Expression Of Related Proteins Research Articles

A new methodology to reveal potential nucleic acid modifications associated with the risk of endometrial cancer through dispersive solid-phase extraction coupled with UHPLC-QE-Orbitrap-MS/MS and HPLC-UV.

Nucleic acid modifications have attracted increasing attention in recent years since they have been found to be related to a number of diseases including cancer. Previous studies have shown that the early development of endometrial cancer (EC) is often accompanied by changes in methylation levels of related genes, and the expression of related proteins that regulate reactive oxygen species (ROS) shows significant differences in EC cells and tissues. However, it has not been reported whether nucleic acid modifications related to methylation or ROS can serve as biomarkers for EC. Accurate quantification of these nucleic acid modifications still has challenges because their amounts in urine are very low and the interferences in urine are complicated. In this study, a novel dispersive solid-phase extraction (DSPE) method based on chitosan-carbon nanotube-Al2O3 (CS-CNT-Al2O3) has been established for the analysis of 5-hydroxymethyluracil (5mU), 5-methyl-2'-deoxycytidine (5-mdC), 5-hydroxymethyl-2'-deoxycytidine (5-hmdC), 5-formyl-2'-deoxycytidine (5-fdC), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in EC patient urine samples coupled with UHPLC-QE-Orbitrap-MS/MS and HPLC-UV. Firstly, the synthesis of the CS-CNT-Al2O3 nanocomposite was conducted by a sono-coprecipitation method and was characterized by scanning electron microscope (SEM), energy dispersive spectrometer (EDS), and Fourier transform infrared (FTIR). Under the optimal extraction conditions of DSPE, we successfully quantified 5mU, 5-mdC, 5-hmdC, 5-fdC, and 8-OHdG in urine samples from 37 EC patients and 39 healthy controls. The results showed that there were significant differences in the levels of 5-mdC, 5-hmdC, 5-fdC, and 8-OHdG in EC patients compared to the healthy control group. The receiver operator characteristic (ROC) curve analysis was carried out to evaluate the potential of 5-mdC, 5-hmdC, 5-fdC, and 8-OHdG to distinguish EC patients from healthy volunteers. The area under the curve (AUC) for 5-mdC, 5-hmdC, 5-fdC, and 8-OHdG was 0.7412, 0.667, 0.8438, and 0.7981, respectively. It indicated that 5-mdC, 5-hmdC, 5-fdC, and 8-OHdG had certain potential in distinguishing between EC patients and healthy volunteers and they could act as potential non-invasive biomarkers for early diagnosis of EC. Moreover, the present study would stimulate investigations of the effects of nucleic acid modifications on the initiation and progression of EC.

Impact of hyperoxia on the phenotype of pulmonary artery smooth muscle cells

Objective: To investigate the influence of varied oxygen (O2) concentration environments on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMC) and the mechanism of pulmonary hypertension. Methods: Primary rat PASMC were isolated and cultured through the process of enzymatic digestion. Following identification, the stable passaged PASMC were subjected to a 6-hour incubation in sealed containers with normal O2 content (group C) and relative O2 content comprising 55% (group H55), 75% (group H75), and 95% (group H95). mRNA and protein expression of α-Actin (α-SMA), smooth muscle 22α (SM22α), osteopontin (OPN), and matrix metalloproteinase-2 (MMP-2) were measured using real-time quantitative PCR and western blot analysis. Results: The H55 group displayed no significant difference from the C group in terms of mRNA and relative protein expression levels for α-SMA, SM22α, OPN, and MMP-2 (all P>0.05). On the other hand, groups H75 and H95 exhibited a reduction in mRNA and relative protein expression of α-SMA and SM22α, along with an increase in mRNA and relative protein expression of OPN and MMP-2 when compared with both the C and H55 groups (all P<0.05). The H95 group showed a higher relative mRNA expression of MMP-2 as compared to the H75 group (P<0.05). Conclusions: Oxygen concentration environments of 75% or higher can serve as the foundation for the pathogenesis of pulmonary hypertension, essentially by inducing a phenotypic transformation in PASMC towards adopting a robust secretory function. This induction is contingent upon the concentration of oxygen present.

Diallyl trisulfide inhibits osteosarcoma 143B cell migration, invasion and EMT by inducing autophagy

BackgroundDiallyl trisulfide (DATS), a compound derived from garlic, has been demonstrated its anti-cancer properties. While it has been shown to inhibit the expression of epidermal growth factor receptor (EGFR) in various cancers, its effects on osteosarcoma (OS) cells remain unclear. This study aimed to investigate the impacts of DATS on OS cells growth, migration, invasion, epithelial-mesenchymal transition (EMT) and autophagy, as well as its underlying mechanisms which was involving in the EGFR/PI3K/AKT/mTOR pathway. MethodsIn this study, human osteosarcoma cells (143B) were treated with different concentrations of DATS (10, 50, 100 and 200 μM) for 24 and 48 h, respectively. Cell viability was measured using CCK8, the half lethal concentration was selected for the following experiments. Wound healing and transwell assays were performed to evaluate migration and invasion abilities, while flow cytometry was used to measure apoptosis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and confocal imaging were employed to analyze the related mRNA and protein expression levels of epithelial-mesenchymal transition (EMT), EGFR/Phosphoinositide 3 kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) signaling pathway and autophagy-related markers. ResultsDATS significantly inhibited proliferation, migration and EMT in osteosarcoma cells. Additionally, DATS promoted cell apoptosis and induced autophagy, which could be rescued by the autophagy inhibitor 3-methyladenine (3-MA). Moreover, DATS treatment led to the inactivation of the EGFR/PI3K/AKT/mTOR pathway in osteosarcoma cells. ConclusionsThis study demonstrated that DATS inhibited osteosarcoma cell growth, migration and EMT, but inducing apoptosis and autophagy. These effects were mediated by the inactivation of the EGFR/PI3K/AKT/mTOR signaling pathway. These findings suggested that DATS could serve as a potential therapeutic agent for osteosarcoma treatment.

Dietary supplementation of osthole and icariin improves the production performance of laying hens by promoting follicular development

Osthole (Ost) and icariin (Ica) are extracted from traditional Chinese medicine Cnidium monnieri and Epimedii Folium, respectively, and both exhibit estrogen-like biological activity. This study aimed to determine the efficacy and safety of combining Ost with Ica on the production performance of laying hens and to explore their possible mechanisms. The production performance, egg quality, residues of Ost and Ica in eggs, serum reproductive hormone levels, expression of ovarian reproductive hormone receptor, proliferation of granulosa cells in small yellow follicles (SYF), and progesterone secretion in large yellow follicles (LYF) related genes and proteins expression were detected. The results showed that adding 2 mg/kg Ost + 2 mg/kg Ica to the feed increased the laying rate, average egg weight, Haugh unit, and protein height of laying hens. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P4) levels increased, and the expression of ovarian estrogen receptor (ER), follicle-stimulating hormone receptor (FSHR), and progesterone receptor (PGR) mRNA was up-regulated. Additionally, the mRNA and protein levels of steroidogenesis acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) increased in LYF. Furthermore, mRNA and protein levels of proliferating cell nuclear antigen (PCNA), cyclin E1, and cyclin A2 were up-regulated in SYF. The residues of Ost and Ica in egg samples were not detected by high-performance liquid chromatography (HPLC). In conclusion, dietary supplementation of Ost and Ica increased granulosa cells proliferation in SYF and increased P4 secretion in granulosa cells of LYF, ultimately improving the production performance of laying hens.

The key proteolytic enzyme analysis of industrial Aspergillus oryzae at solid-state koji fermentation with a local database extension

The productivity and flavor quality of soy sauce are partly determined by the proteolytic enzymes secreted by the industrial microbe Aspergillus oryzae, which break down proteins into peptides and amino acids. Because of how complicated it is to classify proteolytic enzymes and understand how they work, not much is known about what is in the profiles of proteolytic enzymes and which enzymes are most important to A. oryzae during solid-state fermentation. In this study, we collected industrial A. oryzae from Chinese and Japanese soy sauce production, both of which have excellent protein hydrolysis capability but have numerous genetic differences. We systematically analyzed the in vitro enzyme profiles of two industrial A. oryzae based on 4D label-free proteome sequencing technology. In addition, we constructed a local database of proteolytic enzymes from the Aspergillus genus, which helps us analyze the proteomics data more precisely. We found that 96 proteolytic enzymes are excreted by industrial A. oryzae, of which 27 are endopeptidases and 39 are exopeptidases. Based on the relative protein expression and transcription quantity, as well as the catalytic characteristics, we identified 27 potential key proteolytic enzymes associated with high protein breakdown efficiency at solid-state fermentation. Meanwhile, we proposed eight proteolytic enzymes with potential hydrolytic activity. This study deepens our understanding of the proteolytic enzyme profiles excreted by A. oryzae at solid state and provides our knowledge of the candidate key proteolytic enzymes during koji fermentation.

Revealing Melatonin's Mysteries: Receptors, Signaling Pathways, and Therapeutics Applications.

Melatonin (5-methoxy-acetyl tryptamine) is a sleep-inducing hormone, and the pineal gland produces it in response to the circadian clock of darkness. In the body, MT1 and MT2 receptors are mostly found, having an orthosteric pocket and ligand binding determinants. Melatonin acts by binding on melatonin receptors, intracellular proteins, and orphan nuclear receptors. It inhibits adenyl cyclase and activates phospholipase C, resulting in gene expression and an intracellular alteration environment. Melatonin signaling pathways are also associated with other intracellular signaling pathways, i. e., cAMP/PKA and MAPK/ERK pathways. Relative expression of different proteins depends on the coupling profile of G protein, accounting pharmacology of the melatonin receptor bias system, and mediates action in a Gi-dependent manner. It shows antioxidant, antitumor, antiproliferative, and neuroprotective activity. Different types of melatonin agonists have been synthesized for the treatment of sleeping disorders. Researchers have developed therapeutics that target melatonin signaling, which could benefit a wide range of medical conditions. This review focuses on melatonin receptors, pharmacology, and signaling cascades; it aims to provide basic mechanical aspects of the receptor's pharmacology, melatonin's functions in cancer and neurodegenerative diseases, and any treatments and drugs designed for these diseases. This will allow a basic comparison between the receptors in question, highlighting any parallels and differences that may exist and providing fundamental knowledge about these receptors to future researchers.

P65/NLRP3 Inflammasome Mediated Endothelial Cells Pyroptosis: A Novel Mechanism of In-Stent Restenosis

Background: In-stent restenosis (ISR) is one of the key causes of ischemic events after coronary stent implantation, and endothelial cell death and inflammation are considered to be important mechanisms. Pyroptosis is a proinflammatory type of programmed cell death, the effects and underlying mechanisms of endothelial cell (EC) pyroptosis in ISR remains unclear. Method: According to our previous work, an ISR rabbit model was established. Rabbits were divided into sham operation group and stent group. Serum was collected at 0, 4, 8, and 12 weeks to detect interleukin (IL)-1β and IL-18 levels. Rabbits' vascular EC was collected to detect NLRP3, Caspase1, GSDMD and P65 expression by western blot. NLRP3 inhibitor (MCC950) and P65 inhibitor (Helenalin) were used to pretreat EC, cell viability, lactate dehydrogenase (LDH) level of supernatant and pyroptosis related protein expression were measured in different groups. Results: The serum levels of IL-1β and IL-18 gradually increased with time, and the levels at the site of stent implantation were higher than the peripheral level. EC viability decreased significantly in the stent group, and protein levels of NLRP3, caspase1 and GSDMD were higher than those in the sham group. MCC950 and P65 inhibitors can reverse these effects. Conclusions: EC pyroptosis mediated by P65/NLRP3 inflammasome axis may promote ISR, our results provide a potential intervention target for the treatment of ISR.

Grape Seed Proanthocyanidins Protect Pancreatic β Cells Against Ferroptosis via the Nrf2 Pathway in Type 2 Diabetes.

Pancreatic β cell damage is the primary contributor to type 2 diabetes mellitus (T2DM); however, the underlying mechanism remains nebulous. This study explored the role of ferroptosis in pancreatic β cell damage and the protective effects of grape seed proanthocyanidin extract (GSPE). In T2DM model rats, the blood glucose, water intake, urine volume, HbA1c, and homeostasis model assessment-insulin resistance were significantly increased, while the body weight and the insulin level were significantly decreased, indicating the successful establishment of the T2DM model. MIN6 mouse insulinoma β cells were cultured in high glucose and sodium palmitate conditions to obtain a glycolipid damage model, which was administered with GSPE, ferrostatin-1 (Fer-1), or nuclear factor erythroid 2-related factor 2 (Nrf2) small interfering (si) RNA. GSPE and Fer-1 treatment significantly improved pancreatic β-cell dysfunction and protected against cell death. Both treatments increased the superoxide dismutase and glutathione activity, reduced the malondialdehyde and reactive oxygen species levels, and improved iron metabolism. Furthermore, the treatments reversed the expression of ferroptosis markers cysteine/glutamate transporter (XCT) and glutathione peroxidase 4 (GPX4) caused by glycolipid toxicity. GSPE treatments activated the expression of Nrf2 and related proteins. These effects were reversed when co-transfected with si-Nrf2. GSPE inhibits ferroptosis by activating the Nrf2 signaling pathway, thus reducing β-cell damage and dysfunction in T2DM. Therefore, GSPE is a potential treatment strategy against T2DM.

Experimental study of tetramethylpyrazine-loaded electroconductive hydrogel on angiogenesis and neuroprotection after spinal cord injury

To explore the mechanisms for repairing spinal cord injury (SCI) with tetramethylpyrazine-loaded electroconductive hydrogel (hereinafter referred to as "TGTP"). A total of 72 female Sprague-Dawley rats were randomly divided into 4 groups: sham operation group (group A), SCI group (group B), SCI+electroconductive hydrogel group (group C), and SCI+TGTP group (group D). Only the vertebral plate was removed in group A, while the remaining groups were subjected to a whole transection model of spinal cord with a 2 mm gap in the lesions. The recovery of hindlimb motor function was evaluated by Basso, Beattie, Bresnahan (BBB) score and modified Rivlin-Tator inclined plate test before operation and at 1, 3, 7, 14, and 28 days after operation, respectively. Animals were sacrificed at 7 days and 28 days after modeling. Neovascularisation was observed by immunofluorescence staining of CD31 and the expression levels of angiopoietin 1 (Ang-1) and Tie-2 were assessed by Western blot assay. At 28 days postoperatively, the expression levels of pro-angiogenic related proteins, including platelet-derived growth factor B (PDGF-B), PDGF receptor β (PDGFR-β), vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2), were also assessed by Western blot. The fibrous scar in the injured area was assessed using Masson staining, while neuronal survival was observed through Nissl staining. Furthermore, LFB staining was utilized to detect myelin distribution and regeneration. Immunofluorescence and Western blot assay were employed to evaluate the expression of neurofilament 200 (NF200). The hindlimb motor function of rats in each group gradually recovered from the 3rd day after operation. The BBB score and climbing angle in group D were significantly higher than those in group B from 3 to 28 days after operation, and significantly higher than those in group C at 14 days and 28 days after operation ( P<0.05). Masson staining showed that the collagen volume fraction in groups B-D were significantly higher than that in group A, and that in group D was significantly lower than that in groups B and C ( P<0.05); a small amount of black conductive particles were scattered at the broken end in group D, and the surrounding collagen fibers were less than those in group C. Nissl and LFB staining showed that the structure of neurons and myelin sheath in the injured area of spinal cord in group D was relatively complete and continuous, and the number of Nissl bodies and the positive area of myelin sheath in group D were significantly better than those in groups B and C ( P<0.05). NF200 immunofluorescence staining and Western blot assay results showed that the relative expression of NF200 protein in group D was significantly higher than that in groups B and C ( P<0.05). CD31 immunofluorescence staining showed that the fluorescence intensity of group D was better than that of groups B and C at 28 days after operation, and tubular or linear neovascularization could be seen. The relative expressions of Ang-1 and Tie-2 proteins in group D were significantly higher than those in groups B and C at 7 and 28 days after operation ( P<0.05). The relative expressions of PDGF-B and PDGFR-β proteins in group D were significantly higher than those in groups B and C, and group B was significantly higher than group C at 28 days after operation ( P<0.05). The relative expressions of VEGF-A and VEGFR2 proteins in group D were higher than those in groups B and C, showing significant difference when compared with group B ( P<0.05), but only the expression of VEGF-A protein was significantly higher than that in group C ( P<0.05). There was significant difference only in VEGFR-2 protein between groups B and C ( P<0.05). TGTP may enhance the revascularization of the injured area and protect the neurons, thus alleviating the injury of spinal cord tissue structure and promoting the recovery of neurological function after SCI in rats.

Screening and preliminary analysis of differentially expressed genes in quail embryos for feather colour based on RNA-Sequencing

The purpose of this study was to use RNA-Sequencing (RNA-Seq) to screen differentially expressed genes (DEGs) and analyse transcriptomic data from embryonic skin tissues of maroon-feather and white-feather quails. The transcriptome of embryonic skin tissues from quails was sequenced using the Illumina HiSeqTM 2000 sequencing platform. A total of 2 512 DEGs were found. Of these, 550 DEGs were up-regulated and 1 562 DEGs were down-regulated in the skin tissues of white-feather quail embryos. Five hundred and ninety-seven DEGs were enriched and annotated into 50 entries in Gene Ontology (GO) database. The total number of DEGs annotated in each entry in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was 341, enriched in 230 pathways, of which the tight junction pathway and neuroactive ligand-receptor interaction were the most significantly enriched. Candidate genes were analysed using real-time quantitative reverse transcription polymerase chain reaction (to verify the accuracy and reliability of the RNA-Seq results. The relative expression of agouti signalling protein (ASIP) and DOPA decarboxylase (DDC) in white-feather quails were increased. Relative expression of homeobox D1 (HOXD1), cathepsin D (CTSD), keratin 2 (KRT2), methyl-CpG binding domain protein 2 (MBD2), and melatonin receptor 1 (MT1) were decreased. The results of this study provide useful data on the analysis of the feather colour regulatory mechanism in white- and maroon-feather quails.

Quercetin attenuates inflammation in LPS-induced lung epithelial cells via the Nrf2 signaling pathway.

Pneumonia is the leading cause of death among children under five, and kill almost two million children each year. Quercetin, a flavonoid polyphenolic compound, exerts many beneficial biological activities, including anti-inflammatory functions. Our study aimed to investigate the possibility of quercetin as a therapeutic agent for pneumonia and its role in the inflammatory response induced by lipopolysaccharide (LPS). LPS induced human alveolar epithelial cell A549 as a lung inflammation model in vitro. The effects of quercetin on the production of cytokines and the expression of related-proteins were detected by Enzyme-Linked ImmunoSorbent Assay and Western Blot, respectively. Cell Counting Kit-8 assay was used to detect cell viability. flow cytometry was used to measure cell apoptosis. NO levels were also analyzed through NO kit. Our results found that quercetin attenuated the release of IL-1β, IL-6, PGE2, and nitrite in LPS-induced A549 cells. In addition, quercetin inhibits cell apoptosis and relieves ROS generation in LPS-induced A549 cells. Quercetin also inhibits LPS-induced NF-κB activation. They have upregulated the expression of nuclear factor erythroid 2 (Nrf2) and HO-1. In conclusion, these results suggested that quercetin attenuates LPS-induced inflammation in A549 by activating the Nrf2 signaling pathway.

FNDC4 reduces inflammation, proliferation, invasion and migration of rheumatoid synovial cells by inhibiting CCL2/ERK signaling

BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic joint inflammation. Fibronectin type III domain-containing protein 4 (FNDC4) is a secretory factor that can regulate inflammatory diseases. However, the role of FNDC4 in RA has not been reported so far. MethodsThe expression of FNDC4 in synovial tissues of RA was analyzed by GEO database (GSE55235 dataset). Then, the expression of FNDC4 in RA fibroblast-like synoviocytes (RA-FLSs) was detected by RT-qPCR and western blot. After constructing FNDC4 overexpression plasmid, cell proliferation and apoptosis were detected. Wound healing and transwell tests were used to detect cell migration and invasion. Then we examined the expression of cytokines related to cell inflammation. Subsequently, the regulatory mechanism of FNDC4 was further discussed. We detected the expression of CCL2 and ERK signaling pathway related proteins downstream of FNDC4. Finally, the mechanism was discussed through the overexpression of FNDC4 and CCL2 and the addition of ERK pathway activator tBHQ. ResultsGEO database showed that FNDC4 expression decreased in synovial tissues of RA. And FNDC4 expression also decreased in RA-FLSs. Overexpression of FNDC4 inhibited the proliferation, invasion and migration of RA-FLSs and promoted its apoptosis. Overexpression of FNDC4 inhibited the release of RA-FLSs inflammatory factors. The regulatory effect of FNDC4 is achieved by inhibiting the CCL2/ERK signaling pathway. ConclusionFNDC4 reduces inflammation, proliferation, invasion and migration of RA-FLSs in RA by inhibiting CCL2/ERK signaling.

Deciphering relationship between depression and microbial molecules based on multi-omics: A case study of Chaigui Granules

ObjectiveTo decipher the antidepression effect of Chaigui Granules (CGKL) from the relationship between depression and microbial molecules based on multi-omics. MethodsMale SD rats were subjected to chronic unpredictable mild stress (CUMS) for seven weeks. The antidepressants CGKL extract and CGKL were administered for the following four weeks. The behavior test and the content of monoamine neurotransmitters were used to evaluate the efficacy of CGKL. The 16S rRNA sequencing, LC-MS technology and molecular biological techniques were used to explore the pharmacological mechanism of CGKL. ResultsCGKL treatment obviously alleviated the depressive behavioral indicators and regulated the content of monoamine neurotransmitters, and presented dose-dependent manner. CGKL could also improve the arginine metabolism disorder of gut microbiota in the jejunum. Meanwhile, the contents of arginine and its metabolites in the serum and hippocampus were regulated to normal levels. Further investigation indicated that the expression of related rate-limiting enzyme genes and proteins in the hippocampus was validated by qRT-PCR and Western blotting. The results showed that the gut microbiota, metabolites, and genes or proteins of rate-limiting enzymes involved in the arginine pathway were significantly regulated by CGKL. ConclusionThe present study demonstrates that CGKL might exert antidepressant effects through regulating arginine metabolism, and its mechanism may be related to modulating the gut microbiota and related metabolic enzyme.

Abstract WMP1: Neuroprotection by Normobaric Oxygen (NBO): Modulating Inflammasome (NLRP3) Activation and Stress Granule Formation in Ischemic Stroke

Background: NBO has emerged as a promising neuroprotective strategy for ischemic stroke. Key to comprehending these effects is understanding its mechanisms. This study examines the pivotal roles of HIF-1α as an oxygen sensor, the protective emergence of stress granules (SGs) in response to various stimuli, and the inflammatory cell death process known as pyroptosis, triggered by inflammasomes. We elucidated the interplay between HIF-1α, SGs, inflammasomes in ischemia/reperfusion injury and in NBO-induced neuroprotection. Methods: A total of 132 adult male SD rats were subjected to 2 h middle cerebral artery occlusion (MCAO), followed by 2, 6, 24 or 48 h of reperfusion. At the onset of reperfusion, oxygen was inhaled for 2 h at concentration of 95% and flow rate of 2 L/min. YC-1 (HIF-1α inhibitor) was administered 2 h before MCAO. Brain damage was evaluated by infarct volumes (TTC), LDH and ROS levels (ELISA), apoptotic cell death (flow cytometry and TUNEL). Pyroptosis was evaluated by flow cytometry. mRNA and protein levels of HIF-1α and inflammasome related factors, including IL-18, IL-1β, ASC, NLRP3, TXNIP, N-GSDMD, cleaved-Caspase-1, as well as SGs proteins including DDX3X, G3BP1, TIA-1 were examined. Co-IP was used to detect the interaction between DDX3X and G3BP1, as well as DDX3X and NLRP3. Results: Infarct volumes, LDH and ROS levels, apoptotic and pyroptotic cell death, as well as HIF-1α expression were all increased after ischemia/reperfusion. All these increases were reversed by either NBO or YC-1. NBO or YC-1 further reversed the increase in expression of NLRP3 inflammasome related proteins and suppressed the interaction between DDX3X and NLRP3, thereby suppressing inflammasome activation. NBO or YC-1 stimulated the expressions of SGs proteins and facilitated the interaction between DDX3X and G3BP1, thereby promoting the formation of SGs, in association with cell protection. Conclusion: Our findings highlight the neuroprotective effects of NBO in ischemic stroke. This protection is attributed to its dual capacity to inhibit NLRP3 inflammasome activation while simultaneously promoting formation of SGs. Central to these processes, HIF-1α, as an instrumental modulator, sheds light on potential therapeutic targets for stroke intervention.

Abstract WP233: Histone Lactylation in Neuronal Mitochondria Fission: Implications for Stress-Exacerbated Ischemic Stroke Outcomes

Introduction: Mental stress was linked with adverse outcomes following ischemic stroke. Accumulation of lactate due to mental stress is well-documented, especially in skeletal muscles. Importantly, lactate serves as a key substrate for lactylation, a novel post-translational protein modification, which triggers mitochondrial fission. This study investigates the pivotal role of histone lactylation in driving neuronal mitochondrial fission, which may amplify the detrimental outcomes of stroke under mental stress. Methods: A total of 120 C57BL/6J mice were divided into four groups: control, chronic restraint stress (CRS), lactylationantagonist (C646, 10mg/kg, i.p.) and C646+CRS. CRS for mice was induced in a 50mL tube for 6 hours per day for consecutive 28 days. On the day 29 th , Western blot was applied to detect the expression of fission related proteins (DRP1, MFF and FIS1). The effects of lactylation on mitochondria function was determined through JC-1 and mitoSOX™ staining by adding exogenous lactate (10mM) and C646 (20mM) in neuronal cells. The stressed mice then were subjected to 45 minute-middle-cerebral -artery-occlusion (MCAO). At 6 and 24 hours of reperfusion, apoptotic cell death and apoptotic proteins (Bcl-2, BAX and cleaved caspase-3) were measured by TUNEL and Western blot. Infarct volume and neurological deficits were determined by TTC staining and scoring systems at 48 hours of reperfusion. Results: CRS aggravated ischemic injury and apoptosis after stroke. C646, a lactylation antagonist, significantly decreased protein expressions of DRP1, MFF and FIS1 and reversed CRS-associated stroke injury. Furthermore, mitochondria membrane potential (MMP) was decreased and ROS was increased upon exogenous lactate in neuronal cells, accompanied by increased mitochondria fission. Again, C646 reverses mitochondria dysfunction and fission. Conclusions: Our results shed light on how CRS intensifies the ischemic damage post-stroke. Central mechanism is histone lactylation which triggers neuronal mitochondria fission, thereby impacting the outcomes related to stress-associated stroke. Lactylation antagonists offer promising avenues for therapeutic interventions, mitigating the adverse effects of stress on stroke outcomes.

Mechanism of Guizhi Gancao Decoction against myocardial ischemia-reperfusion injury based on network pharmacology and experimental verification

This study employed network pharmacology to investigate the effect of Guizhi Gancao Decoction(GGD) on myocardial ischemia-reperfusion injury(MI/RI) in rats and decipher the underlying mechanism. Firstly, the chemical components and targets of GGD against MI/RI were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), SwissTargetPrediction, and available articles. STRING and Cytoscape 3.7.2 were used to establish the protein-protein interaction(PPI) network for the common targets, and then Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were carried out for the core targets. The &quot;drug-active component-target-pathway&quot; network was built. Furthermore, molecular docking between key active components and targets was conducted in AutoDock Vina. Finally, the rat model of MI/RI was established, and the myocardial infarction area was measured. Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were employed to detect cardiomyocyte pathology and ultrastructural changes. Western blot was employed to determine the expression of related proteins in the myocardial tissue. A total of 75 chemical components of GGD were screened out, corresponding to 318 targets. The PPI network revealed 46 core targets such as tumor protein p53(TP53), serine/threonine kinase 1(AKT1), signal transducer and activator of transcription 3(STAT3), non-receptor tyrosine kinase(SRC), mitogen-activated protein kinase 1(MAPK1), MAPK3, and tumor necrosis factor(TNF). According to GO and KEGG enrichment analyses, the core targets mainly affected the cell proliferation and migration, signal transduction, apoptosis, and transcription, involving advanced glycation end products-receptor(AGE-RAGE), MAPK and other signaling pathways in cancers and diabetes complications. The molecular docking results showed that the core components of GGD, such as licochalcone A,(+)-catechin, and cinnamaldehyde, had strong binding activities with the core target proteins, such as MAPK1 and MAPK3. The results of animal experiments showed that compared with the model group, GGD significantly increase superoxide dismutase, decreased malondialdehyde, lactate dehydrogenase, and creatine kinase-MB, and reduced the area of myocardial infarction. HE staining and TEM results showed that GGD pretreatment restored the structure of cardiomyocytes and alleviated the pathological changes and ultrastructural damage of mitochondria in the model group. In addition, GGD significantly down-regulated the phosphorylation of c-Jun N-terminal kinase and p38 and up-regulate that of extracellular regulated kinases 1/2 in the myocardial tissue. The results suggested that GGD may exert the anti-MI/RI effect by regulating the MAPK signaling pathway via the synergistic effects of Cinnamomi Ramulus and Glycyrrhizae Radix et Rhizoma.

Pirfenidone and nintedanib attenuate pulmonary fibrosis in mice by inhibiting the expression of JAK2.

Pirfenidone and nintedanib were approved by the Food and Drug Administration (FDA) for the treatment of idiopathic pulmonary fibrosis (IPF). These two drugs can slow the progression of the disease, but the specific mechanisms are not fully understood. In the current study, bleomycin (BLM) induced pulmonary fibrosis in mice was accompanied by high p-JAK2 expression in lung tissue, mainly in the nucleus. The expression of p-JAK2 significantly decreased after intragastric administration of pirfenidone and nintedanib. p-JAK2 is reportedly expressed mainly in the cytoplasm and exerts its effect by activating downstream p-STAT3 in the nucleus. In vivo experiments, pulmonary fibrosis was induced in mice with BLM and then treated with pirfenidone and nintedanib. The levels of transforming growth factor-β (TGF-β1), SP-A, SP-D and KL-6 in serum were measured by enzyme-linked immunosorbent assay (ELISA). Pathological staining was performed to assess lung fibrosis in mice, Western blot was performed to detect the expression levels of relevant proteins, and immunofluorescence was performed to observe the fluorescence expression of p-JAK2. In cellular experiments, MLE12 was stimulated with TGF-β1 and intervened with TGF-β1 receptor inhibitor and si-JAK2, pirfenidone and nintedanib, respectively, and the related protein expression levels were detected by Western blot. In both in vivo and in vitro experiments, pirfenidone and nintedanib were found to attenuate the expression of lung fibrosis markers by inhibiting the expression of JAK2, which may reduce the entry of p-JAK2 into the nucleus by downregulating JAK2 phosphorylation through inhibition of the TGF-β receptor. In contrast, inhibition of JAK2 expression greatly reduced the expression of TGF-β receptor and α-smooth muscles actin (a myofibroblast activation marker). In both in vivo and in vitro experiments, the present study demonstrated that TGF-β1 promotes JAK2 phosphorylation through a non-classical pathway, and conversely, inhibition of JAK2 expression affects the TGF-β1 signalling pathway. Therefore, we speculate that TGF-β1 and JAK2 signaling pathways interact with each other and participate in fibrosis.

Exploring the Osteogenic Potential of Zinc-Doped Magnesium Phosphate Cement (ZMPC): A Novel Material for Orthopedic Bone Defect Repair.

In orthopedics, the repair of bone defects remains challenging. In previous research reports, magnesium phosphate cements (MPCs) were widely used because of their excellent mechanical properties, which have been widely used in the field of orthopedic medicine. We built a new k-struvite (MPC) cement obtained from zinc oxide (ZnO) and assessed its osteogenic properties. Zinc-doped magnesium phosphate cement (ZMPC) is a novel material with good biocompatibility and degradability. This article summarizes the preparation method, physicochemical properties, and biological properties of ZMPC through research on this material. The results show that ZMPC has the same strength and toughness (25.3 ± 1.73 MPa to 20.18 ± 2.11 MPa), that meet the requirements of bone repair. Furthermore, the material can gradually degrade (12.27% ± 1.11% in 28 days) and promote osteogenic differentiation (relative protein expression level increased 2-3 times) of rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. In addition, in vivo confirmation revealed increased bone regeneration in a rat calvarial defect model compared with MPC alone. Therefore, ZMPC has broad application prospects and is expected to be an important repair material in the field of orthopedic medicine.

Repair effect of neurotrophic factor III (NT-3) on rats with spinal injury model and its mechanism.

The present study aimed to study the repair effect of neurotrophic factor III (NT-3) on spinal injury model rats and its mechanism. Wistar rats with spinal injury were established by accelerated compression stroke after the operation and divided into control group, model group, and NT-3 intervention group. The motor function of rats in each group was evaluated at different postoperative time points (3, 7, 14 d). HE staining was used to detect the changes in tissue structure and morphology of the injured spinal column in each group. The changes of SOD, MDA and GSH in serum of rats were detected. The concentrations of inflammatory cytokines IL-1β, IL-6, IL-17 and TNF-α in serum were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the expression changes of anti-apoptotic protein (Bcl-2) and pro-apoptotic protein (Bax) in injured spinal tissue of rats in each group. Compared with model group, motor function score of NT-3 intervention group increased gradually, and had statistical significance at 7 and 14 days (5.29±1.62 vs 9.33±2.16, 5.92±1.44 vs 14.56±2.45, T =7.386, 9.294, P =0.004, 0.000). The levels of SOD and GSH in serum of NT-3 intervention group were significantly increased (t=9.117, 12.207, P=0.000, 0.000), while the level of MDA was significantly decreased (t=5.089, P=0.011). Serum levels of inflammatory cytokines IL-1β, IL-6, IL-17 and TNF-α in NT-3 intervention group were significantly decreased (T =6.157, 7.958, 6.339, 6.288, P=0.008, 0.005, 0.005, 0.007). In the NT-3 treatment group, Bax protein was significantly decreased (0.24±0.05 vs 0.89±0.12, T =8.579, P=0.001), and the relative expression of Bcl-2 protein was significantly increased (0.75±0.06 vs 0.13±0.05, T =9.367, P=0.001). Neurotrophic factor III can promote spinal injury repair in spinal injury model rats, and play a role by enhancing antioxidant stress ability, inhibiting inflammatory factors, promoting Bcl-2 and decreasing Bax expression.

Design and synthesis of novel 3-amino-5-phenylpyrazole derivatives as tubulin polymerization inhibitors targeting the colchicine-binding site

As the basic unit of microtubules, tubulin is one of the most important targets in the study of anticarcinogens. A novel series of 3-amino-5-phenylpyrazole derivatives were designed and synthesized, and evaluates for their biological activities. Among them, a majority of compounds exerted excellent inhibitory activities against five cancer cell lines in vitro. Especially, compound 5b showed a strong antiproliferative activity against MCF-7 cells, with IC50 value of 38.37 nM. Further research indicated that compound 5b can inhibit the polymerization of tubulin targeting the tubulin colchicine-binding sites. Furthermore, 5b could arrest MCF-7 cells at the G2/M phase and induce MCF-7 cells apoptotic in a dose-dependent and time-dependent manners, and regulate the level of related proteins expression. Besides, compound 5b could inhibit the cancer cell migration and angiogenesis. In addition, 5b could inhibit tumor growth in MCF-7 xenograft model without obvious toxicity. All these results indicating that 5b could be a promising antitumor agent targeting tubulin colchicine-binding site and it was worth further study.