduced in hearts of DOX-treated male mice only, while the pro-inflammatory marker Il-1α was significantly reduced in livers of DOX-treated female mice only. Gene expression of Tnf-α was reduced in hearts of both DOX-treated male and female mice. Proteomic analysis identified several DEPs after DOX treatment in a sex-specific manner, including anti-inflammatory acute phase proteins. This is the first study to assess sex-specific proteomic changes in a mouse model of delayed DOX-induced cardiotoxicity. Our proteomic analysis identified several sexually dimorphic DEPs, many of which are associated with the anti-inflammatory marker Il-10.

PIM kinase inhibitor AZD1208 in conjunction with Th1 cytokines potentiate death of breast cancer cells in vitro while also maximizing suppression of tumor growth in vivo when combined with immunotherapy

PIM kinases are over-expressed by a number of solid malignancies including breast cancer, and are thought to regulate proliferation, survival, and resistance to treatment, making them attractive therapeutic targets. Because PIM kinases sit at the nexus of multiple oncodriver pathways, PIM antagonist drugs are being tested alone and in conjunction with other therapies to optimize outcomes. We therefore sought to test the combination of pharmacological PIM antagonism and Th1-associated immunotherapy. We show that the pan PIM antagonist, AZD1208, when combined in vitro with Th1 cytokines IFN-γ and TNF-α, potentiates metabolic suppression, overall cell death, and expression of apoptotic markers in human breast cancer cell lines of diverse phenotypes (HER-2pos/ERneg, HER-2pos/ERpos and triple-negative). Interestingly, AZD1208 was shown to moderately inhibit IFN-γ secretion by stimulated T lymphocytes of both human and murine origin, suggesting some inherent immunosuppressive activity of the drug. Nonetheless, when multiplexed therapies were tested in a murine model of HER-2pos breast cancer, combinations of HER-2 peptide-pulsed DCs and AZD1208, as well as recombinant IFN-γ plus AZD1208 significantly suppressed tumor outgrowth compared with single-treatment and control groups. These studies suggest that PIM antagonism may combine productively with certain immunotherapies to improve responsiveness.