Immunofluorescence Technique Research Articles

Huang-Lian-Jie-Du Decoction alleviates diabetic encephalopathy by regulating inflammation and pyroptosis via suppression of AGEs/RAGE/NF-κB pathways

Ethnopharmacological relevanceCognitive dysfunction associated with diabetes, known as diabetic encephalopathy (DE), is a grave neurodegenerative condition triggered by diabetes, and persistent inflammation plays a vital role in its development. The renowned traditional Chinese medicine Huang-Lian-Jie-Du Decoction (HLJDD) is clinically proven to manage diabetes mellitus and Alzheimer's disease and is famous for its heat-clearing and detoxifying effects. However, the underlying mechanisms through which HLJDD affects DE remain to be elucidated. Aim of the studyTo explore the beneficial effects of HLJDD on improving cognitive dysfunction in DE mice. Study design and methodsA diabetic mouse was established through a high-fat diet and subsequent administration of streptozotocin over five consecutive days. After the animals were confirmed to have diabetes, they were treated with HLJDD. After oral administration of HLJDD or metformin for 14 weeks, behavioral tests were used to assess their cognitive capacity. Biochemical analyses were then performed to detect levels of glucose metabolism, followed by histological analyses to assess pathological damage. Furthermore, AGEs/RAGE/NF-κB axis related proteins were detected by Western blot or immunofluorescence techniques. An advanced UPLC-Q-Orbitrap HRMS/MS analytical technique utilizing a chemical derivatization strategy was employed for comprehensive metabolic profiling of carbonyl compounds in the plasma of DE mice. ResultsPharmacological assessment revealed that HLJDD effectively mitigated cognitive dysfunction, normalized glucose metabolic imbalances, and repaired neuronal damage in DE mice. It reduced neuroinflammation by attenuating carbonyl stress, deactivating astrocytes and microglia, and preserving dopaminergic neurons. Additionally, metabolomics analysis revealed 18 carbonyl compounds with marked disparities between DE and control mice, with 12 metabolites approaching normal levels post-HLJDD intervention. Further investigations showed that HLJDD regulated inflammation and pyroptosis through suppressing AGEs/RAGE/NF-κB pathways. ConclusionOur study indicated that HLJDD could ameliorate carbonyl stress via the regulation of carbonyl compound metabolism profiling, and inhibiting the AGEs/RAGE/NF-κB pathway, thereby alleviating inflammation and pyroptosis to exert beneficial effects on DE.

Liquiritin ameliorates painful diabetic neuropathy in SD rats by inhibiting NLRP3-MMP-9-mediated reversal of aquaporin-4 polarity in the glymphatic system.

Despite advancements in diabetes treatment, the management of Painful Diabetic Neuropathy (PDN) remains challenging. Our previous research indicated a significant correlation between the expression and distribution of Aquaporin-4 (AQP4) in the spinal glymphatic system and PDN. However, the potential role and mechanism of liquiritin in PDN treatment remain uncertain. This study established a rat model of PDN using a combination of low-dose Streptozotocin (STZ) and a high-fat, high-sugar diet. Rats were treated with liquiritin and MCC950 (an NLRP3 inhibitor). We monitored fasting blood glucose, body weight, and mechanical allodynia periodically. The glymphatic system's clearance function was evaluated using Magnetic Resonance Imaging (MRI), and changes in proteins including NLRP3, MMP-9, and AQP4 were detected through immunofluorescence and Western blot techniques. The rats with painful diabetic neuropathy (PDN) demonstrated several physiological changes, including heightened mechanical allodynia, compromised clearance function within the spinal glymphatic system, altered distribution of AQP4, increased count of activated astrocytes, elevated expression levels of NLRP3 and MMP-9, and decreased expression of AQP4. However, following treatment with liquiritin and MCC950, these rats exhibited notable improvements. Liquiritin may promote the restoration of AQP4 polarity by inhibiting NLRP3 and MMP-9, thereby enhancing the clearance functions of the spinal cord glymphatic system in PDN rats, alleviating the progression of PDN.

The mechanism of INO80D involved in chromatin remodeling regulating spermatogenesis in Chinese mitten crab (Eriocheir sinensis).

The INO80D protein, a component of the INO80 chromatin remodeling complex, plays a pivotal role in chromatin remodeling, gene expression, and DNA repair within mammalian sperm. In contrast to the condensed nuclear structure of mammalian sperm, Chinese mitten crab, Eriocheir sinensis, exhibits a distinctively decondensed sperm nucleus. The distribution and function of INO80D during the E. sinensis spermatogenesis were previously enigmatic. Our research endeavored to elucidate the distribution and function of INO80D, thereby enhancing our comprehension of sperm decondensation and the process of spermatogenesis in this species. Employing transcriptome sequencing, RT-qPCR, western blot analysis, and immunofluorescence techniques, we observed a pronounced upregulation of INO80D in the adult E. sinensis in comparison to the juvenile. The protein predominantly resides in the cellular nucleus, with high levels in spermatogonia and spermatocytes, less in stage I and III spermatids, and lowest in mature sperm. The results indicated that INO80D is initially instrumental in chromatin decondensation to facilitate gene accessibility and DNA repair during the early phases of spermatogenesis. Its role subsequently shifts to maintaining decondensed chromatin stability and genetic integrity during spermiogenesis. The sustained presence of INO80D during spermiogenesis is essential for the ultimate maturation of the decondensed sperm nucleus, imperative for preserving the unique decondensed state and the protection of genetic material in E. sinensis. Our study concludes that INO80D exerts a multifaceted influence on the spermatogenesis of E. sinensis, impacting chromatin decondensation, genetic integrity, and the regulation of early gene expression. This understanding could potentially improve crab breeding in aquaculture.

Improvement of olfactory function in AD mice mediated by immune responses under 40 Hz light flickering

Background40 Hz light flickering has shown promise as a non-invasive therapeutic approach for alleviating both pathological features and cognitive impairments in Alzheimer’s disease (AD) model mice and AD patients. Additionally, vision may influence olfactory function through cross-modal sensory interactions. ObjectiveTo investigate the impact of 40 Hz light flickering on olfactory behavior in AD model mice and to explore the underlying mechanisms of this intervention. MethodsWe used immunofluorescence techniques to observe the activation of the olfactory bulb (OB) in C57BL/6J mice under 40 Hz light flickering. A buried food test was conducted to evaluate olfactory behavior in AD mice. Additionally, RNA sequencing technology was employed to detect transcriptional alterations in the OBs of AD mice following light stimulation. Results40 Hz light flickering was found to effectively activate the OB. This stimulation led to enhanced olfactory behavior and did not alter P-tau protein mRNA levels within the OBs of AD mice. RNA sequencing revealed significant transcriptional changes in the OBs under flickering, particularly related to immune responses. ConclusionVision can influence olfactory function through cross-modal sensory interactions in rodent models. 40 Hz light stimulation improved olfactory performance in AD mice. However, the improvement in olfaction in AD mice is not related to changes in P-tau mRNA levels. Instead, it may be associated with an altered immune response, providing a scientific basis for the clinical treatment of olfactory disorders in Alzheimer’s disease.

Effects and Mechanisms of the Xianhecao-Huanglian Drug Pair on Autophagy-Mediated Intervention in Acute Inflammatory Bowel Disease via the JAK2/STAT3 Pathway

To explore the effects and mechanisms of the Xianhecao-Huanglian drug pair on autophagy-mediated intervention in acute inflammatory bowel disease (IBD) via the JAK2/STAT3 pathway. The study examined the underlying mechanisms of action of Xianhecao (APL) and Huanglian (CR) using a mouse model of dextran sodium sulfate (DSS)-induced acute inflammatory bowel disease (IBD) and in an in vitro model of IBD induced by lipopolysaccharide (LPS). The assessment of the therapeutic efficacy of the Xianhecao-Huanglian drug combination in a mouse model of IBD caused by DSS included the following parameters: Assessment of weight loss or gain. Measurement of the disease activity index (DAI). Assessment of histological damage. Determination of organ index. Measurement of colon length. Ascertain the levels of inflammatory cytokines in the intestinal tissues and serum of mice. Immunohistochemistry (IHC) for the measurement of tight junction protein concentrations in the colon mucosa, including ZO-1, claudin-1, and occludin. Measurement of mucin levels, specifically Mucin 2 (Muc2). Hematoxylin and eosin (HE) staining for the observation of histopathological alterations in colonic tissues. Examining the effect on goblet cells using periodic acid-Schiff (PAS) labeling. Application of Western blot and immunofluorescence techniques for the detection of autophagy-related markers in colonic tissues and proteins associated with the JAK2/STAT3 pathway. A cell inflammation model of IBD was induced through LPS stimulation, and a serum containing the Xianhecao-Huanglian drug pair (referred to as ACHP-DS) was formulated. Cell viability, anti-proinflammatory cytokines, tight junction proteins, mucins, autophagy-related markers, and the JAK2/STAT3 signaling pathway were assessed. The Xianhecao-Huanglian drug pair significantly ameliorated the symptoms and survival quality of acute IBD mice, reducing the disease activity index score, raising MUC2 secretion and tight junction protein expression to improve the integrity of the intestinal barrier, and preserving goblet cell function; thus, protecting the intestines. It effectively restrained triggering the signaling pathway that involves JAK2 and STAT3, leading to the suppression of inflammation and amelioration of colonic inflammation damage. Additionally, it induced autophagy in mouse colonic tissues.The in vitro experiments demonstrated that the Xianhecao-Huanglian drug combination enhanced the viability of LOVO and NCM460 cells when exposed to LPS stimulation. Furthermore, it suppressed the production of inflammatory cytokines such as IL-6, IL-1β, as well as TNF-α, whilst increasing the production of IL-10, ZO-1, along with MUC2. These effects collectively led to the alleviation of inflammation and the restoration of mucosal integrity. The results were consistent with what was shown in the in vivo trial. Moreover, the medication demonstrated effectiveness in reducing JAK2 along with STAT3 phosphorylation levels in the LPS-induced inflammatory model of IBD cells. The intervention with either the Xianhecao-Huanglian drug combination-containing serum or the JAK2/STAT3 pathway inhibitor AG490 reversed the pro-inflammatory effects and increased autophagy levels in the LPS-stimulated cells. The Xianhecao-Huanglian drug combination modulates the JAK2/STAT3 pathway, leading to the induction of autophagy, which serves as an intervention for IBD.

EEF1B2 regulates the proliferation and apoptosis of human spermatogonial stem cell lines through TAF4B

BackgroundSpermatogonial stem cells (SSCs) are essential for male fertility, maintaining sperm production throughout life. While mouse SSCs have been studied extensively, the mechanisms regulating human SSCs are less understood. ObjectivesTo investigate the role of EEF1B2 in regulating human SSC proliferation and apoptosis. Material and methodsSingle cell RNA sequencing (scRNA-seq) analysis was utilized to investigate the differentially expressed genes of SSC. The distribution of EEF1B2 in the human testis was examined using immunofluorescence and immunohistochemistry techniques. Cell proliferation, DNA replication, and self-renewal were analyzed using CCK8, EdU, Western blot, and flow cytometry. RNA sequencing was employed to analyze the downstream target molecules and signaling pathways of EEF1B2. ResultsIn this study, we analyzed single-cell sequencing data from human testicular samples and identified EEF1B2 as a protein highly expressed in SSCs, with expression decreasing during development. Immunohistochemistry and immunofluorescence confirmed this pattern and showed co-localization with the proliferation marker KI67. Knockdown of EEF1B2 in human SSC lines impaired proliferation and viability, reducing self-renewal proteins like PLZF and CCNE1. RNA sequencing revealed decreased TAF4B following EEF1B2 knockdown, which could be rescued by replenishing TAF4B. Testicular SSCs from non-obstructive azoospermia (NOA) patients also showed reduced EEF1B2. Discussion and conclusionOur findings reveal a novel regulatory mechanism involving EEF1B2 and TAF4B in human SSCs, suggesting EEF1B2 deficiency may contribute to male infertility.

Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D

Immunohistochemistry is a crucial method for detecting specific proteins within tissue samples, yet constrained to one biomarker per tissue section. Multiplexed immunofluorescence, while allowing simultaneous visualization of multiple proteins, faces limitations in the number of simultaneous fluorescent labels due to spectral overlap. Although cyclic immunofluorescence techniques have successfully broadened antibody staining capacities in a single tissue sample, they are plagued by time-consuming and labor-intensive procedures, sample degradation risks, and inability to scale beyond thin sections. In this study, we introduce the use of 3D confocal Fluorescence Lifetime Imaging Microscopy as a high-throughput, multiplexed immunofluorescence platform that can differentiate 11 or more biomarkers in 3D tissue volumes. Leveraging both spectral and lifetime information, this approach allows for practical spatial biology in thin sections that can readily scale to larger volumes of tissue. We believe that this highly multiplexed and versatile biomarker imaging platform will significantly expedite cancer research and enable new translational approaches in the future.

Mechanism of Linggui Zhugan Decoction in inhibiting myocardial fibrosis by regulating Wnt/β-catenin pathway

This study aimed to investigate the regulatory mechanism of Linggui Zhugan Decoction(LGZGD)-medicated serum on the fibrosis of cardiac fibroblasts(CFs) and the protein expression of the Wnt/β-catenin signaling pathway. Blank serum and LGZGD-medicated serum were prepared, and primary CFs were isolated and cultured using trypsin-collagenase digestion and differential adhesion method. Immunofluorescence labeling was used to identify primary CFs. Cells were divided into normal control group, model group, 20% blank serum group, and 5%, 10%, and 20% LGZGD-medicated serum groups. Except for the normal control group, all other groups were stimulated with hydrogen peroxide(H_2O_2) after pretreatment with 20% blank serum or 5%, 10%, 20% LGZGD-medicated serum for 12 hours to establish a model of fibrosis in primary CFs. Scratch healing assay was used to observe cell migration ability. ELISA was used to detect the content of collagen type Ⅰ(Col Ⅰ) and type Ⅲ(Col Ⅲ). Western blot was used to detect the protein expression of α-smooth muscle actin(α-SMA), Wnt1, glycogen synthase kinase 3β(GSK-3β), phosphorylated GSK-3β(p-GSK-3β), β-catenin, and nuclear β-catenin. RT-qPCR was used to detect the gene expression of β-catenin and matrix metalloproteinase 9(MMP9), and immunofluorescence technique was used to detect the expression and localization of key proteins α-SMA and β-catenin. CFs with Wnt1 overexpression were prepared and treated with H_2O_2. The following groups were set up: normal control group, model group, 20% LGZGD-medicated serum group, empty plasmid+20% LGZGD-medicated serum group, and Wnt1 overexpression+20% LGZGD-medicated serum group. ELISA was used to detect the content and ratio of Col Ⅰ and Col Ⅲ. Western blot was used to detect the protein expression of α-SMA, Wnt1, GSK-3β, p-GSK-3β, β-catenin, and nuclear β-catenin. RT-qPCR was used to detect the gene expression of β-catenin and MMP9. Immunofluorescence staining showed that CFs expressed Vimentin positively, appearing green, with blue nuclei and purity greater than 90%, which were identified as primary CFs. RESULTS:: showed that compared with the normal control group, CFs in the model group had enhanced healing rate, increased content of Col Ⅰ and Col Ⅲ, increased ratio of Col Ⅰ/Col Ⅲ, upregulated protein expression of α-SMA, Wnt1, p-GSK-3β, β-catenin, nuclear β-catenin, decreased GSK-3β expression, elevated mRNA expression of β-catenin and MMP9, and enhanced fluorescence intensity and expression of β-catenin and α-SMA. Compared with the model group, 5%, 10%, 20% LGZGD-medicated serum significantly inhibited cell migration ability, reduced the content of Col Ⅰ and Col Ⅲ, decreased ratio of Col Ⅰ/Col Ⅲ, downregulated protein expression of α-SMA, Wnt1, p-GSK-3β, β-catenin, nuclear β-catenin, increased GSK-3β expression, decreased mRNA expression of β-catenin and MMP9, and reduced fluorescence intensity and expression of β-catenin and α-SMA. Compared with the empty plasmid+20% LGZGD-medicated serum group, the effect of LGZGD-medicated serum was significantly reversed after overexpression of Wnt1. LGZGD can reduce excessive deposition of collagen fibers, inhibit excessive proliferation of fibroblasts, and improve the process of myocardial fibrosis. The improvement of myocardial fibrosis by LGZGD is related to the regulation of the Wnt/β-catenin pathway, reduction of collagen deposition, and protection of myocardial cells.

Combined bacterial translocation and cholestasis aggravates liver injury by activation pyroptosis in obstructive jaundice

This study explores the mechanism by which obstructive jaundice (OJ) induces liver damage through pyroptosis. We induced OJ in rats via bile duct ligation and assessed liver damage using serum biochemical markers and histological analysis of liver tissue. Pyroptosis was investigated through immunofluorescence, ELISA, Western blot, and quantitative RT-PCR techniques. Additionally, we examined intestinal function and fecal microbiota alterations in the rats using 16S rDNA sequencing. In vitro experiments involved co-culturing Kupffer cells and hepatocytes, which were then exposed to bile and lipopolysaccharide (LPS). Our findings indicated that OJ modified the gut microbiota, increasing LPS levels, which, in conjunction with bile, initiated a cycle of inflammation, fibrosis, and cell death in the liver. Mechanistically, OJ elevated necrotic markers such as ATP, which in turn activated pyroptotic pathways. Increased levels of pyroptosis-related molecules, including NLRP3, caspase-1, gasdermin D, and IL-18, were confirmed. In our co-cultured cell model, bile exposure resulted in cell death and ATP release, leading to the activation of the NLRP3 inflammasome and its downstream effectors, caspase-1 and IL-18. The combination of bile and LPS significantly intensified pyroptotic responses. This study is the first to demonstrate that LPS and bile synergistically exacerbate liver injury by promoting necrosis and pyroptosis, unveiling a novel mechanism of OJ-associated hepatic damage and suggesting avenues for potential preventive or therapeutic interventions.

Crosstalk between cytokines, inflammation and pulmonary arterial hypertension in heart transplant patients

Background: Heart transplant (HT) is a therapeutic option for patients with advanced heart failure (HF) refractory to optimized treatment. Patients with advanced HF often develop pulmonary arterial hypertension (PAH). PAH is defined as a condition in which the mean pulmonary artery pressure is greater than 20 mmHg. Inflammation is an important aspect of PAH development. In this context, the objective of this work was to evaluate the relationship between the inflammatory process and the development of HAP in patients undergoing HT. Methods: The levels of interleukins IL-6, IL-1β and TNF-α were obtained by ELISA and associated with CD68+ and CD66b neutrophil counts using the immunofluorescence technique in fragments of the pulmonary arteries of donors and patients with or without chagasic cardiomyopathy subjected to HT. Results: The results showed a positive, statistically significant correlation (p < 0.05) between right atrium pressure levels and IL-6. Furthermore, negative, moderate, and statistically significant correlations (p < 0.05) were observed between the variables cardiac index and TNF-α, and between the levels of transpulmonary pressure grandient and TNF-α. The study also revealed the presence of a statistically significant difference (p < 0.05) between patients who died within 30 days and the highest number of CD68 cells per square micrometer in the vessel of the donor and recipient patient. Conclusion: Suggesting the presence of a pro-inflammatory profile in HT patients, independent of measured pulmonary artery pressure levels.

Miamiensis avidus, a Novel Scuticociliate Pathogen Isolated and Identified from Cultured Large Yellow Croaker (Larimichthys crocea).

Scuticociliates are recognized as the causative agents of mass mortalities in certain cultured marine fishes, resulting in enormous economic losses. This study aimed to investigate a fatal infection caused by scuticociliates in farmed large yellow croaker (Larimichthys crocea) in Fujian province, China. Microscopic examinations of focal organs, including the brain, eyes, gills, and skin, revealed the presence of parasites. Active masses of scuticociliates were observed in these organs, and the ciliates were subsequently isolated and maintained in vitro. An immersion challenge experiment revealed that L. crocea experienced cumulative mortalities reaching 73% within 7 d upon exposure to 1.0 × 104 ciliates mL-1. Based on the microscopic and PCR testing of infected fishes, the brain was comprehensively inferred as the main infection organ for the isolated strain. Microscopic and submicroscopic observations of the isolated scuticociliate, coupled with cortical ciliature patterns revealed through α-tubulin indirect immunofluorescence techniques, identified these scuticociliates as Miamiensis avidus. The sequencing of two genetic markers (small subunit ribosomal RNA, SSU rRNA and cytochrome c oxidase subunit I, COI) further confirmed that the isolated strains exhibited the highest sequence similarity to most M. avidus sequences in GenBank. However, significant differences in SSU sequences compared to the M. avidus strain Ma/2, and the lack of published COI and ITS (internal transcribed spacer) sequences for Ma/2, indicate the need for further molecular data to resolve whether there are potential cryptic species within the M. avidus complex.

Protoporphyrin IX metabolism mediated via translocator protein (CgTspO) involved in orange shell coloration of pacific oyster (Crassostrea gigas)

Mollusc shell color polymorphism is influenced by various factors. Pigments secreted in vivo by animals play a critical role in shell coloration. Among the different shell-color hues, orange pigmentation has been partially attributed to porphyrins. However, the detailed causal relationship between porphyrins and orange-shell phenotype in molluscs remains largely unexplored. The various strains of Pacific oyster (Crassostrea gigas) with different shell color provide useful models to study the molecular regulation of mollusc coloration. Accordingly, oysters with orange and gold-shells, exhibiting distinct porphyrin distributions, were selected for analysis of total metabolites and gene expression profile through mantle metabolomic and transcriptomic studies. Translocator protein (TspO) and protoporphyrin IX (PPIX) were identified as potential factors influencing oyster shell-color. The concentration of PPIX was measured using HPLC, while expression profiling of CgTspO was analyzed by qPCR, in situ hybridization, Western blotting, and immunofluorescence techniques. Moreover, the roles of CgTspO in regulating PPIX metabolism and affecting the orange-shell-coloration were investigated in vitro and in vivo. These studies indicate that PPIX and its associated metabolic protein, CgTspO may serve as new regulators of orange-shell-coloration in C. gigas. Data of this study offer new insights into oyster shell coloration and enhancing understandings of mollusc shell color polymorphism.

Curcumin-activated Wnt5a pathway mediates Ca2+ channel opening to affect myoblast differentiation and skeletal muscle regeneration.

Skeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca2+ channel. Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20μL). In addition, we constructed a Wnt5a knockdown C2C12 cell model and a Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80mg/L) and fed to the mice (50mg/kg). Fluorescence microscopy was used to determine the concentration of Ca2+. Western blot and RT-qPCR were used to detect the protein and mRNA levels of Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining. During myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P<0.001) and the Wnt5a signalling pathway was activated. Wnt5a overexpression promoted the expression of MyoD, Myf5, Myogenin, MHC, and Desmin (P<0.05), and conversely, knockdown of Wnt5a inhibited their expression (P<0.001). The Wnt5a pathway mediated the opening of Ca2+ channels, regulated the expression levels of CaN, NFAT2, MyoD, Myf5, Myogenin, MHC, and Desmin (P<0.01) and promoted the differentiation of C2C12 myoblasts and the repair and regeneration of injured skeletal muscle. The expression of Wnt5a, CaN, NFAT2, MyoD, Myogenin, Myf5, and MHC in C2C12 myoblast was significantly increased after curcumin intervention (P<0.05); however, their expression decreased significantly after knocking down Wnt5a on the basis of curcumin intervention (P<0.05). Similarly, in Wnt5a knockout mice, the promotion of muscle regeneration by curcumin was significantly attenuated. Curcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle.

IL-17 signaling pathway: A potential therapeutic target for reducing skeletal muscle inflammation

BackgroundThe interleukin-17 (IL-17) signaling pathway is intricately linked with immunity and inflammation; however, the association between the IL-17 signaling pathway and skeletal muscle inflammation remains poorly understood. The study aims to investigate the role of the IL-17 signaling pathway in skeletal muscle inflammation and to evaluate the therapeutic potential of anti-IL-17 antibodies in reducing muscle inflammation. MethodsA skeletal muscle inflammation model was induced by cardiotoxin (CTX) injection in C57BL6/J mice. Following treatment with an anti-IL-17 antibody, we conducted a comprehensive analysis integrating single-cell RNA sequencing (scRNA-seq), bioinformatics, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and Western blot techniques to elucidate underlying mechanisms. ResultsscRNA-seq analysis revealed a significant increase in neutrophil numbers and activity in inflamed skeletal muscle compared to other cell types, including macrophages, T cells, B cells, endothelial cells, fast muscle cells, fibroblasts, and skeletal muscle satellite cells. The top 30 differentially expressed genes within neutrophils, along with 55 chemokines, were predominantly enriched in the IL-17 signaling pathway. Moreover, the IL-17 signaling pathway exhibited heightened expression in inflamed skeletal muscle, particularly within neutrophils. Treatment with anti-IL-17 antibody resulted in the suppression of IL-17 signaling pathway expression, accompanied by reduced levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, as well as decreased numbers and activity of Ly6g+/Mpo+ neutrophils compared to CTX-induced skeletal muscle inflammation. ConclusionOur findings suggest that the IL-17 signaling pathway plays a crucial role in promoting inflammation within skeletal muscle. Targeting this pathway may hold promise as a therapeutic strategy for ameliorating the inflammatory micro-environment and reducing cytokine production.

Ginsenoside Rg1 regulates astrocytes to promote angiogenesis in spinal cord injury via the JAK2/STAT3 signaling pathway

Ethnopharmacological relevanceGinseng (Panax ginseng C. A. Mey) is a common traditional Chinese medicine used for anti-inflammation, anti-apoptosis, anti-oxidative stress, and neuroprotection. Ginsenosides Rg1, the main active components isolated from ginseng, may be a feasible therapy for spinal cord injury (SCI). Aims of the studySCI causes endothelial cell death and blood vessel rupture, ultimately resulting in long-term neurological impairment. As a result, encouraging spinal angiogenesis may be a feasible therapy for SCI. This investigation aimed to validate the capacity of ginsenoside Rg1 in stimulating angiogenesis within the spinal cord. Materials and methodsRats with SCI were injected intraperitoneally with ginsenoside Rg1. The effectiveness of ginsenoside Rg1 was assessed using the motor function score and the motor-evoked potential (MEP). Immunofluorescence techniques were applied to identify the spinal cord's angiogenesis. Angiogenic factors were examined through Western Blot (WB) and Immunohistochemistry. Oxygen-glucose deprivation (OGD) was employed to establish the hypoxia-ischemia model in vitro, and astrocytes (As) were given ginsenoside Rg1 and co-cultured with spinal cord microvascular endothelial cells (SCMECs). Immunofluorescence, wound healing test, and tube formation assay were used to identify the co-cultured SCMECs' activity. Finally, network pharmacology analysis and siRNA transfection were applied to verify the mechanism of ginsenoside Rg1 promoting angiogenesis. ResultsThe rats with SCI treated with ginsenoside Rg1 indicated more significant functional recovery, more pronounced angiogenesis, and higher levels of angiogenic factor expression. In vitro, the co-culture system with ginsenoside Rg1 intervention improved SCMECs' capacity for proliferating, migrating, and forming tubes, possibly by promoting the expression of vascular endothelial growth factor (VEGF) in As via the janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. ConclusionGinsenoside Rg1 can regulate As to promote angiogenesis, which may help to understand the mechanism of promoting SCI recovery.

P-115 GATAD2B is required for pre-implantation embryo development by delaying zygotic genome activation

Abstract Study question Major zygotic genome activation (ZGA) involving the activation of thousands of genes occurs at the late-2cell stage, but the regulatory mechanisms governing ZGA remain unclear. Summary answer Our results demonstrate that GATAD2B is essential for early embryonic development, in part through facilitating zygotic genome activation (ZGA). What is known already Acetylation modifications of histone tails promote transcriptional activation, and the maternal deletion of H4K16ac leads to failure in ZGA. GATAD2B is one of the core subunits of the Nucleosome remodeling and histone deacetylation complex(NuRD). Study design, size, duration Using mouse embryos as the research model, the study was conducted over a period of approximately two years to ensure a relatively small-scale investigation. Participants/materials, setting, methods We examined the dynamic expression of NURD complex components, including GATAD2B, during pre-implantation embryonic stages. GATAD2B expression was specifically reduced via microinjection of GATAD2B-siRNA during the zygote stage, revealing its impact on early embryonic development in mice. Zygotic genome activation (ZGA), morula compaction, and blastocyst lineage differentiation were investigated using immunofluorescence, western blot, and other techniques. Additionally, we employed RNA-seq, protein immunoprecipitation, and mass spectrometry to uncover underlying molecular mechanisms. Main results and the role of chance Our research has shown that GATAD2B exhibits specific nucleus localization and high protein expression from the late-2-cell to 8-cell. This intriguing phenomenon prompted us to investigate the relationship between GATAD2B and the ZGA. We discovered a distinctive pattern of GATAD2B, starting from the late 2-cell stage with nuclear localization. GATAD2B depletion resulted in defective embryonic development, including increased DNA damage at morula, decreased blastocyst formation rate and abnormal differentiation of ICM/TE lineages. Consistent with the delay during the cleavage stage, the transcriptome analysis of the 2-cell revealed inhibition of the cell cycle G2/M phase transition pathway. Furthermore, the GATAD2B proteomics data provided clear evidence of a certain association between GATAD2B and molecules involved in the cell cycle pathway. As hypothesized, GATAD2B-deficient 2-cell embryos exhibited abnormalities in ZGA during the maternal-to-embryonic transition, with lower expression of the major ZGA mark MERVL. Limitations, reasons for caution Given the limitations of antibody effectiveness and specificity required for the experiments, the exploration of molecular mechanisms is still ongoing. Wider implications of the findings Our findings have elucidated the impact of GATAD2B on early embryonic development through the regulation of zygotic genome activation (ZGA), providing a novel genetic explanation for clinical defects in early embryonic development. Trial registration number not applicable

G-CSF promotes the development of hepatocellular carcinoma by activating the PI3K/AKT/mTOR pathway in TAM.

This investigation seeks to elucidate the role of the Granulocyte Colony-Stimulating Factor (G-CSF) in the progression of hepatocellular carcinoma (HCC), as well as the impact of the substance on related signaling pathways within the disease matrix. Nude mouse tumor-bearing assay was used to detect tumor progression. Levels of Mannose/CD68 and CD34/Mannose within these samples and the concentrations of Mannose and inducible Nitric Oxide Synthase (iNOS) in macrophages were quantified using immunofluorescence techniques. The angiogenic capability was assessed via tube formation assays, and protein expressions of G-CSF, Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-beta (TGF-β), Matrix Metalloproteinases 2 and 9 (MMP2/9), SH2-containing protein tyrosine phosphatase-2 (SHP-2), phosphorylated PI3K/total PI3K (P-PI3K/t-PI3K), phosphorylated AKT/total AKT (P-AKT/t-AKT), and phosphorylated mTOR/total mTOR (P-mTOR/t-mTOR) were measured through Western Blot analysis in both tumor tissues and macrophages. Administration of G-CSF resulted in a marked augmentation of tumor volume. Macrophage Mannose expression was significantly elevated upon G-CSF treatment, while iNOS levels were conspicuously diminished. G-CSF substantially enhanced the secretion of VEGF, TGF-β, and MMPs in tumor tissues. Macrophage parameters, following incubation in G-CSF pre-treated conditioned medium, indicated enhanced tube-forming capabilities relative to the control, an effect mitigated by the introduction of specific inhibitors. Furthermore, the G-CSF group exhibited a notable reduction in SHP-2 expression, alongside a substantial elevation in the phosphorylation levels of the PI3K/AKT/mTOR pathway proteins across all tumor-bearing paradigms. G-CSF ostensibly facilitates the advancement of hepatocellular carcinoma by activating the PI3K/AKT/mTOR signaling cascade within Tumor-Associated Macrophages (TAM).

Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells.

Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.

Dystrophin 71 deficiency causes impaired aquaporin-4 polarization contributing to glymphatic dysfunction and brain edema in cerebral ischemia

ObjectiveThe glymphatic system serves as a perivascular pathway that aids in clearing liquid and solute waste from the brain, thereby enhancing neurological function. Disorders in glymphatic drainage contribute to the development of vasogenic edema following cerebral ischemia, although the molecular mechanisms involved remain poorly understood. This study aims to determine whether a deficiency in dystrophin 71 (DP71) leads to aquaporin-4 (AQP4) depolarization, contributing to glymphatic dysfunction in cerebral ischemia and resulting in brain edema. MethodsA mice model of middle cerebral artery occlusion and reperfusion was used. A fluorescence tracer was injected into the cortex and evaluated glymphatic clearance. To investigate the role of DP71 in maintaining AQP4 polarization, an adeno-associated virus with the astrocyte promoter was used to overexpress Dp71. The expression and distribution of DP71 and AQP4 were analyzed using immunoblotting, immunofluorescence, and co-immunoprecipitation techniques. The behavior ability of mice was evaluated by open field test. Open-access transcriptome sequencing data were used to analyze the functional changes of astrocytes after cerebral ischemia. MG132 was used to inhibit the ubiquitin-proteasome system. The ubiquitination of DP71 was detected by immunoblotting and co-immunoprecipitation. ResultsDuring the vasogenic edema stage following cerebral ischemia, a decline in the efflux of interstitial fluid tracer was observed. DP71 and AQP4 were co-localized and interacted with each other in the perivascular astrocyte endfeet. After cerebral ischemia, there was a notable reduction in DP71 protein expression, accompanied by AQP4 depolarization and proliferation of reactive astrocytes. Increased DP71 expression restored glymphatic drainage and reduced brain edema. AQP4 depolarization, reactive astrocyte proliferation, and the behavior of mice were improved. After cerebral ischemia, DP71 was degraded by ubiquitination, and MG132 inhibited the decrease of DP71 protein level. ConclusionAQP4 depolarization after cerebral ischemia leads to glymphatic clearance disorder and aggravates cerebral edema. DP71 plays a pivotal role in regulating AQP4 polarization and consequently influences glymphatic function. Changes in DP71 expression are associated with the ubiquitin-proteasome system. This study offers a novel perspective on the pathogenesis of brain edema following cerebral ischemia.

A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types.

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience. In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution. Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across differentcontexts.