Western Blot Techniques Research Articles

Increased levels of DNA methyltransferases in the placentas of women affected by Placenta Accreta Spectrum lead to aberrant DNA methylation patterns.

Placenta Accreta Spectrum (PAS) is a serious placental abnormality assosiated with significant maternal death during pregnancy. Due to its invasive characteristics resembling tumor growth, PAS is often associated with tumor-related proteins. While DNA methylation is known to regulate genes involved in development, its potential role in the development of PAS remains unclear. The aim of our study was to investigate the impact of PAS on DNA methylation and DNA methyltransferases (DNMTs). The groups were categorized into three groups: vaginal birth, cesarean delivery, and cesarean delivery with PAS. To measure DNMT protein levels in placental tissue, we employed immunohistochemistry (IHC) and Western blot (WB) techniques. Global DNA methylation levels were assessed using 5-methylcytosine staining. We found that DNMT1, DNMT3A and DNMT3L levels were significantly increased in the placentas of PAS group compared to control counterparts in both WB and IHC analysis. Compatible with DNMTs expressions, global DNA methylation levels were found to be significantly higher in placentas from women with PAS compared to controls. Our results suggest that significant alterations in DNMTs expressions and global DNA methylation within placentas may contribute to the development of Placenta Accreta Spectrum (PAS). To elucidate the precise molecular mechanisms underlying these changes and their role in PAS pathogenesis, further research required.

Esomeprazole inhibits liver inflammation and carcinogenesis by suppressing farnesoid X receptors and NF-κB signaling.

The activity of proton pump inhibitors (PPIs) hinders the function of proton pumps that generate stomach acid. Nuclear factor kappa B (NF-κB) is a transcriptional factor engaged in inflammation, immunity and the formation of cancer. The farnesoid X receptor (FXR) is a nuclear receptor that governs the metabolism of bile acids and the metabolic functioning of the liver. The impact of PPIs on the signaling of FXRs and NF-κB is not well understood. We aimed to study the effects of esomeprazole on FXRs and NF-κB signaling in liver cells. For the liver cell model, we used the human liver cell line HepaG2. Cells were treated with lipopolysaccharides (LPS) and esomeprazole, and then we assessed the effects of esomeprazole on inflammatory and carcinogenic markers, NF-κB and FXR. We applied the techniques of western blotting, reverse-transcription polymerase chain reaction (RT-PCR), confocal microscopic imaging, and electrophoretic mobility shift assay (EMSA). Lipopolysaccharides-induced FXRs and NF-κB signaling upregulated the NF-κB-associated cytokines interleukin 6 (IL-6), cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-α). Esomeprazole inhibited the upregulation of all these cytokines. Additionally, esomeprazole inhibited LPS-induced FXR expression and NF-κB signaling in HepaG2 cells. The net effect on FXRs and NF-κB signaling was the lower levels of the associated inflammatory and carcinogenic cytokines. Our study provides insight into the potential therapeutic effects of esomeprazole on hepatic inflammation and carcinogenesis by inhibiting LPS-induced NF-κB and FXR expression in HepG2 cells.

Metformin attenuates central sensitization by regulating neuroinflammation through the TREM2-SYK signaling pathway in a mouse model of chronic migraine

BackgroundChronic migraine (CM) is a serious neurological disorder. Central sensitization is one of the important pathophysiological mechanisms underlying CM, and microglia-induced neuroinflammation conduces to central sensitization. Triggering receptor expressed on myeloid cells 2 (TREM2) is presented solely in microglia residing within the central nervous system and plays a key role in neuroinflammation. Metformin has been shown to regulate inflammatory responses and exert analgesic effects, but its relationship with CM remains unclear. In the study, we investigated whether metformin modulates TREM2 to improve central sensitization of CM and clarified the potential molecular mechanisms.MethodsA CM mouse model was induced by administration of nitroglycerin (NTG). Behavioral evaluations were conducted using von Frey filaments and hot plate experiments. Western blot and immunofluorescence techniques were employed to investigate the molecular mechanisms. Metformin and the SYK inhibitor R406 were administered to mice to assess their regulatory effects on neuroinflammation and central sensitization. To explore the role of TREM2-SYK in regulating neuroinflammation with metformin, a lentivirus encoding TREM2 was injected into the trigeminal nucleus caudalis (TNC). In vitro experiments were conducted to evaluate the regulation of TREM2-SYK by metformin, involving interventions with LPS, metformin, R406, siTREM2, and TREM2 plasmids.ResultsMetformin and R406 pretreatment can effectively improve hyperalgesia in CM mice. Both metformin and R406 significantly inhibit c-fos and CGRP expression in CM mice, effectively suppressing the activation of microglia and NLRP3 inflammasome induced by NTG. With the administration of NTG, TREM2 expression gradually increased in TNC microglia. Additionally, we observed that metformin significantly inhibits TREM2 and SYK expression in CM mice. Lv-TREM2 attenuated metformin-mediated anti-inflammatory responses. In vitro experiments, knockdown of TREM2 inhibited LPS-induced SYK pathway activation and alleviated inflammatory responses. After the sole overexpression of TREM2, the SYK signaling pathway is activated, resulting in the activation of the NLRP3 inflammasome and an increased expression of pro-inflammatory cytokines; nevertheless, this consequence can be reversed by R406. The overexpression of TREM2 attenuates the inhibition of SYK activity mediated by metformin, and this effect can be reversed by R406.ConclusionsOur findings suggest that metformin attenuates central sensitization in CM by regulating the activation of microglia and NLRP3 inflammasome through the TREM2-SYK pathway.Graphical

Total Alkaloids of Rhizoma Corydalis regulates gut microbiota and restores gut immune barrier to ameliorate cognitive dysfunction in diabetic rats

Background and objectivesGiven the widespread dysbiosis of gut microbiota in patients with T2DM, it has been found that the microbiota-gut-brain axis plays an influential regulatory role in diabetic cognitive dysfunction, and improving gut dysbiosis may be a potential strategy for treating diabetic cognitive dysfunction. Total Alkaloids of Rhizoma Corydalis (TAC) is the main active component extracted from Rhizoma Corydalis. Pharmacological studies have demonstrated its significant pharmacological effects on the cardiovascular and cerebrovascular systems, and berberine, the main component of TAC, has a certain regulatory effect on gut microbiota.Materials and methodsRats were randomly divided into Control group, Model group, TAC-low group, TAC-mid group and TAC-high group. Cognitive function of diabetic rats was evaluated through behavioral testing using the Morris water maze experiment. The relative abundance of gut bacteria in rat feces was determined via 16S rRNA analysis. IHC and Western blot techniques were employed to assess IL-22, IL-23, Reg3g, ZO-1, occludin 1 expression in the colon tissue; GPX4, xCT, NLRP3, Caspase-1 p20, GSDMD-N were detected in the hippocampus.ResultsThe cognitive function of diabetic rats decreased significantly. TAC demonstrated a significant reduction in inflammatory factors in serum, hippocampus, and colon, thus alleviating inflammation. Additionally, it effectively decreased ferroptosis induced by NLRP3 and reduced pathological damage in the hippocampus of diabetic rats. After treatment, the differential microbiota such as Lachnoclotridium and Bacteroides. TAC improved gut barrier permeability and integrity in rats while remodeling gut mucosal homeostasis. Moreover, pyroptosis and ferroptosis caused by the inflammatory cascade in the rat hippocampus were also significantly inhibited.ConclusionThe combination of high lipid and high glucose with STZ can result in gut microbiota disturbance, damage gut immune barrier, decreased gut mucosal permeability and integrity, aggravated gut inflammation, further spread inflammatory factors to brain tissue, cause inflammatory cascade reaction of encephalopathy, and ultimately resulting in neuronal ferroptosis and cognitive dysfunction in diabetes mellitus. Our study suggests that TAC may regulate gut microbiota, restore gut immune homeostasis, improve gut barrier permeability and integrity, inhibit brain tissue inflammatory cascade, reduce neuronal ferroptosis, and thus improve diabetes. This provides new targets for its treatment strategy.

Ginkgo biloba extract alleviates deltamethrin-induced testicular injury by upregulating SKP2 and inhibiting Beclin1-independent autophagy

BackgroundMale infertility is a worldwide concern that is associated with a decline in sperm quality. Environmental pollutants such as deltamethrin (DM) have harmful effects on male reproductive organs. By maintaining intracellular redox homeostasis, ginkgo biloba extract (GBE) can alleviate male reproductive dysfunction. However, research on the mechanisms by which GBE alleviates reproductive toxicity induced by DM is limited. PurposeIn this study, we investigated whether GBE can alleviate DM-induced testicular and Sertoli cell reproductive toxicity by modulating SKP2 and Beclin1, thus providing a theoretical basis for the development of novel therapeutic approaches. Study DesignWe explored the role of GBE in mitigating DM-induced testicular damage, with a specific focus on the intricate involvement of ubiquitination and autophagy. MethodsAn experimental model was constructed using ICR male mice and the TM4 cell line. Tissue, cellular, and sperm morphological changes were observed through methods such as Hematoxylin and Eosin (H&E) staining, Periodate-Schiff (PAS) staining, ultrastructural observation, immunohistochemistry, and immunofluorescence. Enzyme and hormone levels were measured, and gene and protein levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting techniques. ResultsIn vivo experiments showed that DM exposure led to decreased sex hormone levels, increased seminiferous tubule diameter and impaired spermatogenesis. Meanwhile, DM exposure was found to decrease ubiquitination levels, leading to mitochondrial damage and further escalation of mitochondrial autophagy. Furthermore, in the DM-induced cell model, the upregulation of Beclin1 expression was associated with the inhibition of the ubiquitin‒proteasome system (UPS) and SKP2, thereby exacerbating autophagy. However, GBE has demonstrated notable efficacy in alleviating the reproductive toxicity induced by DM. ConclusionOur findings highlighted that SKP2 is a key regulator of Beclin1-independent autophagy and that GBE exerts therapeutic effects by upregulating SKP2 and inhibiting Beclin1 activation, which ameliorates autophagy and reduces DM-induced testicular damage.

Sulfasalazine improves neuronal function in mice with ischemic stroke by inhibiting the STING/NF-κB pathway.

Inflammation plays an essential role in the pathological processes of ischemic stroke (IS). Sulfasalazine is used in clinical practice for the treatment of inflammatory diseases. This study investigated the effects of sulfasalazine in mice with IS and its underlying mechanisms. We employed an in vivo mice model of middle cerebral artery occlusion (MCAO)/reperfusion, investigating the impact of sulfasalazine on MCAO mice using tetrazolium chloride (TTC) staining, behavioral experiments, and pathological staining. Utilizing of network pharmacology methodologies, we speculated that the protective effect of sulfasalazine against IS may be related to inflammation. The effects of sulfasalazine on inflammation and polarization in oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced BV2 cells were examined through immunofluorescence and PCR techniques. Additionally, the influence of sulfasalazine on the STING/NF-κB pathway was assessed using Western blot and immunofluorescence techniques. Sulfasalazine significantly reduced the infarct area in MCAO mice, restored cerebral blood flow, and improved motor function in the MCAO mice. Pathological staining results indicated that sulfasalazine can mitigate neuronal damage in the cerebral cortex of MCAO mice. Immunofluorescence and PCR testing showed that sulfasalazine promotes M1 to M2 polarization, and reduces inflammation in OGD/R-induced BV2 cells. Furthermore, the Western blot and immunofluorescence results both confirmed that sulfasalazine can inhibit the activation of the STING/NF-κB pathway. This study elucidated the potential therapeutic role of sulfasalazine in IS through its anti-inflammatory effects and modulation of STING/NF-κB pathway, offering novel insights into treatment strategies for IS.

Optimized production of a truncated form of the recombinant neuraminidase of influenza virus in Escherichia coli as host with suitable functional activity

BackgroundTo discover effective drugs for treating Influenza (a disease with high annual mortality), large amounts of recombinant neuraminidase (NA) with suitable catalytic activity are needed. However, the functional activity of the full-length form of this enzyme in the bacterial host (as producing cells with a low cost) in a soluble form is limited. Thus, in the present study, a truncated form of the neuraminidase (derived from California H1N1 influenza strain) was designed, then biosynthesized in Escherichia coli BL21 (DE3), Shuffle T7, and SILEX systems. E. coli BL21 (DE3) was selected as a best host for statistical optimization. Using central composite design methodology, neuraminidase expression level was measured at 20 different runs considering most effective factors including; concentration of isopropyl-β-D-thiogalactopyranoside (IPTG), temperature, and induction time.ResultThe recombinant neuraminidase was purified using Ni-affinity chromatography in soluble form. The neuraminidase expression was confirmed by western blot technique with a molecular mass of 48 kDa. The optimum expression condition was at temperature (30°C), induction time (3 h), and concentration of IPTG (0.6 mM) resulting in maximum neuraminidase expression (7.6 µg/mL) with P < 0.05. The analysis of variance with the significant value of R2 (0.97) indicated that the quadratic model utilized for this prediction was highly significant (p < 0.0001). Applying the optimized condition led to a ~ 2.2-fold increase in NA expression level (from 3.4 to 7.6 µg/ml). The kinetic parameters were also confirmed by fluorescent signals (by 2’-(4-Methylumbelliferyl)-α-D-N acetyl neuraminic acid substrate) with specific activity; ~3.5 IU/mg and Km: 86.49 ± 0.1 µ, close to the Vibrio Cholera neuraminidase with specific activity; 4 IU/mg. The neuraminidase inhibition test confirmed the inhibition of the neuraminidase activity by the drug inhibitor (Oseltamivir) compared to the control sample.ConclusionThe high quality and proper functional activity of the truncated neuraminidase described in this research show that E. coli can be a suitable host for a wide range of applications with less cost and risk compared to eukaryotic expression systems.

Melatonin Attenuates Ferritinophagy/Ferroptosis by Acting on Autophagy in the Liver of an Autistic Mouse Model BTBR T+Itpr3tf/J

Autism spectrum disorders (ASDs) are a pool of neurodevelopment disorders in which social impairment is the main symptom. Presently, there are no definitive medications to cure the symptoms but the therapeutic strategies that are taken ameliorate them. The purpose of this study was to investigate the effects of melatonin (MLT) in treating ASDs using an autistic mouse model BTBR T+Itpr3tf/J (BTBR). We evaluated the hepatic cytoarchitecture and some markers of autophagy, ferritinophagy/ferroptosis, in BTBR mice treated and not-treated with MLT. The hepatic morphology and the autophagy and ferritinophagy/ferroptosis pathways were analyzed by histological, immunohistochemical, and Western blotting techniques. We studied p62 and microtubule-associated protein 1 light chain 3 B (LC3B) for evaluating the autophagy; nuclear receptor co-activator 4 (NCOA4) and long-chain-coenzyme synthase (ACSL4) for monitoring ferritinophagy/ferroptosis. The liver of BTBR mice revealed that the hepatocytes showed many cytoplasmic inclusions recognized as Mallory–Denk bodies (MDBs); the expression and levels of p62 and LC3B were downregulated, whereas ACSL4 and NCOA4 were upregulated, as compared to control animals. MLT administration to BTBR mice ameliorated liver damage and reduced the impairment of autophagy and ferritinophagy/ferroptosis. In conclusion, we observed that MLT alleviates liver damage in BTBR mice by improving the degradation of intracellular MDBs, promoting autophagy, and suppressing ferritinophagy/ferroptosis.

Biosynthesis and Characterization of Silver Nanoparticles Using Cleistanthus collinus; Investigation on Molecular Mechanism of Apoptosis Cell Death Against Lung Cancer Cells

Background: The discovery of novel molecular and cellular signaling pathways for cancer medicines would enhance the efficacy of cancer therapy. In the current study, we made an effort to explore the mitochondrial-mediated apoptosis cell death signaling pathway in A549 lung cancer cells by silver nanoparticles (AgNPs) synthesized from leaf extract of Cleistanthus collinus. Methods: In-depth, A549 lung cancer cells were treated with AgNPs, and further studies such as HOECHST 33342, AO/EB, Rhodamine-123 staining, flowcytometry, RT-PCR, and Western blotting techniques (24, 48, and 72 h) evidenced apoptosis pathway in cancer cells. Results: Indeed, the microscopic studies proved that AgNPs-treated lung cancer cells appeared with cell shrinkage, membrane swelling, and apoptotic body formation whereas the untreated cells (control) failed to show any morphological consequences. The flow cytometry analysis revealed the G2/M phase of cell cycle arrest by cells were accumulated in large numbers. The RT-PCR data confirmed the expression (Bax and p53) and suppression (Bcl 2) apoptosis-responsive genes in cancer cells. Finally, immunoblotting proved the increased expression of cytochrome c, initiator caspase 9, and executioner caspase 3, which are effective proteins of intrinsic apoptosis activation. Conclusion: Ultimately, the overall present investigations confirmed the mitochondrial signaling pathway played a key role in inducing apoptosis in A549 cancer cell death and demonstrated that AgNPs are novel therapeutic agents for cancer nanomedicine

IMProve-DURA: Investigations of Molecular Patient profiles and anatomical cadaveric characteristics of the Dura Mater - A Pilot Study Protocol

BackgroundThe dura mater, a fibrous membrane protecting the brain and spinal cord, is prone to lacerations during spinal surgery, often leading to complications such as cerebrospinal fluid (CSF) leakage. Anatomical differences in the spinal dura mater, particularly in regions under high biomechanical stress, suggest that chronic inflammatory processes may compromise its structural integrity. This pilot study investigates the molecular and anatomical characteristics of the spinal dura mater, focusing on its response to inflammation and the challenges of surgical repair. MethodsThe study involves the collection of 40-60 cadaveric spinal dura mater samples and 100 preoperative blood samples from patients undergoing major spinal surgery. Histological and biomolecular analyses will assess tissue integrity, elastin, collagen content, and the expression of inflammatory biomarkers (heat shock proteins [HSPs] and cytokines). Multiplex assays, mass spectrometry, and Western blot techniques will analyze biomarker profiles in both dura mater and blood samples. Comparative and correlation analyses will be performed to identify common molecular patterns between cadaveric tissue and patient blood samples, with statistical tests applied to detect significant differences. ResultsPreliminary power analysis justifies the sample sizes, aiming to detect moderate effect sizes (Cohen’s d = 0.5) with 80% power. Histological and biomolecular results will focus on the impact of chronic inflammation on spinal dura integrity, specifically its thinning and vulnerability to laceration. Key findings will include biomarker profiles correlating with tissue damage and post-surgical complications like CSF leakage. ConclusionThis pilot study aims to enhance our understanding of the inflammatory processes affecting the dura mater and their role in frequent lacerations during spinal surgery. Findings are expected to guide future clinical practices in dura repair strategies, potentially improving surgical outcomes and reducing complications related to CSF leakage.

Curcumin ameliorates aluminum oxide nanoparticle-induced memory deficit by regulating the hippocampal p38 signaling pathway in mice

ABSTRACT Objectives: Exposure to aluminum (Al) has been shown to be strongly associated with the pathogenesis of Alzheimer’s disease (AD). Recent evidence indicates that the toxicity of Al nanoparticle (Al-NP) is far greater than Al itself due to its particle size. Epidemiological studies suggest that curcumin lower the prevalence of AD. MAPKs (ERK, p38 and JNK) were suggested to be involved in AD pathology and memory impairment. The present study aimed to evaluate if curcumin has the ability to protect against behavioral deficits induced by subcutaneously administered Al-NP in mice. Furthermore, the levels of phosphorylated and total hippocampal MAPKs were assessed using western blottechnique. Methods: Al-NP (10 mg/kg/s.c.) was administered to adult male NMRI mice for 10 days with or without curcumin in doses of 2.5 or 25 mg/kg/oral gavage). Memory was assessed using passive avoidance apparatus and anxiety-like behavior was evaluated using elevated plus maze. Following the behavioral tasks, western blot analysis was performed on the hippocampal tissues to detect the levels of phosphorylated and total MAPKs. Results: The results revealed that Al-NP deteriorated memory with no significant effect on anxiety-like behaviors. Additionally, it activated hippocampal p38 signaling pathway with no effect on ERK and JNK. Curcumin treatment at the dose of 25 mg/kg restored memory and p38 activation. Discussion: This study suggests that subcutaneous Al-NP administration impairs memory and hippocampal p38 signaling with no effect on ERK and JNK. Co-administration of curcumin restored Al-NP induced memory impairment and hippocampal p38 phosphorylation.

Pedunculoside alleviates cognitive deficits and neuronal cell apoptosis by activating the AMPK signaling cascade

BackgroundMitochondrial dysfunction emerges as an early pathological hallmark of Alzheimer's disease (AD). The reduction in mitochondrial membrane potential and the elevation of reactive oxygen species (ROS) production are pivotal in the initiation of neuronal cell apoptosis. Pedunculoside(Ped), a novel triterpene saponin derived from the dried barks of Ilex rotunda Thunb, exhibits a potent anti-inflammatory effect. In the course of drug screening, we discovered that Ped offers significant protection against apoptosis induced by Aβ1-42. Nevertheless, the role and mechanism of Ped in AD are yet to be elucidated.MethodsOxidative stress was evaluated by measuring mitochondrial membrane potential and intracellular ROS production. The expression of proteins associated with apoptosis was determined using western blot analysis and flow cytometry. In vivo, the pathological characteristics of AD were investigated through Western blot and tissue immunofluorescence techniques. Cognitive function was assessed using the Morris Water Maze and Novel Object Recognition tests.ResultsWe demonstrated that Ped decreased apoptosis in PC12 cells, reduced the generation of intracellular ROS, and restored mitochondrial membrane potential. Mechanistically, we found that the protective effect of Ped against Aβ-induced neurotoxicity was associated with activation of the AMPK/GSK-3β/Nrf2 signaling pathway. In vivo, Ped alleviated memory deficits and inhibited neuronal apoptosis, inflammation, and oxidative stress in the hippocampus of 3 × Tg AD mice, along with the activation of the AMPK signaling pathway.ConclusionThe findings indicate that Ped exerts its neuroprotective effects against oxidative stress and apoptosis through the AMPK signaling cascade. The results demonstrate that Ped is a potential candidate for the treatment of AD.Graphical

Cardioprotective effects of l-glutamine in an ischemic rat heart model

Introductionl-glutamine has been shown to have cardioprotective effects in models of ischemia-reperfusion injury. Its potential cardioprotective effects when given before and during early reperfusion, however, have not been studied. MethodsThis study hypothesized that l-glutamine administered before and after myocardial ischemia provides better cardioprotection than when administered after ischemia only. Eighteen male rat hearts were exposed to 15 min of ischemia using the Langendorff system and randomly assigned to three groups of six each. Group one received Krebs-Henseleit (KH) buffer over 20 min before ischemia and during 20 min of reperfusion (Control), group two received KH buffer containing 2.5 mmol･L−1 glutamine during reperfusion (Post-Gln) and group three was given KH buffer containing glutamine before and after the ischemic insult (Pre + Post-Gln). Indicators of hemodynamics such as maximum left ventricular derivative of pressure development (LV dP/dt max) were recorded at 5, 10, 15 and 20 min post-reperfusion. Myocardial levels of O-linked β-N-acetylglucosamine (O-GlcNAc) and heat shock protein 70 (HSP70) were measured by Western blotting technique after 20 min of reperfusion. ResultsThe LV dp/dt max in the Pre + Post-Gln group was significantly elevated as compared to the Post-Gln group after 10 min of reperfusion and was significantly higher than in the control group at all-time points. Myocardial expression of O-GlcNAc was increased in the Pre + Post-Gln group (P < 0.01 vs. control) without showing any differences in HSP70. ConclusionIn this model of stunned myocardium, pre- and post-ischemic administration of l-glutamine improved cardiac function indicating cardioprotective effects, possibly mediated by O-GlcNAc.

Unraveling the impact of semaglutide in a diabetic rat model of testicular dysfunction: Insights into spermatogenesis pathways and miRNA-148a-5p

BackgroundDiabetes has been a long-known risk factor for male sexual dysfunction, which may be caused by persistent hyperglycemia, oxidative stress, and spermatogenesis inhibition. This study explored the potential of Semaglutide (Sem) to alleviate testicular dysfunction and spermatogenesis impairment in diabetic rats to understand the molecular mechanism of this protective effect. MethodologyA controlled experiment was conducted where 28 adult male rats were divided into four groups: control, Semaglutide, diabetic, and diabetes + Sem. Diabetes was induced using a single STZ dose (50 mg/kg, i.p.). At the same time, Sem was administered as a daily subcutaneous dose (25 nmol/kg) for four weeks after the confirmed diagnosis of diabetes.Several biochemical and histochemical analyses were performed in addition to mating behavior assessments. The estimation of spermatogenesis-related genes and proteins was conducted using PCR and western blotting techniques. Resultsrevealed promising outcomes, wherein Sem treatment effectively mitigated diabetes-induced sexual and testicular dysfunction. Specifically, it regulated the disrupted redox balance, restored spermatogenesis gene and protein levels, modulated hormonal profiles, and mitigated testicular inflammation. ConclusionSem protects against diabetes-induced testicular and sexual impairments by influencing several pathways and restoring spermatogenesis-related genes and proteins. Future studies may involve a potential investigation of Sem translational applications in clinical settings for treating male infertility associated with diabetes.

P75NTR Modulation Reduces Oxidative Stress and the Expression of Pro-Inflammatory Mediators in a Cell Model of Rett Syndrome.

Background: Rett syndrome (RTT) is an early-onset neurological disorder primarily affecting females, leading to severe cognitive and physical disabilities. Recent studies indicate that an imbalance of redox homeostasis and exacerbated inflammatory responses are key players in the clinical manifestations of the disease. Emerging evidence highlights that the p75 neurotrophin receptor (p75NTR) is implicated in the regulation of oxidative stress (OS) and inflammation. Thus, this study is aimed at investigating the effects of p75NTR modulation by LM11A-31 on fibroblasts derived from RTT donors. Methods: RTT cells were treated with 0.1 µM of LM11A-31 for 24 h, and results were obtained using qPCR, immunofluorescence, ELISA, and Western blot techniques. Results: Our findings demonstrate that LM11A-31 reduces OS markers in RTT fibroblasts. Specifically, p75NTR modulation by LM11A-31 restores protein glutathionylation and reduces the expression of the pro-oxidant enzyme NOX4. Additionally, LM11A-31 significantly decreases the expression of the pro-inflammatory mediators interleukin-6 and interleukin-8. Additionally, LM11A-31 normalizes the expression levels of transcription factors involved in the regulation of the antioxidant response and inflammation. Conclusions: Collectively, these data suggest that p75NTR modulation may represent an effective therapeutic target to improve redox balance and reduce inflammation in RTT.

The role of PDCD6 in stemness maintenance of Glioblastoma

BackgroundGlioblastoma (GBM) poses formidable challenges due to its high malignancy and therapeutic resistance and still exhibits dismal 5-year survival rates, high recurrence propensity, and limited treatment modalities. There is an acute need for innovative treatments for recurrent glioblastoma due to the lack of established protocols. This necessity is driving research into the cellular underpinnings that initiate and drive the disease forward, aiming to discover groundbreaking targets for therapy that could enhance the efficacy of medical interventions. MethodsPatient-derived glioblastoma stem cells (GSCs) were harvested and isolated. Subsequently, PDCD6 expression was quantified through both western blotting (WB) and real-time PCR (RT-PCR) techniques. The stem-like properties of the GSCs were evaluated using sphere-forming assays. All gathered data, inclusive of TCGA datasets, were analyzed using SPSS (IBM) version 23.0. ResultsElevated PDCD6 expression characterized classical GBM tumor tissues. PDCD6 overexpression significantly correlated with diminished overall survival in GBM patients, emerging as an independent prognostic indicator. Notably, primary GBM cells exhibited heightened PDCD6 levels in GSCs compared to NSTCs. Moreover, alterations in stemness markers paralleled PDCD6 modulation, where PDCD6 knockdown attenuated tumor size in GSCs. ConclusionOur findings illuminate PDCD6's role in fostering stemness within classical GBM, hinting at its potential as a therapeutic target.

Treatment of Melanoma Cells with Chloroquine and Everolimus Activates the Apoptosis Process and Alters Lipid Redistribution.

The balance between apoptosis and autophagy plays a key role in cancer biology and treatment strategies. The aim of this study was to assess the effect of the mTOR kinase inhibitor everolimus and chloroquine on the regulation of proliferation, caspase-3 activation, and apoptosis in melanoma cells. We studied the activity of caspase-3 and the levels of caspase-3 and -9 using the Western blot technique. Cellular apoptosis was examined using a DNA fragmentation assay, and changes in the cell nucleus and cytoskeleton were examined using fluorescence microscopy DAPI, OA/IP. We also studied the rearrangement of lipid structures using fluorescent dyes: Nile Red and Nile Blue. A low nanomolar concentration of the mTOR kinase inhibitor everolimus in combination with chloroquine activated the apoptosis process and decreased cell proliferation. These changes were accompanied by an obvious change in cell morphology and rearrangement of lipid structures. Alterations in lipid redistribution accompanying the process of apoptosis and autophagy are among the first to occur in the cell and can be easily monitored in in vitro studies. The combination of mTOR inhibitors and chloroquine represents a promising area of research in cancer therapy. It has the potential to enhance treatment efficacy through complementary mechanisms.

The role of CISD1 reduction in macrophages in promoting COPD development through M1 polarization and mitochondrial dysfunction

BackgroundThe mitochondrial dysfunction and oxidative stress imbalance caused by macrophage polarization play a role in the progression of COPD, with CDGSH iron–sulfur domain-containing protein 1 (CISD1) playing a key role. This study revealed the role and mechanism of CISD1 in smoke-induced macrophages.MethodsUsing a pure cigarette smoke exposure-induced COPD mouse model, stimulation of Raw264.7 macrophages with cigarette smoke extract mimics the COPD environment. Knocking down CISD1 expression in macrophages and combining it with high-throughput sequencing to obtain subsequent differentially expressed genes and pathways. Macrophage polarization tendency under different treatments was determined using flow cytometry. Meanwhile, Mitosox, JC-1, DCFH-DA fluorescence intensity was measured to detect mitochondrial function and cellular oxidative stress levels. Western Blot technique was employed to validate autophagy (mitochondrial autophagy) pathway-related proteins. In addition, Elisa technique was used to measure inflammatory factors (IL-6, TNF-a) in the cell supernatant after co-culturing macrophages (Raw264.7) with epithelial cells (MLE12).ResultsCISD1 is underexpressed in peripheral blood monocytes of COPD patients. Under in vitro conditions, we verified that cigarette smoke (smoke extract) indeed inhibits CISD1 expression in macrophages. Subsequently, we found that macrophages with knocked-down CISD1 tend to polarize towards M1 phenotype, and exhibit signs of mitochondrial dysfunction and oxidative stress imbalance. In addition, we observed significant activation of the autophagy pathway in CISD1-inhibited macrophages, with upregulation of LC3A/B and downregulation of p62 protein, as well as increased expression of mitochondrial autophagy-related proteins (PINK1, PARKN). Furthermore, co-culturing CISD1-knockdown macrophages (Raw264.7) with epithelial cells (MLE12) resulted in upregulation of inflammatory factors in the supernatant.ConclusionsSmoke-induced reduction of CISD1 in macrophages promotes M1 polarization and mitochondrial dysfunction by activating the autophagy pathway, thereby promoting the occurrence and development of COPD.

Protective effect of metformin on the NG-nitro-l-arginine methyl ester (l-NAME)-induced rat models of preeclampsia

BackgroundPreeclampsia (PE) is a complex multi-organ disorder characterized by systemic inflammation, endothelial dysfunction, and vasoconstriction, which manifests as hypertension, with or without proteinuria. Effective preventive strategies for PE are currently lacking in clinical practice, leading to significant morbidity and mortality among mothers and newborns. ObjectiveThis study aims to investigate the impact of metformin (MET) on the l-NAME-induced PE rat model, focusing on the mechanisms through which MET may exert its effects. MethodsThirty pregnant Sprague-Dawley (SD) rats were randomly assigned to three groups: Control, PE, and PE + MET, on gestational day 0 (GD0). Regularly measure blood pressure and 24-h proteinuria, and collect tissue samples on GD20. Enzyme-linked immunosorbent assay (ELISA) was used to analyze inflammatory factors, endothelial function biomarkers, angiogenic factors, and apoptosis-related factors in the rat plasma. Western blot, RT-qPCR, and immunohistochemistry techniques were employed to determine the expression levels of key apoptotic proteins in placental tissue. ResultsThe study findings demonstrate that MET administration improves blood pressure and 24-h proteinuria, alleviates fetal growth restriction, ameliorate inflammation cytokines, and restores the balance of angiogenic factors and endothelial function. Moreover, MET inhibits the expression levels of critical apoptotic proteins in the plasma and placental tissue of PE-like rats. ConclusionThe results suggest that MET shows promise in alleviating symptoms associated with l-NAME-induced PE in rats, preserving endothelial function, enhancing angiogenesis, reducing inflammation, inhibiting placental apoptosis, improving placental function, and promoting fetal growth. These findings highlight MET as a potential therapeutic agent for the prevention and treatment of preeclampsia.

A comprehensive genome-based analysis identifies the anti-cancerous role of the anoikis-related gene ADH1A in modulating the pathogenesis of breast cancer.

Breast cancer (BC), a widespread and lethal neoplasm, is irrespective of the subtype of BC. Metastasis remains a crucial determinant for unfavorable outcome. The identification of novel diagnostic markers is instrumental in optimizing the treatment regime for BC. The direct correlation between anoikis and the progression/outcome of BC is well established. Nevertheless, the contribution of anoikis-related genes (ARGs) in BC remains obscure at present. We implemented the METABRIC dataset to scrutinize and assess differentially expressed ARGs in BC versus healthy breast tissues. An unsupervised consensus clustering approach for ARGs was employed to classify patients into diverse subtypes. ESTIMATE algorithms were utilized to assess immune infiltrative patterns. Prognostic gene expression patterns were derived from LASSO regression and univariate COX regression analysis. Subsequently, these signatures underwent examination via use of the Kaplan-Meier survival curve. 6 pairs of fresh tissue specimens (tumor and adjacent non-tumor) were employed to assess the expression of 7 ARGs genes via qPCR. Notably, DCN and FOS were not expressed in BC tissue, which had been excluded in our subsequent experiments. Also, among remaining 5 ARGs, solely the expression of ADH1A demonstrated a statistically remarkable disparity between freshly collected cancer tissues and the adjacent ones. ADH1A-overexpressed and ADH1A-sh vectors were transfected into MCF-7 and MCF-7-AR cell lines, respectively. The expression status of FABP4, CALML5, ADH1A, C1orf106, CIDEC, β-catenin, N-cadherin, and Vimentin in the clinical samples were scrutinized using RT-qPCR and western blotting techniques. Migration and invasion through transwell chambers were employed to assess the migratory and invasive potential of the cells. Detailed evaluation of cell proliferation was conducted utilizing a Cell Counting Kit-8 (CCK-8) assay. The apoptotic index of the cells was determined by flow cytometry analysis. An innovative anoikis-associated signature consisting of seven genes, namely ADH1A, DCN, CIEDC, FABP4, FOS, CALML5, and C1orf106, was devised to stratify BC patients into high- and low-risk cohorts. This unique risk assessment model, formulated via the distinctive signature approach, has been validated as an independent prognostic indicator. Additional analysis demonstrated that distinct risk subtypes manifested variances in the tumor microenvironment and drug sensitivities. Suppression of ADH1A enhanced the migratory and invasive capacities and reduced these tumorigenesis-related protein levels, underscoring the prognostic role of ADH1A in the progression of BC. Through our meticulous study, we have elucidated the possible molecular markers and clinical implications of ARGs in BC. Our model, which incorporate seven ARGs, has proven to accurately forecast the survival outcomes of BC patients. Moreover, the thorough molecular study of ADH1A has augmented our comprehension of ARGs in BC and opened a novel avenue for guiding personalized and precise therapeutic interventions for BC patients.