Expression Of Alpha-smooth Muscle Actin Research Articles

#207 Early single session of hyperbaric oxygen therapy mitigates renal apoptosis in lipopolysaccharides-induced endotoxemia in rats

Abstract Background and Aims Sepsis-associated acute kidney injury (SA-AKI) is a prominent sepsis complication, often having adverse clinical outcomes. Hyperbaric oxygen therapy (HBOT), known for its anti-inflammatory characteristics, antioxidant effects, and ability to deliver high oxygen tension to hypo-perfused tissues, may offer potential benefits for SA-AKI. This study investigated whether HBOT improved renal injury in sepsis and elucidated its underlying mechanisms. Method An lipopolysaccharides (LPS)-induced endotoxemia model was established using eight-week-old C57BL/6 mice. Thirty minutes post-LPS administration, a subset of mice underwent HBOT at a 2.5 atmospheric pressure absolute with 100% oxygen for 60 minutes. After 24 hours, all mice were euthanized for measurements. Results Our results demonstrated that HBOT effectively mitigated renal tubular cell apoptosis, as evidenced by decreased apoptosis-associated proteins, including Bax/Bcl-2 and cleaved caspase 3/caspase 3 ratios. HBOT significantly reduced phosphorylated p53 proteins and cytochrome C levels, suggesting that HBOT may attenuate renal apoptosis through decreased p53 activation and cytochrome C release. Notably, HBOT preserved manganese-dependent levels of superoxide dismutase, an antioxidant enzyme, compared to the LPS group. Furthermore, transforming growth factor-β (TGF- β)/Smad4 and alpha-smooth muscle actin expressions were significantly reduced in the LPS+HBOT group. Conclusion An early single session of HBOT exhibited renoprotective effects in LPS-induced endotoxemia mice models by mitigating apoptosis by suppressing p53 activation and cytochrome C levels. The observed TGF-β decrease, downstream Smad expression reduction, and antioxidant capacity preservation following HBOT may contribute to these effects.

Inhibiting Effect and Mechanism of Aconitum tanguticum (Maxim.) Stapf on Intestinal Fibrosis of CCD-18Co Cells

Abstract Objective: The objective of the study was to explore the potential signaling mechanism of Aconitum tanguticum (Maxim.) Stapf (ATS) and its impact on intestinal fibrosis in vitro. Methods: Network pharmacology was used to screen the active components of ATS and predict their potential targets in intestinal fibrosis. The protein–protein interaction network graph was constructed using drug–disease intersection targets retrieved from the Search Tool for Retrieval of Interacting Genes/Proteins database. The network diagram was analyzed using Cytoscape 3.6.1’stopology function. The gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted using the database for annotation, visualization, and integrated discovery platform. Intestinal fibroblast model in vitro was constructed using transforming growth factor-β1 (TGF-β1)-induced CCD-18Co cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the network pharmacology-predicted antifibrotic signaling pathway of ATS and the traditional antifibrotic signaling pathway. Results: Network pharmacology revealed that there were 19 active components in ATS, suggesting that ATS could be involved in the regulation of mitogen-activated protein kinase 1 (MAPK1), PIK3CA, MAPK3, and other important targets that are present in cancer pathways, including proteoglycans, influenza A, and phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) signaling pathways. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) demonstrated that 0.1–1 μg/mL ATS may suppress tissue inhibitor of metalloproteinase 1, collagen I, and alpha-smooth muscle actin expression levels. The expression of the TGF-β/suppressor of the mother against decapentaplegic (Smad) and PI3K/Akt signaling pathways was controlled by ATS. In addition, ATS inhibited the PI3K/Akt pathway by reducing the expression of MAPK1, HSP90AA1, and PIK3CA. Conclusions: It has been confirmed that ATS is a multipathway and multitarget treatment for intestinal fibrosis. This study suggested that ATS might alleviate intestinal fibrosis by blocking both the TGF-β/Smad and the PI3K/Akt pathway.

Biodegradable material for glaucoma drainage devices - A pilot study in rabbits.

To study the morphological and histological characteristics of the fibrous capsule formed around a novel biodegradable glaucoma drainage device (GDD) implant made of oxidized regenerated cellulose (ORC) after 1 and 3 months of its implantation. It was a prospective, interventional, preclinical experimental study using New Zealand white rabbits. All animals underwent GDD implantation with a novel biodegradable device. The device's body was made of ORC and attached to a silicone tube, connecting the body to the anterior chamber. Histopathology (hematoxylin and eosin/Masson's trichrome staining) and immunohistochemistry (Alpha-Smooth Muscle Actin expression) characteristics of the bleb formed around the novel device were noted at 1 and 3 months. One month post implantation, the biodegradable material produced a significant foreign body type of reaction evidenced by the exuberant infiltration by macrophages, lymphocytes, and multinucleated giant cells. The granulomatous response subsided by 3 months with disorganized collagen deposition on Masson's trichrome staining. The silicone tube was an internal control, and histopathology demonstrated well-organized collagen deposition around it at 3 months. Immunohistochemistry for α-smooth muscle actin also demonstrated more myofibroblast transformation at the site of the tube than the biodegradable implant. Our results indicate that the tissue response around a biodegradable GDD was different from the response to conventional devices. A diffuse, loose vascularized mesh was observed to develop, which may be more compatible with sustained IOP control over a longer period, in contrast to the usual thick and dense capsule formed around the non-biodegradable devices.

Ameliorative Potential of Bone Marrow-Derived Mesenchymal Stem Cells Versus Prednisolone in a Rat Model of Lung Fibrosis: A Histological, Immunohistochemical, and Biochemical Study.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease of unknown origin with limited treatment options and poor prognosis. The encouraging findings from preclinical investigations utilizing mesenchymal stem cells (MSCs) indicated that they could serve as a promising therapeutic alternative for managing chronic lung conditions, such as IPF. The objective of this study was to compare the efficiency of bone marrow-derived MSCs (BM-MSCs) versus prednisolone, the standard anti-inflammatory medication, in rats with bleomycin (BLM)-induced lung fibrosis. Four groups were created: a control group, a BLM group, a prednisolone-treated group, and a BM-MSCs-treated group. To induce lung fibrosis, 5 mg/kg of BLM was administered intratracheally. BLM significantly increased serum levels of pro-inflammatory cytokines and oxidative stress markers. The disturbed lung structure was also revealed by light and transmission electron microscopic studies. Upregulation in the immune expression of alpha-smooth muscle actin, transforming growth factor beta-1, and Bax was demonstrated. Interestingly, all findings significantly regressed on treatment with prednisolone and BM-MSCs. However, treatment with BM-MSCs showed better results than with prednisolone. In conclusion, BM-MSCs could be a promising approach for managing lung fibrosis.

Canine Visceral Leishmaniasis: A Histological and Immunohistochemical Study of Fibropoiesis in Chronic Interstitial Pneumonitis.

We studied some fibrotic aspects of chronic interstitial pneumonitis in the lungs of dogs infected with Leishmania infantum. The lungs of eleven naturally infected dogs, twelve experimentally infected with two distinct strains of L. infantum (BH401 and BH46), and six uninfected (controls) dogs, were analyzed by histological, parasitological, and immunohistochemical studies. Conventional histology (HE), collagen deposition (Gomori's silver staining for reticulin collagen fibers), and immunohistochemistry for myofibroblast characterization were carried out based on the cellular expression of alpha-smooth muscle actin, vimentin, cytokeratin, E-cadherin, snail antigen homologue 1 (SNAI1) (Snail), and the cytokine expression of transforming growth factor-beta (TGF-β). Parasitological screening was carried out using conventional polymerase chain reaction (PCR) and the immunohistochemical reaction of streptavidin-peroxidase for visualizing Leishmania amastigotes. Dogs naturally infected with L. infantum and experimentally infected with L. infantum BH401 strains showed intense interstitial pneumonitis characterized by thickening of the alveolar septa as a consequence of an intense diffuse and focal (plaques) chronic exudate of mononuclear cells associated with fibrogenesis. The expression of alpha-actin, vimentin, and TGF-β was higher in the lung interstitium of all infected dogs than in the other two groups (BH46 strain and controls). Moreover, in both the naturally and experimentally infected dog (BH401 strain) groups, the expression of Snail was moderate to intense in contrast to the other groups. Based on these immunohistochemical results, we concluded that mesenchymal cells are active in promoting changes in the extracellular matrix in the lungs of dogs naturally and experimentally infected with L. infantum, but it depends on the virulence of the parasite.

Effects of different preparation protocols of blood serum eyedrops on corneal healing after alkali burn: Clinical evaluation and α-SMA expression

Background: In cases of alkali burn, blood serum eyedrops may supply protein and growth factors. A standard preparation protocol is necessary for consistency in epitheliotropic capacity. Objective: We aim to evaluate how post-alkali-burn administration of blood serum eyedrops from different preparation protocols results in different outcomes on rabbits. Methods: This randomized experimental study used three treatments: placebo, non-diluted serum, and 25% concentration serum. We analyzed the corneal haziness, neovascularization degree, histopathological examination of neovascularization scores, and alpha-smooth muscle actin (α-SMA) expression. Results: Twelve rabbits (24 eyes) were split evenly into three treatment groups. On day 14, the group receiving the 25% concentration serum had the least corneal haziness (p = 0.005). No adverse effect was observed on the treated eyes. Histopathologically, no significant difference in neovascularization scores and α-SMA expression was observed. Conclusions: Treatment using the 25% concentration serum resulted in less corneal haziness, smaller defect size, and greater healing rate. There was also no significant difference in histopathological outcomes among the three treatment groups.

The circAno6/miR-296-3p/TLR4 signaling axis mediates the inflammatory response to induce the activation of hepatic stellate cells

BackgroundCircular RNA (circRNA) is a novel functional non-coding RNA(ncRNA) that plays a role in the occurrence and development of multiple human liver diseases, including liver fibrosis (LF). LF is a reversible repair response after liver injury, and the activation of hepatic stellate cells (HSCs) is the core event. However, the regulatory mechanisms by which circRNAs induce the activation of HSCs in LF are still poorly understood. The circAno6/miR-296-3p/toll-like receptor 4 (TLR4) signaling axis that mediates the inflammatory response and causes the activation of HSCs was investigated in this study. MethodsFirst, a circAno6 overexpression plasmid and small interfering RNA were transfected into cells to determine whether circAno6 can affect the function of HSCs. Second, real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), western blotting (WB) and immunofluorescence (IF) were used to detect the effects of circAno6 plasmid/siRNA transfection on HSC activation indices, inflammatory markers and the circAno6/miR-296-3p/TLR4 signaling axis. The subcellular position of circAno6 was then examined by nucleo-cytoplasmic separation and fluorescence in situ hybridization (FISH). Finally, a luciferase reporter gene assay was used to identify the relationship between circAno6 and miR-296-3p as well as the relationship between miR-296-3p and TLR4. ResultsCircAno6 was considerably upregulated in HSCs and positively correlated with cell proliferation and alpha-smooth muscle actin (α-SMA), collagen I, NOD-likereceptorthermalproteindomainassociatedprotein 3 (NLRP3), interleukin-1β (IL-1β) and interleukin-18 (IL-18) expression. Overexpression of circAno6 increased the inflammatory response and induced HSC activation, whereas interference resulted in the opposite effects. FISH experiments revealed the localization of circAno6 in the cytoplasm. Then, a double luciferase reporter assay confirmed that miR-296-3p significantly inhibited luciferase activity in the circAno6-WT and TLR4-WT groups. ConclusionThis study suggests that circAno6 and miR-296-3p/TLR4 may form a regulatory axis and regulate the inflammatory response, which in turn induces HSC activation. Targeting circAno6 may be a potential therapeutic strategy to treat LF.

The Dynamic Role Of Myofibroblasts In Oral Tissue Homeostasis And Disease

Myofibroblasts are specialized cells that play a crucial role in maintaining the structural integrity and function of oral tissues. These cells exhibit a unique phenotype marked by the expression of alpha-smooth muscle actin (α-SMA), and possess the ability to contract and produce extracellular matrix components. In healthy oral tissues, myofibroblasts contribute to tissue homeostasis by mediating wound healing, tissue remodeling, and matrix turnover. However, the dysregulation of myofibroblast function can lead to pathological conditions such as fibrosis, scarring, and oral cancer. This review aims to explore the dynamic role of myofibroblasts in oral tissue homeostasis and disease. It begins by examining the origin of myofibroblasts in the oral cavity, their phenotypic characteristics, and their interactions with other cell types, including epithelial cells, immune cells, and endothelial cells. The review further highlights the role of myofibroblasts in various oral diseases, such as oral submucous fibrosis, periodontal disease, and oral cancer, and discusses potential therapeutic strategies targeting myofibroblast function. Understanding the dynamic role of myofibroblasts in oral tissue homeostasis and disease is essential for developing innovative therapeutic approaches to modulate myofibroblast function and improve the management of oral diseases.

Overexpressed miR‐486 in bone marrow mesenchymal stem cells represses urethral fibrosis and targets Col13a1 in urethral stricture rats

AbstractUrethral stricture (US) is a challenging problem in urology and its pathogenesis of US is closely related to the fibrotic process. Previous evidence has indicated the downregulation of microRNA (miR)‐486 in injured urethral specimens of rats. This study aimed to explore the effects of miR‐486‐overexpressed bone marrow mesenchymal stem cells (BMSCs) on US. BMSCs were identified by detecting their multipotency and surface antigens. Lentivirus virus expressing miR‐486 was transduced into rat BMSCs to overexpress miR‐486. Transforming growth factor (TGF)‐β1 induced fibrotic phenotypes in urethral fibroblasts (UFs) and rat models. Western blotting showed protein levels of collagen I/III and collagen type XIII alpha 1 chain (Col13a1). Real time quantitative polymerase chain reaction was utilized for messenger RNA level evaluation. Hematoxylin‐eosin, Masson's trichrome, and Von Willebrand Factor staining were conducted for histopathological analysis. Immunofluorescence staining was employed for detecting alpha smooth muscle actin (α‐SMA) expression. Luciferase reporter assay verified the interaction between miR‐486 and Col13a1. The results showed that miR‐486‐overexpressed BMSCs suppressed collagen I/III and α‐SMA expression in TGF‐β1‐stimulated UFs. miR‐486‐overexpressed BMSCs alleviated urethral fibrosis, collagen deposition, and epithelial injury in the urethral tissue of US rats. miR‐486 targeted and negatively regulated Col13a1 in US rats. In conclusion, overexpression of miR‐486 in BMSCs targets Col13a1 and attenuates urethral fibrosis in TGF‐β1‐triggered UFs and US rats.

Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels.

In obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), the extracellular matrix (ECM) protein amount and composition of the airway smooth muscle (ASM) is often remodelled, likely altering tissue stiffness. The underlying mechanism of how human ASM cell (hASMC) mechanosenses the aberrant microenvironment is not well understood. Physiological stiffnesses of the ASM were measured by uniaxial compression tester using porcine ASM layers under 0, 5 and 10% longitudinal stretch above in situ length. Linear stiffness gradient hydrogels (230 kPa range) were fabricated and functionalized with ECM proteins, collagen I (ColI), fibronectin (Fn) and laminin (Ln), to recapitulate the above-measured range of stiffnesses. Overall, hASMC mechanosensation exhibited a clear correlation with the underlying hydrogel stiffness. Cell size, nuclear size and contractile marker alpha-smooth muscle actin (αSMA) expression showed a strong correlation to substrate stiffness. Mechanosensation, assessed by Lamin-A intensity and nuc/cyto YAP, exhibited stiffness-mediated behaviour only on ColI and Fn-coated hydrogels. Inhibition studies using blebbistatin or Y27632 attenuated most mechanotransduction-derived cell morphological responses, αSMA and Lamin-A expression and nuc/cyto YAP (blebbistatin only). This study highlights the interplay and complexities between stiffness and ECM protein type on hASMC mechanosensation, relevant to airway remodelling in obstructive airway diseases.

Interferon-γ Induces Interleukin-6 Production and Alpha-smooth Muscle Actin Expression in Systemic Sclerosis Fibroblasts.

Systemic sclerosis (SSc) is an autoimmune systemic disease that is characterized by immune dysregulation, inflammation, vasculopathy, and fibrosis. Tissue fibrosis plays an important role in SSc and can affect several organs such as the dermis, lungs, and heart. Dysregulation of interferon (IFN) signaling contributes to the SSc pathogenesis and interferon regulatory factor 1 (IRF1) has been indicated as the main regulator of type I IFN. This study aimed to clarify the effect of IFN-gamma (-γ) and dexamethasone (DEX) on the IRF1, extracellular signal-regulated kinase 1/2 (ERK1/2), and the expression of alpha-smooth muscle actin (α-SMA) in myofibroblasts and genes involved in the inflammation and fibrosis processes in early diffuse cutaneous systemic sclerosis (dcSSc). A total of 10 early dcSSc patients (diffuse cutaneous form) and 10 unaffected control dermis biopsies were obtained to determine IFNγ and DEX effects on inflammation and fibrosis. Fibroblasts were treated with IFNγ and DEX at optimum time and dose. The expression level of genes and proteins involved in the fibrosis and inflammation processes have been quantified by quantitative real-time PCR (RT-qPCR) and western blot, respectively. IFNγ could up-regulate some of the inflammation-related genes (Interleukin-6; IL6) and down-regulate some of the fibrosis-related genes (COL1A1) in cultured fibroblasts of patients with early dcSSc compared to the untreated group. Besides, it has been revealed that IFNγ can induce fibroblast differentiation to the myofibroblast that expresses α-SMA. Concerning the inhibitory effect of IFNγ on some fibrotic genes and its positive effect on the inflammatory genes and myofibroblast differentiation, it seems that IFNγ may play a dual role in SSc.

Endothelial-to-Mesenchymal Transition in Human and Murine Models of Congenital Diaphragmatic Hernia

Introduction: Congenital diaphragmatic hernia (CDH) is a complex congenital disorder, characterized by pulmonary hypertension (PH) and hypoplasia. PH secondary to CDH (CDH-PH) features devastating morbidity and mortality (25–30%) among neonates. An unmet need is determining mechanisms triggering CDH-PH to save infants. Prior data suggest abnormal remodeling of the pulmonary vascular extracellular matrix (ECM), presumed to be driven by endothelial-to-mesenchymal transition (EndoMT), hinders postnatal vasodilation and limits anti-PH therapy in CDH. There are limited data on the role of EndoMT in CDH-PH. Methods: The purpose of the study was to investigate how EndoMT contributes to CDH-PH by identifying cells undergoing EndoMT noted by alpha smooth muscle actin (α-SMA) expression in human umbilical vein endothelial cells (HUVECs) and lung tissue obtained from murine pups using the nitrofen model. N = 8 CDH, N = 8 control HUVECs were stained for α-SMA and CD31 after being exposed for 24 h to TGFB, a known EndoMT promoter. N = 8 nitrofen, N = 8 control murine pup lungs were also stained for α-SMA and CD31. α-SMA and CD31 expression was quantified in HUVECs and murine tissue using Fiji imaging software and normalized to the total number of cells per slide noted by DAPI staining. Results: CDH HUVECs demonstrated a 1.1-fold increase in α-SMA expression (p = 0.02). The murine model did not show statistical significance between nitrofen and control pup lungs; however, there was a 0.4-fold increase in α-SMA expression with a 0.8-fold decrease in CD31 expression in the nitrofen pup lungs when compared to controls. Conclusion: These results suggest that EndoMT could potentially play a role in the ECM remodeling seen in CDH-PH.

Effects of Bone Marrow Sparing and TGF-β3 Treatment in Total Body Irradiation of C57BL/6J Mice

Introduction: Mortality from acute radiation syndrome is frequently caused by hematopoietic or gastrointestinal radiotoxicity, the latter of which currently has no effective treatment. Transforming growth factor-beta 3 (TGF-β3) may decrease the severity of radiation-induced gastrointestinal damage in mice. In addition, treatment with TGF-β3 may alleviate radiation-induced fibrosis. Objectives: The current study aimed to investigate the effect of TGF-β3 treatment on acute and late radiotoxicity in whole body irradiated mice. Methods: C57BL/6J mice were total body irradiated with 8.5 Gy X-rays with or without shielding of one hind leg to alleviate hematopoietic radiotoxicity. The effects of intravenous TGF-β3 treatment were investigated. Body weight and pain expression were monitored. Intestine, lung, and liver tissues were preserved and analyzed. Alpha smooth muscle actin (α-SMA) expression in MRC-5 cells after 3.5 Gy X-irradiation combined with TGF-β3 treatment was analyzed using flow cytometry. Results: All total body irradiated animals died within ten days after irradiation. Ninety-three percent of femur-shielded mice survived until sampling or termination. No effect of TGF-β3 treatment was observed in either group. No increase in collagen content was detected in the lungs or liver from irradiated mice regardless of TGF-β3 treatment. In vitro, α-SMA expression increased synergistically after irradiation and TGF-β3 treatment. Conclusions: Shielding of the femur during total body irradiation decreased acute gastrointestinal radiation toxicity and increased survival. TGF-β3 treatment did not impact symptoms or survival. TGF-β3 treatment and irradiation increased α-SMA expression in MRC-5 cells synergistically.

Effects of Mesenchymal Stem Cells-Derived Extracellular Vesicles on Inhibition of Hepatic Fibrosis by Delivering miR-200a.

Hepatic fibrosis (HF) is a common pathological feature of chronic hepatic diseases. We aimed to illuminate the significance of amniotic mesenchymal stem cells (AMSCs)-derived extracellular vesicles (AMSCs-EVs) in HF. Human AMSCs-EVs were isolated and identified. HF mice were constructed and treated with EVs. The fibrosis was observed by staining experiments and Western blot (WB) assay. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and hepatic hydroxyproline (Hyp) were detected to confirm liver function. For the in vitro experiments, human hepatic stellate cells were induced with transforming growth factor-β and treated with EVs. To measure the degree of HF, the expression of alpha-smooth muscle actin (α-SMA) and Collagen I was detected by WB assay, and cell proliferation was detected by cell counting kit 8 assay. The levels of miR-200a, Zinc finger E-box binding homeobox 1 (ZEB1), and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were detected by WB and real-time quantitative polymerase chain reaction. The binding of ZEB1 to PIK3R3 and miR-200a to ZEB1 was analyzed by chromatin immunoprecipitation and dual luciferase assays to validate their relationships. Human AMSCs and AMSCs-EVs were obtained. Serum ALT, AST, TBIL, and hepatic Hyp were increased, implying the fibrosis degree was aggravated in HF mice, which was decreased again after EV treatment. EVs inhibited HF degree by reducing α-SMA and Collagen I and promoting cell proliferation. AMSCs-EVs delivered miR-200a into hepatocytes, which up-regulated miR-200a expression, inhibited ZEB1 expression, and reduced its enrichment on the PIK3R3 promoter, therefore inhibiting PIK3R3 expression and alleviating HF. Overexpression of ZEB1 or PIK3R3 attenuated the anti-fibrotic effect of AMSCs-EVs. Human AMSCs-derived EVs mediated miR-200a delivery and inhibition of intracellular ZEB1/PIK3R3 axis to exert anti-fibrosis effects.

Inula britannica ameliorates alcohol-induced liver injury by modulating SIRT1-AMPK/Nrf2/NF-κB signaling pathway

ObjectiveInula britannica is a traditional Chinese medicinal and functional food with various effects such as anti-liver injury, hypoglycemia, antioxidants, and anti-tumor. This study aimed to investigate the protective effects and mechanisms of the ethanolic extract of I. britannica (EEIB) on alcohol-induced liver injury in mice. MethodsFifty-six female C57BL/6 mice were randomly divided into seven groups: control group (Con), ethanol feeding model group (EtOH), Silibinin positive treatment group (EtOH + Silibinin 100 mg/kg), EEIB treatment group (EtOH + EEIB 100, 200, and 400 mg/kg), and EEIB control group (EEIB 400 mg/kg). The National Institute on Alcohol Abuse and Alcoholism (NIAAA) ethanol-feeding model was used to study the effects of EEIB on alcohol-induced lipid metabolism, inflammation, oxidative stress, and fibril formation in mice by histopathological evaluation, immunofluorescence staining, Western blotting analysis and molecular docking. ResultsEEIB reduced liver indices to different degrees to normal levels and improved liver morphology in mice. EEIB inhibited alcohol-induced liver injury by activating the sirtuin 1 (SIRT1)-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway in the liver of alcohol-fed mice, in which sesquiterpenes may be the potential active ingredients, and also down-regulated the expression of alpha-smooth muscle actin (α-SMA), collagen alpha (Collagen I), tumor necrosis factor-alpha (TNF-α) and attenuated alcohol-induced liver injury. In addition, EEIB also activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, which alleviated alcohol-induced liver injury at the level of oxidative stress. Notably, the EEIB control group demonstrated that EEIB had no toxic effects in mice. EEIB reduced alcoholic liver injury in a dose-dependent manner. Its therapeutic efficacy was comparable to, if not better than, that of Silibinin when administered at a dose of 400 mg/kg. ConclusionEEIB showed significant therapeutic effects on alcohol-induced liver injury in mice, and its mechanism of action was related to the SIRT1-AMPK, nuclear factor-kappa B (NF-κB), and Nrf2 signaling pathways, in which sesquiterpenes may be the potential active ingredients.

Abstract 3016: MCPIP1 endoribonuclease protects from liver fibrosis and regulates hepatocellular development

Abstract Introduction: MCPIP1 (Monocyte Chemotactic Protein-1 Induced Protein), also known as Regnase-1, belongs to the CCCH zinc finger protein family, featuring a PilT N-terminus (PIN) domain-like RNase domain. Utilizing its intrinsic endonuclease activity, MCPIP1 functions as a precise modulator of cytokines, exhibiting an anti-inflammatory effect through the targeted degradation of specific mRNA transcripts. While recent studies have highlighted MCPIP1 as a potential tumor suppressor with decreased levels in breast cancer and clear cell renal cell carcinoma, its role in hepatocellular carcinoma (HCC) development remains unexplored. Material and Methods: To investigate the role of MCPIP1 in HCC development, we used tissue-specific knockout mice with the deletion of Mcpip1 in hepatocytes. HCC was induced by administering diethylnitrosamine (DEN) to two-week-old mice. We conducted a comprehensive analysis, including the assessment of EMT and fibrosis markers, proinflammatory cytokines, and immune cell infiltration, using Western blot (WB), mass spectrometry, quantitative PCR (qPCR), and multiple immunohistochemistry (IHC) stainings. To identify differences between the wild-type (wt) and Mcpip1 knockout (KO) mice groups more effectively, hepatocytes were isolated using the perfusion method, and RNA-Seq analysis was performed. Results and Discussion: The tissue-specific knockout of Mcpip1 in the liver has notable molecular consequences. Transcriptomic profiling revealed an upregulation of transcripts integral to tumor development, including Cxcr4, Met, Cxcl12, Hgf, and Src, alongside factors characteristically associated with cancer stem cells, Myc, Klf4, and CD133. The absence of MCPIP1 triggered immune cell activation in the liver, as evidenced by an augmented expression of markers specific to myeloid and lymphoid cell populations, indicative of an intensified immunological response. IHC examination of liver tissue from Mcpip1-deficient mice revealed significant fibrotic alterations, characterized by increased collagen deposition, and enhanced expression of alpha-smooth muscle actin (αSMA). The lack of Mcpip1 influences the increased levels of vimentin, fibronectin, and connective tissue growth factor (cTGF), indicating enhanced tissue remodeling. Conclusions: MCPIP1, recognized for its anti-inflammatory functions, appears to function as a potential tumor suppressor in HCC development. The lack of MCPIP1 results in the upregulation of transcripts linked to tumorigenesis, immune cell activation, and notable fibrotic changes, highlighting its involvement in the complex molecular dynamics of HCC progression. This study was supported by National Science Center grants no. 2017/26/E/NZ5/00691, 2021/41/N/NZ4/04187. Citation Format: Katarzyna Miekus, Oliwia Kwapisz, Paulina Marona, Judyta Gorka, Jerzy Kotlinowski, Ewelina Pospiech, Jolanta Jura. MCPIP1 endoribonuclease protects from liver fibrosis and regulates hepatocellular development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3016.

Empagliflozin and pirfenidone confer renoprotection through suppression of glycogen synthase kinase-3β and promotion of tubular regeneration in rats with induced metabolic syndrome

Metabolic syndrome (MetS) is largely coupled with chronic kidney disease (CKD). Glycogen synthase kinase-3β (GSK-3β) pathway drives tubular injury in animal models of acute kidney injury; but its contribution in CKD is still elusive. This study investigated the effect empagliflozin and/or pirfenidone against MetS-induced kidney dysfunction, and to clarify additional underpinning mechanisms particularly the GSK-3β signaling pathway. Adult male rats received 10%w/v fructose in drinking water for 20 weeks to develop MetS, then treated with either drug vehicle, empagliflozin (30 mg/kg/day) and/or pirfenidone (100 mg/kg/day) via oral gavage for subsequent 4 weeks, concurrently with the high dietary fructose. Age-matched rats receiving normal drinking water were used as controls. After 24 weeks, blood and kidneys were harvested for subsequent analyses. Rats with MetS showed signs of kidney dysfunction, structural changes and interstitial fibrosis. Activation of GSK-3β, decreased cyclinD1 expression and enhanced apoptotic signaling were found in kidneys of MetS rats. There was abundant alpha-smooth muscle actin (α-SMA) expression along with up-regulation of TGF-β1/Smad3 in kidneys of MetS rats. These derangements were almost alleviated by empagliflozin or pirfenidone, with evidence that the combined therapy was more effective than either individual drug. This study emphasizes a novel mechanism underpinning the beneficial effects of empagliflozin and pirfenidone on kidney dysfunction associated with MetS through targeting GSK-3β signaling which can mediate the regenerative capacity, anti-apoptotic effects and anti-fibrotic properties of such drugs. These findings recommend the possibility of using empagliflozin and pirfenidone as promising therapies for management of CKD in patients with MetS.

PM2.5 exposure-induced senescence-associated secretory phenotype in airway smooth muscle cells contributes to airway remodeling

Fine particulate matter (PM2.5) has been linked to increased severity and incidence of airway diseases, especially chronic obstructive pulmonary disease (COPD) and asthma. Airway remodeling is an important event in both COPD and asthma, and airway smooth muscle cells (ASMCs) are key cells which directly involved in airway remodeling. However, it was unclear how PM2.5 affected ASMCs. This study investigates the effects of PM2.5 on airway smooth muscle and its mechanism. We first showed that inhaled particulate matter was distributed in the airway smooth muscle bundle, combined with increased airway smooth muscle bundle and collagen deposition in vivo. Then, we demonstrated that PM2.5 induced up-regulation of collagen-I and alpha-smooth muscle actin (α-SMA) expression in rat and human ASMCs in vitro. Next, we found PM2.5 led to rat and human ASMCs senescence and exhibited senescence-associated secretory phenotype (SASP) by autophagy-induced GATA4/TRAF6/NF-κB signaling, which contributed to collagen-I and α-SMA synthesis as well as airway smooth muscle remodeling. Together, our results provided evidence that SASP induced by PM2.5 in airway smooth muscle cells prompted airway remodeling.

Protective Effect of CD137 Deficiency against Post-infarction Cardiac Fibrosis and Adverse Cardiac Remodeling via ERK1/2 Signaling Pathways.

Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart following MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared to wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin (α-SMA) and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings, as CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. Additionally, the administration of CD137L recombinant protein further promoted α-SMA expression and collagen I synthesis, suggesting a pro-fibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the pro-fibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function.

Human umbilical cord mesenchymal stem cells improve uterine incision healing after cesarean delivery in rats by modulating the TGF-β/Smad signaling pathway

ObjectiveAlthough human umbilical cord-derived mesenchymal stem cells (HU-MSCs) have attracted increasing attention because of their pivotal functions in the process of wound healing, the underlying molecular mechanisms have been poorly understood. It has been shown that the TGF-β/Smad signaling pathway plays an important role in the process of scar formation. The present study focused on exploring whether HU-MSCs improve uterine incision healing after cesarean delivery in rats via the TGF-β/Smad signaling pathway.Study DesignPregnant rats were randomly assigned to three groups, including the NP group, incision-injected group (HU-MSCs1 group), and tail vein-injected group (HU-MSCs2 group), and 30 days after cesarean section, sampling was carried out to further explore the specific mechanisms from tissue and protein levels.ResultsHU-MSCs secretion could inhibit the fibrosis of scar tissue. We observed that the TGF-β induced expression of TGF-β1, Smad2, and Smad3 was attenuated upon HU-MSCs treatment in scar tissue, while the decrease in TGF-β3 expression was enhanced by HU-MSCs. Furthermore, HU-MSCs treatment accelerated wound healing and attenuated collagen deposition in a damaged uterine rat model, leading to the promoting of uterine incision scarring. In addition, the expression of alpha-smooth muscle actin (a-SMA) was enhanced by HU-MSCs treatment.ConclusionHU-MSCs transplantation promotes rat cesarean section uterine incision scar healing by modulating the TGF-β/Smad signaling pathway.