Relative Protein Levels Research Articles

Lentiviral Injection of Inter-α-Trypsin Inhibitor Heavy Chain 4 Promotes Female Spinal Cord Injury Mice Recuperation by Diminishing Peripheral and Central Inflammation.

Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) acts as a mediator of inflammation and extracellular matrix stabilization. The current study intended to delve into the impact of ITIH4 on locomotor performance, nerve injury, neuroinflammation, systemic inflammation, and the downstream pathway in spinal cord injury (SCI) mice. Overexpression lentivirus of ITIH4 (LV-ITIH4) and negative control lentivirus (LV-NC) were intravenously injected into adult C57BL/6 mice on 7days before SCI surgery. All mice were euthanized on day 28 after SCI surgery, and their blood samples and spinal cord tissues were collected. Decreased relative gene expression and protein levels of ITIH4 were observed in SCI mice. LV-ITIH4 improved the locomotor performance compared to LV-NC in SCI mice. In spinal cord of SCI mice, LV-ITIH4 reduced apoptosis and increased survival of neurons compared to LV-NC. By comparison with LV-NC, LV-ITIH4 also reduced relative gene expressions of interleukin (IL)-6 and tumor necrosis factor-α in spinal cord of SCI mice. Moreover, LV-ITIH4 reduced microglia M1 polarization compared with LV-NC in spinal cord of SCI mice. In the serum, LV-ITIH4 decreased the protein levels of IL-6 and IL-1β compared to LV-NC in SCI mice. LV-ITIH4 also inhibited the nuclear factor kappa-B (NF-κB) pathway compared to LV-NC in spinal cord of SCI mice. ITIH4 enhances locomotor performance in SCI mice, and it inhibits nerve injury, neuroinflammation, systemic inflammation, and the NF-κB pathway in SCI mice.

WTAP mediates IL-1β-induced chondrocyte injury by enhancing CA12 mRNA stability depending on m6A modification

BackgroundOsteoarthritis (OA) poses a significant risk to the mobility of patients. Carbonic anhydrase 12 (CA12) can boost apoptosis and inflammation in several cancers, but its role in OA is unknown.MethodsDifferentially expressed genes in OA were analyzed using the GEO database (GSE169077). RT-qPCR and western blot estimated relative mRNA and protein levels of CA12. Cell viability and apoptosis were estimated by cell counting and flow cytometry assays. Oxidative stress (OxS) was determined by detecting with ROS and MDA levels, as well as CAT and SOD activities. Cytokine levels of IL-6 and TNF-α were detected by ELISA. Parameters associated with apoptosis and extracellular matrix (ECM) were detected by western blot. The m6A modification profile was determined by methylated RNA immunoprecipitation assays.ResultsRelative CA12 and wilms’ tumor 1-associating protein (WTAP) mRNA and protein levels were overexpressed in OA articular cartilages and IL-1β-challenged chondrocytes CHON-001. CA12 silencing impaired IL-1β-induced cell apoptosis, inflammation, OxS, and ECM degradation in chondrocytes. Yet, CA12 overexpression exerted an opposing function. WTAP reinforced the stability of CA12 mRNA depending on the m6A modification. Furthermore, WTAP knockdown weakened cell apoptosis, inflammation, OxS, and ECM degradation in chondrocytes induced by IL-1β, but these changes were impaired after CA12 overexpression. In addition, WTAP knockdown mitigates cartilage degeneration in DMM-induced mouse models.ConclusionIL-1β-induced WTAP enhances CA12 mRNA stability depending on m6A modification, thus promoting chondrocyte apoptosis, inflammatory response, OxS, and ECM degradation, providing evidence to support the possibility of WTAP and CA12 as potential targets for OA treatment.

Prognostic value of nomogram model based on clinical risk factors and CT radiohistological features in hypertensive intracerebral hemorrhage.

To construct a nomogram model based on clinical risk factors and CT radiohistological features to predict the prognosis of hypertensive intracerebral hemorrhage (HICH). A total of 148 patients with HICH from April 2022 to July 2024 were retrospectively selected as the research subjects. According to the modified Rankin scale at the time of discharge, they were divided into good group (Rankin scale score 0-2) and bad group (Rankin scale score 3-6). To compare the clinical data and the changes of CT radiographic characteristics in patients with different prognosis. Relevant factors affecting the prognosis were analyzed, and nomogram model was established based on the influencing factors. The fitting degree, prediction efficiency and clinical net benefit of the nomogram model were evaluated by calibration curve, ROC curve and clinical decision curve (DCA). Compared with the good group, the hematoma volume in the poor group was significantly increased, the serum thromboxane 2(TXB2) and lysophosphatidic acid receptor 1(LPAR1) levels were significantly increased, and the energy balance related protein (Adropin) level was significantly decreased. The proportions of irregular shape, promiscuous sign, midline displacement, island sign and uneven density were all significantly increased (p < 0.05). In Logistic multivariate analysis, hematoma volume, Adropin, TXB2, LPAR1 and CT radiological features were all independent factors influencing the poor prognosis of HICH (p < 0.05). A nomogram prediction model was established based on the influencing factors. The calibration curve showed that the C-index was 0.820 (95% CI: 0.799-0.861), the goodness of fit test χ2 = 5.479, and p = 0.391 > 0.05, indicating a high degree of fitting. The ROC curve showed that the AUC was 0.896 (95% CI: 0.817-0.923), indicating that this model had high prediction ability. The DCA curve shows that the net benefit of the nomogram model is higher when the threshold probability is 0.1-0.9. The nomogram prediction model established based on hematoma volume, Adropin, TXB2, LPAR1 and other clinical risk factors as well as CT radiographic characteristics has high accuracy and prediction value in the diagnosis of poor prognosis in patients with HICH.

Circ-TTC17 Promotes Esophagus Squamous Cell Carcinoma Cell Growth, Metastasis, and Inhibits Autophagy-Mediated Radiosensitivity Through miR-145-5p/SIRT1 Axis.

Circular RNA (circRNA) plays a significant role in esophagus squamous cell carcinoma (ESCC) progression. Nevertheless, circ-TTC17 roles in ESCC have not fully understood. The levels of circ-TTC17, miR-145-5p and sirtuin 1 (SIRT1) were determined using qRT-PCR. ESCC cell functions were examined by CCK8 assay, flow cytometry, transwell assay and colony formation assay. The relative protein levels of autophagy marker and SIRT1 were determined by western blot (WB). The interactions among circ-TTC17, miR-145-5p, and SIRT1 were verified by dual-luciferase reporter assay and RIP assay. circ-TTC17 was overexpressed and miR-145-5p was underexpressed in ESCC. circ-TTC17 knockdown restrained ESCC cell proliferation and metastasis, while enhance apoptosis and autophagy-mediated radiosensitivity. Circ-TTC17 could sponge miR-145-5p, and its inhibitor reversed the inhibitory effect of circ-TTC17 knockdown on ESCC cell progression. Additionally, SIRT1 was targeted by miR-145-5p, and SIRT1 overexpression abolished miR-145-5p-mediated the suppressive effect on ESCC cell progression. Also, circ-TTC17 interference reduced ESCC tumor growth via miR-145-5p/SIRT1 axis. circ-TTC17 promoted ESCC cell growth, metastasis and inhibited autophagy-mediated radiosensitivity by miR-145-5p/SIRT1 axis.

Salidroside facilitates neuroprotective effects in ischemic stroke by promoting axonal sprouting through promoting autophagy

BackgroundIschemic stroke is a common cerebrovascular disease characterized by high incidence, disability, mortality, and recurrence. The limitations of current pharmacological treatments, which have primarily single neuroprotective action and a narrow therapeutic time window, lead to unsatisfactory therapeutic efficacy. Activation of autophagy can facilitate neural regeneration. ObjectiveTo clarify whether salidroside can promote axonal sprouting through autophagy resulting in protecting neurons. MethodsIn vivo, a Middle Cerebral Artery Occlusion/reperfusion (MCAO/IR) model was used, and in vitro, an Oxygen-Glucose Deprivation/Reoxygenation (OGD/R)-induced primary neuronal cell model was employed to evaluate the neuroprotective effects of salidroside. BDA neurotracer, immunofluorescence, and Western blot (WB) were utilized to determine its impact on axonal sprouting and the levels of related proteins (MAP2, GAP43, and PSD-95). Proteomics, transmission electron microscopy (TEM), and WB were applied to identify the effects on autophagy-related proteins (beclin1, LC3, p62, and LAMP2), autophagosomes and lysosomes. The mechanism of salidroside in promoting axonal sprouting through inducing autophagy was further confirmed by blocking with the autophagy inhibitor 3-MA. ResultsSalidroside reduced neurologic deficits and infarct volume induced by MCAO/IR in vivo and protected OGD/R induced primary neuronal cells in vitro. Both in vivo and in vitro, it increased the number and length of axons and upregulated the expression of key axonal proteins (MAP2, GAP43, and PSD-95) and mediated autophagy-related proteins. Mechanistic studies showed that the promoting effects of salidroside on autophagy and axonal sprouting disappeared after the blockade by 3-MA. ConclusionThis study reports for the first time that the neuroprotective effect of salidroside in ischemic stroke can be executed through mediating autophagy-related protein (beclin1, LC3, p62, and LAMP2), resulting in induced axonal sprouting or mature protein (MAP2, GAP43, and PSD-95).

Kaixinsan regulates neuronal mitochondrial homeostasis to improve the cognitive function of Alzheimer's disease by activating CaMKKβ-AMPK-PGC-1α signaling axis

BackgroundAlzheimer's disease (AD) is a neurodegenerative disease primarily characterized by cognitive impairments. With the intensification of population aging, AD has become a major health issue faced by the elderly. Kaixinsan, a classical traditional Chinese prescription consists of Panax ginseng C. A. Mey., Polygala tenuifolia Willd., Poria cocos (Schw.) Wolf and Acori Tatarinowii rhizoma, is commonly used in clinical for treating memory decline. However, its mechanism remains unclear, which hinders its popularization and application. MethodMorris water maze (MWM) was performed to evaluate the effect of Kaixinsan on improving learning and memory ability in SAMP8 (senescence-accelerated mouse prone 8, an AD model mice) mice. Nissl staining, TdT-mediated dUTP Nick End Labeling (TUNEL) and western blotting (Bax and Bcl-2) were used to confirm the effect of Kaixinsan on the neuronal structure and apoptosis of SAMP8 mice. ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to identify the distribution components of the brain tissue after receiving Kaixinsan extraction. Based on the identified brain distribution components, the mechanism of Kaixinsan improving the cognitive function was predicted by network pharmacology. Then, using HSP60 as mitochondrial marker and RBFOX3 as neuron marker, immunofluorescence co-localization was used to confirm the effect of Kaixinsan on neuronal mitochondria quantity in SAMP8 mice. Using western blotting to detect the expression of predicted proteins (AMPK, CaMKKβ, PGC-1α and HSP90) affecting mitochondrial homeostasis. To further confirm the mechanism of Kaixinsa. The authors replicated SH-SY5Y cell injury model induced by Amyloid β - protein fragment 25-35 (Aβ25-35) and compared the effects of serum containing Kaixinsan on apoptosis of injured SH-SY5Y cells (detected by flow cytometer) and the expression level of apoptosis-associated protein (Bax and Bcl-2) and mitochondrial homeostasis related proteins (AMPK, CaMKKβ, PGC-1α and HSP90), with or without the addition of CaMKKβ inhibitor (STO-609). ResultsThe results indicate that Kaixinsan can improve the cognitive function of SAMP8 mice, alleviate the hippocampal tissue lesions and inhibit neuron apoptosis. Seventeen brain distribution components of Kaixinsan were identified. Based on the brain distribution components of Kaixinsan, the results of network pharmacology suggest that Kaixinsan may regulate mitochondrial homeostasis through the CaMKKβ-AMPK-PGC-1α signaling axis. In vivo experiments, The authors observed that Kaixinsan could reverse neuronal mitochondrial loss in SAMP8 mice by upregulating CaMKKβ, AMPK, HSP90 and PGC-1α to promote mitochondrial biogenesis and increase the number of neuronal mitochondria. Additionally, the in vitro experiments demonstrated that Kaixinsan can inhibit apoptosis of Aβ25-35 injured SH-SY5Y cells and upregulate mitochondrial homeostasis-related proteins CaMKKβ, AMPK and PGC-1α. However, in addition to CaMKKβ inhibitors, the neuroprotective effect disappeared. ConclusionThe results indicate that Kaixinsan can improve the cognitive function of SAMP8 mice by regulating CaMKKβ-AMPK-PGC-1α signaling axis to maintain mitochondrial homeostasis and inhibit neuronal apoptosis.

FTO Facilitates Cervical Cancer Malignancy Through Inducing m6A-Demethylation of PIK3R3 mRNA.

The incidence rate and mortality of cervical cancer rank the fourth in the global female cancer. N6-methyladenosine (m6A) always plays an important role in tumor progression, and fat mass and obesity-associated gene (FTO) works as the m6A demethylase. Our study aimed to narrate the biological function and potential mechanisms for FTO in cervical cancer malignancy. We analyzed potential clinical value of FTO in cervical cancer patients. The relative protein levels of FTO in cervical cancerous tissue and paracancerous tissue were verified by IHC. After changing the FTO expression level by lentivirus transfection, the proliferation and metastasis ability of cervical cancer cells were detected both invitro and invivo. Further, Merip-seq and Merip-qPCR are used to profile m6A transcriptome-wide. Finally, western blot were performed to identify the regulatory mechanism. Based on TCGA-CESC cohort and GEO dataset, FTO expression levels in HPV-positive cancer patients were significantly higher than those in HPV-negative cancer patients and could predict advanced FIGO stage. The protein level of FTO in cervical cancerous tissue was higher than that in paracancerous tissue. Functional assays indicated that FTO promoted the proliferation, migration and invasion of cervical cancer cells both invitro and invivo. The Merip-seq and Merip-qPCR evoked that relative PIK3R3 m6A level was significantly increased after FTO knockdown, which effected the activation of FoxO pathway. After knocking down FTO, upregulation of PIK3R3 can restore the malignancy of cervical cancer. All in all, these data suggest a vital role for FTO in occurrence and development of cervical cancer.

Xiao-xu-ming Decoction Improved Synaptic Damage after Acute Cerebral Ischemia and Reperfusion via JAK2/STAT3 Pathway in Rats.

Ischemic stroke is an important cause of death and disability worldwide. Xiao-xu-ming Decoction (XXMD) is a classic prescription for the treatment of stroke patients, which has been widely used in China and has significant therapeutic effect, but the therapeutic target and mechanism are still unclear. The current study aimed to investigate temporal alternation of synaptic damage and the protective effects of XXMD on synaptic damage following cerebral ischemia and reperfusion in vivo. Adult male Sprague-Dawley (SD) rats subjected to 90 mins of middle cerebral artery occlusion (MCAO) and subsequent reperfusion at various time points. Neurobehavioral function was assessed using modified neurological severity scores (mNSS), while pro-inflammatory cytokine levels were measured using ELISA kits. Histological assessment involved silver staining and Luxol Fast Blue (LFB) staining, and the ultrastructural alterations in neurons and synapses were examined using a transmission electron microscope (TEM). Golgi-cox staining was used to evaluate the density of dendritic spines. Levels of synapse-related proteins were quantified via immunofluorescence staining and Western blotting. Additionally, JAK2/STAT3 pathway related protein levels were assessed using Western blotting. In the second part, the rats were randomly divided into sham operation group, 24 hours of reperfusion group, and XXMD group. The ultrastructural alterations and dendrite spine density of synapses were observed by TEM and Golgi-Cox staining respectively, and the expression levels of SYN, PSD95, GAP43, p-JAK2 and p-STAT3 were evaluated by WB. Findings included deteriorated neurobehavioral function, increased release of IL-6, IL-1β, and TNFα, and time-dependent neuronal and synaptic damages during the initial phase of ischemia and reperfusion. At the ultrastructural level, neurons and synapses exhibited structural failure in the peri-infarct cortex. In addition, golgi-cox staining showed dendritic density in ischemic cortex significantly reduced after cerebral ischemia and reperfusion. Moreover, significant reductions in SYN, PSD95, and GAP43 expression levels, along with increases in p-JAK2 and p-STAT3 expression levels, were observed after cerebral ischemia and reperfusion. Meantime, XXMD significantly reduced synaptic impairment and down-regulated SYN, GAP43, PSD95 expression and phosphorylation expression of JAK2 and STAT3 following MCAO and 24 hours of reperfusion. Collectively, these results indicates XXMD may play a neuroprotective role in reducing synaptic damage via JAK2/STAT3 signaling pathway.

BMSCs Downregulate CXCL12 by Secreting Exosomal miR-20a-5p to Promote Macrophage M2 Polarization and Alleviate the Development of Sepsis

ABSTRACT Objective Sepsis is a syndrome of the systemic inflammatory response caused by infection that can endanger a patient’s life. The aim of this study was to explore the molecular mechanism by which bone marrow mesenchymal stem cells-derived exosomes (BMSCs-exo) carrying miR-20a-5p regulate the progression of sepsis. Methods Clinical samples from sepsis patients were collected. Mouse and cell models of sepsis were induced by lipopolysaccharide (LPS). The levels of related genes and proteins were determined by RT‒qPCR, Western blotting and ELISA. CCK-8 and flow cytometry assays were used to assess cell viability, apoptosis, and markers of macrophage polarization. Results In septic patients, miR-20a-5p levels were significantly lower and CXCL12 expression was significantly increased. After LPS induction, M2 polarization of macrophages was significantly reduced, the level of inflammatory factors was increased, and apoptosis was increased. The addition of BMSCs-exo increased the miR-20a-5p level and decreased the expression of CXCL12 in macrophages, thereby promoting macrophage M2 polarization and reducing the levels of inflammatory factors. Conclusion This study demonstrated for the first time that BMSCs-exo promoted the polarization of M2 macrophages through the miR-20a-5p/CXCL12 axis, thus alleviating the development of sepsis. These findings provide a new theoretical basis for the targeted treatment of sepsis with exosomes or miR-20a-5p.

TNF-α Regulated Bidirectional Interaction Between Bone Marrow Mesenchymal Stem Cells and Articular Chondrocytes.

Articular chondrocytes (ACs) secrete a variety of extracellular matrix components to maintain the functions of articular cartilage. Degeneration of ACs leads to the degeneration of articular cartilage and consequently to osteoarthritis. The secretion of bone marrow mesenchymal stem cells (BMSCs) is capable of protecting ACs from degeneration, and thus BMSCs are widely applied to treat osteoarthritis. This study aims to explore whether BMSCs and ACs will affect the functions of each other through their secretions in the context of osteoarthritis. BMSCs and ACs isolated from rabbits were identified using flow cytometry and immunocytochemistry. Conditioned medium of BMSCs and ACs treated with 0, 5, 10, 20, and 40 ng/ml of tumor necrosis factor-alpha (TNF-α) were collected and used to treat ACs and BMSCs, respectively. The viabilities of ACs and BMSCs treated with condition medium were assessed using a Cell Count Kit-8 (CCK-8) kit. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA) methods were employed to evaluate the relative expression levels of genes and proteins, as well as the cytokine concentrations in the supernatant. Immunofluorescence and flow cytometry results indicated that the purity of isolated cells exceeded 95%. CCK-8 analysis showed that 6 hours of treatment with a conditioned medium did not affect the viability of BMSCs and ACs. However, treatment for 12 hours or longer significantly increased the viability of BMSCs (p < 0.05) and significantly decreased the viability of ACs (p < 0.01). RT-qPCR results demonstrated that the relative expression levels of Runx2 (1.15-3.91), Alp (1.06-2.84), TNF (BMSCs: 0.94-2.54; ACs: 1.03-2.64), IL6 (BMSCs: 0.98-2.78; ACs: 0.96-3.71), IL17A (BMSCs: 1.08-5.91; ACs: 0.90-4.20), and IL10 (BMSCs: 0.93-2.82; ACs: 0.89-2.25) genes in conditioned medium-treated BMSCs and ACs were dose-dependently elevated (p < 0.001) by TNF-α treatment. Immunoblotting analysis revealed that the expression levels of RUNX2 (0.53-0.86) and ALP (0.49-0.85) proteins were also dose-dependently elevated (p < 0.001) by TNF-α treatment. ELISA results showed similar TNF-α dose-dependent increases (p < 0.001) in the supernatant concentrations of pro-inflammatory cytokines TNF-α (BMSCs: 36.90 ± 0.75 to 199.38 pg/ml; ACs: 29.76 to 293.99 pg/ml), interleukin (IL)-6 (BMSCs: 4.96-48.24 pg/ml; ACs: 6.12-38.15 pg/ml), IL-17 (BMSCs: 3.06-28.99 pg/ml; ACs: 3.08-28.51 pg/ml), as well as the anti-inflammatory cytokine IL-10 (BMSCs: 6.34-65.02 pg/ml; ACs: 5.30-34.85 pg/ml). Together, these results indicate a TNF-α-regulated bidirectional interaction between BMSCs and ACs, deepening our understanding of the pathogenesis of osteoarthritis and aiding in its prevention and treatment.

The mRNA N6-Methyladenosine Response to Dehydration in Xenopus laevis.

The African clawed frog, Xenopus laevis, exhibits remarkable adaptations to survive in its arid habitat, including behavioral and metabolic changes during periods of drought. During extreme dehydration, X. laevis undergoes estivation, a state characterized by increased urea and ammonia levels, depression of the metabolic rate, and tissue hypoxia. To understand the molecular mechanisms underlying these adaptations, we investigated the potential role of N6-methyladenosine (m6A), a widespread mRNA modification, in X. laevis during extreme dehydration. We analyzed the protein levels of key components in the m6A pathway, including writers (METTL3, METTL14, and WTAP), erasers (ALKBH5 and FTO), and readers (SRSF3, YTHDF1, YTHDF2, YTHDF3, and eIF3a), in the liver and kidneys of control frogs and frogs that had lost 35 ± 0.93% of their total body water. The relative protein levels generally decreased or remained unchanged, with the exception of YTHDF3, which depicted a protein level increase in the liver. Notable changes included eIF3a, which was downregulated by 26 ± 8% and 80 ± 8% in the dehydrated liver and kidney tissues, respectively. Additionally, the total m6A increased by 353 ± 30% and 177 ± 17% in dehydrated liver and kidney RNA samples, respectively. This study highlights the importance of epigenetic mechanisms in stress tolerance and provides a foundation for further exploration of the role of epigenetics in dehydration tolerance.

Melatonin attenuates cardiac dysfunction and inflammation in dilated cardiomyopathy via M2 macrophage polarization.

Melatonin is a neuroendocrine hormone that exerts protective effects on the heart. Increasing evidence suggests that macrophage M2-type polarization improves myocardial regeneration and repair. Therefore, this study investigated whether melatonin ameliorates dilated cardiomyopathy (DCM) by modulating M2-type polarization. DCM mice were established by induction with doxorubicin and then treated with melatonin. Cardiac dysfunction was determined by measuring left ventricular ejection fraction (LVEF) and left ventricular internal dimensions at end-diastole and end-systole. Heart injury and fibrosis were determined by hematoxylin and eosin staining and Sirius Red staining, respectively. Serum concentrations of melatonin, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) were measured through enzyme-linked immunosorbent assays. M2-type macrophages were analyzed by flow cytometry. Relative mRNA and protein levels were determined by reverse transcription quantitative polymerase chain reaction and western blotting, respectively. Circulating melatonin levels were significantly decreased in DCM mice and were associated with LVEF. Treatment with melatonin markedly ameliorated cardiac dysfunction, improved survival, and alleviated pathological changes and collagen deposition in DCM mice. Furthermore, melatonin-treated DCM mice displayed lower serum and cardiac levels of IL-1β, IL-6, and TNF-α, as well as higher numbers of M2-type macrophages in cardiac tissue, indicating that melatonin treatment could decrease inflammatory responses and facilitate M2 macrophage polarization in DCM mice. Thus, melatonin treatment alleviated cardiac dysfunction and inflammatory responses by promoting M2 macrophage polarization in the DCM mouse model.

STIM1 promotes cervical cancer progression through autophagy activation via TFEB nuclear translocation

BackgroundAutophagy plays an important role in maintaining the stability of intracellular environment, abnormal autophagy is associated with the occurrence and progression of cancer, the role of STIM1 in regulating cancer autophagy remains controversial, and its clinical relevance is unclear. Our study aimed to investigate the effect and mechanism of STIM1 on cervical cancer, thus to provide new molecular therapeutic targets for cervical cancer in clinic. MethodsWe collected CIN III, FIGO IB and IIA fresh Specimens without chemotherapy from patients in Renmin Hospital of Hubei University of Medicine (n = 10). STIM1, TFEB and autophagy related proteins of different stage tissues were detected. In vitro, SKF96365 and AncoA4 were used to inhibit STIM1-administrated Ca2+ entry of SiHa cells, Cyclosporine A (calcineurin inhibitors) were used to inhibit CaN/TFEB pathway, Ad-mCherry-GFPLC3B was used to detect autophagy flux, shSTIM1 was used to knockdown STIM1 expression. ResultsThe expression levels of STIM1, TFEB and autophagy related proteins were positively correlated with the progression of cervical cancer. Inhibition of STIM1-mediated SOCE can decrease proliferation and migration, and promoted the apoptosis of cervical cancer cells. Knockdown STIM1 can inhibit autophagy and TFEB nuclear translocation. ConclusionSTIM1 can promote autophagy and accelerate cervical cancer progression by increasing TFEB nuclear translocation of cervical cancer cells.

Limosilactobacillus Fermentun ZS09 Can Improve Antibiotic-Induced Motor Dysfunction in Mice by Regulating the Brain-Gut Functions.

When consumed in appropriate quantities, probiotics, which are live microorganisms, are good for health. In this study, a mouse model of antibiotic-induced dyskinesia was established using a sterile mixed antibiotic solution to investigate the preventive impact of Limosilactobacillus fermentum ZS09 (LFZS09) on this condition in mice. Following modeling, alterations in the serum and brain tissue of mice were assessed for motor measures such as running and swimming, malondialdehyde (MDA), superoxide dismutase, reduced glutathione, interleukin (IL-6, IL-10), and tumor necrosis factor (TNF)-α. The mouse cecum was used to evaluate the relative mRNA expression levels of the intestinal barrier genes, namely occludin-1, zona occludens-1 (ZO-1), and claudin-1. The relative mRNA expression levels of cAMP-response element binding protein (CREB), extracellular signal-regulated kinase (ERK), and brain-derived neurotrophic factor (BDNF) genes in the mouse brain tissue were also evaluated. Compared with the model group, LFZS09 considerably increased the swimming and running duration of mice, significantly decreased the levels of the inflammatory factors TNF-α and IL-6 and increased SOD expression in the mouse brain, and decreased MDA accumulation in the mouse brain and serum. Furthermore, LFZS09 upregulated occludin-1 gene expression in the cecal tissue to maintain the intestinal barrier, which in turn maintained the normal physiological function of the body. LFZS09 also enhanced the effect of BDNF and increased the expression of BDNF metabolic pathway-related genes, namely CREB, ERK1/2, and BDNF, in the mouse brain tissue. LFZS09 increased the number of Lactobacillus and Bifidobacterium in the gut of mice with motor dysregulation, and decreased the number of Enterococcus and Clostridium perfringens. The findings indicate that LFZS09 regulates antibiotic-induced motor impairment in mice, thereby offering a theoretical foundation for future studies and probiotic or parabiotics production aimed at augmenting motor function.

(-)-Epicatechin regulates endoplasmic reticulum stress and promotes ferroptosis in lung cancer cells via the PERK/eIF2α/ATF4 signaling pathway.

(-)-Epicatechin (EC) is an active ingredient of Fagopyrum dibtrys (D. Don) Hara and can regulate lung cancer progression. However, the specific regulatory mechanism is poorly understood. This study explored the specific mechanism of EC in the treatment of lung cancer. H460 cells were injected subcutaneously into the left dorsal sides of nude mice to establish an animal model of lung cancer. H460 and H1299 cells and nude mice were treated with different concentrations of EC. The expression levels of related proteins were detected by Western blotting. Cell proliferation, migration, and invasion were detected by CCK-8, colony formation, and Transwell assays. Flow cytometry was used to detect the Ca2+ level in lung cancer cells. Immunohistochemistry was used to detect the expression of Ki-67 in tumor tissues. This study revealed that ferroptosis in lung cancer cells was inhibited during lung cancer development. EC treatment promotes ferroptosis, inhibits the proliferation, migration and invasion of lung cancer cells, and inhibits the formation of tumors in vivo. Ferroptosis inhibitors (Fer-1) weaken the effects of EC on lung cancer cells, whereas a ferroptosis inducer (erastin) further promotes the effects of EC. In addition, endoplasmic reticulum (ER) stress is involved in the EC-induced ferroptosis of lung cancer cells, and treatment with GSK, an inhibitor of the ER stress protein PERK, can reverse the effect of EC. EC therapy activates the PERK-eIF2α-ATF4 signaling pathway to increase ER stress, thereby promoting ferroptosis in lung cancer cells and inhibiting the occurrence and development of lung cancer. Our research suggests that EC may become a drug candidate for treating lung cancer.

Clinical phenotype and functional influence of GRIN2A variants in epilepsy-aphasia syndrome.

N-methyl-D-aspartate receptors are glutamate-gated ion channels that play a crucial role in brain function. Numerous inherited or de novo variants in the GRIN2A gene, encoding the GluN2A subunit of the receptor, have been identified in patients with epilepsy. In addition, it is worth noting that GRIN2A variants exhibit a strong correlation with epilepsy-aphasia syndromes, a group of age-dependent epileptic, cognitive, and language disorders with a characteristic electroencephalographic pattern. Whole exome sequencing was conducted in enrolled patients with epilepsy-aphasia syndromes, and GRIN2A variants were screened. The conservation of substituted residues, conformational changes of mutant subunits, and in silico predictions of pathogenicity were thoroughly assessed in our study. Functional alterations of the variants were examined using whole-cell voltage-clamp current recordings while the relative surface expression levels of subunit proteins were assessed via immunofluorescence assays. A summary of previously published GRIN2A missense variants was conducted to investigate the genotypic-phenotypic-functional correlations. Two missense GRIN2A variants (c. 2482A >G/p. M828V, c.2627 T >C/p. I876T) were identified, which are located in the transmembrane helix M4 and C-terminus domain of the GluN2A subunit, respectively. Both variants exhibited reduced current density of NMDARs and surface/total expression levels of GluN2A subunits, while M828V showed a decreased extent of desensitization as well. A further summary of the previously reported GRIN2A variants demonstrated that more variable phenotypes were observed for variants situated in the C-terminus domain or those with loss-of-function effects. Our study expands the phenotypic and functional range of GRIN2A-related disorders. In order to optimally establish the domain-function-phenotype correlations in GRIN2A variants, it is imperative to gather a more extensive set of clinical and functional data. This study has identified two genetic variants of the GRIN2A gene in patients with epilepsy-aphasia syndrome. We assess the variants' harmfulness through a variety of functional experiments, including evaluating the expression level of the mutated protein and the resulting changes in electrophysiological activities. Also, we reviewed previously published papers about GRIN2A variants in epilepsy to learn more about the correlations between their locations, functional changes, and clinical manifestations.

ZC3H13-induced the m6A modification of hsa_circ_0081723 promotes cervical cancer progression via AMPK/p53 pathway.

N6-methyladenosine (m6A) modification and circular RNAs (circRNAs) have been confirmed to participate in cervical cancer (CC) progression. However, the function of a novel circRNA, hsa_circ_0081723, has not yet been explored in CC. Therefore, this study aimed to investigate the potential role of hsa_circ_0081723 and its m6A modification in CC. The hsa_circ_0081723 and ZC3H13 expressions were examined by qRT-PCR in the CC tissues, and their prognostic significance was evaluated via Kaplan-Meier Plotter. The role of hsa_circ_0081723 in CC progression was checked by loss-of-function assays. The relative protein levels of AMPK/p53 pathway were determined by western blotting. The interactions of hsa_circ_0081723 and ZC3H13 were verified via MeRIP and RNA stability assays. The hsa_circ_0081723 expression was elevated in CC samples, and its higher levels indicated high histological grade, high FIGO stage, poor differentiation, and poor prognosis. Functionally, silencing hsa_circ_0081723 impaired the malignant behavior of CC cells and enhanced the protein levels of key molecules of the AMPK signaling pathway. Moreover, ZC3H13 was also elevated in CC samples and demonstrated a positive association with hsa_circ_0081723. The relative enrichment of hsa_circ_0081723 m6A and its stability were enhanced in ZC3H13 overexpressed CC cells. Mechanically, ZC3H13 overexpression partially reversed the antitumor effects caused by hsa_circ_0081723 knockdown in CC cells. This study innovatively demonstrates that ZC3H13-mediated m6A modification of hsa_circ_0081723 promotes CC progression by modulating AMPK/p53 pathway. Our findings may contribute to the understanding of the molecular mechanisms underlying CC and offer potential therapeutic targets for clinical treatment.

Nanochitosan-encapsulated melatonin: an eco-friendly strategy to delay petal senescence in cut gerbera flowers

BackgroundThe preservation of cut flowers, particularly Gerbera jamesonii, is crucial for maintaining their aesthetic value and extending vase life in the floriculture industry. To address this challenge, this study investigated the effects of melatonin (Mel) and encapsulated melatonin with nanochitosan (nCS-Mel) as preservative solutions on cut Gerbera jamesonii cv. ‘Terra kalina’ flowers. In research, we examined various physiological and biochemical parameters, including relative water content, membrane stability index, carbohydrate content, and antioxidant enzyme activities, to evaluate the efficacy of these treatments in prolonging the vase life and quality of cut gerbera flowers under controlled environmental conditions.ResultsOur results demonstrated that cut Gerbera jamesonii flowers maintained in vase solutions containing 0.1 and 0.5 mM nCS-Mel exhibited enhanced preservation of cell membrane integrity and anthocyanin content, while also maintaining higher levels of carbohydrates and total flavonoids in petals at the conclusion of their vase life. A decline in petal relative water content and protein levels was observed concomitantly with petal senescence, whereas total phenolic compounds showed an increase. The hydrogen peroxide (H2O2) content in petals exhibited an upward trend during vase life in control specimens, but this effect was mitigated in treatments containing melatonin. Although malondialdehyde (MDA) content generally increased throughout the vase life period, flowers subjected to either Mel or nCS-Mel treatments displayed reduced MDA accumulation. The activity of catalase (CAT) demonstrated an increasing trend during vase life, with the maximum activity observed in Gerbera flowers treated with 0.1 mM nCS-Mel. A similar upward trend was noted for superoxide dismutase (SOD) activity, with flowers in 0.5 mM nCS-Mel treatment exhibiting peak SOD values on day 12 relative to control and other treatments. Peroxidase (POD) activity also increased across all treatments, with particularly pronounced effects in vase solutions containing 0.1 mM Mel and nCS-Mel. Notably, flowers placed in vase solutions containing 0.1 mM nCS-Mel, followed by 0.5 mM nCS-Mel and 0.1 mM Mel, exhibited the most prolonged vase life, extending up to 12, 10.66, and 10.33 days, respectively, under room temperature conditions.ConclusionsThe application of nanoencapsulated melatonin as a vase solution for cut Gerbera jamesonii flowers demonstrates significant potential in extending vase life and maintaining flower quality through enhanced preservation of cellular integrity, antioxidant activity, and biochemical parameters. This innovative approach not only outperforms conventional treatments but also presents a more environmentally friendly alternative to traditional antimicrobial preservatives and sugars, offering a promising solution for the floriculture industry to improve cut flower longevity and reduce ecological impact.

CXCL12/CXCR4 Axis Promotes the Chemotaxis and Phagocytosis of B Cells through the PI3K-AKT Signaling Pathway in an Early Vertebrate.

Chemokines play crucial roles in the regulation of immune cell migration and development. The CXCL12/CXCR4 axis has been extensively studied in mammals, but its regulatory mechanism in teleost fish remains unclear. In this study, we used Nile tilapia (Oreochromis niloticus) as a teleost model to investigate the mediation of the CXCL12/CXCR4 axis in IgM+ B cells. Our findings demonstrate that the CXCL12/CXCR4 axis exhibits chemotactic activity on IgM+ B cells and promotes the phagocytosis of IgM+ B cells. Blocking CXCR4 severely impairs the chemotaxis and phagocytosis of IgM+ B cells invitro and reduces the percentages and numbers of IgM+ B cells that migrate to peripheral blood after pathogen infection invivo. This reduction in migration leads to a decrease in the inflammatory response, an increase in tissue bacterial load, and a decrease in survival rate. We also discovered that the evolutionarily conserved PI3K-AKT signaling pathway and Girdin are involved in the immune response during Streptococcus agalactiae infection. Inhibitors of the PI3K-AKT signaling pathway prevent the chemotaxis and phagocytosis of IgM+ B cells, impair the expression and phosphorylation levels of related proteins invitro, and prevent IgM+ B cells chemotaxis into the peripheral blood after pathogen infection invivo. Furthermore, CXCR4 blocking significantly downregulates the expression of AKT and Girdin. Overall, our study reveals the regulatory mechanism of the CXCL12/CXCR4 axis on IgM+ B cells via the PI3K-AKT signaling pathway in tilapia, suggesting that the functions of the CXCL12/CXCR4 axis in B cells may be conserved between mammals and teleost fish.

Role and mechanism of Actein on condylar bone metabolism in APOE deletion-induced osteoporotic mice

AimsTo investigate the effects of Actein from Cimicifugae Rhizoma on condylar bone and cartilage in APOE deletion-induced osteoporotic mice, and to preliminarily explore the underlying mechanism. MethodsSixty 8-week-old female mice were used, which underwent APOE−/− and ovariectomy procedures, followed by oral administration of Actein (15 mg/kg) and Atorvastatin Calcium (AC, 3 mg/kg) for eight weeks. Body weight, uterine weight, and systemic indexes related to bone metabolism and lipid metabolism were assessed in each group. Changes in condylar bone histomorphometric parameters were evaluated using Micro-CT. Morphological changes in the condyle were observed with Hematoxylin-Eosin (H&E), Safranin O/Fast Green, and Alcian Blue Hematoxylin/Orange G (ABH/OG) staining, with OARSI pathology scoring performed. Sirius red staining and immunofluorescence were used to determine the expression levels of Collagen I (Col I) and Collagen III (Col III) in bone matrix, and Col II in cartilage matrix. Immunohistochemistry assessed the relative expression levels of ALP and proteins associated with the Wnt/β-catenin/RUNX2 signaling pathway. ResultsAPOE−/− exacerbates ovariectomy -induced osteoporosis (OP) in condylar of mice. Actein and AC significantly reversed OP, improved bone mineral density (BMD), increased bone microarchitecture, and restored abnormal calcium and phosphorus metabolism in the blood and urine. Morphologically, APOE−/− and ovariectomy reduced condylar cartilage thickness, disrupted chondrocyte arrangement, chondrocyte cleavage, and clustered aggregation, resembling osteoarthritis (OA)-like changes. Actein and AC partially restored the disrupted chondrocyte arrangement, smoothed chondrocyte cleavage, and up-regulated the levels of chondrocyte matrix (Col II, aggrecan) and bone matrix (Col III, ALP). Actein reversed the OA process, potentially through the Wnt/β-catenin/RUNX2 signaling pathway. ConclusionAPOE−/− and ovariectomy induced OP, leading to OA-like lesions in condylar of mice. Actein promoted cartilage repair and trabecular bone recovery by increasing extracellular matrix synthesis (Col II, Col III, aggrecan), reversing the OA process, possibly through the Wnt/β-catenin/RUNX2 signaling pathway.