TGF-ß/Smad signaling pathway in fatty liver disease: a case-control study.

BACKGROUND: Fatty liver disease is a metabolic disorder that recently has been classified into two categories: metabolic dysfunction-associated fatty liver disease (MAFLD) and non-MAFLD. TGF-ß signaling pathway is likely a significant factor in the pathogenesis of this condition, exerting its effects through its downstream signaling proteins, Smad2/3. Accordingly, this study aimed to investigate the TGF-ß signaling pathway in the white blood cells (WBCs) of patients with MAFLD compared to those with non-MAFLD and control groups. METHODS AND RESULTS: In this study, 41 patients with fatty liver were evaluated, comprising 22 patients with MAFLD and 19 patients with non-MAFLD, and compared to 22 healthy controls. Gene expression of TGF-ß1, TGF-ß3, and CTGF were quantified using qRT-PCR, and the protein expressions of Smad2/3 and P-Smad2/3 were analyzed using western blotting. Gene expression analysis revealed a significant decrease in the gene expressions of the TGF-ß1 and TGF-ß3 and an increase in CTGF gene expression in patients with MAFLD and non-MAFLD compared to the control group. Notably, the Smad2/3 protein expression was significantly higher in the non-MAFLD group compared to the control group (P < 0.05). On the other hand, the P-smad2/3 protein expression was significantly elevated in the MAFLD group compared to the control group (P < 0.001). CONCLUSIONS: TGF-ß signaling pathway in WBCs of patients with fatty liver are affected by a complex signaling pathway. However, metabolic factors most probably affect TGF-ß1 gene expression and its downstream signaling proteins more than TGF-ß3.

SULF1 expression is increased and promotes fibrosis through the TGF-ß1/SMAD pathway in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology. Despite the increasing global incidence and poor prognosis, the exact pathogenic mechanisms remain elusive. Currently, effective therapeutic targets and treatment methods for this disease are still lacking. This study tried to explore the pathogenic mechanisms of IPF. We found elevated expression of SULF1 in lung tissues of IPF patients compared to normal control lung tissues. SULF1 is an enzyme that modifies heparan sulfate chains of heparan sulfate proteoglycans, playing a critical role in biological regulation. However, the effect of SULF1 in pulmonary fibrosis remains incompletely understood. Our study aimed to investigate the impact and mechanisms of SULF1 in fibrosis. METHODS: We collected lung specimens from IPF patients for transcriptome sequencing. Validation of SULF1 expression in IPF patients was performed using Western blotting and RT-qPCR on lung tissues. ELISA experiments were employed to detect SULF1 concentrations in IPF patient plasma and TGF-ß1 levels in cell culture supernatants. We used lentiviral delivery of SULF1 shRNA to knock down SULF1 in HFL1 cells, evaluating its effects on fibroblast secretion, activation, proliferation, migration, and invasion capabilities. Furthermore, we employed Co-Immunoprecipitation (Co-IP) to investigate the regulatory mechanisms involved. RESULTS: Through bioinformatic analysis of IPF transcriptomic sequencing data (HTIPF) and datasets GSE24206, and GSE53845, we identified SULF1 may potentially play a crucial role in IPF. Subsequently, we verified that SULF1 was upregulated in IPF and predominantly increased in fibroblasts. Furthermore, SULF1 expression was induced in HFL1 cells following exposure to TGF-ß1. Knockdown of SULF1 suppressed fibroblast secretion, activation, proliferation, migration, and invasion under both TGF-ß1-driven and non-TGF-ß1-driven conditions. We found that SULF1 catalyzes the release of TGF-ß1 bound to TGFßRIII, thereby activating the TGF-ß1/SMAD pathway to promote fibrosis. Additionally, TGF-ß1 induces SULF1 expression through the TGF-ß1/SMAD pathway, suggesting a potential positive feedback loop between SULF1 and the TGF-ß1/SMAD pathway. CONCLUSIONS: Our findings reveal that SULF1 promotes fibrosis through the TGF-ß1/SMAD pathway in pulmonary fibrosis. Targeting SULF1 may offer a promising therapeutic strategy against IPF.

Inhibition of Ubiquitin C-Terminal Hydrolase L1 Facilitates Cutaneous Wound Healing via Activating TGF-ß/Smad Signalling Pathway in Fibroblasts.

Ubiquitin C-terminal hydrolase L1 (UCHL1) plays vital roles in cell proliferation, angiogenesis, inflammation and oxidative stress. Nevertheless, it is unclear whether UCHL1 could regulate the biologic behaviour of cells and ultimately influences wound healing. We aim to illustrate the roles and the underlying mechanism of UCHL1 in cutaneous wound healing. Murine full-thickness excisional wound model was utilised to study the effects of UCHL1 on wound healing through topical administration of the UCHL1 inhibitor LDN57444, followed by assessment of wound areas and histological alterations. Subsequently, ethynyldeoxyuridine, scratch and transwell assays were performed to examine fibroblast migration and proliferation. The extracellular matrix (ECM)-related genes expression and transforming growth factor-ß (TGF-ß)/Smad signalling pathways activation were investigated by immuno-fluorescent staining, Western blots and quantitative reverse transcription polymerase chain reaction. We identified elevated UCHL1 expression in non-healing wound tissues. The UCHL1 expression displayed a dynamic change and reached a peak on Day-7 post-wounding during the healing process in mice. Cutaneous administration of LDN57444 promoted wound healing by facilitating collagen deposition, myofibroblast activation and angiogenesis. In vitro experiments demonstrated that UCHL1 concentration dependently inhibited migration, ECM synthesis and activation of human dermal fibroblasts, which was mechanistically related to downregulation of TGF-ß/Smad signalling. Furthermore, these effects could be reversed by TGF-ß inhibitor SB431542. Our findings reveal that UCHL1 is a negative regulator of cutaneous wound healing and considered as a novel prospective therapeutic target for effective wound healing.

Oroxylin A suppressed colorectal cancer metastasis by inhibiting the activation of the TGF-ß/SMAD signal pathway.

Metastatic colorectal cancer continues to have a high fatality rate, with approximately only 14% of patients surviving more than 5 years. To improve the survival rate of these patients, the development of new therapeutic drugs is a priority. In this study, we investigated the effects of Oroxylin A on the metastasis of human colorectal cancer cells and its potential molecular mechanism. This study utilised CCK8 assay, transwell assay, flow cytometry, western blot analysis, molecular docking, HE staining, immunofluorescence staining, and xenograft models. The proliferation, migration, and invasion of colon cancer cells were effectively suppressed by Oroxylin A in a dose-dependent manner. Oroxylin A has the potential to inhibit the process of epithelial‒mesenchymal transition (EMT) by upregulating the expression of E-cadherin, a marker associated with epithelial cells, while downregulating the levels of N-cadherin, Snail, vimentin, and slug, which are markers associated with mesenchymal cells. In addition, 200 mg/kg of Oroxylin A inhibited the growth of colorectal tumours. Molecular docking technology revealed that Oroxylin A can bind to TGFß and inhibit the activation of the TGFß-smad signalling pathway. The overexpression of TGFß weakened the inhibitory effect of Oroxylin A on the proliferation, migration, and invasion of human colorectal cancer cells, as well as the promoting effect on apoptosis. Oroxylin A inhibited the activation of the TGF-smad signalling pathway and the EMT process, thereby suppressing the migration and invasion of human colorectal cancer cells.

Lumican/Lumikine Promotes Healing of Corneal Epithelium Debridement by Upregulation of EGFR Ligand Expression via Noncanonical Smad-Independent TGFß/TBRs Signaling.

The synthetic peptide of lumican C-terminal 13 amino acids with the cysteine replaced by an alanine, hereafter referred to as lumikine (LumC13C-A: YEALRVANEVTLN), binds to TGFß type I receptor/activin-like kinase5 (TBR1/ALK5) in the activated TGFß receptor complex to promote corneal epithelial wound healing. The present study aimed to identify the minimum essential amino acid epitope necessary to exert the effects of lumikine via ALK5 and to determine the role of the Y (tyrosine) residue for promoting corneal epithelium wound healing. This study also aimed to determine the signaling pathway(s) triggered by lumican-ALK5 binding. For such, adult Lum knockout (Lum-/-) mice (~8-12 weeks old) were subjected to corneal epithelium debridement using an Agerbrush®. The injured eyes were treated with 10 µL eye drops containing 0.3 µM synthetic peptides designed based on the C-terminal region of lumican for 5-6 h. To unveil the downstream signaling pathways involved, inhibitors of the Alk5 and EGFR signaling pathways were co-administered or not. Corneas isolated from the experimental mice were subjected to whole-mount staining and imaged under a ZEISS Observer to determine the distance of epithelium migration. The expression of EGFR ligands was determined following a scratch assay with HTCE (human telomerase-immortalized cornea epithelial cells) in the presence or not of lumikine. Results indicated that shorter LumC-terminal peptides containing EVTLN and substitution of Y with F in lumikine abolishes its capability to promote epithelium migration indicating that Y and EVTLN are essential but insufficient for Lum activity. Lumikine activity is blocked by inhibitors of Alk5, EGFR, and MAPK signaling pathways, while EGF activity is only suppressed by EGFR and MAPK inhibitors. qRT-PCR of scratched HTCE cells cultures treated with lumikine showed upregulated expression of several EGFR ligands including epiregulin (EREG). Treatment with anti-EREG antibodies abolished the effects of lumikine in corneal epithelium debridement healing. The observations suggest that Lum/lumikine binds Alk5 and promotes the noncanonical Smad-independent TGFß/TBRs signaling pathways during the healing of corneal epithelium debridement.

Lidocaine combined with general anesthetics impedes metastasis of breast cancer cells via inhibition of TGF-ß/Smad-mediated EMT signaling by reprogramming tumor-associated macrophages.

Surgical resection is the best-known approach for breast cancer treatment. However, post-operative metastases increase the rate of death. The potential effect of anesthetic drugs on long-term tumor growth, risk of metastasis, and recurrence after surgery has been investigated in cancer patients. However, the underlying mechanisms remain unclear. Therefore, we aimed to elucidate the anti-metastatic effect of lidocaine combined with common anesthetics and its mechanisms of action on lung metastasis in breast cancer models. The combination of lidocaine with propofol or sevoflurane inhibited the growth of TNBC cells compared to treatment alone. In addition, the combination effectively inhibited cancer cell migration and invasion. It suppressed tumor growth and increased the survival rate in breast 4 T1 orthotopic models. More importantly, it inhibited lung metastasis and recurrence compared with groups treated with a single anesthetic. In co-culture with TAMs and TNBC cells, lidocaine not only reduced M2-tumor-associated macrophages (TAM) that were increased by sevoflurane or propofol but also increased M1 macrophage polarization, impeding tumor growth in TNBC. Also, we found that the transforming growth factor-ß (TGF-ß) derived from TAMs increased EMT signaling in TNBC cells, and that lidocaine affected cancer cells as well as M2-TAMs, inducing M2 to M1 reprogramming and decreasing TGF-ß/Smads-mediated EMT signaling in TNBC cells, leading to inhibition of cancer metastasis and recurrence. These findings suggest lidocaine combined with general anesthetics as a potential therapeutic approach for the inhibition of recurrence and metastasis of breast cancer patients undergoing curative resection.

GAMG ameliorates silica-induced pulmonary inflammation and fibrosis via the regulation of EMT and NLRP3/TGF-ß1/Smad signaling pathway.

Silicosis is an occupational disease caused by exposure to silica characterized by pulmonary inflammation and fibrosis, for which there is a lack of effective drugs. Glycyrrhetinic acid 3-O-ß-D-glucuronide (GAMG) can treat silicosis due to its anti-inflammatory and anti-fibrotic properties. Here, the effect of therapeutic interventions of GAMG was evaluated in early-stage and advanced silicosis mouse models. GAMG significantly improved fibrotic pathological changes and collagen deposition in the lungs, alleviated lung inflammation in the BALF, reduced the expression of TNF-α, IL-6, NLRP3, TGF-ß1, vimentin, Col-Ⅰ, N-cadherin, and inhibited epithelial-mesenchymal transition (EMT), thereby ameliorating pulmonary fibrosis. Moreover, the dose of 100 mg/kg GAMG can effectively prevent early-stage silicosis, while that of 200 mg/kg was recommended for advanced silicosis. In vitro and in vivo study verified that GAMG can suppress EMT through the NLRP3/TGF-ß1/Smad2/3 signaling pathway. Therefore, GAMG could be a promising preventive (early-stage silicosis) and therapeutic (advanced silicosis) strategy, which provides a new idea for formulating prevention and treatment strategies.

Optimal performance objectives in the highly conserved bone morphogenetic protein signaling pathway.

Throughout development, complex networks of cell signaling pathways drive cellular decision-making across different tissues and contexts. The transforming growth factor ß (TGF-ß) pathways, including the BMP/Smad pathway, play crucial roles in determining cellular responses. However, as the Smad pathway is used reiteratively throughout the life cycle of all animals, its systems-level behavior varies from one context to another, despite the pathway connectivity remaining nearly constant. For instance, some cellular systems require a rapid response, while others require high noise filtering. In this paper, we examine how the BMP-Smad pathway balances trade-offs among three such systems-level behaviors, or "Performance Objectives (POs)": response speed, noise amplification, and the sensitivity of pathway output to receptor input. Using a Smad pathway model fit to human cell data, we show that varying non-conserved parameters (NCPs) such as protein concentrations, the Smad pathway can be tuned to emphasize any of the three POs and that the concentration of nuclear phosphatase has the greatest effect on tuning the POs. However, due to competition among the POs, the pathway cannot simultaneously optimize all three, but at best must balance trade-offs among the POs. We applied the multi-objective optimization concept of the Pareto Front, a widely used concept in economics to identify optimal trade-offs among various requirements. We show that the BMP pathway efficiently balances competing POs across species and is largely Pareto optimal. Our findings reveal that varying the concentration of NCPs allows the Smad signaling pathway to generate a diverse range of POs. This insight identifies how signaling pathways can be optimally tuned for each context.

Development of an automated high-content immunofluorescence assay of pSmads quantification: Proof-of-concept with drugs inhibiting the BMP/TGF-ß pathways.

INTRODUCTION: Bone morphogenetic proteins (BMPs) and transforming growth factors (TGF-ß) are members of the TGF-ß superfamily, known for their roles in several physiological and pathological processes. These factors are known to bind in vivo to BMP and TGF-ß receptors, respectively, which induces the phosphorylation of Smad (pSmad) transcription factors. This pathway is generally studied with Western blot and luciferase bioluminescence assay, which presents some limitations. PURPOSE: In this work, we developed and optimized a high-throughput assay to study pSmad pathways using immunofluorescence (IF) as an alternative to Western blot. We aimed to overcome the technical challenges usually faced in the classical IF assay in image acquisition, analysis, and quantification. METHODS: We used C2C12 cells as a cellular model. The cells were stimulated with BMP-2 and TGF-ß1 that were delivered either in solution (soluble) or via a biomaterial presenting the growth factor (GF), that is in a "matrix-bound" manner. Image acquisition parameters, analysis methods, and quantification of pSmads using IF were optimized for cells cultured on two types of supports: on bare glass and on a biomimetic coating made by self-assembly of the biopolymers hyaluronic acid and poly(l-lysine), which was crosslinked and then loaded with the GFs. RESULTS: We performed high-content kinetic studies of pSmad expression for cells cultured in 96-well microplates in response to soluble and matrix-bound BMP-2 and TGF-ß1. The detection limit of the IF-based assay was found to be similar to Western blot. Additionally, we provide a proof-of-concept for drug testing using inhibitors of BMP and TGF-ß receptors, under conditions where specific signaling pathways are engaged via the ligand/receptor interactions. Altogether, our findings offer perspectives for future mechanistic studies on cell signaling and for studies at the single cell level using imaging methods.

miRNAs in Signal Transduction of SMAD Proteins in Breast Cancer.

The aim of this study was to identify miRNAs that could potentially influence the activity of SMAD proteins involved in TGFß signal transduction in five types of breast cancer in Polish women. Patients with five breast cancer subtypes were included in the study: luminal A (n = 130), luminal B HER2- (n = 100), luminal B HER2+ (n = 96), non-luminal HER2+ (n = 36), and TNBC (n = 43). During surgery, tumor tissue was removed along with a margin of healthy tissue (control). Molecular analysis included determination of the expression of genes related to SMAD protein signal transduction using mRNA microarrays and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Protein expression was determined using an enzyme-linked immunosorbent assay (ELISA). The miRNA profiling was performed using miRNA microarrays and the miRDB database. SMAD3 and SMAD5 were overexpressed in all types of breast cancer, which could be related to the reduced expression of miR-145, and the findings for SMAD4 and miR-155 were similar. Additionally, the level of SMAD7 was reduced, which may be due to the low activity of miR-15b and miR21b. This study determined the gene expression profiles involved in SMAD protein signal transduction across five different types of breast cancer and identified the miRNAs potentially regulating their activity. Overexpression of SMAD3, SMAD4, and SMAD5 suggests excessive activation of the TGFß pathway, potentially promoting tumor growth and development. Concurrently, a significant reduction in SMAD7 expression removes inhibitory control in the TGFß pathway, a phenomenon that is particularly evident in more aggressive breast cancer types.

N6-methyladenosine enhances the expression of TGF-ß-SMAD signaling family to inhibit cell growth and promote cell metastasis.

TGF-ß-SMAD signaling pathway plays an important role in the progression of various cancers. However, posttranscriptional regulation such as N6-methyladenosine (m6A) of TGF-ß-SMAD signaling axis remains incompletely understood. Here, we reveal that insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) is low expression as well as associated with poor prognosis in clear cell renal cell carcinoma (ccRCC) patients and inhibits proliferation as well as promotes metastasis of ccRCC cells. Mechanistically, IGF2BP2 systematically regulates TGF-ß-SMAD signaling family, including TGF-ß1/2, TGF-ßR1/2 and SMAD2/3/4, through mediating their mRNA stability in an m6A-dependent manner. Furthermore, the functional effects of IGF2BP2 on ccRCC cells is mediated by TGF-ß-SMAD signaling downstream effector SMAD4, which is identified three m6A sites in 5'UTR and CDS. Our study establishes IGF2BP2-TGF-ß-SMAD axis as a new regulatory effector in ccRCC, providing new insights for developing novel therapeutic strategies.

Pilose Antler Protein Relieves UVB-Induced HaCaT Cells and Skin Damage.

Extended exposure to UVB (280-315 nm) radiation results in oxidative damage and inflammation of the skin. Previous research has demonstrated that pilose antler extracts have strong anti-inflammatory properties and possess antioxidant effects. This study aimed to elucidate the mechanism of pilose antler protein in repairing photodamage caused by UVB radiation in HaCaT cells and ICR mice. Pilose antler protein (PAP) was found to increase the expression of type I collagen and hyaluronic acid in HaCaT cells under UVB irradiation while also inhibiting reactive oxygen species (ROS) production and oxidative stress in vitro. In vivo, the topical application of pilose antler protein effectively attenuated UVB-induced skin damage in ICR mice by reducing interleukin-1ß (IL-ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) and inhibiting skin inflammation while alleviating UVB-induced oxidative stress. It was shown that pilose antler protein repaired UVB-induced photodamage through the MAPK and TGF-ß/Smad pathways.

[Conbercept reverses TGF-ß2-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-ß/Smad signaling pathway].

OBJECTIVE: To investigate the mechanism by which conbercept reverses transforming growth factor-ß2 (TGF-ß2)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs). METHODS: Cultured HLEC SRA01/04 cells were treated with TGF-ß2, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α-SMA, and Snail), extracellular matrix components, and genes related to the TGF-ß/Smad signaling pathway. RESULTS: Conbercept significantly reduced TGF-ß2-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P <0.05). Transwell assay showed significantly lower cell migration ability in TGF-ß2+conbercept group than in TGF-ß2 group (P <0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-ß2-induced EMT (P <0.01). CONCLUSION: Conbercept inhibits TGF-ß2 induced EMT by downregulating the expression of pSmad2/3 in TGF-ß/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.

LncRNA XIST/miR-455-3p/HOXC4 axis promotes breast cancer development by activating TGF-ß/SMAD signaling pathway.

Breast cancer is the second primary cause of cancer death among women. Long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) is a central regulator for X chromosome inactivation, and its abnormal expression is a primary feature of breast cancer. So far, the mechanism of XIST in breast cancer has not been fully elucidated. We attempted to illustrate the mechanism of XIST in breast cancer. The expressions of XIST, microRNA-455-3p (miR-455-3p) in breast cancer were measured using quantitative real-time PCR. The expressions of homeobox C4 (HOXC4) were assessed with immunohistochemical and Western blot. Also, the functions of XIST in breast cancer were assessed by Cell Counting Kit-8 analysis, colony formation assay, flow cytometry, Western blot, Transwell, and cell scratch assays. Meanwhile, the mechanism of XIST in breast cancer was validated using database analysis and dual-luciferase reporter assay. Furthermore, the function of XIST in breast cancer in vivo was estimated by tumor xenograft model, immunohistochemical assay, and hematoxylin-eosin staining. XIST and HOXC4 expressions were increased, but miR-455-3p expressions were decreased in breast cancer tissues and cells. Knocking down XIST restrained breast cancer cell proliferation, invasion, migration, epithelial-mesenchymal transformation (EMT), and induced cell cycle arrest at G0/G1. Meanwhile, XIST interacted with miR-455-3p, while miR-455-3p interacted with HOXC4. XIST knockdown repressed breast cancer cell proliferation, invasion, and EMT, while miR-455-3p inhibitor or HOXC4 overexpression abolished those impacts. HOXC4 overexpression also blocked the impacts of miR-455-3p mimic on breast cancer cell malignant behavior. In vivo experimental data further indicated that XIST knockdown repressed breast cancer cell tumorigenic ability, and decreased HOXC4 and p-SMAD3 (TGF-ß/SMAD-related protein) expressions.XIST/miR-455-3p/HOXC4 facilitated breast cancer development by activating the TGF-ß/SMAD pathway.

Geniposide ameliorates bleomycin-induced pulmonary fibrosis in mice by inhibiting TGF-ß/Smad and p38MAPK signaling pathways.

Pulmonary fibrosis (PF) is an interstitial lung disease characterized by inflammation and fibrotic changes, with an unknown cause. In the early stages of PF, severe inflammation leads to the destruction of lung tissue, followed by upregulation of fibrotic factors like Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CTGF), which disrupt normal tissue repair. Geniposide, a natural iridoid glycoside primarily derived from the fruits of Gardenia jasminoides Ellis, possesses various pharmacological activities, including liver protection, choleretic effects, and anti-inflammatory properties. In this study, we investigated the effects of Geniposide on chronic inflammation and fibrosis induced by bleomycin (BLM) in mice with pulmonary fibrosis (PF). PF was induced by intratracheal instillation of bleomycin, and Geniposide(100/50/25mg•kg-1) was orally administered to the mice once a day until euthanasia(14 day/28 day). The Raw264.7 cell inflammation induced by LPS was used to evaluate the effect of Geniposide on the activation of macrophage. Our results demonstrated that Geniposide reduced lung coefficients, decreased the content of Hydroxyproline, and improved pathological changes in lung tissue. It also reduced the number of inflammatory cells and levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid (BALF) of bleomycin-induced PF mice. At the molecular level, Geniposide significantly down-regulated the expression of TGF-ß1, Smad2/3, p38, and CTGF in lung tissues of PF mice induced by bleomycin. Molecular docking results revealed that Geniposide exhibited good binding activity with TGF-ß1, Smad2, Smad3, and p38. In vitro study showed Geniposide directly inhibited the activation of macrophage induced by LPS. In conclusion, our findings suggest that Geniposide can ameliorate bleomycin-induced pulmonary fibrosis in mice by inhibiting the TGF-ß/Smad and p38MAPK signaling pathways.

PARP9 affects myocardial function through TGF-ß/Smad axis and pirfenidone.

Cardiac arrhythmias are often linked to the overactivity of cardiac fibroblasts (CFs). Investigating the impact of poly (ADP-ribose) polymerase 9 (PARP9) on Angiotensin II (Ang II)-induced fibroblast activation and the therapeutic effects of pirfenidone (PFD) offers valuable insights into cardiac arrhythmias. This study utilized weighted gene co-expression network analysis (WGCNA), differential gene expression (DEG) analysis, protein-protein interaction (PPI), and receiver operating characteristic (ROC) analysis on the GSE42955 dataset to identify the hub gene with a significant diagnostic value. The ImmuCellAI tool revealed an association between PARP9 and immune cell infiltration. Our in vitro assessments focused on the influence of PFD on myofibroblast differentiation, transforming growth factor-beta (TGF-ß) expression, and Ang II-induced proliferation and migration in CFs. Additionally, we explored the impact on fibrosis markers and the TGF-ß/Smad signaling pathway in the context of PARP9 overexpression. Analysis of the GSE42955 dataset revealed PARP9 as a central gene with high clinical diagnostic value, linked to seven types of immune cells. The in vitro studies demonstrated that PFD significantly mitigates Ang II-induced CF proliferation, migration, and fibrosis. It also reduces Ang II-induced PARP9 expression and decreases fibrosis markers, including TGF-ß, collagen I, collagen III, and α-SMA. Notably, PARP9 overexpression can partially counteract PFD's inhibitory effects on CFs and modify the expression of fibronectin, CTGF, α-SMA, collagen I, collagen III, MMP2, MMP9, TGF-ß, and p-Smad2/3 in the TGF-ß/Smad signaling pathway. In summary, our findings suggest that PFD effectively counteracts the adverse effects of Ang II-induced CF proliferation and fibrosis, and modulates the TGF-ß/Smad signaling pathway and PARP9 expression. This identifies a potential therapeutic approach for managing myocardial fibrosis.

Pirfenidone inhibits TGF-ß1-induced fibrosis via downregulation of Smad and ERK pathway in MDCK cells.

The prevalence of chronic kidney disease (CKD) in dogs increases with age, and renal fibrosis is an important pathophysiological mechanism in this process. However, only a few drugs that can effectively inhibit fibrosis in the kidneys of dogs are currently available. In this study, we aimed to determine whether pirfenidone, a drug that has shown antifibrotic effects in various clinical studies, also exerts antifibrotic effects on canine renal tubular epithelial cells, Madin-Darby canine kidney cells (MDCK). To this end, we treated MDCK cells with various concentrations of pirfenidone, followed by transforming growth factor-beta1 (TGF-ß1) to stimulate fibrotic conditions. A cell viability assay was performed to determine the effect of pirfenidone on cell survival. Fibrosis-related markers and TGF-ß1 fibrotic pathway-related markers were assessed using qPCR, Western blot analysis and immunocytochemistry. A one-way analysis of variance (ANOVA) was performed, followed by Tukey's post-hoc test for multiple comparisons. Pirfenidone treatment significantly reduced the expression of profibrotic markers such as α-smooth muscle actin, fibronectin, and collagen. Additionally, it upregulated the expression of E-cadherin, an epithelial marker. Furthermore, pirfenidone effectively inhibited the phosphorylation of key factors involved in the TGF-ß1 signaling pathway, including Smad2/3 and ERK1/2. These results demonstrate that pirfenidone suppresses TGF-ß1-induced fibrosis in MDCK cells by attenuating epithelial-mesenchymal transition and the relevant signaling pathways.

The novel peptide PEP-Z-2 potentially treats renal fibrosis in vivo and in vitro by regulating TGF-ß/Smad/AKT/MAPK signaling.

Renal fibrosis is a process in which excessive deposition of extracellular matrix leads to an increase in tissue hardness and gradual destruction of the renal parenchyma. Chronic kidney disease (CKD) commonly progresses to end-stage renal disease (ESRD), ultimately leading to renal failure. This disease has high incidence and mortality rates, but to date, effective treatment options are lacking. PEP-Z-2 is a collagen peptide isolated from redlip croaker scales and may have potential fibroprotective activity. In this study, PEP-Z-2 was found to alleviate unilateral ureteral obstruction (UUO)- and folic acid (FA)-induced kidney injury in a mouse model, reduce collagen deposition in tissues, normalize renal function, reduce the expression of fibrosis markers, reduce reactive oxygen species (ROS) production, and restore the balance of the oxidant/antioxidant system. In vitro experiments also demonstrated that PEP-Z-2 inhibits the TGF-ß-induced differentiation of fibroblasts and renal tubular epithelial cells into myofibroblasts and reduces the production of extracellular matrix (ECM) proteins such as fibronectin, Col I, and α-SMA, demonstrating notable therapeutic effects on renal fibrosis. This effect is achieved by regulating the TGF-ß/Smad/AKT/MAPK pathway. Our research suggested that PEP-Z-2 is a potential therapeutic drug for renal fibrosis, and peptides from aquatic organisms may constitute a new class of candidate drugs for the treatment of renal fibrosis and even other types of organ fibrosis.

Apelin-13 alleviates intrauterine adhesion by inhibiting epithelial-mesenchymal transition of endometrial epithelial cells and promoting angiogenesis.

Intrauterine adhesion (IUA) is a common complication of surgical manipulation of the uterine cavity such as abortion. The pathology of IUA is characterized by fibrosis, but the pathogenesis is not fully understood. The function of Apelin-13 in IUA and related mechanisms were investigated in this study. The IUA rat model was established. The pathological changes and fibrosis degree of rat uterine tissues were detected by HE and Masson staining after intraperitoneal injection of Apelin-13. Epithelial-mesenchymal transition (EMT) of endometrial epithelial cells and endothelial-mesenchymal transition (EnMT) of vein endothelial cells were induced by TGF-ß1. Tube-forming assay using HUVEC was implemented to detect the effect of Apelin-13 upon angiogenesis. IHC staining, immunofluorescence staining, and Western blot were conducted to detect the expression levels of EMT markers, angiogenesis, and key proteins of the TGF-ß1/Smad signaling. Apelin-13 significantly alleviated IUA and fibrosis, and increased endometrial thickness and gland number in IUA rats. In addition, Apelin-13 significantly reversed EMT and EnMT induced by IUA modeling and TGF-ß1, promoted the tube-forming ability of HUVEC, and up-regulated the expression of angiogenesis-related proteins. Mechanistically, Apelin-13 significantly suppressed smad2/3 phosphorylation and inhibited the TGF-ß1/Smad signaling via its receptor APJ. Apelin-13 might alleviate IUA via repressing the TGF-ß1/Smad pathway and is expected to be a potent therapeutic option for the clinical treatment of IUA.

The effects of BMP2 and the mechanisms involved in the invasion and angiogenesis of IDH1 mutant glioma cells.

PURPOSE: This study investigated the effect of an isocitrate dehydrogenase 1 (IDH1) mutation (mutIDH1) on the invasion and angiogenesis of human glioma cells. METHODS: Doxycycline was used to induce the expression of mutIDH1 in glioma cells. Transwell and wound healing assays were conducted to assess glioma cell migration and invasion. Western blotting and cell immunofluorescence were used to measure the expression levels of various proteins. The influence of bone morphogenetic protein 2 (BMP2) on invasion, angiogenesis-related factors, BMP2-related receptor expression, and changes in Smad signaling pathway-related proteins were evaluated after treatment with BMP2. Differential gene expression and reference transcription analysis were performed. RESULTS: Successful infection with recombinant lentivirus expressing mutIDH1 was demonstrated. The IDH1 mutation promoted glioma cell migration and invasion while positively regulating the expression of vascularization-related factors and BMP2-related receptors. BMP2 exhibited a positive regulatory effect on the migration, invasion, and angiogenesis of mutIDH1-glioma cells, possibly mediated by BMP2-induced alterations in Smad signaling pathway-related factors.After BMP2 treatment, the differential genes of MutIDH1-glioma cells are closely related to the regulation of cell migration and cell adhesion, especially the regulation of Smad-related proteins. KEGG analysis confirmed that it was related to BMP signaling pathway and TGF-ß signaling pathway and cell adhesion. Enrichment analysis of gene ontology and genome encyclopedia further confirmed the correlation of these pathways. CONCLUSION: Mutation of isocitrate dehydrogenase 1 promotes the migration, invasion, and angiogenesis of glioma cells, through its effects on the BMP2-driven Smad signaling pathway. In addition, BMP2 altered the transcriptional patterns of mutIDH1 glioma cells, enriching different gene loci in pathways associated with invasion, migration, and angiogenesis.