Expression Of Fibrotic Markers Research Articles

Dietary Antrodia cinnamomea Polysaccharide Intervention Modulates Clinical Symptoms by Regulating Ovarian Metabolites and Restructuring the Intestinal Microbiota in Rats with Letrozole-Induced PCOS.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine and metabolic disorder. This study investigated the mitigating effects of the Antrodia cinnamomea polysaccharide (ACP) on a letrozole-induced PCOS rat model. Results demonstrated that ACP reduced obesity and ameliorated dyslipidemia in PCOS rats. Moreover, ACP restored estrous cycle regularity, suppressed polycystic ovarian changes, and regulated serum levels of sex hormones, SOD, and MDA. Furthermore, ACP increased the α-diversity and modulated the abundance of phyla (Bacteroidetes, Firmicutes, and Verrucomicrobia) and genera (Lactobacillus, Helicobacter, Akkermansia, Oscillospira, Coprococcus, Roseburia, Blautia, and Allobaculum) in the gut microbiota. ACP also restored compromised intestinal barriers by upregulating the expression of ZO1, Occludin, Claudin1, and Claudin7 in the colon. ACP mitigated ovarian fibrosis by preventing activation of the NLRP3 inflammasome, decreasing the expression of fibrotic markers (TGF-β1, collagen-I, α-SMA, and CTGF), and regulating four ovarian fibrosis-associated metabolomics pathways. Generally, dietary ACP effectively ameliorated clinical symptoms and inhibited ovarian fibrosis in PCOS rats.

The promotive role of reticulocalbin 3 (RCN3) in the pathogenesis of keloid via TGFβ1/Smad2/Smad7 signaling pathway in vitro

Abstract Objectives Keloids, characterized by an excessive accumulation of fibrous tissue, remain a significant dermatological challenge. This study elucidates the role of Reticulocalbin 3 (RCN3) in modulating keloid fibroblasts (KFs) in vitro. Methods The RCN3 expression levels in human KFs and normal dermal fibroblast cells were examined using RT-qPCR and Western blot methods. RCN3 expression was knocked down in keloid fibroblasts after cells were transfected with shRNA targeting RCN3. On the other hand, recombinant RCN3 was added to treat the cells. RT-qPCR, Western blot, flow cytometry, CCK8 assays, and wound healing assays were performed to analyze the fibrotic markers (collagen I, III, MMP2, α-SMA) and TGFβ1/Smad2/6/7 pathway, apoptosis, proliferation and migration. Results RCN3 was upregulated in KFs. Its knockdown reduced fibrotic marker expression (collagen I, III, MMP2, α-SMA), cell proliferation, and migration, while increasing apoptosis. Conversely, recombinant RCN3 treatment enhanced the fibrotic responses. Changes in TGFβ1/Smad2 pathway, especially in Smad2 phosphorylation and Smad7, were evident following RCN3 modulation. Conclusions The study reveals RCN3 as a regulator in keloid pathology, affecting fibrosis, cellular behavior through TGFβ1/Smad2/Smad7 signaling.

Yohimbine treatment improves pulmonary fibrosis by attenuating the inflammation and oxidative stress via modulating the MAPK pathway

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disorder characterized by the accumulation of extracellular matrix and collagen, resulting in significant parenchymal scarring and respiratory failure that leads to mortality. Yohimbine (YBH) is an α-2 adrenergic receptor antagonist with anti-oxidant and anti-inflammatory properties. In the current study, we aimed to investigate the anti-inflammatory, anti-oxidant and anti-fibrotic activity of YBH against LPS/TGF-β-induced differentiation in BEAS-2B/LL29 cells and bleomycin (BLMN) induced pulmonary fibrosis model in rats. Network pharmacology, gene expression, Western-blot analysis, immune-cytochemistry/immunohistochemistry, lung function and histology techniques were used to assess the fibrotic marker expression/levels in cells or rat lung tissues. YBH treatment significantly attenuated the LPS-induced pro-inflammatory (identified through a network-pharmacology approach) and oxidative stress markers expression in lung epithelial cells. TGF-β stimulation significantly elevated the fibrotic cascade of markers and treatment with YBH attenuated these markers’ expression/levels. Intra-tracheal administration of BLMN caused a significant elevation of various inflammatory/oxidative stress and fibrotic markers expression in lung tissues and treatment with YBH significantly mitigated the same. Ashcroft score analysis revealed that BLMN exhibited severe distortion of the lungs, elevation of thickness of the alveolar walls and accumulation of collagen in tissues, further treatment with YBH significantly suppressed these events and improved the lung architecture. Lung functional parameters demonstrated that BLMN-induced stiffness and resistance were reduced considerably upon YBH treatment and restored lung function dose-dependently. Overall, this study reveals that YBH treatment significantly attenuated the BLMN-induced fibrosis by regulating the MAPK pathway and provided insightful information for progressing towards translational outcomes.

C1q/tumor necrosis factor-related protein-6 suppresses the angiotensin II-induced differentiation of cardiac fibroblasts to myofibroblasts via activation of the AMPK pathway.

C1q/tumor necrosis factor-related protein-6 (CTRP6) has multiple protective effects against cardiovascular diseases. Myofibroblast differentiation plays a critical role in cardiac fibrosis under various cardiac pathological conditions. The aim of the present study was to determine the effects of CTRP6 on cardiac fibrosis, and to identify the possible mechanisms of action. Toward this end, we measured the expression of fibrotic markers, including collagen I, collagen III, CTGF, and TGFβ1, and assessed the effects of CTRP6 on cardiac fibroblast differentiation into myofibroblasts. CTRP6 inhibited the expression of the angiotensin II (Ang II)-induced myofibroblast markers α-SMA and SM22, and of profibrotic molecules, including collagen I, collagen III, CTGF, TGFβ1, MMP2, MMP9, and TIMP1. Furthermore, CTRP6 significantly attenuated the proliferation and migration of cardiac fibroblasts incubated with Ang II and activated the phosphorylation of AMP-activated protein kinase (AMPK). Incubation with an AMPK inhibitor reversed the subsequent inhibitory effects of CTRP6 on Ang II-induced myofibroblast differentiation. Therefore, CTRP6 suppresses cardiac fibrosis by inhibition of myofibroblast differentiation via AMPK pathway activation, suggesting CTRP6 as a target for the treatment of cardiac fibrosis.

GLP-1 and GIP agonism has no direct actions in human hepatocytes or hepatic stellate cells

The use of incretin agonists for managing metabolic dysfunction-associated steatohepatitis (MASH) is currently experiencing considerable interest. However, whether these compounds have a direct action on MASH is still under debate. This study aims to investigate whether GLP-1R/GIPR agonists act directly in hepatocytes and hepatic stellate cells (HSCs). For this, human hepatocyte and HSCs lines, as well as primary human hepatocytes and HSCs treated with Liraglutide, Acyl-GIP or the GLP-1/GIP dual agonist (MAR709) were used. We show that the concentrations of each compound, which were effective in insulin release, did not induce discernible alterations in either hepatocytes or HSCs. In hepatocytes displaying elevated fatty acid content after the treatment with oleic acid and palmitic acid, none of the three compounds reduced lipid concentration. Similarly, in HSCs activated with transforming growth factor-β (TGFb), Liraglutide, Acyl-GIP and MAR709 failed to ameliorate the elevated expression of fibrotic markers. The three compounds were also ineffective in phosphorylating CREB, which mediates insulinotropic actions, in both hepatocytes and HSCs. These findings indicate that incretin agonists have no direct actions in human hepatocytes or hepatic stellate cells, suggesting that their beneficial effects in patients with MASH are likely mediated indirectly, potentially through improvements in body weight, insulin resistance and glycemic control.

A novel ex vivo approach for investigating profibrotic macrophage polarization using murine precision-cut lung slices

Idiopathic pulmonary fibrosis (IPF) is fatal interstitial lung disease characterized by excessive scarring of the lung tissue and declining respiratory function. Given its short prognosis and limited treatment options, novel strategies to investigate emerging experimental treatments are urgently needed. Macrophages, as the most abundant immune cell in the lung, have key implications in wound healing and lung fibrosis. However, they are highly plastic and adaptive to their surrounding microenvironment, and thus to maximize translation of research to lung disease, there is a need to study macrophages in multifaceted, complex systems that are representative of the lung. Precision-cut lung slices (PCLS) are living tissue preparations derived from the lung that are cultured ex vivo, which bypass the need for artificial recapitulation of the lung milieu and architecture. Macrophage programming studies are traditionally conducted using isolated cells in vitro, thus our objective was to establish and validate a moderate-throughput, biologically-translational, viable model to study profibrotic polarization of pulmonary-resident macrophages using murine PCLS. To achieve this, we used a polarization cocktail (PC), consisting of IL-4, IL-13, and IL-6, over a 72-h time course. We first demonstrated no adverse effects of the PC on PCLS viability and architecture. Next, we showed that multiple markers of macrophage profibrotic polarization, including Arginase-1, CD206, YM1, and CCL17 were induced in PCLS following PC treatment. Through tissue microarray-based histological assessments, we directly visualized and quantified Arginase-1 and CD206 staining in PCLS in a moderate-throughput manner. We further delineated phenotype of polarized macrophages, and using high-plex immunolabelling with the Iterative Bleaching Extends Multiplexity (IBEX) method, showed that the PC effects both interstitial and alveolar macrophages. Substantiating the profibrotic properties of the system, we also showed expression of extracellular matrix components and fibrotic markers in stimulated PCLS. Finally, we demonstrated that clodronate treatment diminishes the PC effects on profibrotic macrophage readouts. Overall, our findings support a suitable complex model for studying ex vivo profibrotic macrophage programming in the lung, with future capacity for investigating experimental therapeutic candidates and disease mechanisms in pulmonary fibrosis.

Anti-fibrotic effects of the sirt6-activator mdl-800 in cardiac stromal cells

Abstract Background Epigenetic changes are among the molecular substrates of cellular stress responses and tissue remodeling in heart failure progression. The sirtuin family of NAD+-dependent histone and non-histone protein deacetylases exert a protective anti-fibrotic role by negatively modulating the inflammatory response and by positive regulation of autophagy-related genes. Sirtuin6 (SIRT6) stands out as a key cardiac protector that can mitigate aging-induced cardiomyocyte senescence and cardiac hypertrophy. Cardiac stromal cells (CSCs) play a pivotal role in fibrosis and remodeling, regulating the composition and stiffness of the extracellular matrix (ECM). Autophagy enhancement has anti-fibrotic effects in CSCs, but the specific role of SIRT6 modulation in CSCs remains unexplored. Purpose To investigate the biological effects of MDL-800, a synthetic allosteric SIRT6 activator, on the stress response and fibrotic activation of CSCs. Methods CSCs were isolated from 4-week-old C57Bl6J mice by explant culture and stimulated with 10ng/ml of TGF-beta1 (TGFb1) up to 72h to induce fibrotic activation. CSCs were treated with MDL-800 up to 10µM and up to 72h, analyzed for cell viability, gene and protein expression levels. Results TGFb1 treatment led to a 0.64-fold reduction in SIRT6 gene expression after 24h. The MTS assay was used to assess a viability dose-response to MDL-800 up to 72h of treatment. The non-toxic concentrations of 2.5µM and 5µM were selected for further experiments (2.5µM vs CTR 0.97-fold and 5µM vs CTR 0.92-fold, normalized OD vs t0). Co-treatment of 5µM MDL-800 with TGFb1 for 72h resulted in a dose-dependent decrease in the expression of TGFb1-induced fibroblast activation markers, as confirmed by immunofluorescence staining for aSMA (TGFb1 vs CTR 2.21-fold, p<0.05; 5µM vs TGFb1 0.46-fold, p<0.05, N=4; mean fluorescence intensity). The anti-fibrotic effect mediated by MDL-800 was further validated by real-time PCR indicating a reduction in the mRNA expression of multiple fibrotic markers, including ACTA2 (TGFb1 vs CTR 1.67-fold, 5µM vs TGFB 0.29-fold; N=2), COL1a1 (TGFb1 vs CTR 1.32-fold, 5µM vs TGFb1 0.87-fold; N=2) COL3a1 (TGFb1 vs CTR 1.06-fold, 5µM vs TGFb1 0.43-fold; N=2) and POSTN (TGFb1 vs CTR 6.37-fold, 5µM vs TGFb1 0.23-fold; N=2). Treatment with 5µM MDL-800 demonstrated an increase in SIRT6 gene expression, suggesting a transcriptional modulation (TGFb1 vs CTR 0.95-fold, 5µM vs TGFb1 1.68-fold; N=2). Western Blot analyses showed an increase in protein levels of the autophagosome marker LC3-II following treatment with 5µM MDL-800 (TGFb1 vs CTR 0.95-fold; 5µM vs TGFb1 3.01-fold), suggesting that autophagy enhancement may be a potential mechanism for the observed anti-fibrotic effects. Conclusion MDL-800 treatment in CSCs is associated with a dose-dependent reduction of TGFb1-induced fibrotic markers and to increased levels of the autophagy marker, suggesting its potential use as an anti-fibrotic molecule for the heart.

Comparing the anti-remodeling effect of sacubitril/valsartan and angiotensin-converting enzyme inhibition on myocardial samples of advanced heart failure patients undergoing heart transplantation

Abstract Background In decreasing heart failure (HF) hospitalization and cardiovascular death in HF patients with reduced ejection fraction, Sacubitril/Valsartan (SAC/VAL) treatment was proved to be superior to angiotensin-converting enzyme inhibition (ACEi). According to recent experimental results and clinical data based on echocardiography and circulating biomarkers, this benefit might be at least partially attributed to a greater anti-remodeling effect. However, there is a lack of direct evidence from human myocardial samples for this assumption. Purpose Therefore, we aimed to compare the anti-remodeling effect of SAC/VAL treatment to ACEi on myocardial samples of HF patients undergoing heart transplantation (HTX). Methods We studied left ventricular (LV) samples of seventy advanced HF patients who underwent HTX and received either SAC/VAL (SAC/VAL group, N=32) or ACEi treatment (ACEi group, N=38) for at least three months prior to HTX. Furthermore, all patients received beta-blockers and mineralocorticoid receptor antagonists titrated to maximally tolerated or target dose. Our groups were matched for age, sex, ejection fraction and other clinical parameters. Patients with liver or kidney failure, mechanical circulatory support, systemic inflammatory/fibrotic diseases or positive inotrope treatment were excluded. Myocardial mRNA expression of fibrotic markers and B-type natriuretic peptide (BNP) were measured with qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase expression. Interstitial fibrosis area was assessed histologically. Results Preoperative plasma N-terminal pro-BNP levels were similar in the two groups (SAC/VAL: 4363±752 pg/mL vs. ACEi: 4616±1020 pg/mL; p=0.911). In contrast, the mRNA level of BNP in the myocardium was lower in patients treated with SAC/VAL (SAC/VAL: 2.2±1.05 vs. ACEi: 5.2±1.74; p=0.006). TIMP metallopeptidase inhibitor 1 (SAC/VAL: 0.07±0.01 vs. ACEi: 0.13±0.02; p=0.011), TIMP metallopeptidase inhibitor 2 (SAC/VAL: 0.06±0.01 vs. ACEi: 0.11±0.01; p<0.001), collagen type I alpha 1 chain (SAC/VAL: 0.02±0.00 vs. ACEi: 0.08±0.02; p<0.001) and collagen type III alpha 1 chain (SAC/VAL: 0.12±0.02 vs. ACEi: 0.27±0.04; p<0.001) relative gene expressions were also decreased in the LV of SAC/VAL-treated patients. Histology analysis confirmed reduced interstitial myocardial fibrosis area in the SAC/VAL group as well (SAC/VAL: 7.2±0.4% vs. ACEi: 8.6±0.5%; p=0.031). Conclusions SAC/VAL treatment decreased the myocardial expression of BNP and fibrosis-related genes to a greater extent compared with ACEi in LV samples of patients undergoing HTX. Histological analysis also verified the differences in fibrosis. Thus, the anti-remodeling effect of SAC/VAL might be superior to ACEi even in advanced HF.

Time-Restricted Feeding Attenuates Adipose Tissue Inflammation and Fibrosis in Mice Under Chronic Light Exposure.

Time-restricted feeding (TRF) has emerged as a promising dietary approach for improving metabolic parameters associated with obesity. However, it remains largely unclear whether TRF offers benefits for obesity related to exposure to light at night. This study examined whether lean and obese mice under chronic light exposure could benefit from TRF intervention. Six-week-old C57BL/6 male mice were fed either a low-fat diet or a high-fat diet under a 12 h light/12 h dark cycle for 6 weeks. They were then divided into three subgroups: control light, chronic 24 h light, and chronic light with a daily 10 h TRF. Chronic light exposure led to increased weight gain and higher expression of inflammatory and fibrotic markers in the adipose tissue of both lean and obese mice. It also increased hepatic triglyceride content in mice, regardless of their weight status. TRF protected both lean and obese mice from weight gain, normalized inflammatory and fibrotic gene expression, and reduced adipose tissue collagen and liver triglyceride accumulation caused by light exposure alone or in combination with obesity. These results suggest that TRF could have clinical implications for preventing obesity associated with night shift work, regardless of current weight status.

ApaI Polymorphism in the Vitamin D Receptor Gene Decreases the Risk of Perianal Fistulas in Crohn’s Disease

Background: Vitamin D, through the activation of its receptor (VDR), plays an immunomodulatory role in the gastrointestinal tract. Single-nucleotide polymorphisms (SNPs) in the VDR gene have been associated with Crohn’s disease (CD) risk, and patients carrying the TaqI polymorphism in this gene run a higher risk of developing a penetrating behavior. Aims: We analyzed the association of BsmI, ApaI, TaqI, and FokI SNPs in the VDR gene with the clinical characteristics of CD. Methods: Four polymorphisms identified in the VDR gene (BsmI, FokI, ApaI, and TaqI) were genotyped in blood samples from CD patients (n = 115) by using PCR-RFLP. The disease’s location and behavior and the presence of perianal fistulas were collected from each patient. Intestinal fibroblasts from ileal resections of CD patients (n = 10) were genotyped, and the expression of fibrotic and inflammatory markers was analyzed by RT-PCR. Results: The data reveal no association between any of the polymorphisms and CD risk. A strong linkage disequilibrium was detected between TaqI and both ApaI and BsmI, which in turn were strongly associated. Homozygosis or heterozygosis for the a allele of the ApaI SNP or b allele of the BsmI SNP was significantly associated with a lower risk of a penetrating behavior, while the aa genotype was associated with a lower risk of perianal fistulas. Fibroblasts carrying the aa genotype expressed lower levels of fibrotic and inflammatory markers. Conclusion: The aa genotype of the ApaI SNP in the VDR gene is associated with a lower risk of perianal fistulas in CD and a reduced expression of fibrotic and inflammatory markers in intestinal fibroblasts.

β-Mangostin Alleviates Renal Tubulointerstitial Fibrosis via the TGF-β1/JNK Signaling Pathway.

The epithelial-to-mesenchymal transition (EMT) plays a key role in the pathogenesis of kidney fibrosis, and kidney fibrosis is associated with an adverse renal prognosis. Beta-mangostin (β-Mag) is a xanthone derivative obtained from mangosteens that is involved in the generation of antifibrotic and anti-oxidation effects. The purpose of this study was to examine the effects of β-Mag on renal tubulointerstitial fibrosis both in vivo and in vitro and the corresponding mechanisms involved. As shown through an in vivo study conducted on a unilateral ureteral obstruction mouse model, oral β-Mag administration, in a dose-dependent manner, caused a lesser degree of tubulointerstitial damage, diminished collagen I fiber deposition, and the depressed expression of fibrotic markers (collagen I, α-SMA) and EMT markers (N-cadherin, Vimentin, Snail, and Slug) in the UUO kidney tissues. The in vitro part of this research revealed that β-Mag, when co-treated with transforming growth factor-β1 (TGF-β1), decreased cell motility and downregulated the EMT (in relation to Vimentin, Snail, and N-cadherin) and phosphoryl-JNK1/2/Smad2/Smad3 expression. Furthermore, β-Mag co-treated with SB (Smad2/3 kinase inhibitor) or SP600125 (JNK kinase inhibitor) significantly inhibited the TGF-β1-associated downstream phosphorylation and activation of JNK1/2-mediated Smad2 targeting the Snail/Vimentin axis. To conclude, β-Mag protects against EMT and kidney fibrotic processes by mediating the TGF-β1/JNK/Smad2 targeting Snail-mediated Vimentin expression and may have therapeutic implications for renal tubulointerstitial fibrosis.

Lens capsule advanced glycation end products induce senescence in epithelial cells: Implications for secondary cataracts.

Posterior capsule opacification (PCO) is a common complication after cataract surgery. Residual lens epithelial cells (LECs) on the anterior lens capsule, after cataract surgery, migrate to the posterior lens capsule and undergo transdifferentiation into myofibroblast-like cells. Those cells synthesize excessive amounts of extracellular matrix and contribute to fibrosis during PCO. Cellular senescence, a phenomenon that increases with aging, has been implicated in several fibrotic diseases. Here, we have investigated the prevalence of senescent LECs within the lens posterior capsule and the ability of advanced glycation end products (AGEs) in lens capsules to induce senescence, contributing to PCO. Aged lens capsules from pseudophakic human cadaver eyes showed the presence of senescent LECs. In human capsular bags, LECs showed an age-dependent increase in senescence after 28 days of culture. Human LECs cultured on aged lens capsules for 3 days underwent senescence; this effect was not seen in LECs cultured on young lens capsules. Human LECs cultured on an AGE-modified extracellular matrix (ECM-AGEs) showed an AGE-concentration-dependent increase in the expression of senescence markers and reactive oxygen species (ROS) levels. Treatment with a RAGE antagonist and ROS inhibitor reduced the expression of senescence and fibrotic markers. Additionally, conditioned media from ECM-AGEs-treated cells induced the expression of fibrotic markers in naïve LECs. Together, these suggest that AGEs in the capsule induce senescence of LECs, which triggers the mesenchymal transition of neighboring non-senescent LECs and contributes to PCO.

Inhibition of intestinal inflammation and fibrosis by Scutellaria Baicalensis georgi and Boswellia serrata in human epithelial cells and fibroblasts.

Inflammatory bowel disease, including Crohn's disease and ulcerative colitis, manifests with chronic intestinal inflammation and frequent sequential fibrosis. Current pharmacological therapies may show harmful side effects and are not useful for prevention or resolution of fibrosis. Thus, the use of alternative therapies is emerging as a novel useful approach. Previous results suggest that Scutellaria baicalensis Georgi (SBG) and Boswellia serrata (BS) display anti-inflammatory properties. The aim of this study was to investigate in intestinal epithelial cells and fibroblasts the anti-inflammatory and anti-fibrotic potential of SBG and BS, alone or in combination. Human colorectal adenocarcinoma cells (HT29), human intestinal epithelial cells (HIEC6) and human colon fibroblasts (CCD-18Co) were used. Cells were pretreated with SBG and BS and then exposed to pro-inflammatory and pro-fibrotic cytokines. SBG and BS extracts significantly decreased pro-inflammatory cytokine expression and improved epithelial restitution in HT29 and HIEC6 cells. Besides, fibrotic marker expression, including SNAIL, ACTA2, ZNF281, was strongly reduced. Colon myofibroblasts treated with SBG and BS showed a significant decrease of fibrotic markers as well. SBG and BS extracts significantly reduce inflammation and impair fibrosis in intestinal epithelial cells and colon myofibroblasts. No cooperative effect is observed.

C2C12 myoblasts differentiate into myofibroblasts via the TGF-β1 signaling pathway mediated by Fibulin2

Myoblast cells play a critical role in the regeneration of skeletal muscle following injury. It has been reported that local elevation of transforming growth factor-β1(TGF-β1) after skeletal muscle injury induces differentiation of myoblast cells into myofibroblasts.However, the mechanisms underlying this differentiation process remain incompletely understood. In this study, we found that Fibulin2 expression significantly increases in myoblast cells in response to TGF-β1 stimulation.Elevated Fibulin2 levels enhance the expression of fibrotic markers, mediated through phosphorylation of Smad2.Conversely, downregulation of Fibulin2 in myoblast cells inhibits the upregulation of fibrotic markers induced by TGF-β1 stimulation.Extracellular secretion of Fibulin2 activates the TGF-β1-Smad2 pathway, thereby promoting the upregulation of fibrotic markers.Hence, Fibulin2 and TGF-β1 form a positive feedback loop that facilitates differentiation of myoblast cells into myofibroblasts.

Imperatorin ameliorates ferroptotic cell death, inflammation, and renal fibrosis in a unilateral ureteral obstruction mouse model

BackgroundImperatorin is a naturally occurring furocoumarin derivative found in traditional Chinese medicine Angelica dahurica for its anticancer, antihypertensive, and antidiabetic properties. Chronic kidney disease (CKD) is a global health issue, characterized by a high prevalence, significant morbidity and mortality, and a range of related complications. ObjectiveThis study aims to investigate the protective effects of imperatorin treatment and the specific underlying mechanisms in progressive CKD. MethodsImperatorin was orally administrated for 14 consecutive days to mice with unilateral ureteral obstruction (UUO) to investigate the renal pathological alternations, pro-inflammatory mediators, antioxidant response, and ferroptotic death signaling. Imperatorin was also tested in the erastin-induced injury of renal proximal tubular cells (NRK-52E). Cell viability, ferroptosis protein markers, erastin-induced oxidative stress, and lipid peroxidation were assessed. ResultsIn vivo, imperatorin treatment alleviated kidney histology alternations and attenuated the protein expression of fibrotic markers. Furthermore, imperatorin administration reduced inflammatory cell infiltration, and alleviated the oxidative stress burden by downregulating protein markers such as catalase, superoxide dismutase 2 (SOD-2), NADPH oxidase 4 (NOX-4), and thioredoxin reductase 1 (Trxr-1). It also mitigated ferroptosis markers such as glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11/cystine transporter (SLC7A11/xCT), and transferrin receptor 1 (TFR-1), and attenuated renal cell apoptosis. In vitro, imperatorin treatment effectively decreased erastin-induced feroptotic cell death, restored the antioxidant enzyme levels, and mitigated lipid peroxidation as well as the expression of ferroptosis-related markers (XCT, GPX4, and p-p53) in a dose-dependent manner. ConclusionOur finding demonstrated for the first time, that imperatorin treatment holds therapeutic potential in a UUO mouse model of CKD and inhibits the erastin-induced oxidative stress, ferroptosis, and subsequent lipid peroxidation in vitro. This highlights the potential of imperatorin as a future therapeutic target for ferroptosis to improve the progression of CKD.

Ethanol with thioacetamide murine model of alcoholic liver disease identifies hepatic pathways as targets for the human disease

Introduction and ObjectivesHepatic proteome and gut microbiota alterations are known in alcohol-associated hepatitis (AAH). Current animal models sparsely mimic human AAH. We aimed to develop an murine model that closely resembled human AAH. Materials and MethodsMale C57BL/6N mice were pair-fed control/incremental ethanol Lieber-DeCarli diets and thioacetamide (TAA) for 12-weeks to induce AAH. Hepatic proteome was analyzed using LC-MS/MS. Gut-bacteria was determined using 16s-rRNA sequencing. ResultsMice exposed to EtOH+TAA displayed higher expression of liver triglycerides (1.5-fold, p = 0.001), pro-inflammatory (IL6, 1.5-fold, p = 0.002 and TNFα, 1.7-fold, p = 0.01), fibrotic (TGF-β, 2.7-fold, p = 0.01 and Col1α1, 2-fold, p = 0.01) and oxidative markers (GSH and SOD (-1.5 fold, p = 0.004 & 0.005 respectively)) as compared to EtOH alone. Histology of EtOH+TAA liver displayed pericellular liver fibrosis, increased steatosis, and neutrophil infiltration, which resembled human AAH. In the 12wk EtOH+TAA group, Desulfobacteria, Campylobacteria, and Patescibacteria increased by 2-fold (p = 0.02). Pathway combined score (CS, log10) in EtOH+TAA treatment showed upregulated hepatic ethanol oxidation (CS=1.93), fatty acid biosynthesis (CS=2.48), necrosis (CS=1.59), collagen formation (CS=1.28) and hypoxia (CS=0.68) and downregulated fatty acid beta-oxidation (CS=2.37), PPAR signaling (CS=1.35) fatty acid degradation (CS=2.35), bile acid metabolism (CS=1.87), and oxidative phosphorylation (CS=1.50), as observed in human disease. ConclusionsUsing an ethanol-thioacetamide combination in mice results in a faster establishment of AAH with fibrosis than previously known models. Differential protein expression strongly correlates with pathways found altered in human AAH, thus making the model mimic human disease better than other known models., respectively. Thioacetamide (TAA) was administered to enhance liver fibrosis and mimic human AAH.

Oridonin Attenuates Diabetes‑induced Renal Fibrosis via the Inhibition of TXNIP/NLRP3 and NF‑κB Pathways by Activating PPARγ in Rats.

Oridonin possesses remarkable anti-inflammatory, immunoregulatory properties. However, the renoprotective effects of oridonin and the underlying molecular mechanisms in diabetic nephropathy (DN). We hypothesized that oridonin could ameliorate diabetes‑induced renal fibrosis. Streptozocin (STZ)-induced diabetic rats were provided with a high-fat diet to establish a type 2 diabetes mellitus (T2DM) animal model, and then treated with Oridonin (10, 20 mg/kg/day) for two weeks. Kidney function and renal fibrosis were assessed. High glucose-induced human renal proximal tubule epithelial cells (HK-2) were also treated with oridonin. The expression of inflammatory factors and fibrotic markers were analyzed. Oridonin treatment preserved kidney function and markedly limited the renal fibrosis size in diabetic rats. The renal fibrotic markers were inhibited in the oridonin 10 mg/kg/day and 20 mg/kg/day groups compared to the T2DM group. The expression of thioredoxin-interacting proteins/ nod-like receptor protein-3 (TXNIP/NLRP3) and nuclear factor (NF)‑κB pathway decreased, while that of peroxisome proliferator-activated receptor-gamma (PPARγ) increased in the oridonin treatment group compared to the non-treated group. In vitro, PPARγ intervention could significantly regulate the effect of oridonin on the high glucose-induced inflammatory changes in HK-2 cells. Oridonin reduces renal fibrosis and preserves kidney function via the inhibition of TXNIP/NLRP3 and NF‑κB pathways by activating PPARγ in rat T2DM model, which indicates potential effect of oridonin in the treatment of DN.

Trimetazidine Alleviates Bleomycin-Induced Pulmonary Fibrosis by Targeting the Long Noncoding RNA CBR3-AS1-Mediated miRNA-29 and Resistin-Like Molecule alpha 1: Deciphering a Novel Trifecta Role of LncRNA CBR3-AS1/miRNA-29/FIZZ1 Axis in Lung Fibrosis.

Pulmonary fibrosis (PF) and tissue remodeling can greatly impair pulmonary function and often lead to fatal outcomes. In the present study, we explored a novel molecular interplay of long noncoding (Lnc) RNA CBR3-AS1/ miRNA-29/ FIZZ1 axis in moderating the inflammatory processes, immunological responses, and oxidative stress pathways in bleomycin (BLM)-induced lung fibrosis. Furthermore, we investigated the pharmacological potential of Trimetazidine (TMZ) in ameliorating lung fibrosis. Our results revealed that the BLM-treated group exhibited a significant upregulation in the expression of epigenetic regulators, lncRNA CBR3-AS1 and FIZZ1, compared to the control group (P<0.0001), along with the downregulation of miRNA-29 expression. Furthermore, Correlation analysis showed a significant positive association between lnc CBR3-AS1 and FIZZ1 (R=0.7723, p<0.05) and a significant negative association between miRNA-29 and FIZZ1 (R=-0.7535, p<0.05), suggesting lnc CBR3-AS1 as an epigenetic regulator of FIZZ1 in lung fibrosis. BLM treatment significantly increased the expression of Notch, Jagged1, Smad3, TGFB1, and hydroxyproline. Interestingly, the administration of TMZ demonstrated the ability to attenuate the deterioration effects caused by BLM treatment, as indicated by biochemical and histological analyses. Our investigations revealed that the therapeutic potential of TMZ as an antifibrotic drug could be ascribed to its ability to directly target the epigenetic regulators lncRNA CBR3-AS1/ miRNA-29/ FIZZ1, which in turn resulted in the mitigation of lung fibrosis. Histological and immunohistochemical analyses further validated the potential antifibrotic effects of TMZ by mitigating the structural damage associated with fibrosis. Taken together, our study showed for the first time the interplay between epigenetic lncRNAs CBR3-AS1 and miRNA-29 in lung fibrosis and demonstrated that FIZZ1 could be a downregulatory gene for lncRNA CBR3-AS1 and miRNA-29. Our key findings demonstrate that TMZ significantly reduces the expression of fibrotic, oxidative stress, immunomodulatory, and inflammatory markers, along with epigenetic regulators associated with lung fibrosis. This validates its potential as an effective antifibrotic agent by targeting the CBR3-AS1/miRNA-29/FIZZ1 axis.

IKKε-deficient macrophages impede cardiac repair after myocardial infarction by enhancing the macrophage–myofibroblast transition

The regulatory role of the inhibitor of NF-kB kinase ε (IKKε) in postmyocardial infarction (MI) inflammation remains uncertain. Using an MI mouse model, we examined the cardiac outcomes of IKKε knockout (KO) mice and wild-type mice. We employed single-cell RNA sequencing (scRNA-seq) and phosphorylated protein array techniques to profile cardiac macrophages. IKKε KO mice exhibited compromised survival, heightened inflammation, pronounced cardiac fibrosis, and a reduced ejection fraction. A distinct cardiac macrophage subset in IKKε KO mice exhibited increased fibrotic marker expression and decreased phosphorylated p38 (p-p38) levels, indicating an enhanced macrophage–myofibroblast transition (MMT) post-MI. While cardiac inflammation is crucial for initiating compensatory pathways, the timely resolution of inflammation was impaired in the IKKε KO group, while the MMT in macrophages accelerated post-MI, leading to cardiac failure. Additionally, our study highlighted the potential of 5-azacytidine (5-Aza), known for its anti-inflammatory and cardioprotective effects, in restoring p-p38 levels in stimulated macrophages. The administration of 5-Aza significantly reduced the MMT in cardiac macrophages from the IKKε KO group. These findings underscore the regulation of the inflammatory response and macrophage transition by the IKKε-p38 axis, indicating that the MMT is a promising therapeutic target for ischemic heart disease.

Interleukin-33 promotes intrauterine adhesion formation in mice through the mitogen-activated protein kinase signaling pathway

IL-33 belongs to the inflammatory factor family and is closely associated with the inflammatory response. However, its role in the development of intrauterine adhesions (IUAs) remains unclear. In this study, the role of IL-33 in the formation of IUAs after endometrial injury was identified via RNA sequencing after mouse endometrial organoids were transplanted into an IUA mouse model. Major pathological changes in the mouse uterus, consistent with the expression of fibrotic markers, such as TGF-β, were observed in response to treatment with IL-33. This finding may be attributed to activation of the phosphorylation of downstream MAPK signaling pathway components, which are activated by the release of IL-33 in macrophages. Our study provides a novel mechanism for elucidating IUA formation, suggesting a new therapeutic strategy for the prevention and clinical treatment of IUAs.