Exploring the wound healing potential of Lobostemon fruticosus using in vitro and in vivo bioassays.

ETHNOPHARMACOLOGICAL RELEVANCE: Lobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. AIM OF THE STUDY: Aligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. MATERIALS AND METHODS: An aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. RESULTS: Six major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 µg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 µg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. CONCLUSIONS: The study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.

[Mechanism of Linggui Zhugan Decoction in inhibiting myocardial fibrosis by regulating Wnt/ß-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/ß-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 3ß(GSK-3ß), phosphorylated GSK-3ß(p-GSK-3ß), ß-catenin, and nuclear ß-catenin. RT-qPCR was used to detect the gene expression of ß-catenin and matrix metalloproteinase 9(MMP9), and immunofluorescence technique was used to detect the expression and localization of key proteins α-SMA and ß-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-3ß, p-GSK-3ß, ß-catenin, and nuclear ß-catenin. RT-qPCR was used to detect the gene expression of ß-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-3ß, ß-catenin, nuclear ß-catenin, decreased GSK-3ß expression, elevated mRNA expression of ß-catenin and MMP9, and enhanced fluorescence intensity and expression of ß-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-3ß, ß-catenin, nuclear ß-catenin, increased GSK-3ß expression, decreased mRNA expression of ß-catenin and MMP9, and reduced fluorescence intensity and expression of ß-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/ß-catenin pathway, reduction of collagen deposition, and protection of myocardial cells.

The role of the PI3K/AKT/mTOR pathway in mediating PD-L1 upregulation during fibroblast transdifferentiation.

Silicosis is a progressive interstitial lung disease characterized by diffuse pulmonary fibrosis. The transdifferentiation of lung fibroblasts into myofibroblasts is a key cellular event driving the progression of silicosis fibrosis. Recent studies have shown that PD-L1 expression is significantly upregulated in activated fibroblasts, and PD-L1 plays a crucial role in mediating fibroblast transdifferentiation. This study aims to elucidate the molecular mechanisms regulating PD-L1 expression in fibroblasts and analyze the functional significance of PD-L1 upregulation in fibroblast activity and silicosis fibrosis. In this research, an in vitro model of TGF-ß1-induced NIH-3 T3 fibroblast transdifferentiation was established. Small molecule inhibitors, siRNA, and plasmids were used to interfere with the PI3K/AKT/mTOR signaling pathway and PD-L1 expression. It was found that TGF-ß1 stimulation increased PD-L1 expression in fibroblasts, while blocking the PI3K/AKT/mTOR pathway inhibited this upregulation. Knockdown of PD-L1 significantly inhibited fibroblast transdifferentiation and impeded TGF-ß1-induced activation of the PI3K/AKT/mTOR pathway, whereas PD-L1 overexpression had the opposite effect. Additionally, PD-L1 protein in fibroblasts undergoes ubiquitin-proteasome-mediated degradation, negatively regulating PD-L1 upregulation. In vivo, adeno-associated virus was used to specifically knockdown PD-L1 in mouse lung fibroblasts, resulting in significantly reduced lung tissue damage and fibrosis in silicosis mice. This effect was associated with the involvement of the PI3K/AKT/mTOR pathway. In summary, PD-L1 expression in fibroblasts is upregulated during transdifferentiation, a process regulated by the PI3K/AKT/mTOR pathway. Upregulated PD-L1 enhances PI3K/AKT/mTOR signaling through positive feedback, sustaining fibroblast activation. Ubiquitin-proteasome-mediated protein degradation may serve as a negative feedback mechanism maintaining PD-L1 homeostasis.

SNAI2 as a Prognostic Biomarker Based on Cancer-Associated Fibroblasts in Patients With Lung Adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is a common type of malignant tumor with therapeutic challenges. Cancer-associated fibroblasts (CAFs) promote LUAD growth and metastasis, regulate the tumor immune response, and influence tumor treatment responses and drug resistance. However, the molecular mechanisms through which CAFs control LUAD progression are largely unknown. In this study, we aimed to determine the correlations between CAF-related genes and overall survival (OS) in patients with LUAD. Methods: We acquired the gene expression data and clinical information of 522 patients with LUAD patients from The Cancer Genome Atlas (TCGA) and 442 patients with LUAD from the Gene Expression Omnibus (GEO) databases. CAF infiltration levels were assessed using the Microenvironment Cell Population (MCP) counter, the Estimating the Proportions of Immune and Cancer cells (EPIC) algorithm, and Tumor Immune Dysfunction and Exclusion (TIDE) scores. A CAF-related gene network was constructed using the Weighted gene co-expression network analysis (WGCNA). Based on the CAF-related genes, univariate Cox regression and Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression analyses were performed to identify prognostic genes. Gene expression levels within the prognostic model were validated using the Cancer Cell Line Encyclopedia (CCLE) databases and Western blotting. Results: Our results demonstrated that high CAF scores were associated with lower survival rates in patients with LUAD. Gene modules that were highly correlated with high CAF scores were closely associated with tissue characteristics and extracellular matrix structures in LUAD. In addition, correlations between CAF scores and responses to immunotherapy and chemotherapy were observed. Finally, we found that SNAI2 expression was higher in lung cancer tissues than in normal tissues. Conclusion: Deepening our understanding of the influence of CAFs on tumor progression and treatment response at the molecular level can aid the development of more effective therapeutic strategies. This study provides important insights into the functional mechanisms of action of CAFs in LUAD and highlights their clinical implications.

Extracellular vesicles derived from melanoma cells induce carcinoma-associated fibroblasts via miR-92b-3p mediated downregulation of PTEN.

In melanoma, carcinoma-associated fibroblasts (CAFs) are important cellular components in the tumour microenvironment due to their potential to promote tumour growth and metastatic spread of malignant cells. Melanoma cells have the ability to affect non-tumour cells in the microenvironment by releasing extracellular vesicles (EVs). The mechanisms responsible for reprogramming normal dermal fibroblasts (NHDFs) into CAFs remain incompletely understood. However, it is likely thought to be mediated by melanoma-specific miRNAs, which are transported by EVs derived from melanoma cells. Therefore, we wondered if one of the most enriched miRNAs in EVs secreted by melanoma cells, miR-92b-3p, is involved in the conversion of normal fibroblasts into CAFs. We observed that melanoma cell-derived EVs indeed delivered miR-92b-3p into NHDFs and that its accumulation correlated with CAF formation, as demonstrated by enhanced expression of CAF marker genes and increased proliferation and migration. Overexpression of miR-92b-3p in NHDFs revealed similar results, while EVs deficient of miR-92b-3p did not induce a CAF phenotype. As a target we identified PTEN, whose repression led to increased expression of CAF markers. We thus provide a novel pathway of intercellular communication by which melanoma cells control the transformation of CAFs by virtue of EV-transported miRNAs.

MIR4435-2HG: A novel biomarker for triple-negative breast cancer diagnosis and prognosis, activating cancer-associated fibroblasts and driving tumor invasion through EMT associated with JNK/c-Jun and p38 MAPK signaling pathway activation.

BACKGROUND: Breast cancer has the highest incidence rate and causes the most fatalities among all female cancers worldwide. Triple-negative breast cancer (TNBC) is known for its strong invasiveness and higher rates of recurrence. In this research, we aimed to identify MIR4435-2HG as a promising long non-coding RNA (lncRNA) biomarker and therapeutic target for TNBC. METHODS: Utilizing clinicopathological information and transcriptome data from The Cancer Genome Atlas (TCGA) database, we assessed the clinical relevance of MIR4435-2HG in breast cancer through univariate and multivariate COX regression, receiver operating characteristic (ROC) analysis, as well as Kaplan-Meier survival analysis. To investigate the biological role of MIR4435-2HG in TNBC, we conducted gene set enrichment analysis (GSEA), as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Additionally, we constructed and validated a nomogram to predict disease-free survival (DFS). Both the R package "pRRophetic" and the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm were employed to forecast the sensitivity to different therapeutics between the high- and low-MIR4435-2HG groups. We employed single-cell RNA sequencing analysis and tumor microenvironment infiltration analysis to investigate the potential involvement of MIR4435-2HG in the TNBC tumor microenvironment. Cellular biological behaviors were assessed utilizing CCK-8, transwell assays, and wound-healing assays. Furthermore, we performed RNA-seq, qRT-PCR, and western blotting analyses to elucidate and confirm the specific mechanisms underlying the role of MIR4435-2HG in TNBC. RESULTS: In our study, we have identified MIR4435-2HG as a significant diagnostic and prognostic factor for TNBC. We observed that MIR4435-2HG is widely expressed and might have a significant impact on the reshaping of the TNBC tumor microenvironment. Patients with TNBC in the high-MIR4435-2HG group may show reduced sensitivity to cisplatin, doxorubicin, and gemcitabine and have an increased propensity for immune escape. Knockdown of MIR4435-2HG inhibits cancer-associated fibroblasts (CAFs) activation. Notably, MIR4435-2HG predominantly enhances the migratory and invasive capabilities of TNBC cells through the epithelial-mesenchymal transition (EMT) process. Mechanistically, we validated that MIR4435-2HG activates the JNK/c-Jun and p38 non-classical MAPK signaling pathway in TNBC cells. CONCLUSIONS: Our findings highlight the significant potential of MIR4435-2HG as a highly promising biomarker for TNBC. Targeting MIR4435-2HG could represent an appealing therapeutic approach for TNBC.

Corilagin Alleviates Ang II-Induced Cardiac Fibrosis by Regulating the PTEN/AKT/mTOR Pathway.

This research aimed to evaluate the therapeutic effect of corilagin (Cor) against angiotensin II (Ang II)-induced cardiac fibrosis and its underlying mechanisms. C57BL/6 mice (male, 8-10 weeks) received saline or Ang II (2.0 mg/kg/day) via subcutaneous infusion and intraperitoneal injection of Cor (30 mg/kg) for 28 days. Ang II induction increased the fibrotic area, whereas Cor treatment inhibited the fibrotic area significantly. Cor markedly reduced the Ang II-induced cardiac fibroblasts. Cor significantly inhibited Ang II-induced increase in expressions of smooth muscle alpha-actin (α-SMA), collagen I, collagen III, transforming growth factor beta 1 (TGF-ß1), fibronectin, and connective tissue growth factor (CTGF). Cor suppressed the intracellular reactive oxygen species (ROS) production. Cor therapy reduced Ang II-induced malondialdehyde (MDA) content, whereas superoxide dismutase (SOD) and catalase (CAT) activities were increased (all, P < .001). Moreover, Ang II induction elevated the expression of phosphorylated phosphatase and tensin homolog (p-PTEN), phosphorylated protein kinase B (p-AKT) (Ser473) and phosphorylated mammalian target of rapamycin (p-mTOR) (Ser 2448), whereas Cor reduced their expressions. Cor treatment inhibited the migration ability of the cardiac fibroblast, whereas a PTEN inhibitor, VO-ohpic, increased the migration capability. Cor could have a protective effect against Ang II-induced cardiac fibrosis via inhibition of the PTEN/AKT/mTOR pathway.

SFRP1 mediates cancer-associated fibroblasts to suppress cancer cell proliferation and migration in head and neck squamous cell carcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAFs), as key cell populations in the tumor microenvironment (TME), play a crucial role in tumor regulation. Previous studies on a prognostic signature of 8 CAF-related genes in head and neck squamous cell carcinoma (HNSCC) revealed that Secreted frizzled-related protein 1 (SFRP1) is one of the hub genes closely related to CAFs. SFRP1 is deficiently expressed in numerous types of cancer and is classified as a tumor suppressor gene. However, the role of SFRP1 in TME regulation in HNSCC remains unclear. This study aimed to explore the role of SFRP1 in the proliferation and migration of HNSCC cells by mediating CAFs and their regulatory mechanisms. METHODS: The expression differences, prognosis, and immune infiltration of SFRP1 in HNSCC were analyzed using the TIMER and GEPIA2 databases. The expression of SFRP1 in HNSCC tumor tissues, as well as the expression and secretion of SFRP1 in CAFs and tumor cells, were examined. An indirect co-culture system was constructed to detect the proliferation, migration, and apoptosis of HNSCC cells, and to clarify the effect of SFRP1 on tumor cells by mediating CAFs. Furthermore, the expression and secretion of 10 cytokines derived from CAFs that act on immune cells were verified. RESULTS: SFRP1 was differently expressed in HNSCC tumor tissues and highly expressed in CAFs. SFRP1 inhibited the proliferation and migration of tumor cells and promoted apoptosis by mediating CAFs. The detection of CAFs-derived factors suggested that the mechanism of action of SFRP1 was associated with the regulation of immune cells. CONCLUSION: SFRP1 inhibits the proliferation and migration of HNSCC cells by mediating CAFs, and the mechanism of action is related to the regulation of immune cells, which may provide new research directions and therapeutic targets for HNSCC.

Increased susceptibility of human limbal aniridia fibroblasts to oxidative stress.

Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6+/-). In the corneal epithelium of PAX6+/- mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (p≤0.008), while the decrease in PHD1 was limited to AN-LFCs (p=0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (p=0.013). As a result of OS, the mRNA levels of Nrf2 (p<0.0001) and the antioxidant enzymes CAT (p=0.005), SOD1 (p=0.005), GPX1 (p=0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (p=0.019) and PRDX6 (p=0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (p≥0.151) and protein expression (p≥0.629) of antioxidant enzymes, except for the GPX1 mRNA level (p=0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl2 treatment.

Melatonin increases Olaparib sensitivity and suppresses cancer-associated fibroblast activation via suppressing the LAMB3-CXCL2 axis in TNBC.

Triple-negative breast cancer (TNBC) is the most malignant breast cancer subtype, characterized with high aggressiveness and a high recurrence rate. Olaparib is the first US Food and Drug Administration-approved poly(ADP ribose) polymerase (PARP) inhibitor (PARPi) to treat breast cancer patients with a germline BRCA1 or BRCA2 mutation. However, resistance to Olaparib treatment restricts the therapeutic effects, and thus novel therapeutics are urgently required. In the present study, we identified that melatonin combined with Olaparib synergistically enhanced the sensitivity of TNBC cells. Moreover, melatonin exerted promising antitumor activities in Olaparib-resistant cells, implying the potential for its clinical application. An RNA-sequencing analysis revealed that melatonin treatment downregulated laminin subunit beta 3 (LAMB3) expression. Genetic ablation of LAMB3 significantly increased Olaparib sensitivity, and subsequently suppressed proliferation, epithelial-to-mesenchymal transition (EMT)-related gene expressions, and aggressiveness of breast cancer cells. Accordingly, LAMB3 expression was positively correlated with C-X-C motif chemokine ligand 2 (CXCL2), and they collaboratively promoted cancer-associated fibroblast (CAF) infiltration. An in vivo study demonstrated that combined treatment with melatonin and Olaparib showed enhanced inhibitory efficacy against tumor growth, LAMB3 expression, CXCL2 levels, and CAF infiltration compared to single treatment groups, and combined treatment with melatonin and Olaparib significantly ameliorated the immunosuppressive tumor microenvironment. These findings illustrated a promising therapeutic strategy using melatonin to overcome Olaparib resistance and activate antitumor immunity via attenuating the LAMB3-CXCL2 axis in breast cancer patients.

Effect of Ritanserin and Duloxetine on the Gene Expression of Primary Aniridia and Healthy Human Limbal Stromal Cells, In Vitro.

INTRODUCTION: In congenital aniridia caused by mutations in paired box 6 (PAX6), PAX6 influences the migration and differentiation of limbal epithelial cells (LECs), thereby playing a pivotal role in aniridia-associated keratopathy. The antidepressants ritanserin and duloxetine affect PAX6 expression in LECs. Limbal stromal cells, which support limbal epithelial stem cells, are crucial in the limbal stem cell niche. This study explores how ritanserin and duloxetine influence gene expression in primary human limbal stromal cells from subjects with congenital aniridia and from healthy subjects, in vitro. METHODS: Primary human limbal stromal cells from corneas affected by aniridia (AN-LSCs) (n = 8) and from healthy corneas (LSCs) (n = 8) were isolated and cultured in either low-glucose serum-free (LGSF) or normal-glucose serum-containing (NGSC) media. Cells were treated with 4 µM ritanserin or duloxetine for 24 h. Quantitative PCR (qPCR) and western blot were used to assess the expression of PAX6, FOSL2, TGF-ß1, ACTA2A1, LUM, COL1A1, COL5A1, DSG1, FABP5 and ADH7. RESULTS: In AN-LSCs with LGSF-medium, ritanserin increased PAX6 messenger RNA (mRNA) (p = 0.007) and decreased TGF-ß1 and FOSL2 mRNA levels (P = 0.005, P = 0.038). In addition, TGF-ß1 protein levels decreased with both treatments (P = 0.02, P = 0.007), and FABP5 protein level increased, using ritanserin (P = 0.019). In LSCs with LGSF-medium, ACTA2A1 mRNA levels decreased using ritanserin and duloxetine (P = 0.028; P = 0.031), while FABP5 mRNA levels increased with ritanserin treatment (P = 0.003). Also, duloxetine use reduced α-SMA protein (P = 0.013) and increased FABP5 protein levels (P = 0.029). In LSCs with NGSC-medium, ritanserin elevated LUM, FABP5 and ADH7 mRNA and protein levels (P = 0.025, P = 0.003, P = 0.047, P = 0.024, P = 0.013, P = 0.039). CONCLUSIONS: The results of our study confirmed that the antipsychotropic drugs ritanserin and duloxetine alter PAX6 and TGF-ß1 gene expression in AN-LSCs cultured in LGSF-medium. These drugs were found to have an impact on retinoic acid signaling pathways and keratocyte characteristic markers both in LSCs and AN-LSCs, using different culture media.

Ischemic cardiac stromal fibroblast-derived protein mediators in the infarcted myocardium and transcriptomic profiling at single cell resolution.

This article focuses on screening the major secreted proteins by the ischemia-challenged cardiac stromal fibroblasts (CF), the assessment of their expression status and functional role in the post-ischemic left ventricle (LV) and in the ischemia-challenged CF culture and to phenotype CF at single cell resolution based on the positivity of the identified mediators. The expression level of CRSP2, HSP27, IL-8, Cofilin-1, and HSP90 in the LV tissues following coronary artery bypass graft (CABG) and myocardial infarction (MI) and CF cells followed the screening profile derived from the MS/MS findings. The histology data unveiled ECM disorganization, inflammation and fibrosis reflecting the ischemic pathology. CRSP2, HSP27, and HSP90 were significantly upregulated in the LV-CABG tissues with a concomitant reduction ion LV-MI whereas Cofilin-1, IL8, Nrf2, and Troponin I were downregulated in LV-CABG and increased in LV-MI. Similar trends were exhibited by ischemic CF. Single cell transcriptomics revealed multiple sub-phenotypes of CF based on their respective upregulation of CRSP2, HSP27, IL-8, Cofilin-1, HSP90, Troponin I and Nrf2 unveiling pathological and pro-healing phenotypes. Further investigations regarding the underlying signaling mechanisms and validation of sub-populations would offer novel translational avenues for the management of cardiac diseases.

A comprehensive evaluation of dermal fibroblast therapy in clinical trials for treating skin disorders and cosmetic applications: a scoping review.

BACKGROUND: Fibroblast cells have the ability to improve skin conditions through regenerative medicine and cell-based therapies. The purpose of this scoping review is to assess the contribution of fibroblast cells to skin homeostasis and extracellular matrix deposition in clinical trials involving skin disorders and cosmetic applications. METHODS: Using targeted search terms, published publications from January 2000 to August 2023 that addressed fibroblast uses in clinical trials of skin conditions were obtained from bibliographic databases like PubMed, Scopus, and Web of Science (WoS). Precise inclusion and exclusion criteria were used during the screening process. The potential benefits of induction treatment with fibroblasts lead to the choosing of clinical trials for this kind of treatment. RESULTS: Out of the 820 published ppapers initially identified, only 35 studies fulfilled our meticulous eligibility criteria after careful screening. To ensure clarity, we methodically eliminated any duplicate or irrelevant published papers, thereby offering a transparent account of our selection process. CONCLUSION: This study highlights the advantages of fibroblast therapy in treating skin conditions such as diabetic foot, venous leg ulcers, and cosmetic reasons. Fibroblasts possess remarkable regenerating capabilities, making dermal fibroblast therapy crucial in cell-based and skin regenerative treatments. Nevertheless, additional research is required for more disorders and cosmetic applications.

Matairesinol blunts adverse cardiac remodeling and heart failure induced by pressure overload by regulating Prdx1 and PI3K/AKT/FOXO1 signaling.

BACKGROUND: Pathological cardiac remodeling is a critical process leading to heart failure, characterized primarily by inflammation and apoptosis. Matairesinol (Mat), a key chemical component of Podocarpus macrophyllus resin, exhibits a wide range of pharmacological activities, including anti-hydatid, antioxidant, antitumor, and anti-inflammatory effects. PURPOSE: This study aims to investigate whether Matairesinol alleviate cardiac hypertrophy and remodeling caused by pressure overload and to elucidate its mechanism of action. METHODS: An in vitro pressure loading model was established using neonatal rat cardiomyocytes treated with angiotensin Ⅱ, while an in vivo model was created using C57 mice subjected to transverse aortic constriction (TAC). To activate the PI3K/Akt/FoxO1 pathway, Ys-49 was employed. Moreover, small interfering RNA (siRNA) and short hairpin RNA (shRNA) were utilized to silence Prdx1 expression both in vitro and in vivo. Various techniques, including echocardiography, wheat germ agglutinin (WGA) staining, HE staining, PSR staining, and Masson trichrome staining, were used to assess cardiac function, cardiomyocyte cross-sectional area, and fibrosis levels in rats. Apoptosis in myocardial tissue and in vitro was detected by TUNEL assay, while reactive oxygen species (ROS) content in tissues and cells was measured using DHE staining. Furthermore, the affinity of Prdx1 with Mat and PI3K was analyzed using computer-simulated molecular docking. Western blotting and RT-PCR were utilized to evaluate Prdx1 levels and proteins related to apoptosis and oxidative stress, as well as the mRNA levels of cardiac hypertrophy and fibrosis-related indicators. RESULTS: Mat significantly alleviated cardiac hypertrophy and fibrosis induced by TAC, preserved cardiac function, and markedly reduced cardiomyocyte apoptosis and oxidative damage. In vitro, mat attenuated ang Ⅱ - induced hypertrophy of nrvms and activation of neonatal rat fibroblasts. Notably, activation of the PI3K/Akt/FoxO1 pathway and downregulation of Prdx1 expression were observed in TAC mice; however, these effects were reversed by Mat treatment. Furthermore, Prdx1 knockdown activated the PI3K/Akt/FoxO1 pathway, leading to exacerbation of the disease. Molecular docking indicated that Molecular docking indicated that Mat upregulated Prdx1 expression by binding to it, thereby inhibiting the PI3K/Akt/FoxO1 pathway and protecting the heart by restoring Prdx1 expression levels. CONCLUSION: Matairesinol alleviates pressure overload-induced cardiac remodeling both in vivo and in vitro by upregulating Prdx1 expression and inhibiting the PI3K/Akt/FoxO1 pathway. This study highlights the therapeutic potential of Matairesinol in the treatment of cardiac hypertrophy and remodeling, providing a promising avenue for future research and clinical application.

Overexpression of Basonuclin Zinc Finger Protein 2 in stromal cell is related to mesenchymal phenotype and immunosuppression of mucinous colorectal adenocarcinoma.

BACKGROUND: Mucinous carcinoma (MC) is a distinct histologic subtype of colorectal cancer (CRC) that is less studied and associated with poor prognosis. This study aimed to identify MC-specific therapeutic targets and biomarkers to improve the prognosis of this aggressive disease. METHODS: CRC samples from The Cancer Genome Atlas (TCGA) were categorized into MC and non-MC (NMC) groups based on histologic type. A multi-scale embedded gene co-expression network analysis (MEGENA) was constructed to identify gene modules associated with the MC group. The potential functions of Basonuclin Zinc Finger Protein 2 (BNC2) were further analyzed using the Biomarker Exploration for Solid Tumors (BEST) database. In vivo and in vitro experiments were conducted to validate the predicted results. RESULTS: We identified the stromal component-related gene, BNC2, in the MC population. This gene is associated with a shorter progression-free interval (PFI) in CRC patients. BNC2 promotes FAP (encoding Fibroblast Activation Protein Alpha) transcription in cancer-associated fibroblasts (CAFs) and is involved in angiogenesis through two pathways. Additionally, BNC2 enhances tumor cell invasiveness in a CAF-dependent manner. Patients with high BNC2 expression benefited less from immunotherapy compared to those with low BNC2 expression. CONCLUSIONS: Our study highlights the clinical importance of BNC2 in MC, and targeting BNC2 on stromal cells (fibroblasts and endothelial cells) may be an effective strategy for treating MC.

The healing effects of moderate exercise on acetic acid-induced gastric ulcer in male rats.

Aim: This study aimed to evaluate the effect of moderate exercise on the healing of acetic acid-induced gastric ulcers in male rats. Background: Gastric ulcers include benign mucosal and submucosal lesions of the gastric wall. Exercise regulates a wide range of physiological processes. Methods: 48 male Wistar rats were randomly divided into three experimental groups (n=16 per group) as follows: control, which was left untreated after causing stomach ulcers; experimental group 1, the rats were first exercised and then received acetic acid; experimental group 2, the rats received acetic acid, and then exercised. The ulcer was caused by injecting 0.12 ml of a 60% acetic acid solution after 24 hours of not eating. The rats had a period of moderate treadmill activity either before or after the development of ulcers, lasting for a duration of 30 days. On the seventh and fourteenth days after the experiment, the rats were sacrificed, their stomach was removed, and the wound healing parameters, and wound depth were determined. Results: Exercise before and after inducing gastric ulcers significantly decreased the depth of gastric ulcers in the experimental groups. The average number of PMN in the control group decreased in comparison to the seventh and fourteenth days following the experiment. Conversely, the number of fibroblasts, epithelialization, and new vessels increased. It seems that exercise before the appearance of ulcers has a greater effect on gastric ulcers compared to exercise after inducing gastric ulcers. Conclusion: Exercise can prepare the gastric mucosa for forthcoming injuries, and heal gastric ulcers. Moderate aerobic exercise has significant restorative effects on gastric ulcers caused by acetic acid and is recommended.

Integrative Analysis and Validation of a Cancer-associated Fibroblasts Senescence-related Signature for Risk Stratification and Therapeutic Prediction in Esophageal Squamous Cell Carcinoma.

Cellular senescence is closely associated with cancer development and progression. There is ample evidence that tumor stromal cells, especially cancer-associated fibroblasts (CAFs) undergo senescence in response to various stimuli. However, the possible biological roles and prognostic significance of senescent CAFs in esophageal squamous cell carcinoma (ESCC) remain unexplored. In this study, we found that CAFs exhibited a significantly higher level of cellular senescence than other cell clusters at the single-cell level. Then, we constructed a CAFs senescence-associated risk model with 7 genes (GEM, SLC2A6, CXCL14, STX11, EFHD2, PTX3, and HCK) through Cox regression and LASSO analysis. Kaplan-Meier survival analysis revealed that the risk model was significantly correlated with worse prognosis in training and validation cohorts. Subsequent analysis indicated that the risk model was an independent prognostic factor. In addition, the signature showed a distinct negative correlation with immune cell infiltration and immunotherapy responses. In vitro experiments showed remarkably higher mRNA and protein levels of prognosis-related genes (STX11 and EFHD2) in senescent CAFs than control group, consistent with the bioinformatics analysis results. Moreover, senescent CAFs significantly promoted ESCC cell proliferation and migration as shown by CCK-8 and scratch assays. In conclusion, our study identified a novel CAFs senescence-based classifier that may help predict prognosis of ESCC, and a thorough characterization of the signature could also be helpful in evaluating the response of ESCC to anti-tumor therapies and provide meaningful clinical options for cancer treatment.

Immunological signatures from irradiated cancer-associated fibroblasts.

Introduction: Cancer-associated fibroblasts (CAFs) are abundant and influential elements of the tumor microenvironment (TME), giving support to tumor development in multiple ways. Among other mechanisms, CAFs are important regulators of immunological processes occurring in tumors. However, CAF-mediated tumor immunomodulation in the context of radiotherapy remains poorly understood. In this study, we explore effects of radiation on CAF-derived immunoregulatory signals to the TME. Methods: Primary CAF cultures were established from freshly collected human NSCLC lung tumors. CAFs were exposed to single-high or fractionated radiation regimens (1x18Gy or 3x6Gy), and the expression of different immunoregulatory cell-associated and secreted signaling molecules was analyzed 48h and 6 days after initiation of treatment. Analyses included quantitative measurements of released damage-associated molecular patterns (DAMPs), interferon (IFN) type I responses, expression of immune regulatory receptors, and secretion of soluble cytokines, chemokines, and growth factors. CAFs are able to survive ablative radiation regimens, however they enter into a stage of premature cell senescence. Results: Our data show that CAFs avoid apoptosis and do not contribute by release of DAMPs or IFN-I secretion to radiation-mediated tumor immunoregulation. Furthermore, the secretion of relevant immunoregulatory cytokines and growth factors including TGF-ß, IL-6, IL-10, TNFα, IL-1ß, VEGF, CXCL12, and CXCL10 remain comparable between non-irradiated and radiation-induced senescent CAFs. Importantly, radiation exposure modifies the cell surface expression of some key immunoregulatory receptors, including upregulation of CD73 and CD276. Discussion: Our data suggest that CAFs do not participate in the release of danger signals or IFN-I secretion following radiotherapy. The immune phenotype of CAFs and radiation-induced senescent CAFs is similar, however, the observed elevation of some cell surface immunological receptors on irradiated CAFs could contribute to the establishment of an enhanced immunosuppressive TME after radiotherapy.

Development of a Simvastatin-Loaded Copolymer Acid-Sensitive Nanocarrier and Its Experimental Use in the Treatment of Keloids.

OBJECTIVE: The lipid-lowering simvastatin (SIM) has been shown to be an effective inhibitor of keloid proliferation. However, due to its low water solubility and short half-life, simvastatin aggregates to the liver and does not reach the skin lesions after oral administration, which restricts its widespread clinical use. The development of nanomedicine provides the possibility for us to break through this bottleneck problem clinically. The objective of this study was to investigate the feasibility of using complex nanocontrolled delivery system (CNDS), simvastatin-loaded polyethylene glycol-poly lactic-co-glycolic acid (PEG-PLGA), in the treatment of keloids. METHODS: In the in vitro study, the release of simvastatin in fibroblasts by CNDS@Simvastatin and its effect on inhibition of the proliferation of fibroblasts, Col Ι, and CTGF by the slow release of simvastatin were assessed. The efficacy of CNDS@Simvastatin in improving keloids and the biocompatibility and safety of CNDS@Simvastatin were examined in vivo by establishing a murine ear keloid model. RESULTS: CNDS@Simvastatin showed sustained and uniform inhibition of the proliferation of fibroblasts, Col Ι, and CTGF via the gradual release of simvastatin over 72 h. It was efficient in the treatment of the murine ear keloid with no observable toxic side effects on various organs. CONCLUSION: Simvastatin-loaded copolymer acid-sensitive nanocarriers, CNDS@Simvastatin nanospheres, were successfully developed in this study, and these were characterized by favorable physicochemical properties and biocompatibility.

Sacsin levels in PBMCs: A diagnostic assay for SACS variants in peripheral blood cells - A PROSPAX study.

BACKGROUND: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a common recessive ataxia that is still underdiagnosed worldwide. An easily accessible diagnostic biomarker might help to diagnostically confirm patients presenting SACS variants of unknown significance (VUS) or atypical phenotypes. OBJECTIVES: To detect sacsin in peripheral blood mononuclear cells (PBMCs) and to validate its diagnostic biomarker quality to discriminate biallelic SACS patients (including patients with VUS and/or atypical phenotypes) against healthy controls, non-ARSACS spastic ataxia patients, and heterozygous SACS carriers. METHODS: Sacsin protein levels in PBMCs were assessed in patients versus controls and validated in skin-derived fibroblasts. RESULTS: Patients with biallelic SACS variants - including patients with VUS and/or atypical phenotypes - showed loss of sacsin in PBMCs, with discriminative performance against healthy, heterozygous, and non-ARSACS controls. This included all investigated SACS missense variants. Also, C-terminal variants escaping nonsense-mediated decay, while not differing from controls in expression level, showed lower molecular weight in this assay. CONCLUSIONS: Assessing sacsin levels using PBMCs offers an easy, peripherally accessible diagnostic biomarker for ARSACS, with PBMCs being much less invasive and easier to handle than fibroblasts. Additionally, this might be a potential target-engagement blood biomarker for sacsin-increasing therapies. © 2024 International Parkinson and Movement Disorder Society.