Actin Levels Research Articles

Urinary miR-185-5p is a biomarker of renal tubulointerstitial fibrosis in IgA nephropathy.

For IgA nephropathy (IgAN), tubular atrophy/interstitial fibrosis is the most important prognostic pathological indicator in the mesangial and endocapillary hypercellularity, segmental sclerosis, interstitial fibrosis/tubular atrophy, and presence of crescents (MEST-C) score. The identification of non-invasive biomarkers for tubular atrophy/interstitial fibrosis would aid clinical monitoring of IgAN progression and improve patient prognosis. The study included 188 patients with primary IgAN in separate confirmation and validation cohorts. The associations of miR-92a-3p, miR-425-5p, and miR-185-5p with renal histopathological lesions and prognosis were explored using Spearman correlation analysis and Kaplan-Meier survival curves. Bioinformatics analysis and dual luciferase experiments were used to identify hub genes for miR-185-5p. The fibrotic phenotypes of tubular epithelial cells were evaluated in vivo and in HK-2 cells. miRNA sequencing and cohort validation revealed that the expression levels of miR-92a-3p, miR-425-5p, and miR-185-5p in urine were significantly increased among patients with IgAN; these levels could predict the extent of tubular atrophy/interstitial fibrosis in such patients. The combination of the three biomarkers resulted in an area under the receiver operating characteristic curve of 0.742. The renal prognosis was significantly worse in the miR-185-5p high expression group than in the low expression group (P=0.003). Renal tissue in situ hybridization, bioinformatics analysis, and dual luciferase experiments confirmed that miR-185-5p affects prognosis in patients with IgAN mainly by influencing expression of the target gene tight junction protein 1 (TJP1) in renal tubular epithelial cells. In vitro experiment revealed that an miR-185-5p mimic could reduce TJP1 expression in HK-2 cells, while increasing the levels of α-smooth muscle actin, fibronectin, collagen I, and collagen III; these changes promoted the transformation of renal tubular epithelial cells to a fibrotic phenotype. An miR-185-5p inhibitor can reverse the fibrotic phenotype in renal tubular epithelial cells. In a unilateral ureteral obstruction model, the inhibition of miR-185-5p expression alleviated tubular atrophy/interstitial fibrosis. Urinary miR-185-5p, a non-invasive biomarker of tubular atrophy/interstitial fibrosis in IgAN, may promote the transformation of renal tubular epithelial cells to a fibrotic phenotype via TJP1.

Effects of inhibition of Rho/ROCK pathway on proliferation and migration of airway smooth muscle cells and related mechanisms

To investigate the effects and molecular mechanisms of inhibition of the Ras homolog gene (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway on the proliferation and migration of airway smooth muscle cells involving myocardin (MYOCD). Human airway smooth muscle cells were infected with the adenoviral vector Ad-ZsGreen-shRNA-hROCK1 in vitro. The cells were randomly divided into four groups: ROCK1 gene silencing control (shNC) group, shNC + arachidonic acid (AA, Rho/ROCK pathway activator) group, ROCK1 gene silencing (shROCK1) group, and shROCK1 + AA group (n=3 each). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK1 and MYOCD mRNA and protein. ELISA was employed to measure the levels of globular actin and filamentous actin, while immunofluorescent staining and scratch assays were utilized to assess cell proliferation and migration. Compared to the shNC + AA group, the shROCK1 + AA group exhibited decreased levels of ROCK1 and MYOCD mRNA and protein expression, reduced expression levels of globular actin and filamentous actin, and diminished cell proliferation and migration capabilities (P<0.05). Inhibition of the Rho/ROCK pathway suppresses the proliferation and migration of airway smooth muscle cells, which may be associated with the downregulation of MYOCD.

Improvement of idiopathic pulmonary fibrosis through a combination of Astragalus radix and Angelica sinensis radix via mammalian target of rapamycin signaling pathway-induced autophagy in rat.

There is a major need for effective, well-tolerated treatments for idiopathic pulmonary fibrosis (IPF) in clinic. Astragalus radix (AR; Huangqi) and Angelica sinensis radix (AS; Danggui) have been frequently used in the treatment of IPF. This study aimed to reveal the pharmacological effects and the mechanisms of the action of an AR-AS combination in treating IPF. Sprague-Dawley rats were randomly divided into six groups (n=5): control, bleomycin (BLM) model, AR, AS, AR + AS, and prednisone (PDN) groups. A transforming growth factor-β1 (TGF-β1)-induced MRC-5 cell model were also used. Pulmonary fibrosis, inflammation, oxidative stress, and autophagy were evaluated by performing hematoxylin and eosin (H&E) staining, Masson staining, immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and hydroxyproline assay following the treatment of AR, AS, and the AR-AS herb pair. Rats administered the AR-AS herb pair had lower α-smooth muscle actin (α-SMA), collagen I, fibronectin, and vimentin levels in lung tissues, and lower inflammatory cytokine levels in rat serum. In addition, the AR-AS herb pair induced mammalian target of rapamycin (mTOR)-mediated autophagy and reduced oxidative stress in BLM-induced rats. The effects of the AR and AS combination were confirmed in MRC-5 cells treated with TGF-β1. Specifically, the combination of AR and AS attenuated MRC-5 cell fibrosis, inflammation, and oxidative stress while inducing autophagy. The combination of AR and AS protects against IPF by inducing autophagy via inhibiting the mTOR signaling pathway. The synergistic action of AR and AS is superior to that of either AR or AS alone.

Abstract B057: An organoids-on-a-chip model to recapitulate and dissect the tumor microenvironment of PDAC

Abstract Abstract Body: Characteristics of the PDAC tumor microenvironment (TME) such as desmoplasia and an immunosuppressive landscape contribute to its resistance to chemotherapy and immunotherapy approaches. To recapitulate and dissect the in vivo PDAC TME, we bioengineered a PDAC organoid-on-a-chip model by integrating patient-derived PDAC organoids on a microfluidic-based organ-on-a-chip. While the patient-derived organoids (PDOs) maintain the characteristics of the in vivo counterparts with high fidelity, the organ-on-a-chip provides a controllable and reproducible environment with stromal and immune niche cells, creating an in vitro model that may providing insights into PDAC drug resistance and allow testing for new therapies. The multicellular organoid-on-a-chip model is composed of 1) a central PDAC niche including patient-derived PDAC organoids, patient-derived cancer-associated macrophages (CAFs), tumor-associated macrophages (TAMs) and blood vessels, and 2) surrounding vascular networks for nutrient transport and drug delivery. We reproduced the hypovasculariy of PDAC stromal niche on the chip and found a lower level of CD31 expression in the PDAC organoid niche than in the normal pancreatic organoid niche (p&amp;lt;0.0001). Comparing with the niche without PDOs, the PDAC niche on chip demonstrated a higher expression level of α-smooth muscle actin (α-SMA) on CAFs (fold-change=2.32, p&amp;lt;0.0001) and a more extensive deposition of ECM components (e.g., collagen I, collagen III, collagen IV and hyaluronic acid, fold-change&amp;gt;=2.25, p⇐0.0002), indicating the existence of desmoplasia on chip. Profiling of cytokine levels in the media revealed that, compared to models used with normal pancreatic epithelial cells with the other cellular components, the PDAC organoid TME induced the upregulation of a series of pro-tumor cytokines including MIP-3α, IL-8, CXCL5, IL-13 (fold-change&amp;gt;=5.02), the inflammation-related cytokines MIF, IL-5, MCP-1 (fold-change&amp;gt;=1.82), and CAF-derived cytokines like CXCL10, HGF, CXCL1, CXCL12 (fold-change&amp;gt;=1.82). The immunosuppressive cytokine TGF-β was increased after PDOs were loaded on the chip (762.3 pg/mL vs. 222.6 pg/mL in conditioned medium), as was the proinflammatory cytokine IL-6 (1089.5 pg/mL vs. 81.3 pg/mL), was maintained at a higher level than that in the niche with normal organoids (380.5 pg/mL vs. 60.4 pg/mL at day 13). We also found that PDOs induced TAMs inclined to be M2-like (CD68+CD163+) phenotype on chip. In turn niche cells (TAMs and CAFs) contributed to the immunosuppression in PDAC TME: the secretion of cytokines like IL-6, IL-13, MIP-1β and HGF were downregulated without niche cells (fold-change⇐0.646). In conclusion, we have developed a PDAC organoid-on-a-chip recapitulates the PDAC TME and is promising to serving as a platform to study mechanisms of drug resistance in PDAC as well as test novel therapies. Citation Format: Lunan Liu, Diane M. Simeone, Weiqiang Chen. An organoids-on-a-chip model to recapitulate and dissect the tumor microenvironment of PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B057.

Leech extract alleviates idiopathic pulmonary fibrosis by TGF-β1/Smad3 signaling pathway

Ethnopharmacological relevanceLeech, as a traditional Chinese medicine for the treatment of blood circulation and blood stasis, was also widely used to cure pulmonary fibrosis in China. In clinical practice, some traditional Chinese medicine preparation such as Shui Zhi Xuan Bi Hua Xian Tang and Shui Zhi Tong Luo Capsule composed of leech, could improve the clinical symptoms and pulmonary function in patients with idiopathic pulmonary fibrosis (IPF). However, the material basis of the leech in the treatment of IPF were not yet clear. Aim of the studyScreen out the components of leech that have the anti-pulmonary fibrosis effects, and further explore the therapeutic mechanism of the active components. Materials and methodsIn this study, the different molecular weight components of leech extract samples were prepared using the semi-permeable membranes with different pore sizes. The therapeutic effects of the leech extract groups with molecular weight greater than 10 KDa (>10 KDa group), between 3 KDa and 10 KDa (3–10 KDa group), and less than 3 KDa (<3 KDa group) on pulmonary fibrosis were firstly investigated by cell proliferation and cytotoxicity assay (MTT), cell wound healing assay, immunofluorescence staining (IF) and Western blot (WB) assay through the TGF-β1-induced fibroblast cell model. Then bleomycin-induced pulmonary fibrosis (BML-induced PF) mouse model was constructed to investigate the pharmacological activities of the active component group of leech extract in vivo. Pathological changes of the mouse lung were observed by hematoxylin-eosin staining (H&E) and Masson's trichrome staining (Masson). The hydroxyproline (HYP) content of lung tissues was quantified by HYP detection kit. The levels of extracellular matrix-related fibronectin (FN) and collagen type Ⅰ (Collagen Ⅰ), pyruvate kinase M2 (PKM2) monomer and Smad7 protein were determined via WB method. PKM2 and Smad7 protein were further characterized by IF assays. ResultsUsing TGF-β1-induced HFL1 cell line as a PF cell model, the in vitro results demonstrated that the >10 KDa group could significantly inhibited the cell proliferation and migration, downregulated the expression level of cytoskeletal protein vimentin and α-smooth muscle actin (α-SMA), and reduced the deposition of FN and Collagen Ⅰ. In the BML-induced PF mouse model, the >10 KDa group significantly reduced the content of HYP, downregulated the expression levels of FN and Collagen Ⅰ in lung tissues, and delayed the pathological changes of lung tissue structure. The results of WB and IF assays further indicated that the >10 KDa group could up-regulate the expression level of PKM2 monomer and Smad7 protein in the cellular level, thereby delaying the progression of pulmonary fibrosis. ConclusionsOur study revealed that the >10 KDa group was the main material basis of the leech extract that inhibited pulmonary fibrosis through TGF-β1/Smad3 signaling pathway.

The Inhibition Effect and Mechanism of Staurosporine Isolated from Streptomyces sp. SNC087 Strain on Nasal Polyp.

This study aims to explore the potential inhibition effects of staurosporine isolated from a Streptomyces sp. SNC087 strain obtained from seawater on nasal polyps. Staurosporine possesses antimicrobial and antihypertensive activities. This research focuses on investigating the effects of staurosporine on suppressing the growth and development of nasal polyps and elucidating the underlying mechanisms involved. The experimental design includes in vitro and ex vivo evaluations to assess the inhibition activity and therapeutic potential of staurosporine against nasal polyps. Nasal polyp-derived fibroblasts (NPDFs) were stimulated with TGF-β1 in the presence of staurosporine. The levels of α-smooth muscle actin (α-SMA), collagen type-I (Col-1), fibronectin, and phosphorylated (p)-Smad 2 were investigated using Western blotting. VEGF expression levels were analyzed in nasal polyp organ cultures treated with staurosporine. TGF-β1 stimulated the production of Col-1, fibronectin, and α-SMA and was attenuated by staurosporine pretreatment. Furthermore, these inhibitory effects were mediated by modulation of the signaling pathway of Smad 2 in TGF-β1-induced NPDFs. Staurosporine also inhibits the production of VEGF in ex vivo NP tissues. The findings from this study will contribute to a better understanding of staurosporine's role in nasal polyp management and provide insights into its mechanisms of action.

FOXM1 Participates in Scleral Remodeling in Myopia by Upregulating APOA1 Expression Through METTL3/YTHDF2.

Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in scleral remodeling in myopia and its underlying mechanisms. A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1. Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis. FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.

Plac8 regulates the fibrogenic ability of cardiac fibroblasts in mice with myocardial infarction

Cardiac fibroblasts (CFs) are of vital importance for post-myocardial infarction (MI) remodeling. This study explored the role of Placenta specific 8 (Plac8) in MI on the basis of single-cell RNA-Seq (scRNA-Seq) data (GSE136088) and micro-array data (GSE153485). Through bioinformatics analysis, Plac8 was finally identified as a key gene related to fibroblasts. In addition, primary CFs of the neonatal mice were isolated, cultured and treated with hypoxia for in vitro cell model construction. Hydroxyproline assay demonstrated enhanced collagen production in hypoxic CFs, and qRT-PCR revealed increased relative contents of type I and type III collagen (Col1a1 and Col3a1). Western blot showed increased protein expression level of α-smooth muscle actin (α-SMA), and transwell assay and wound-healing assay collectively suggested enhanced cell migration. Moreover, Plac8 was found to be upregulated in hypoxic CFs. Then, after transfection with siNC or siPlac8, it was revealed that down-regulating Plac8 protein expression in hypoxic CFs reduced the levels of collagen secretion, the number of activated CFs and the degree of cell migration, indicating the regulation of Plac8 on hypoxic CFs’ transformation into myofibroblasts, cell migration and collagen deposition. Excessive myocardial fibrosis can cause adverse cardiac remodeling, leading to malignant arrhythmia and even heart failure.

Effect of total astragalosides on diabetic non-healing wound by regulating immune microenvironment.

The aim of the research was to analyse the regulatory effect of astragaloside (AST) on the immune microenvironment of diabetic non-healing wound (DNHW), and to analyse the clinical efficacy and mechanism of wound repair in multiple layers. Ninety adult male Wistar rats, which were kept healthy (SPF) under natural infection, were randomly divided into three groups, namely, blank, control and observation groups, with 30 rats in each group. After adaptive feeding for 7 days, the diabetes model was established. After the model was formed, the wounds were uniformly prepared, and then the blank group only was shaved. Both the control group and the observation group were treated with moist exposure therapy. The control group was covered with physiological saline gauze, while the observation group was covered with AST gauze. The healing status of the wounds in both groups was observed and recorded on the 1st, 7th, and 14th days after formation. And the levels of α-smooth muscle actin (α-SMA) and collagen I (COL-1) in the wound tissue were measured. On the 1st day after wound formation, the wound healing area, α-SMA, and COL-1 levels in the three groups were consistent (p > 0.05). On the 7th and 14th days after wound formation, the wound healing area in the three groups increased compared within the group, but only the control and observation groups had significantly higher wound healing area than on the 1st day after wound formation (p < 0.05). In addition, the blank group had lower levels of α-SMA and COL-1, while the control and observation groups had higher levels of α-SMA and COL-1 (p < 0.05). In the comparison between groups, the wound healing area, α-SMA, and COL-1 levels in the control and observation groups were higher than those in the blank group, while the wound healing area, α-SMA, and COL-1 levels in the observation group were higher than those in the control group (p < 0.05). AST can regulate the immune microenvironment of DNHW, improve α-SMA and COL-1, and accelerate the wound healing of DNHW.

Self-generating electricity system driven by aqueous humor flow and trabecular meshwork contraction motion activated BCKa for glaucoma intraocular pressure treatment.

Primary open-angle glaucoma (POAG) is the most common form of glaucoma and the leading cause of irreversible vision loss and blindness worldwide. Intraocular pressure (IOP) is the only modifiable risk factor, and prompt treatment to lower IOP can effectively slow the rate of vision loss due to glaucoma. Trabecular meshwork (TM) cells can maintain IOP homeostasis by correcting and adjusting the resistance to aqueous humor outflow in response to sustained pressure changes. TM cells' function is reduced, and membrane ion channels are impaired in POAG. The dysfunction of Large conductance Ca2+-activated K+ (BKCa) plays a central role in the pathogenesis of POAG. In this work, we targeted MXene nanoparticles (MXene-RGD) with piezoelectric response to TM cells in a 3D model of glaucoma in vitro as well as in the rabbit Transient Ocular Hypertension (OHT) Model in vivo. MXene-RGD gives the TM electromechanical transfer properties, while the self-enhancing and self-generated electricity properties of the TM are determined by the aqueous humor flow rate and the size of the deformation of the TM. MXene-RGD is nontoxic, as illustrated by a cell toxicity study and histological examination. In a 3D in vitro model of high-pressure glaucoma, whole-cell patch-clamp confirmed that piezoelectric stimulation turns on BKCa, which reduces the volume of the cell. MXene-RGD was injected into the anterior chamber with minimal trauma, i.e., anterior chamber injection, and specifically targeted to TM cells. The OHT model in vivo confirmed the potential IOP-lowering ability of MXene-RGD. We evaluated the ion channels involved in the reduction of IOP by MXene-RGD by pre-treatment with a BKCa channel blocker (iberiotoxin, IbTX) and a voltage-gated Ca2+channel blocker (nifedipine). Quantitative qPCR analysis showed that MXene-RGD inhibited the upregulation of mRNA expression levels of the myofibroblast marker α-smooth muscle actin (α-SMA) and the inflammatory response marker interleukin-6 (IL-6) induced by IOP. Histology confirmed that MXene-RGD attenuated IOP-induced proliferation and collagen production in the TM. Taken together, we present for the first time a minimally invasive surgical approach for targeting TM cells for POAG by utilizing piezoresponse nanomaterials to target BKCa to repair or awaken the ability of TM cells to regulate IOP homeostasis on their own.

Immunohistochemical FAP Expression Reflects 68Ga-FAPI PET Imaging Properties of Low- and High-Grade Intraductal Papillary Mucinous Neoplasms and Pancreatic Ductal Adenocarcinoma.

Pancreatic intraductal papillary mucinous neoplasms (IPMNs) are grossly visible (typically > 5 mm) intraductal epithelial neoplasms of mucin-producing cells, arising in the main pancreatic duct or its branches. According to the current 2-tiered grading scheme, these lesions are categorized as having either low-grade (LG) dysplasia, which has a benign prognosis, or high-grade (HG) dysplasia, which formally represents a carcinoma in situ and thus can transform to pancreatic ductal adenocarcinoma (PDAC). Because both entities require different treatments according to their risk of becoming malignant, a precise pretherapeutic diagnostic differentiation is inevitable for adequate patient management. Recently, our group has demonstrated that 68Ga-fibroblast activation protein (FAP) inhibitor (FAPI) PET/CT shows great potential for the differentiation of LG IPMNs, HG IPMNs, and PDAC according to marked differences in signal intensity and tracer dynamics. The purpose of this study was to biologically validate FAP as a target for PET imaging by analyzing immunohistochemical FAP expression in LG IPMNs, HG IPMNs, and PDAC and comparing with SUV and time to peak (TTP) measured in our prior study. Methods: To evaluate the correlation of the expression level of FAP and α-smooth muscle actin (αSMA) in neoplasm-associated stroma depending on the degree of dysplasia in IPMNs, 98 patients with a diagnosis of LG IPMN, HG IPMN, PDAC with associated HG IPMN, or PDAC who underwent pancreatic surgery at the University Hospital Heidelberg between 2017 and 2023 were identified using the database of the Institute of Pathology, University Hospital Heidelberg. In a reevaluation of hematoxylin- and eosin-stained tissue sections of formalin-fixed and paraffin-embedded resection material from the archive, which was originally generated for histopathologic routine diagnostics, a regrading of IPMNs was performed by a pathologist according to the current 2-tiered grading scheme, consequently eliminating the former diagnosis of "IPMN with intermediate-grade dysplasia." For each case, semithin tissue sections of 3 paraffin blocks containing neoplasm were immunohistologically stained with antibodies directed against FAP and αSMA. In a masked approach, a semiquantitative analysis of the immunohistochemically stained slides was finally performed by a pathologist by adapting the immunoreactive score (IRS) and human epidermal growth factor receptor 2 (Her2)/neu score to determine the intensity and percentage of FAP- and αSMA-positive cells. Afterward, the IRS of 14 patients who underwent 68Ga-FAPI-74 PET/CT in our previous study was compared with their SUVmax, SUVmean, and TTP for result validation. Results: From 98 patients, 294 specimens (3 replicates per patient) were immunohistochemically stained for FAP and αSMA. Twenty-three patients had LG IPMNs, 11 had HG IPMNs, 10 had HG IPMNs plus PDAC, and 54 had PDAC. The tumor stroma was in all cases variably positive for FAP. The staining intensity, percentage of FAP-positive stroma, IRS, and Her2/neu score increased with higher malignancy. αSMA expression could be shown in normal pancreatic stroma as well as within peri- and intraneoplastic desmoplastic reaction. No homogeneous increase in intensity, percentage, IRS, and Her2/neu score with higher malignancy was observed for αSMA. The comparison of the mean IRS of FAP with the mean SUVmax, SUVmean, and TTP of 68Ga-GAPI-74 PET/CT showed a matching value increasing with higher malignancy in 68Ga-FAPI-74 PET imaging and immunohistochemical FAP expression. Conclusion: The immunohistochemical staining of IPMNs and PDAC validates FAP as a biology-based stromal target for invivo imaging. Increasing expression of FAP in lesions with a higher degree of malignancy matches the expectation of a stronger FAP expression in PDAC and HG IPMNs than in LG IPMNs and corroborates our previous findings of higher SUVs and a longer TTP in PDAC and HG IPMNs than in LG IPMNs.

Marein Ameliorates Myocardial Fibrosis by Inhibiting HIF-1α and TGF-β1/Smad2/3 Signaling Pathway in Isoproterenol-stimulated Mice and TGF-β1-stimulated Cardiac Fibroblasts.

Myocardial fibrosis significantly contributes to the pathogenesis and progression of heart failure. We probe into the impact of marein, a key bioactive compound in functional food Coreopsis tinctoria, on isoproterenol-stimulated myocardial fibrotic mice and transforming growth factor β1 (TGF-β1)-stimulated cardiac fibroblasts (CFs). Isoproterenol was administered to the experimental mice via subcutaneous injection, and simultaneous administration of marein (25-100 mg/kg) was performed via oral gavage. CFs were stimulated with TGF- β1 to trigger differentiation and collagen synthesis, followed by treatment with marein at concentrations of 5-20 μM. Treatment with marein in mice and CFs resulted in a significant reduction in the protein expression levels of α-smooth muscle actin, collagen type I, and collagen type III. Additionally, marein treatment decreased the protein expression levels of TGF-β1, hypoxia-inducible factor-1α (HIF-1α), p-Smad2/3, and Smad2/3. Notably, molecular docking analysis revealed that marein directly targets HIF-1α. Marein might exert a protective function in isoproterenol-stimulated myocardial fibrotic mice and TGF-β1-stimulated CFs, which might result from the reduction of TGF-β1 induced HIF-1α expression, then inhibiting p-Smad2/3 and Smad2/3 expressions.

A study of the anti-fibrotic effect of ghrelin on mice with nonalcoholic steatohepatitis

Objective: To study the anti-fibrotic effect of ghrelin on high-fat diet-induced non-alcoholic steatohepatitis (NASH) in mice. Methods: 24 male C57BL/6 mice were randomly divided into a normal diet group, a normal diet + ghrelin group, a high-fat diet group, and the high-fat diet + ghrelin group. The HFD and HFD+ghrelin groups were fed high-fat diet for 16 weeks to induce non-alcoholic steatohepatitis. Among them, the NCD+ghrelin group and HFD+ghrelin group were continuously given ghrelin intervention (11nmol·kg(-1)·d(-1)) for 2 weeks after feeding for 14 weeks. 16 mice were euthanized on weekends. The plasma levels of alanine aminotransferase (ALT) and hyaluronic acid (HA) were measured in mice. The content of hydroxyproline (Hyp) was determined in liver tissue. RT-qPCR was used to detect the mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, and IV in liver tissue. A Western blot was used to detect the expression level of the α-smooth muscle actin (α-SMA) protein in liver tissue. HE staining was used to observe the morphological changes in liver tissue. VG staining was used to observe the fibrotic condition in liver tissue. Results: Compared with the NCD group, plasma ALT (266.80±146.80)U/L, HA (219.00±39.47) ng/ml levels, Hyp content (0.35±0.05)μg/mg prot (P < 0.05), mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, IV (P < 0.05), and the expression level of α-SMA protein in the HFD group were significantly increased (P < 0.05), with congestion in the hepatic central lobular veins, hepatocytes swelling, and deposition of a large amount of collagen fibers in liver tissue. Compared with the HFD group, plasma ALT (57.17±20.88)U/L, HA (75.68±8.40)μg/mg levels, Hyp content (0.19±0.07)μg/mg prot, mRNA expression levels of transforming growth factor β1 (TGF-β1) and collagen types I, III, IV (P < 0.05), and the expression level of α-SMA protein in the HFD+ghrelin mice group was significantly reduced (P < 0.05), with only mild sinusoidal congestion in the liver tissue but significant improvement and reduction in liver injury and collagen fiber deposition. Conclusion: Ghrelin has a significant improvement effect on liver fibrosis in NASH mice.

WAVE2 Is a Vital Regulator in Myogenic Differentiation of Progenitor Cells through the Mechanosensitive MRTFA-SRF Axis.

Skeletal myogenesis is an intricate process involving the differentiation of progenitor cells into myofibers, which is regulated by actin cytoskeletal dynamics and myogenic transcription factors. Although recent studies have demonstrated the pivotal roles of actin-binding proteins (ABPs) as mechanosensors and signal transducers, the biological significance of WAVE2 (Wiskott-Aldrich syndrome protein family member 2), an ABP essential for actin polymerization, in myogenic differentiation of progenitor cells has not been investigated. Our study provides important insights into the regulatory roles played by WAVE2 in the myocardin-related transcription factor A (MRTFA)-serum response factor (SRF) signaling axis and differentiation of myoblasts. We demonstrate that WAVE2 expression is induced during myogenic differentiation and plays a pivotal role in actin cytoskeletal remodeling in C2C12 myoblasts. Knockdown of WAVE2 in C2C12 cells reduced filamentous actin levels, increased globular actin accumulation, and impaired the nuclear translocation of MRTFA. Furthermore, WAVE2 depletion in myoblasts inhibited the expression and transcriptional activity of SRF and suppressed cell proliferation in myoblasts. Consequently, WAVE2 knockdown suppressed myogenic regulatory factors (i.e., MyoD, MyoG, and SMYD1) expressions, thereby hindering the differentiation of myoblasts. Thus, this study suggests that WAVE2 is essential for myogenic differentiation of progenitor cells by modulating the mechanosensitive MRTFA-SRF axis.

Role of reactive oxygen species in epithelial-mesenchymal transition and apoptosis of human lens epithelial cells.

To investigate the role of reactive oxygen species (ROS) in epithelial-mesenchymal transition (EMT) and apoptosis of human lens epithelial cells (HLECs). Flow cytometry was used to assess ROS production after transforming growth factor β2 (TGF-β2) induction. Apoptosis of HLECs after H2O2 and TGF-β2 interference with or without ROS scavenger N-acetylcysteine (NAC) were assessed by flow cytometry. The corresponding protein expression levels of the EMT marker α-smooth muscle actin (α-SMA), the extracellular matrix (ECM), marker fibronectin (Fn), and apoptosis-associated proteins were detected by using Western blotting in the presence of an ROS scavenger (NAC). Wound-healing and Transwell assays were used to assess the migration capability of HLECs. TGF-β2 stimulates ROS production within 8h in HLECs. Additionally, TGF-β2 induced HLECs cell apoptosis, EMT/ECM synthesis protein markers expression, and pro-apoptotic proteins production; nonetheless, NAC treatment prevented these responses. Similarly, TGF-β2 promoted HLECs cell migration, whereas NAC inhibited cell migration. We further determined that although ROS initiated apoptosis, it only induced the accumulation of the EMT marker α-SMA protein, but not COL-1 or Fn. ROS contribute to TGF-β2-induced EMT/ECM synthesis and cell apoptosis of HLECs; however, ROS alone are not sufficient for EMT/ECM synthesis.

Mechanism of adipose tissue-derived stromal cell-extracellular vesicles in treating oral submucous fibrosis by blocking the TGF-β1/Smad3 pathway via the miR-760-3p/IGF1R axis.

Oral submucous fibrosis (OSF) is a prevalent chronic condition, and understanding its pathogenesis is crucial for developing effective therapeutic strategies. This study explores the potential of adipose tissue-derived stromal cell-extracellular vesicles (ADSC-EVs) in mitigating OSF and investigates the underlying molecular mechanisms. OSF was induced in mice by arecoline feeding. Adipose tissue-derived stromal cells (ADSCs), fibrotic buccal mucosal fibroblasts (fBMFs) isolated from OSF mice, and ADSC-EVs were comprehensively characterized. The treatment effects of extracellular vesicles (EVs) and pcDNA3.1-IGF1R on fBMF proliferation, migration, and invasion were assessed using Cell Counting Kit-8 (CCK-8) assay, transwell assay, and flow cytometry assay. The expression levels of alpha-smooth muscle actin (α-SMA), collagen I, collagen III, and insulin-like growth factor 1 receptor (IGF1R) were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. The interaction between miR-760-3p and IGF1R was investigated. In fBMFs and OSF mice treated with a miR-760-3p inhibitor and/or EVs, the expression patterns of miR-760-3p, IGF1R, and proteins related to the TGF-β1/Smad3 pathway were determined. ADSC-EVs demonstrated the ability to upregulate miR-760-3p, impede cell proliferation, migration, and invasion, and reduce α-SMA, collagen I, and collagen III levels in fBMFs. The expression of miR-760-3p was diminished in ADSC-EVs treated with a miR-760-3p inhibitor. However, silencing miR-760-3p or overexpressing IGF1R partially counteracted the beneficial effects of ADSC-EVs on fBMF fibrosis. miR-760-3p directly targets IGF1R. Significantly, ADSC-EVs exert their suppressive effects on the TGF-β1/Smad3 pathway through the miR-760-3p/IGF1R axis. In summary, ADSC-EVs, by transferring miR-760-3p and inhibiting IGF1R expression, effectively block the TGF-β1/Smad3 pathway, thereby alleviating fibrosis in fBMFs and preventing the progression of OSF.

Yeast profilin mutants inhibit classical nuclear import and alter the balance between actin and tubulin levels.

Profilin is a small protein that controls actin polymerization in yeast and higher eukaryotes. In addition, profilin has emerged as a multifunctional protein that contributes to other processes in multicellular organisms. This study focuses on profilin (Pfy1) in the budding yeast Saccharomyces cerevisiae. The primary sequences of yeast Pfy1 and its metazoan orthologs diverge vastly. However, structural elements of profilin are conserved among different species. To date, the full spectrum of Pfy1 functions has yet to be defined. The current work explores the possible involvement of yeast profilin in nuclear protein import. To this end, a panel of well-characterized yeast profilin mutants was evaluated. The experiments demonstrate that yeast profilin (i) regulates nuclear protein import, (ii) determines the subcellular localization of essential nuclear transport factors, and (iii) controls the relative abundance of actin and tubulin. Together, these results define yeast profilin as a moonlighting protein that engages in multiple essential cellular activities.

Protective effect and mechanism of Maiwei Yangfei Decoction on pulmonary fibrosis mice based on Nrf2 regulation of oxidative stress

This study explored the effect and mechanism of Maiwei Yangfei Decoction(MWYF) on pulmonary fibrosis(PF) mice. MWYF was prepared, and its main components were detected by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLC-MS/MS). Male C57BL/6J mice were randomly divided into a control group, a model group, a pirfenidone(PFD) group, and low-, medium-, and high-dose MWYF groups, with 10 mice in each group. The PF model was induced in mice except for those in the control group by intratracheal instillation of bleomycin(BLM), and model mice were treated with saline or MWYF or PFD by gavage the next day. The water consumption, food intake, hair, and activity of mice were observed daily. The pathological changes in lung tissues were observed by hematoxylin-eosin(HE) staining, Masson staining, and CT scanning. The level of hydroxyproline(HYP) in lung tissues was detected by alkaline hydrolysis. Immunohistochemistry was used to observe the expression of collagen type Ⅲ(COL3) and fibronectin. The mRNA expression levels of α-smooth muscle actin(α-SMA), type Ⅰ collagen α1(COL1α1), COL3, and vimentin were detected by reverse transcription real-time fluorescence quantitative polymerase chain reaction(RT-qPCR). Superoxide dismutase(SOD) and malondialdehyde(MDA) kits were used to detect oxidative stress indicators in lung tissues and serum. The nuclear translocation of nuclear factor E2-related factor 2(Nrf2) protein was detected by immunofluorescence. The protein and mRNA expression levels of Nrf2, catalase(CAT), and heme oxygenase 1(HO-1) in lung tissues were detected by Western blot and RT-qPCR. Twelve chemical components were detected by UPLC-MS/MS. Animal experiments showed that MWYF could improve alveolar inflammation, collagen deposition, and fibrosis in PF mice, increase body weight of mice, and down-regulate the expression of fibrosis indexes such as HYP, α-SMA, COL1α1, COL3, fibronectin, and vimentin in lung tissues. In addition, MWYF could potentiate the activity of SOD in lung tissues and serum of PF mice, up-regulate the expression level of Nrf2, and promote its transfer to the nucleus, up-regulate the levels of downstream antioxidant target genes CAT and HO-1, and then reduce the accumulation of lipid metabolite MDA. In summary, MWYF can significantly improve the pathological damage and fibrosis of lung tissues in PF mice, and its mechanism may be related to the activation of the Nrf2 pathway to regulate oxidative stress.

Aloin Attenuates Oxidative Stress, Inflammation, and CCl4-Induced Liver Fibrosis in Mice: Possible Role of TGF-β/Smad Signaling.

Liver fibrosis refers to the excessive buildup of extracellular matrix (ECM) components in liver tissue. It is considered a pathological response to liver damage for which there is no effective treatment. Aloin, an anthraquinone compound isolated from the aloe plant, has shown good pharmacological effects in the treatment of gastric cancer, ulcerative colitis, myocardial hypertrophy, traumatic brain injury, and other diseases; however, its specific impact on liver fibrosis remains unclear. To address this gap, we conducted a study to explore the mechanisms underlying the potential antifibrotic effect of aloin. We constructed a mouse liver fibrosis model using carbon tetrachloride (CCl4) dissolved in olive oil as a modeling drug. Additionally, a cellular model was developed by using transforming growth factor β1 (TGF-β1) as a stimulus applied to hepatic stellate cells. After aloin intervention, serum alanine aminotransferase, hepatic hydroxyproline, and serum aspartate aminotransferase were reduced in mice after aloin intervention compared to CCl4-mediated liver injury without aloin intervention. Aloin relieved the oxidative stress caused by CCl4 via reducing hepatic malondialdehyde in liver tissue and increasing the level of superoxide dismutase. Aloin treatment decreased interleukin (IL)-1β, IL-6, and tumor necrosis factor-α and increased the expression of IL-10, which inhibited the inflammatory response in liver injury. In addition, aloin inhibited the activation of hepatic stellate cells and reduced the level of α-smooth muscle actin (α-SMA) and collagen type I. In cell and animal experiments, aloin attenuated liver fibrosis, acting through the TGF-β/Smad2/3 signaling pathway, and mitigated CCl4- and TGF-β1-induced inflammation. Thus, the findings of this study provided theoretical data support and a new possible treatment strategy for liver fibrosis.

Trimethylamine N-Oxide Induces Renal Fibrosis Through the PI3K/AKT/SREBP1 Pathway

To investigate the role and mechanism of trimethylamine N-oxide (TMAO), a uremic toxin, in renal fibrosis. A total of 20 male BALB/c mice were randomly and evenly assigned to a Control group and a TMAO group. Mice in the Control group received intraperitoneal injection of normal saline, while mice in the TMAO group received intraperitoneal injection of TMAO (20 mg/[kg·d]). The injection was given once a day for 8 weeks. Histopathology and fibrosis of kidney were observed by H&E staining and Masson staining. Immunohistochemistry was performed to determine the levels of alpha smooth muscle actin (α-SMA), recombinant human fibronectin fragment (Fibronectin), and sterol-regulatory element binding protein 1 (SREBP1). Western blot was performed to determine α-SMA, SREBP1, phosphatidylinositol 3 kinase (PI3K), phospho-phosphatidylinositol 3 kinase (p-PI3K), protein kinase B (PKB, also known as AKT), and phospho-AKT (p-AKT) protein levels. HK2 cells were treated with SREBP1 small interfering RNA (siRNA) and PI3K/AKT inhibitor, respectively, and the reversal of the effects of TMAO was examined. Animal experiments showed that, compared with the Control group, the mice treated with TMAO experienced pathological damage and fibrosis of the kidney tissue and the expression levels of fibrosis markers, α-SMA and Fibronectin, in the kidney were increased (all P<0.05). According to the findings from further investigation, the TMAO-treatment group showed increased expression of SREBP1 and an up-regulation of PI3K phosphorylation ratio and AKT phosphorylation ratio compared with those of the Control group (all P<0.05). Cell experiments produced results similar to those of the animal experiment. After siRNA interference with SREBP1 expression, the expression levels of fibrosis marker proteins decreased (P<0.05). Besides, the high expression of SREBP1 caused by TMAO was inhibited after HK2 cells were incubated with LY294002, a PI3K-AKT pathway inhibitor (P<0.05). TMAO may induce renal fibrosis by promoting the PI3K/AKT/SREBP1 pathway.