TGF-β1 Expression Research Articles

Mutations in fibronectin dysregulate chondrogenesis in skeletal dysplasia.

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER. Massive amounts of these vesicles were not cleared from the cytosol, and a smaller subset showed the presence of lysosomal markers. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGFβ1) expression, crucial for mesenchymal condensation. Exogenous FN or TGFβ1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis.

Interleukin-2 improves insulin sensitivity through hypothalamic sympathetic activation in obese mice

BackgroundIL-2 regulates T cell differentiation: low-dose IL-2 induces immunoregulatory Treg differentiation, while high-dose IL-2 acts as a potent activator of cytotoxic T cells and NK cells. Therefore, high-dose IL-2 has been studied for use in cancer immunotherapy. We aimed to utilize low-dose IL-2 to treat inflammatory diseases such as obesity and insulin resistance, which involve low-grade chronic inflammation.Main bodySystemic administration of low-dose IL-2 increased Treg cells and decreased inflammation in gonadal white adipose tissue (gWAT), leading to improved insulin sensitivity in high-fat diet-fed obese mice. Additionally, central administration of IL-2 significantly enhanced insulin sensitivity through the activation of the sympathetic nervous system. The sympathetic signaling induced by central IL-2 administration not only decreased interferon γ (IFNγ) + Th1 cells and the expression of pro-inflammatory cytokines, including Il-1β, Il-6, and Il-8, but also increased CD4 + CD25 + FoxP3 + Treg cells and Tgfβ expression in the gWAT of obese mice. These phenomena were accompanied by hypothalamic microgliosis and activation of pro-opiomelanocortin neurons. Furthermore, sympathetic denervation in gWAT reversed the enhanced insulin sensitivity and immune cell polarization induced by central IL-2 administration.ConclusionOverall, we demonstrated that IL-2 improves insulin sensitivity through two mechanisms: direct action on CD4 + T cells and via the neuro-immune axis triggered by hypothalamic microgliosis.

Short- and long-term immunosuppressive effects of melanoma influence the prognostic value of the sentinel lymph node status

BackgroundPresence of micrometastases in the sentinel lymph node (SLN) is currently used to assess prognosis of melanoma patients. The immunoactivity within the SLN is known to be influenced by the primary tumor (PT), which may in turn impact the SLNs’ metastatic state. AimWe characterize the temporal dependence and underlying mechanisms of the immunological effects of the PT on the SLN. MethodsThe prognostic value of SLN state as a function of PT removal time was evaluated. To put the results into a functional context, selected PTs and corresponding SLNs were analyzed for gene and protein expression patterns. ResultsIn a cohort of 202 patients with known distant metastasis and similar PT prognostic characteristics, SLNs removed before or within one week after the PT (IM-SLN) had a higher incidence of micrometastases than those removed at least one week after the PT (DEL-SLN).The immunoactivity in IM-SLN was found to be lower than in DEL-SLN. Specifically, in IM-SLNs, T helper 17 / regulatory T-cells were predominant, whereas in DEL-SLNs, cytotoxic γδT-cells were more frequent. The higher immune activity in DEL-SLNs was probably facilitated by CD209+ antigen-presenting cells. Indeed, in PT with high TGFβ expression CD209+ cells appear to be trapped and no increased immunoactivity was observed in DEL-SLN. ConclusionsPresence of micrometastases in DEL-SLNs have a higher negative prognostic value as in IM-SLNs since they indicate not only a melanoma’s propensity to metastasize, but possibly also its capacity to escape immune responses.

Modification of transvaginal polypropylene mesh with co-axis electrospun nanofibrous membrane to alleviate complications following surgical implantation

BackgroundSurgeries for treating pelvic organ prolapse involving the utilization of synthetic mesh have been associated with complications such as mesh erosion, postoperative pain, and dyspareunia. This work aimed to reduce the surgical implantation-associated complications by nanofibrous membranes on the surface of the polypropylene mesh. The nanofiber of the nanofibrous membrane, which was fabricated by co-axial electrospinning, was composed of polyurethane as fiber core and gelatin as the fiber out layer. The biocompatibility of the modified mesh was evaluated in vitro by cell proliferation assay, immunofluorescence stain, hematoxylin-eosin (HE) staining, and mRNA sequencing. Polypropylene mesh and modified mesh were implanted in a rat pelvic organ prolapse model. Mesh-associated complications were documented. HE and Picro-Sirius red staining, immunohistochemistry, and western blotting were conducted to assess the interactions between the modified mesh and vaginal tissues.ResultsThe modified mesh significantly enhanced the proliferation of fibroblasts and exerted a positive regulatory effect on the extracellular matrix anabolism in vitro. When evaluated in vivo, no instances of mesh exposure were observed in the modified mesh group. The modified mesh maintained a relatively stable histological position without penetrating the muscle layer or breaching the epidermis. The collagen content in the vaginal wall of rats with modified mesh was significantly higher, and the collagen I/III ratio was lower, indicating better tissue elasticity. The expression of metalloproteinase was decreased while the expression levels of tissue inhibitor of metalloproteinase were increased in the modified mesh group, suggesting an inhibition of collagen catabolism. The expression of TGF-β1 and the phosphorylation levels of Smad3, p38 and ERK1/2 were significantly increased in the modified mesh group. NM significantly improved the biocompatibility of PP mesh, as evidenced by a reduction in macrophage count, decreased expression levels of TNF-α, and an increase in microvascular density.ConclusionsThe nanofibrous membrane-coated PP mesh effectively reduced the surgical implantation complications by inhibiting the catabolism of collagen in tissues and improving the biocampibility of PP mesh. The incorporation of co-axial fibers composed of polyurethane and gelatin with polypropylene mesh holds promise for the development of enhanced surgical materials for pelvic organ prolapse in clinical applications.

HACE2 upregulation and participation of macrophages and clear cells in the immune response of epididymis to SARS-CoV-2 in K18-hACE2 mice.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus caused the coronavirus disease 2019 pandemic, and the prevalence of deaths among men is higher than among women. The epididymis, divided into caput, corpus, and cauda, shows a region-specific immunity. The K18-hACE2 mouse expresses human angiotensin-converting enzyme 2 (hACE2), the receptor that allows SARS-CoV-2 infection. However, studies using this transgenic mouse to evaluate the impact of this viral infection in epididymis have not yet been performed. We evaluated the expression of hACE2 in the epididymis of SARS-CoV-2-infected K18-hACE2 mice, and assessed the epididymal immune response, focusing on F4/80+ mononuclear phagocytes and tumor necrosis factor-alpha expression. The following analyses were performed in the epididymal sections of infected mice: epithelial height and duct diameter, birefringent collagen, Terminal deoxynucleotidyl Transferase-mediated dUTP Nick End Labelling, immunoreactions for detection of hACE2, spike, FGF, V-ATPase, F4/80, tumor necrosis factor-alpha, and iNOS. Viral particles were identified under electron microscopy. hACE2, Rigi, Tgfb1 and Tnfa expression were also evaluated by real-time quantitative polymerase chain reaction. All epididymal regions expressed hACE2, which increased in all epididymal regions in the infected mice. However, the caput appeared to be the most infected region. Despite this, the caput region showed minimal changes while the cauda showed significant epithelial changes associated with increased iNOS immunoexpression. The F4/80+ mononuclear phagocyte area increased significantly in both stroma and epithelium. In addition to the epithelial and stromal mononuclear phagocytes, tumor necrosis factor-alpha was also detected in clear cells, whose cytoplasm showed a significant increase of this cytokine in the infected animals. The K18-hACE2 mouse is a useful model for evaluating the impact of SARS-CoV-2 infection in the epididymis. The infection induced hACE2 upregulation, favoring the virulence in the epididymis. The epididymal regions responded differentially to infection, and the activation of F4/80+ mononuclear phagocytes associated with the increased tumor necrosis factor-alpha immunolabeling in clear cells indicates a role of clear cells/mononuclear phagocytes immunoregulatory mechanisms in the epididymal immune response to SARS-CoV-2 infection.

Expression of HMGB1, TGF-β1, BIRC3, ADAM17, CDKN1A, and FTO in Relation to Left Ventricular Remodeling in Patients Six Months after the First Myocardial Infarction: A Prospective Study.

Background: After myocardial infarction (MI), adverse left ventricular (LV) remodeling may occur. This is followed by LV hypertrophy and eventually heart failure. The remodeling process is complex and goes through multiple phases. The aim of this study was to investigate the expression of HMGB1, TGF-β1, BIRC3, ADAM17, CDKN1A, and FTO, each involved in a specific step of LV remodeling, in association with the change in the echocardiographic parameters of LV structure and function used to assess the LV remodeling process in the peripheral blood mononuclear cells (PBMCs) of patients six months after the first MI. The expression of selected genes was also determined in PBMCs of controls. Methods: The study group consisted of 99 MI patients, who were prospectively followed-up for 6 months, and 25 controls. Cardiac parameters, measured via conventional 2D echocardiography, were evaluated at two time points: 3-5 days and 6 months after MI. The mRNA expression six-months-post-MI was detected using TaqMan® technology (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Results:HMGB1 mRNA was significantly higher in patients with adverse LV remodeling six-months-post-MI than in patients without adverse LV remodeling (p = 0.04). HMGB1 mRNA was significantly upregulated in patients with dilated LV end-diastolic diameter (LVEDD) (p = 0.03); dilated LV end-diastolic volume index (LVEDVi) (p = 0.03); severely dilated LV end-systolic volume index (LVESVi) (p = 0.006); impaired LV ejection fraction (LVEF) (p = 0.01); and LV enlargement (p = 0.03). It was also significantly upregulated in PBMCs from patients compared to controls (p = 0.005). TGF-β1 and BIRC3 mRNA were significantly lower in patients compared to controls (p = 0.02 and p = 0.05, respectively). Conclusions: Our results suggest that HMGB1 is involved in adverse LV remodeling six-months-post-MI, even on the mRNA level. Further research and validation are needed.

Macrophage erythropoietin signaling promotes macrophage-myofibroblast transformation and fibroblast-myofibroblast differentiation

While myofibroblasts are the key cause of abnormal extracellular matrix accumulation, the origin of which has not yet been fully elucidated. Recently, it has been found that macrophage-myofibroblast transformation (MMT) defined by the expression of both macrophage markers (F4/80 or CD68) and myofibroblast markers (α-SMA) is one of its important sources. In the process of MMT, it is unclear whether epor is involved. In this study, when BMDM was induced by tgf-β1, the number of F4/80+α-SMA+ cells increased, the cells polarized toward M2, and the expression of tgf-β1 increased. After the activation of epor, the number of F4/80 +α-SMA + cells and the polarization level of M2 were further increased. At the same time, we found that the conditioned medium from MMT cells could induce the activation of 3T3 cells with increased the expression of α-SMA and col-1. In contrast, the number of F4/80+α-SMA + cells, the polarization of M2, and the expression of Tgf-β1 decreased after epor was inhibited by siRNA. Our results demonstrate that the activation of epor in BMDMs could promote the transformation of macrophage-myofibroblast induced by TGF-β1.

Preconditioning of Mesenchymal Stem Cells Enhances the Neuroprotective Effects of Their Conditioned Medium in an Alzheimer's Disease In Vitro Model.

Alzheimer's disease (AD) develops as a result of oxidative damage to neurons and chronic inflammation of microglia. These processes can be influenced by the use of a conditioned medium (CM) derived from mesenchymal stem cells (MSCs). The CM contains a wide range of factors that have neurotrophic, antioxidant, and anti-inflammatory effects. In addition, the therapeutic potential of the CM can be further enhanced by pretreating the MSCs to increase their paracrine activity. The current study aimed to investigate the neuroprotective effects of CM derived from MSCs, which were either activated by a TLR3 ligand or exposed to CoCl2, a hypoxia mimetic (pCM or hCM, respectively), in an in vitro model of AD. We have developed a novel in vitro model of AD that allows us to investigate the neuroprotective and anti-inflammatory effects of MSCs on induced neurodegeneration in the PC12 cell line and the activation of microglia using THP-1 cells. This study demonstrates for the first time that pCM and hCM exhibit more pronounced immunosuppressive effects on proinflammatory M1 macrophages compared to CM derived from untreated MSCs (cCM). This may help prevent the development of neuroinflammation by balancing the M1 and M2 microglial phenotypes via the decreased secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased secretion of IL-4, as well as the expression of IL-10 and TGF-β by macrophages. Moreover, a previously unknown increase in the neurotrophic properties of hCM was discovered, which led to an increase in the viability of neuron-like PC12 cells under H2O2-induced oxidative-stress conditions. These results are likely associated with an increase in the production of growth factors, including vascular endothelial growth factor (VEGF). In addition, the neuroprotective effects of CM from preconditioned MSCs are also mediated by the activation of the Nrf2/ARE pathway in PC12 cells. TLR3 activation in MSCs leads to more potent immunosuppressive effects of the CM against pro-inflammatory M1 macrophages, while the use of hCM led to increased neurotrophic effects after H2O2-induced damage to neuronal cells. These results are of interest for the potential treatment of AD with CM from preactivated MSCs.

Endometriotic lesions and their recurrence: A Study on the mediators of immunoregulatory (TGF-β/miR-20a) and stemness (NANOG/miR-145)

Endometriosis is a common estrogen-dependent disease that involves various cellular processes. Additionally, miRNAs play a crucial role in the development of the disease as an important component of the microenvironment. In this study, tissue specimens of eutopic and ectopic lesions of 20 women, whose endometriosis was later approved by the pathology laboratory, were biopsied through laparoscopy. As a control group, endometrial tissue specimens were collected from 20 women who underwent curettage for reasons unrelated to endometriosis. The expression levels of miR-20A and miR-145 and their target genes, TGF-β and NANOG, were measured in these samples as markers of stemness and immunomodulatory properties, respectively. The study also aimed to compare the expression levels of target genes and miRNAs in ectopic lesions regarding endometriosis recurrence post-surgery. The study revealed that the expression of TGF-β and NANOG genes was significantly upregulated in endometriotic tissues compared to the control group. There was also a notable increase in miR-20A and miR-145 expression in the endometriotic tissues compared to the control group. While there was no significant correlation between the expression of miR-20a and TGF-β, we observed a negative correlation between the expression level of miR-145 and NANOG. Additionally, the ROC curve analysis emphasized miR-14 as a potential biomarker for endometriosis over miR-20a. However, our findings on disease recurrence underscore the importance of miR-20a in the early detection of endometriosis recurrence.

Expression Of TGF-Β In Oral Papillary Squamous Cell Carcinoma and Different Histological Grades of Oral Squamous Cell Carcinoma: An Immunohistochemical Comparative Study

Expression Of TGF-Β In Oral Papillary Squamous Cell Carcinoma and Different Histological Grades of Oral Squamous Cell Carcinoma: An Immunohistochemical Comparative Study

Single-cell and spatial transcriptomics reveal apelin/APJ pathway's role in microvessel formation and tumour progression in hepatocellular carcinoma.

The apelin receptor (APJ) is a key player in tumour angiogenesis, but its role in hepatocellular carcinoma (HCC) remains unclear. This study aims to elucidate the function of the apelin/APJ pathway in HCC using a multi-omics approach and identify potential therapeutic biomarkers. Differentially expressed genes related to the apelin/APJ axis were identified from bulk transcriptomics to reveal HCC-associated disparities. Single-cell and spatial transcriptomics were used to localize and analyse the function of these genes. Machine learning models were constructed to predict outcomes based on apelin/APJ expression, and experimental validation was conducted to explore the pathway's impact on HCC angiogenesis. Single cell analysis revealed an overexpression of APJ/Aplin in the endothelium. The stemness of endothelial cell (EC) with high apelin/APJ was enhanced, as well as the expression of TGFb, oxidative stresses and PI3K/AKT pathway genes. Spatial transcriptomics confirmed that EC populations with high APJ scores were enriched within the tumour. Machine learning models showed high prognostic accuracy. High APJ expression was linked to worse outcomes (p = 0.001), and AUC values were high (1 year, 3 year, 5 year) (0.95, 0.97, 0.98). Immune suppression and non-responsiveness of immune therapy were also seen in high-risk groups. The experimental validation showed that silencing apelin reduced angiogenesis (p < 0.05), endothelial proliferation, decreased expression of ANG2, KLF2, VEGFA and lower ERK1/2 phosphorylation. Apelin may serve as a potential therapeutic target in HCC, given its role in promoting tumour angiogenesis and poor patient outcomes.

Inhibition of TGF-β signaling in bone marrow endothelial cells promotes hematopoietic recovery in acute myeloid leukemia patients

Although it is an effective treatment for acute myeloid leukemia (AML), chemotherapy leads to myelosuppression and poor hematopoietic reconstruction. Hematopoiesis is regulated by bone marrow (BM) endothelial cells (ECs), and BM ECs are dysfunctional in acute leukemia patients with poor hematopoietic reconstitution after allogenic hematopoietic stem cell transplantation. Thus, it is crucial to explore the underlying mechanism of EC impairment and establish strategies for targeted therapy. TGF-β signaling was found to be upregulated in ECs from AML patients in complete remission (CR ECs) and led to CR EC damage. Administration of a TGF-β inhibitor rescued the dysfunction of ECs caused by TGF-β1 expression in vitro, especially their hematopoiesis-supporting ability. Moreover, inhibition of TGF-β expression repaired the BM EC damage triggered by chemotherapy in both AML patients in vitro and in an AML-CR murine model, and restored normal hematopoiesis without promoting AML progression. Mechanistically, our data reveal alterations in the transcriptomic pattern of damaged BM ECs, accompanied by the overexpression of downstream molecules TGF-βRI, pSmad2/3, and functional genes related to adhesion, angiogenesis suppression and pro-apoptosis. Collectively, our findings reveal for the first time that the activation of TGF-β signaling leads to BM EC dysfunction and poor hematopoietic reconstitution. Targeting TGF-β represents a potential therapeutic strategy to promote multilineage hematopoiesis, thereby benefiting more cancer patients who suffer from myelosuppression after chemotherapy.

Chitosan/siRNA nanoparticles targeting PARP-1 attenuate Neuroinflammation and apoptosis in hyperglycemia-induced oxidative stress in Neuro2a cells

Hyperglycemia induces an excessive production of superoxide by the mitochondria's electron-transport chain triggers several pathways of injury contributing to the development of diabetic complications. This increase in oxidative and nitrosative stress triggers the activation of PARP-1, a nuclear enzyme, through mechanisms such as DNA damage. siRNA-chitosan nanoparticles were formed based on electrostatic interaction, their particle size, zeta potential, STEM, and cellular uptake were characterized. Neuro2a cells were treated with low glucose (LG) and high glucose (HG) for 24 and 48 h. Neuro2a cells were pre-treated with negative siRNA, naked siRNA, siRNA-Lipofectamine™300, and ChNPs-5. qRT-PCR was used to analyze the expression of regulatory, inflammatory, and apoptotic biomarkers. The siRNA-chitosan complex at the weight ratio 1:3000 were approximately uniform spheres with particle size 150.5 nm and a positive zeta potential of about +41.5 mV. The uptake of FITC-labeled nanoparticles into Neuro2a cells was visualized using fluorescence microscopy with no significant cytotoxicity compared to the control cells. High glucose stimulation of Neuro2a cells increased PARP1 expression, and with siRNA-ChNP (1:3000) treatment, significant inhibition of PARP1 expression is observed that consequently reversed the expression of regulatory genes like SIRT1, FOXO1, FOXO3, and p53. PARP-1 inhibition reduced HG-induced inflammatory response, including NF-kB, IL6, IL1β, TNFα, iNOS, and TGF-β expression, and HG-induced apoptosis response, such as Cas-3, Cas-9, BAK, BAX, and AIF expression. This study highlights the crucial role of siRNA delivery via ChNPs and PARP-1 inhibition in hyperglycemia-induced oxidative stress in Neuro2a cells and PARP-1 inhibition may be a feasible strategy for the treatment of hyperglycemia-induced oxidative stress.

Weitiao No. 3 (3) enhances the efficacy of anti-programmed cell death protein-1 immunotherapy by modulating the intestinal microbiota in an orthotopic model of gastric cancer mice.

To explore the effects of Weitiao No. 3 (3, WD-3) on anti-programmed cell death protein-1 (PD-1) immunotherapy in gastric cancer (GC). The intestinal microbiota was analyzed by 16S rDNA sequencing of fecal samples from three groups: healthy people (Health), GC patients (GC), and WD-3-treated GC patients (WD-3). Next, we established an orthotopic model of GC mice, which were treated with anti-PD-1, WD-3, or an inoculation of intestinal bacteria. Immune markers CD3, CD4, CD8, and forkhead box protein P3 (FOXP3), and the cell proliferation marker Ki67, were evaluated by immunohistochemistry. Cell apoptosis in GC tumors was assessed by terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling staining. Enzyme-linked immunosorbent assays (ELISAs) were performed to analyze the serum levels of the following cytokines in GC mice: tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-6, IL-10, interferon (IFN)-γ, and transforming growth factor (TGF)-β. Sequencing data showed that there were significant differences in the composition of the gut microbial community among the three human groups. The gut bacteria in the three groups mainly comprised the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. At the genus level, the relative abundances of Bifidobacterium and Coprococcus showed significant decreases in the GC group, and an obvious increase in the WD-3 group, compared with the Health group. Interestingly, the relative abundance of Saccharopolyspora was only detected in the WD-3 group. The results of in vivo experiments in GC mice showed that WD-3 or anti-PD-1 treatment increased the levels of CD3+, CD4+, and CD8+ T cells, but decreased the levels of FOXP3+ regulatory T cells. Furthermore, WD-3 or PD-1 antibody treatment inhibited proliferation and promoted apoptosis of GC tumor cells. ELISA analysis showed that WD-3 or PD-1 antibody treatment facilitated TNF-α, IL-2, and IFN-γ expression, while suppressing IL-6, IL-10, and TGF-β expression. Combination therapy with WD-3 and anti-PD-1 intensified all of these effects. WD-3 enhanced the immunotherapeutic efficacy of anti-PD-1 by modulating the intestinal microbiota in an orthotopic model of GC mice.

Hydrogen sulfide donor NaHS inhibits formaldehyde-induced epithelial-mesenchymal transition in human lung epithelial cells via activating TGF-β1/Smad2/3 and MAPKs signaling pathways

Formaldehyde (FA) long term exposure leads to abnormal pulmonary function and small airway obstruction of the patients. Hydrogen sulfide (H2S) is one of the recognized gaseous transmitters involved in a wide range of cellular functions. It is unknown the involvement of H2S in FA-induced lung injury. The purpose of this study is to investigate the therapeutic potential and mechanism of H2S on FA-induced epithelial-mesenchymal transition (EMT) of human lung epithelial cells. The cell viability of Beas2B and A549 cells after FA treatment were assessed using MTT assay. The endogenous H2S was visualized by fluorescence microscopy using of the 7-azido-4-methylcoumarin (AzMC). Cell morphology was observed under phase contrast microscope. The mRNAs and proteins level were evaluated by reverse transcription-polymerase chain reaction and western blotting assays. FA treatment downregulated the endogenous H2S levels and the mRNAs and proteins level of H2S synthesizing enzymes, such as cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in Beas2B and A549 cells. FA treatment changed the cell morphology of Beas2B cells from cuboid to a spindle-shape, while declined the protein level of E-cadherin and increased the protein level of Vimentin. Moreover, FA treatment increased the proteins level of transforming growth factor-β1 (TGF-β1), phosphorylated-Smad2 (p-Smad2), phosphorylated-Smad3 (p-Smad3), phosphorylated-extracellular signal-regulated kinase (p-ERK), phosphorylated-c-Jun N-terminal kinase (p-JNK), and phosphorylated-P38 (p-P38). Furthermore, the inhibitors of TGF-β receptor type 1 (TGFβRI) and mitogen-activated protein kinases (MAPKs) signaling pathways reversed FA-induced decrease in E-cadherin expression and increase in Vimentin expression in Beas2B cells. Sodium hydrogen sulfide (NaHS) increased the level of H2S, while reversed FA-induced the low expression of E-cadherin and the high expression of Vimentin, TGF-β1, p-Smad2, p-Smad3, p-ERK, p-JNK, and p-P38. These findings indicates FA treatment downregulating the endogenous H2S in human lung epithelial cells. NaHS may inhibit FA-induced EMT in human lung epithelial cells via modulating TGF-β1/Smad2/3 and MAPKs signaling pathways. Therefore, we demonstrated that supplementation of exogenous H2S may inhibit FA-induced lung injury.

Transarterial Embolization Using an Inorganic Phosphate Binder Modulates Immunity and Angiogenesis-related Factors in a Rat Model of Liver Cancer

PurposeTo determine how low inorganic phosphate stress (LIPS) induced by sevelamer transartieral embolization (S-TAE) affects immune regulation and angiogenesis in hepatocellular carcinoma (HCC). Material and MethodsTranscatheter arterial embolization (TAE) using conventional lipiodol plus Poly (vinyl alcohol) (PVA) microsphere and S-TAE were conducted on a McA-RH7777 orthotopic liver tumor model in rats, followed by the assessment of alterations in immunity- and angiogenesis-related factors. The cells were cultured under hypoxic conditions and stimulated with LIPS to analyze the modulation of programmed cell death 1 ligand 1 (PD-L1), vascular endothelial growth factor (VEGFα), and transforming growth factor-β1 (TGF-β1) expression through Western blotting, qRT‒PCR, and immunofluorescence assays. Cell migratory capacity and angiogenesis were also evaluated. ResultsTAE increased the expression of neoplastic PD-L1 and VEGFα, and S-TAE, which depletes intratumoral Pi, downregulated the expression of PD-L1, VEGFα and TGF-β1, and augmented the infiltration of CD8+ T-cells, thereby inhibited angiogenesis and activated anticancer immunity. In vitro, the study demonstrated that LIPS inhibits hypoxia-induced upregulation of PD-L1 expression and the HIF-1α/VEGFα axis. Moreover, LIPS inhibited the tube formation ability of Human Umbilical Vein Endothelial Cells (HUVECs) and the migration ability and epithelial–mesenchymal transition (EMT) process of cancer cells under hypoxic conditions. ConclusionsS-TAE inhibited the expression of PD-L1 and VEGFα, thereby activated anti-tumor immunity and suppressing tumor angiogenesis. All the findings reveal the biology of tumors under low Pi stress and suggest the potential therapeutic value of S-TAE.

Chronic PPARα/γ and CB2R agonist treatments attenuated visceral adipose tissue (VAT)-derived extracellular vesicle-related VAT and intestinal abnormalities in NASH mice

This study explores the mechanisms and combined effects of chronic peroxisome proliferator-activated receptor (PPAR)α/γ and cannabinoid receptor 2 (CB2R) agonists on visceral adipose tissue (VAT)-derived extracellular vesicle (EVs) release and associated systemic/VAT inflammation, decreased VAT capillary density/fibrosis, and intestinal inflammation/hyperpermeability in nonalcoholic steatohepatitis (NASH) mice. NASH mice receiving 1 month of PPARα/γ agonist aleglitazar (10 mg/kg/day), CB2R agonist JWH015 (3 mg/kg/day) alone or combined. High EV release from VAT of NASH mice was associated with severe systemic/VAT/intestinal inflammation, reduced capillary network of VAT, and intestinal hyperpermeability. Combined JWH015 with aleglitazar treatment significantly suppressed HFD-induced obesity/adiposity, inhibited VAT expansion, reduced VAT inflammation/fibrosis, normalized VAT capillary network, and attenuated intestinal mucosal injury, inflammation, and hyperpermeability in NASH+aleg+JWH015 mice. The inhibition of AT-derived EV release and hypoxia inducible factor (HIF)1α levels in AT-derived EV, normalization of CB2R, PPARα, PPARγ, PPARγ1, PPARγ2, tight junction proteins, VEGF/CD31 expression, and downregulations of HIF1α, MCP-1 and TGFβ1 were observed in the VAT and intestine of the NASH+aleg+jwh015 group. In vitro experiments revealed that PPARα/γ and CB2R activation attenuated NASH AT-derived EV (EVnash)-induced pathogenic changes in the J774/SVEC4-10/Caco2 /3T3-L1 cell system. This study suggested that VAT-derived EVs contribute to the pathogenesis of NAFLD and that combined PPARα/γ and CB2R agonist treatment reduces VAT-released EV release and HIF1/MCP-1 signals to ameliorate hepatic steatosis and VAT/intestine abnormalities of NASH mice.

Potential Oncogenic and Immunosuppressive Roles of Multiple EGF-Like Domains 6 (MEGF6) in Colon Cancer: Insights from Bioinformatic Analysis

Multiple epidermal growth factor-like domains protein 6 (MEGF6) is a high molecular weight protein with several EGF-like domains. Although it has been demonstrated to promote metastasis and chemoresistance in colon cancer cells, its specific oncogenic and immunologic functions remain largely unexplored. This study aims to comprehensively analyze publicly available datasets to define the role of MEGF6 in colon cancer development and immune response modulation. The expression of MEGF6 in colon adenocarcinoma (COAD) and normal tissues at both mRNA and protein levels was assessed using the GEPIA2 and HPA databases, respectively. The UALCAN database was employed to examine the association between MEGF6 expression and clinicopathological outcomes in COAD patients, and survival analysis was conducted using the KM plotter database. Functional enrichment analysis of MEGF6-correlated genes was performed using the ShinyGO online tool. Additionally, the correlation of MEGF6 with immune cell infiltration and immunosuppressive molecules was explored through the TIMER and TISIDB databases. Our analysis revealed significantly higher mRNA and protein expressions of MEGF6 in COAD tissues compared to normal tissues, with associations to cancer stage, nodal metastasis status, and histological subtype. High MEGF6 expression was correlated with poorer survival outcomes, and genes correlated with MEGF6 were enriched in biological processes related to cellular component organization, cell adhesion, and extracellular matrix interaction. Furthermore, MEGF6 expression demonstrated a significant correlation with immune cell infiltration and the expression of immunosuppressive molecules, particularly CTLA-4, PD-1, PD-L1, TGF-β, and IL-10. In conclusion, our findings suggest that MEGF6 may play an oncogenic role by influencing cell and extracellular matrix interactions. Its overexpression may indicate immunosuppressive properties within the cancer microenvironment. Therefore, MEGF6 holds promise as a potential biomarker for predicting both prognosis and therapeutic responses in colon cancer. HIGHLIGHTS MEGF6 has been shown to involve colon cancer growth and chemotherapeutic resistance. Bioinformatics confirmed a prognostic significance of MEGF6 in colon cancer. MEGF6 may exert its oncogenic effects by influencing cell-matrix interactions. MEGF6 expression is correlated with immunosuppressive properties of cancers. GRAPHICAL ABSTRACT

Effects of Shaofu Zhuyu decoction on intestinal flora and fibrosis in a mouse model of endometriosis

Shaofu Zhuyu decoction has been widely used to treat gynecological diseases; however, its mechanism of action in endometriosis remains unclear. We analyzed Shaofu Zhuyu decoction’s chemical composition using ultra-high performance liquid chromatography-mass spectrometry. In an endometriosis mouse model, ectopic lesions weight measurements and hematoxylin and eosin staining were used to assess the therapeutic efficacy of Shaofu Zhuyu decoction. Effects on intestinal microflora were analyzed using 16S ribosomal ribonucleic acid sequencing, and impacts on focal fibrosis were analyzed using Masson’s trichrome staining. Moreover, fibrosis- and metabolism-related proteins were assessed using immunohistochemistry and enzyme-linked immunosorbent assay. The study identified 157 chemical constituents within Shaofu Zhuyu decoction. Shaofu Zhuyu decoction treatment in mice with endometriosis resulted in a reduction in ectopic lesions weight (P<0.05) and delayed disease progression. Moreover, it improved the diversity and abundance of intestinal flora, and decreased the expression of Lachnospiraceae (P<0.05), Rikenellaceae (P<0.01), Ruminococcaceae (P<0.01), Lachnoclostridium (P<0.05), and unclassified_f__Ruminococcaceae (P<0.05). Kyoto Encyclopedia of Genes and Genomes analysis revealed enrichment in carbohydrate, amino acid, and lipid metabolism pathways. Masson’s trichrome staining revealed that compared to the untreated group, the Shaofu Zhuyu decoction group exhibited significantly reduced collagen deposition areas (P<0.001), lower TGF-β1 (P<0.001), COL1A1 (P<0.05), and α-SMA (P<0.01) expression in ectopic lesions, along with increased serum adiponectin (P<0.05), decreased serum leptin (P<0.05), TGF-β1 (P<0.001), and CTGF (P<0.05). Shaofu Zhuyu decoction regulates the intestinal flora of mice with endometriosis while also reducing fibrosis at the lesion site. These findings highlight novel mechanisms for its efficacy in alleviating endometriosis.

Activation of SIRT3 by Tanshinone IIA ameliorates renal fibrosis by suppressing the TGF-β/TSP-1 pathway and attenuating oxidative stress.

Although doxorubicin (DOX) is a common chemotherapeutic drug, the serious nephrotoxicity caused by DOX-induced renal fibrosis remains a considerable clinical problem. Tanshinone IIA (Tan IIA), a compound extracted from Salvia miltiorrhiza, has been reported to have an anti-fibrotic effect. Therefore, this study investigated the molecular pathway whereby Tan IIA protects the kidneys from DOX administration. DOX (3mg/kg body weight) was intraperitoneally administered every 3 d for a total of 7 injections (cumulative dose of 21mg/kg) to induce nephrotoxicity. Then, Tan IIA (5 or 10mg/kg/d) was administered by intraperitoneal injection for 28 d. In an in vitro study, 293T cells were cultured and treated with DOX and Tan IIA for 24h. Tan IIA reduced the blood urea nitrogen levels elevated by DOX while increasing superoxide dismutase activity, down-regulating reactive oxygen species, ameliorating renal-tubule thickening, and rescuing mitochondrial morphology. Additionally, Tan IIA reduced the renal collagen deposition, increased ATP production and complex-I activity, down-regulated transforming growth factor-β1 (TGF-β1) and thrombospondin-1 (TSP-1), and up-regulated sirtuin 3 (SIRT3). Tan IIA significantly increased cell viability. Additionally, RNA interference was employed to silence the expression of SIRT3, which eliminated the effect of Tan IIA in suppressing the expression of TGF-β1 and TSP-1. In conclusion, Tan IIA ameliorated DOX-induced nephrotoxicity by attenuating oxidative injury and fibrosis. The Tan IIA-induced rescue of mitochondrial morphology and function while alleviating renal fibrosis may be associated with the activation of SIRT3 to suppress the TGF-β/TSP-1 pathway.