α-SMA Expression Research Articles

Scleral remodeling in early adulthood: the role of FGF-2

Emmetropization, a natural process of ocular elongation, is closely associated with scleral remodeling. The Fibroblast growth factor-2 (FGF-2) was reported involved in scleral remodeling in myopia models. Herein, we aimed to investigate the role of scleral fibroblast-to-myofibroblast differentiation and FGF-2 in scleral remodeling during maturation. Our findings revealed that the posterior scleral fibroblasts (SFs) from mature guinea pigs exhibit increased stiffness compared to those from young guinea pigs. Moreover, mature SFs displayed decreased cell proliferation but increased levels of α-SMA, matrix metalloproteinase 2 (MMP2), and collagen 1, when compared to young SFs. Additionally, the mRNA expression of scleral Fgf-2, Fgf receptor 1 (Fgfr1), Fgfr2, Fgfr3, and Fgfr4 was increased in mature SFs. Notably, exogenous FGF-2 showed increased cell proliferation and led to decreased expression of α-SMA, MMP2, and collagen 1 in mature SFs. Overall, our findings highlight the influence of maturation on SFs from posterior scleral shells, resulting in increased stiffness and the manifestation of fibroblast-to-myofibroblast differentiation during development. Exogenous FGF-2 increased cell proliferation and reversed the age-related fibroblast-to-myofibroblast differentiation, suggesting a potential role of FGF-2 in regulating scleral remodeling.

Neutralizing tumor-related inflammation and reprogramming of cancer-associated fibroblasts by Curcumin in breast cancer therapy

Tumor-associated inflammation plays a vital role in cancer progression. Among the various stromal cells, cancer-associated fibroblasts are promising targets for cancer therapy. Several reports have indicated potent anti-inflammatory effects attributed to Curcumin. This study aimed to investigate whether inhibiting the inflammatory function of cancer-associated fibroblasts (CAFs) with Curcumin can restore anticancer immune responses. CAFs were isolated from breast cancer tissues, treated with Curcumin, and co-cultured with patients' PBMCs to evaluate gene expression and cytokine production alterations. Blood and breast tumor tissue samples were obtained from 12 breast cancer patients with stage II/III invasive ductal carcinoma. Fibroblast Activation Protein (FAP) + CAFs were extracted from tumor tissue, treated with 10 μM Curcumin, and co-cultured with corresponding PBMCs. The expression of smooth muscle actin-alpha (α-SMA), Cyclooxygenase-2(COX-2), production of PGE2, and immune cell cytokines were evaluated using Real-Time PCR and ELISA, respectively. Analyzes showed that treatment with Curcumin decreased the expression of genes α-SMA and COX-2 and the production of PGE2 in CAFs. In PBMCs co-cultured with Curcumin-treated CAFs, the expression of FoxP3 decreased along with the production of TGF-β, IL-10, and IL-4. An increase in IFN-γ production was observed that followed by increased T-bet expression. According to our results, Curcumin could reprogram the pro-tumor phenotype of CAFs and increase the anti-tumor phenotype in PBMCs. Thus, CAFs, as a component of the tumor microenvironment, are a suitable target for combination immunotherapies of breast cancer.

Accelerated Oral Healing by Angelica gigas Nakai from Hot Melt Extrusion Technology: An In Vitro Study.

Background and Objectives: In spite of the oral environment being healing-prone, its dynamic changes may affect wound healing. The purpose of this study was to assess the oral wound healing effect of Angelica gigas Nakai (AG) prepared by hot-melt extrusion. Materials and Methods: Human gingival fibroblast (HGF) cells were treated with AG or AG via hot-melt extrusion (AGH) for 24 h to determine the optimal concentration. For evaluating the anti-inflammatory effect of AG and AGH, a nitric oxide assay was performed under lipopolysaccharide (LPS) stimulation. The wound-healing effects of AG and AGH were evaluated using cell proliferation/migration assays and wound-healing marker expression through qRT-PCR. Results: Both AG and AGH showed no cytotoxicity on HGH cells. Regarding nitric oxide production, AGH significantly decreased LPS-induced nitric oxide production (p < 0.05). AGH showed a significantly positive result in the cell proliferation/cell migration assay compared with that in AG and the control. Regarding wound healing marker expression, AGH showed significantly greater VEGF and COL1α1 expression levels than those in the others (p < 0.05), whereas α-SMA expression was significantly different among the groups. Conclusions: Within the limits of this study, AGH accelerated oral wound healing in vitro.

Stevioside attenuates bleomycin-induced pulmonary fibrosis by activating the Nrf2 pathway and inhibiting the NF-κB and TGF-β1/Smad2/3 pathways

Objective: This study aimed to investigate the effects of stevioside (STE) on pulmonary fibrosis (PF) and the potential mechanisms. Methods: In this study, a mouse model of PF was established by a single intratracheal injection of bleomycin (BLM, 3 mg/kg). The experiment consisted of four groups: control group, BLM group, and STE treatment groups (STE 50 and 100 mg/kg). ELISA and biochemical tests were conducted to determine the levels of TNF-α, IL-1β, IL-6, NO, hydroxyproline (HYP), SOD, GSH, and MDA. Histopathological changes and collagen deposition in lung tissues were observed by HE and Masson staining. Immunohistochemistry was performed to determine the levels of collagen I-, collagen III-, TGF-β1- and p-Smad2/3-positive cells. Western blot analysis was used to measure the expression of epithelial-mesenchymal transition (EMT) markers, including α-SMA, vimentin, E-cadherin, and ZO-1, as well as proteins related to the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, nuclear transcription factor-κB (NF-κB) pathway, and TGF-β1/Smad2/3 pathway in lung tissues. Results: STE significantly alleviated BLM-induced body weight loss and lung injury in mice, decreased HYP levels, and reduced the levels of collagen I- and collagen III-positive cells, thereby decreasing extracellular matrix (ECM) deposition. Moreover, STE markedly improved oxidative stress (MDA levels were decreased, while SOD and GSH activity were enhanced), the inflammatory response (the levels of TNF-α, IL-1β, IL-6, and NO were reduced), and EMT (the expression of α-SMA and vimentin was downregulated, and the expression of E-cadherin and ZO-1 was upregulated). Further mechanistic analysis revealed that STE could activate the Nrf2 pathway and inhibit the NF-κB and TGF-β1/Smad2/3 pathways. Conclusion: STE may alleviate oxidative stress by activating the Nrf2 pathway, suppress the inflammatory response by downregulating the NF-κB pathway, and inhibit EMT progression by blocking the TGF-β1/Smad2/3 pathway, thereby improving BLM-induced PF.

H4K20me3 upregulated by reactive oxygen species is associated with tumor progression and poor prognosis in patients with hepatocellular carcinoma

Epigenetic alteration by oxidative stress is vitally involved in carcinogenesis and cancer progression. Previously, we demonstrated that oxidative stress was increased in hepatocellular carcinoma (HCC) patients and associated with tumor aggressiveness. Herein, we immunohistochemically investigated whether histone methylation, specifically H4K20me3, was upregulated in human hepatic tissues obtained from HCC patients (n = 100). Also, we experimentally explored if the H4K20me3 was upregulated by reactive oxygen species (ROS) and contributed to tumor progression in HCC cell lines. We found that H4K20me3 level was increased in HCC tissues compared with the adjacent noncancerous liver tissues. H3K9me3 and H3K4me3 levels were also increased in HCC tissues. Cox regression analysis revealed that the elevated H4K20me3 level was associated with tumor recurrence and short survival in HCC patients. Experimentally, H2O2 provoked oxidative stress and induced H4K20me3 formation in HepG2 and Huh7 cells. Transcript expression of histone methyltransferase Suv420h2 (for H4K20me3), Suv39h1 (for H3K9me3), and Smyd3 (for H3K4me3) were upregulated in H2O2-treated HCC cells. H2O2 also induced epithelial-mesenchymal transition (EMT) in HCC cells, indicated by decreased E-cadherin but increased α-SMA and MMP-9 mRNA expression. Migration, invasion, and colony formation in HCC cells were markedly increased following the H2O2 exposure. Inhibition of H4K20me3 formation by A196 (a selective inhibitor of Suv420h2) attenuated EMT and reduced tumor migration in H2O2-treated HCC cells. In conclusion, we demonstrated for the first time that H4K20me3 level was increased in human HCC tissues, and it was independently associated with poor prognosis in HCC patients. ROS upregulated H4K20me3 formation, induced mRNA expression of EMT markers, and promoted tumor progression in human HCC cells. Inhibition of H4K20me3 formation reduced EMT and tumor aggressive phenotypes in ROS-treated HCC cells. Possibly, ROS-induced EMT and tumor progression in HCC cells was epigenetically mediated through an increased formation of repressive chromatin H4K20me3.

Smooth Muscle Actin as a Criterion for Gravisensitivity of Stomach and Jejunum in Laboratory Rodents.

Smooth muscle tissue (SMT) is one of the main structural components of visceral organs, acting as a key factor in the development of adaptive and pathological conditions. Despite the crucial part of SMT in the gastrointestinal tract activity, the mechanisms of its gravisensitivity are still insufficiently studied. The study evaluated the content of smooth muscle actin (α-SMA) in the membranes of the gastric fundus and jejunum in C57BL/6N mice (30-day space flight), in Mongolian gerbils Meriones unguiculatus (12-day orbital flight) and after anti-orthostatic suspension according to E.R. Morey-Holton. A morphometric analysis of α-SMA in the muscularis externa of the stomach and jejunum of mice and Mongolian gerbils from space flight groups revealed a decreased area of the immunopositive regions, a fact indicating a weakening of the SMT functional activity. Gravisensitivity of the contractile structures of the digestive system may be due to changes in the myofilament structural components of the smooth myocytes or myofibroblast actin. A simulated antiorthostatic suspension revealed no significant changes in the content of the α-SMA expression level, a fact supporting an alteration in the functional properties of the muscularis externa of the digestive hollow organs under weightless environment. The data obtained contribute to the novel mechanisms of the SMT contractile apparatus remodeling during orbital flights and can be used to improve preventive measures in space biomedicine.

Variants in exon 2 of MED12 gene causes uterine leiomyoma’s through over-expression of MMP-9 of ECM pathway

AimsTo study the impact of Mediator complex subunit 12 (MED12) gene variants on the encoded protein’s function and pathogenic relevance for genesis of uterine leiomyoma’s (ULs). MethodsMutational analysis in exon-2 of MED12 gene was performed by PCR amplification and DNA sequencing in 89 clinically diagnosed ULs tissues. Pathogenicity prediction of variation was performed by computational analysis. The functional effects of missense variation were done by quantity RT-PCR and western blot analysis. Result(s)Out of 89 samples, 40 (44.94%) had missense variation in 14 different CDS position of exon-2 of MED12 gene. Out of 40 missense variation, codon 44 had 25 (62.5%) looking as a hotspot region for mutation for ULs, because CDS position c130 and c131present at codon 44 that have necleotide change G>A, T, C at c130 and c131 have necleotide change G>A and C. We also find somenovel somatic mutations oncodon 36 (T > C), 38 (G>T) of exon-2 and 88 (G>C) of intron-2. No mutations were detected in uterine myometrium samples. Our computational analysis suggests that change in Med12c .131 G>A leads to single substitution of amino acid [Glycine (G) to Aspartate (D)] which has a pathogenic and lethal impact and may cause instability of MED12 protein. Further, analysis of extracellular matrix (ECM) component (MMP-2 & 9, COL4A2 and α-SMA) mRNA and protein expression levels in the set of ULs having MED12 mutation showed significantly higher expression of MMP-9 and α-SMA. Conclusion(s)The findings of present study suggest that missense variation in codon 44 of MED12 gene lead to the genesis of leiomyoma’s through over-expression of MMP-9 of ECM pathway which could be therapeutically targeted for non-surgical management of ULs.

Triptolide attenuates CCL4-induced liver fibrosis by regulating the differentiation of CD4+ T cells in mice

Liver fibrosis is a major global health issue, and immune dysregulation is a main contributor. Triptolide is a natural immunosuppressive agent with demonstrated effectiveness in ameliorating liver fibrosis, but whether it exerts anti-liver fibrotic effects via immunoregulation remains obscure. In this study, first, by employing a CCL4-induced liver fibrosis mouse model, we demonstrated that triptolide could alleviate pathological damage to liver tissue and attenuate liver function damaged by CCL4. In addition, triptolide inhibited the expression of liver fibrotic markers such as hydroxyproline, collagen type IV, hyaluronidase, laminin, and procollagen type III, and the protein expression of α-SMA in CCL4-induced liver fibrosis. Second, with the help of network pharmacology, we predicted that triptolide’s anti-liver fibrotic effects might occur through the regulation of Th17, Th1, and Th2 cell differentiation, which indicated that triptolide might mitigate liver fibrosis via immunoregulation. Finally, multiplex immunoassays and flow cytometry were adopted to verify this prediction. The results suggested that triptolide could reverse the aberrant expression of inflammatory cytokines caused by CCL4 and regulate the differentiation of Th1, Th2, Th17, and Treg cells. In conclusion, triptolide could attenuate CCL4-induced liver fibrosis by regulating the differentiation of CD4+ T cells. The results obtained in this study extended the application of triptolide and introduced a new mechanism of triptolide’s anti-liver fibrotic effects.

Circulating large Extracellular Vesicles (EV-L) from STEMI patients contribute to post-injury adverse myocardial remodeling

Abstract Background While primary percutaneous coronary intervention (PCI) is the treatment of choice in patients with ST-segment elevation myocardial infarction (STEMI), this treatment does not fully prevent late adverse cardiac remodeling. Infarcted myocardial tissue releases damage-associated extracellular vesicles (EV) that mediate tissue inflammation and may regulate myocardial remodeling. Here, we investigated the role of circulating EV in STEMI patients, with a focus on endothelial cell (EC) injury and myocardial fibrosis. Methods Peripheral blood samples were obtained from STEMI patients (n=30) within 4 hrs of onset of pain before PCI and from age- and gender-matched healthy control (CTRL; n=30) with documented absence of coronary artery disease. Raw serum was characterized by atomic force microscopy (AFM) single-particle morphometry. Large EV (EV-L) were isolated by differential centrifugation and used for in vitro experiments on human aortic endothelial cells (hAEC), and human cardiac fibroblast (hCF). Results AFM analysis showed a 7.5-fold increase in EV-L in STEMI vs. CTRL serum. In vitro treatment of hAEC with EV-L from STEMI patients, but not CTRL-EV-L, induced an increase in reactive oxygen species (ROS; 1.15-Fold increase), p66SHC and oxidized low-density lipoprotein receptor 1 (LOX-1) expression (1.34 and 20-Fold increase, respectively), and cell death (1.40-Fold increase). Interestingly, ELISA analysis on EV-L showed an enrichment of LOX-1 in STEMI-EV-L compared to CTRL-EV-L (3.01-Fold increase). Moreover, STEMI-EV-L, but not CTRL-EV-L, stimulated in vitro proliferation (1.4-Fold increase) of hCF and their activation in myofibroblasts, as assessed by alpha smooth muscle actin (αSMA) expression (1.8-fold increase), resulting in increased extracellular matrix (ECM) secretion (1.5-Fold increase). Conclusions Our preliminary data showed a pathological role of circulating EV-L derived from STEMI patients. In particular, results from in vitro experiments evidence their function in inducing endothelial damage and activation of cardiac fibroblast into myofibroblast; two important features in the progression of pathological myocardial remodeling.Graphical abstract

Abstract 13116: Profiling the Role of Gut Microbiota-Derived Trimethylamine- N -Oxide in Cardiac Fibrosis — Evidence From a Primary Human in vitro Model

Introduction: The gut derived trimethylamine- N -oxide (TMAO) is correlated with increased atrial inflammation and fibrosis and associated with atrial fibrillation, heart failure, and stroke. However, the mechanisms mediating these links remain unresolved. Hypothesis: TMAO and its precursor metabolite L-carnitine (LCART) induce the transformation of atrial fibroblasts into pro-fibrotic phenotypes. Aim: To characterise the association between TMAO and cardiac fibrosis. Methods: Primary human cardiac (atrial) fibroblasts (hCFs) were sourced from healthy donors. hCFs were starved for 24 h and treated with PBS (control), 20 ng/mL transforming growth factor beta 1 (TGFβ1), 10 mM TMAO, or 1.0 mM LCART for 72 h (n=6 in each), then analysed by flow cytometry for α-smooth muscle actin (αSMA) expression. Unbiased proteomics was performed using LC-MS/MS to determine protein expression profile. Results: Analysis of αSMA expression revealed 3 hCF states: quiescent (Fbs), quiescent-to-myofibroblast (intermediate [Fbs-myoFbs]), and fully activated myofibroblast (myoFbs), (Fig-A). Compared to controls, there was no difference in the intermediate state after TGFβ1, TMAO, or LCART treatment (p=0.30). TMAO and LCART resulted in a significant induction of myofibroblast states compared to controls and TGFβ1 group. Unbiased proteomics identified 92, 65, and 43 proteins to be overexpressed and 84, 46, and 78 proteins downregulated following TGFβ1, TMAO, and LCART treatments (Fig-B). Both TGFβ1 and TMAO demonstrated shared enrichment for oxidative stress-regulated ferroptosis pathway (fold enrichment [FE] 31.8 and 22.2, p &lt;.01 and 0.03); LCART showed enrichment for cell motility (FE 3.8, p=0.03). Conclusions: TMAO and its precursor L-carnitine are associated with activation of pro-fibrotic states, which may be due to oxidative stress and abnormal cell migration related mechanisms.

Abstract 17441: Non-Invasive Quantification of Peripheral Angioplasty-Induced Vascular Inflammation in a Porcine Model Using PET/CT Imaging

Background: Similarities in peripheral vascular anatomy between swine and humans make this animal an ideal model for evaluating the effects of lower extremity angioplasty procedures commonly performed to improve limb blood flow in peripheral artery disease (PAD). Hypothesis: We hypothesized that PET/CT imaging would provide a non-invasive strategy for quantifying the inflammatory response to balloon overdilation injury in a swine model of peripheral angioplasty. Methods: Five Yorkshire pigs (weight = 25 kg) underwent overdilation of the right femoral artery. A balloon catheter (7 mm x 40 mm) was introduced via the carotid artery and guided to the femoral artery under fluoroscopy. The balloon was then inflated to a pressure 1.5 times greater the recommended nominal pressure to induce arterial injury. 18 F-fluorodeoxyglucose (FDG) PET/CT imaging was performed 14 days after angioplasty to quantify in vivo inflammation in the injured and control femoral arteries, which was expressed as the maximum standardized uptake value (SUV max ). Pigs were euthanized 14 days post-angioplasty and arteries were harvested and sectioned for gamma counting ( 18 F-FDG uptake), and stained with hematoxylin and eosin histology (H&amp;E), Masson’s trichrome, and alpha-smooth muscle actin (α-SMA) for evaluation of intimal remodeling and smooth muscle cell proliferation. Results: 18 F-FDG PET/CT imaging quantified a significant increase in inflammation for the injured versus control femoral artery 14 days post-angioplasty (p=0.04). Gamma counting confirmed a significant increase in 18 F-FDG uptake in the injured artery (p=0.02). H&amp;E revealed a significant increase in intimal thickening in the injured artery (p=0.03). α-SMA expression was also significantly increased in the injured versus control femoral artery (p=0.01), demonstrating an increase in smooth muscle cell proliferation. Conclusions: 18 F-FDG PET/CT quantifies arterial inflammation resulting from overdilation injury that is associated with intimal remodeling and smooth muscle cell proliferation. PET/CT imaging may provide a non-invasive strategy for in vivo testing of the efficacy of emerging drug-coated endovascular peripheral devices.

3D Airway Epithelial-Fibroblast Biomimetic Microfluidic Platform to Unravel Engineered Nanoparticle-Induced Acute Stress Responses as Exposome Determinants.

Insights into how biological systems respond to high- and low-dose acute environmental stressors are a fundamental aspect of exposome research. However, studying the impact of low-level environmental exposure in conventional in vitro settings is challenging. This study employed a three-dimensional (3D) biomimetic microfluidic lung-on-chip (μLOC) platform and RNA-sequencing to examine the effects of two model anthropogenic engineered nanoparticles (NPs): zinc oxide nanoparticles (Nano-ZnO) and copier center nanoparticles (Nano-CCP). The airway epithelium exposed to these NPs exhibited dose-dependent increases in cytotoxicity and barrier dysregulation (dominance of the external exposome). Interestingly, even nontoxic and low-level exposure (10 μg/mL) of the epithelium compartment to Nano-ZnO triggered chemotaxis of lung fibroblasts toward the epithelium. An increase in α smooth muscle actin (α-SMA) expression and contractile activity was also observed in these cells, indicating a bystander-like adaptive response (dominance of internal exposome). Further bioinformatics and network analysis showed that a low-dose Nano-ZnO significantly induced a robust transcriptomic response and upregulated several hub genes associated with the development of lung fibrosis. We propose that Nano-ZnO, even at a no observable effect level (NOEL) dose according to conventional standards, can function as a potent nanostressor to disrupt airway epithelium homeostasis. This leads to a cascade of profibrotic events in a cross-tissue compartment fashion. Our findings offer new insights into the early acute events of respiratory harm associated with environmental NPs exposure, paving the way for better exposomic understanding of this emerging class of anthropogenic nanopollutants.

Simvastatin attenuates diabetes mellitus erectile dysfunction in rats by miR-9-5p-regulated PDCD4.

DMED is a common complication of diabetes, for which new treatment methods are urgently required. Focused on DMED, the pharmacological mechanism of simvastatin (Sim) was probed. A model of DMED was made in rats with streptozotocin and orally medicated with Sim. Lentiviral vectors that interfere with miR-9-5p or PDCD4 were injected, and the erectile function, histopathology of cavernous tissue, and α-SMA expression were evaluated. Cavernous smooth muscle cells (CMSCs) obtained from DMED rats were treated with Sim and transfected with the plasmid vector that interferes with miR-9-5p or PDCD4 to observe cell viability and apoptosis. The binding relationship between miR-9-5p and PDCD4 was checked. After 8-week treatment with Sim, erectile function was improved and the corpus cavernosum injury was alleviated. Upregulating miR-9-5p or downregulating PDCD4 further improved erectile function and cavernous injury in rats. miR-9-5p targeted regulation of PDCD4. In vitro cell experiment results showed that Sim induced proliferation and reduced apoptosis of CSMCs by enhancing miR-9-5p-targeted regulating PDCD4 in vitro. Sim attenuates DMED in rats via miR-9-5p/PDCD4.

TGFβ1-driven SMAD2/3 phosphorylation and myofibroblast emergence are fully dependent on the TGFβ1 pre-activation of MAPKs and controlled by maternal leucine zipper kinase

Following wounding, endogenously secreted TGFβs drive resident and bone marrow-derived cells to convert into α-smooth actin (SMA)-rich, contractile myofibroblasts. The TGFβ effect is initiated by the phosphorylation of SMADs 2 and 3 (SMAD2/3). This event has been referred to as the canonical response to TGFβ. TGFβ also elicits other responses viewed as parallel events not directly connected to the SMAD activation, and thus referred to as noncanonical. A recognized response is the phosphorylation of the -activated kinase (TAK1/MAP3K), an upstream component of the mitogen-activated protein kinase (MAPK) cascade. We have now examined the relationship between these two effects of TGFβ1 at their earliest stages. The bulk of the studies were carried out with primary fibroblasts derived from the human cornea. The results' widespread relevance was confirmed in critical experiments with dermal-, and Tenon's capsule-derived fibroblasts. Cells were treated with kinase inhibitors or targeting siRNAs followed by induction by 2 ng/ml TGFβ1, and/or 10 ng/ml TNF-α. Cells were collected after 1 to 30 min for Western blot analysis and assayed for the accumulation of phosphorylated TAK1, ASK1, JNK1/2, p38, HPS27, MELK, SMAD2/3, and GAPDH. The effect of the kinase inhibitors on α-SMA expression and α-SMA stress fiber organization was also tested. For the immediate response to TGFβ1 we found that a) activation of the MAPK pathway was completed within 1 min after the addition of TGFβ1; b) phosphorylation of JNK1/2 was fully dependent on TAK1 and ASK1 activity, c) phosphorylation of MELK was fully dependent on JNK1/2 activity; d) phosphorylation of ASK1 depends on MELK activity, indicating the existence of an ASK1-MELK positive activation feedback loop; e) phosphorylation of SMAD2/3 started only after a 5 min period and reached a nadir after 10–15 min, f) the latter phosphorylation was fully blocked by inhibition of TAK1, ASK1, JNK1/2, and MELK, and siRNA-driven MELK downregulation; g) the inhibitors equally blocked the α-SMA protein expression, stress fiber development, and cell morphology changes at 72 h. These results demonstrate that the activation of the canonical pathway is fully subordinate to the activity of the MAPK pathway, challenging the concept of canonical and noncanonical TGFβ pathways and that SMAD2/3 activation is mediated by MELK, a kinase not previously associated with rapid pharmacological responses.

Research on the mechanism of mechanical ventilation induced endoplasmic reticulum stress promoting mechanical ventilation-induced pulmonary fibrosis

To demonstrate the mechanism of mechanical ventilation (MV) induced endoplasmic reticulum stress (ERS) promoting mechanical ventilation-induced pulmonary fibrosis (MVPF), and to clarify the role of angiotensin receptor 1 (AT1R) during the process. The C57BL/6 mice were randomly divided into four groups: Sham group, MV group, AT1R-shRNA group and MV+AT1R-shRNA group, with 6 mice in each group. The MV group and MV+AT1R-shRNA group mechanically ventilated for 2 hours after endotracheal intubation to establish MVPF animal model (parameter settings: respiratory rate 70 times/minutes, tidal volume 20 mL/kg, inhated oxygen concentration 0.21). The Sham group and AT1R-shRNA group only underwent intubation after anesthesia and maintained spontaneous breathing. AT1R-shRNA group and MV+AT1R-shRNA group were airway injected with the adeno-associated virus one month before modeling to inhibit AT1R gene expression in lung tissue. The expressions of AT1R, ERS signature proteins [immunoglobulin heavy chain-binding protein (BIP), protein disulfide isomerase (PDI)], fibrosis signature proteins [collagen I (COL1A1), α-smooth muscle actin (α-SMA)] in lung tissues were detected by immunofluorescence and Western blotting. Hematoxylin-eosin (HE) staining was used to evaluate lung injury and Masson staining was used to evaluate pulmonary fibrosis. Compared with the Sham group, the degree of pulmonary fibrosis and lung injury were more significant in the MV group. In the MV group, the protein expressions of AT1R, BIP, PDI, COL1A1 and α-SMA were increased (AT1R/β-actin: 1.40±0.02 vs. 1, BIP/β-actin: 2.79±0.07 vs. 1, PDI/β-actin: 2.07±0.02 vs. 1, COL1A1/α-Tubulin: 2.60±0.15 vs. 1, α-SMA/α-Tubulin: 2.80±0.25 vs. 1, all P < 0.01). The number of E-cad+/AT1R+ and E-cad+/BIP+ cells in lung tissue increased, and the fluorescence intensity of COL1A1 and α-SMA increased. Compared with the MV group, the degree of pulmonary fibrosis and lung injury were significantly relieved in the MV+AT1R-shRNA group. In the MV+AT1R-shRNA group, the protein expressions of AT1R, BIP, PDI, COL1A1 and α-SMA were decreased (AT1R/β-actin: 0.53±0.03 vs. 1.40±0.02, BIP/β-actin: 1.73±0.15 vs. 2.79±0.07, PDI/β-actin: 1.04±0.07 vs. 2.07±0.02, COL1A1/α-Tubulin: 1.29±0.11 vs. 2.60±0.15, α-SMA/α-Tubulin: 1.27±0.10 vs. 2.80±0.25, all P < 0.01). The number of E-cad+/AT1R+ and E-cad+/BIP+ cells in lung tissue decreased, and the fluorescence intensity of COL1A1 and α-SMA decreased. There was no statistically significant difference in the indicators between AT1R-shRNA group and Sham group. MV up-regulate the expression of AT1R in alveolar epithelial cells, activate the AT1R pathway, induce ERS and promote the progression of MVPF.

ATF3 Modulates the Proliferation, Migration, and Apoptosis of Synovial Fibroblasts after Arthroscopy by Promoting RGS1 Transcription.

Osteoarthritis (OA) is a degenerative joint disease involving both cartilage and synovium. Activating transcription factor 3 (ATF3) and regulator of G protein signaling 1 (RGS1) have been reported to be up-regulated in OA. However, little is known regarding the relationship between these two genes and the mechanism of this relationship in OA development. Therefore, the present study explores the mechanism of ATF3-mediated RGS1 in the proliferation, migration, and apoptosis of synovial fibroblasts. After the OA cell model was constructed with TGF-β1 induction, human fibroblast-like synoviocytes (HFLSs) were transfected with ATF3 shRNA or RGS1 shRNA alone or co-transfected with ATF3 shRNA and pcDNA3.1-RGS1. Then, proliferation, migration, apoptosis, and the expression of ATF3, RGS1, α-SMA, BCL-2, caspase3, and cleaved-caspase3 were measured. Meanwhile, the potential relationship between ATF3 and RGS1 was predicted and validated. Analysis of the GSE185059 dataset suggested that RGS1 was up-regulated in OA synovial fluid exosomes. Moreover, ATF3 and RGS1 were both highly expressed in TGF-β1-induced HFLSs. Transfection of ATF3 shRNA or RGS1 shRNA significantly reduced proliferation and migration and promoted apoptosis of TGF- β1-induced HFLSs. Mechanistically, ATF3 bound to the RGS1 promoter and elevated RGS1 expression. Silencing ATF3 repressed proliferation and migration and enhanced apoptosis of TGF-β1-induced HFLSs by down-regulating RGS1. ATF3 binds to the RGS1 promoter and enhances RGS1 expression to accelerate cell proliferation and block cell apoptosis in TGF-β1-induced synovial fibroblasts.

Improvement on renal interstitial fibrosis in rats with renal failure via TLR4/NF-κB signaling pathway by means of panax notoginseng saponins

Panax notoginseng saponins (PNS) have excellent effects on treating cardiovascular and cerebrovascular diseases, tumors and organ interstitial diseases. This study assessed the effect of PNS on renal interstitial fibrosis (RIF). Four groups of models were firstly created, which included; renal failure model, healthy group, model group, PNS group, and then established TLR4 inhibitor group (TAK-242 group), TLR4 activator group (LPS group), combined PNS and TLR4 inhibitor group (PNS+ TAK-242 group) and combined PNS and TLR4 agonist (PNS+LPS group). TLR4/NF-κB signaling pathway and regulatory mechanism for PNS were observed along with analysis of TGF-β1, α-SMA, Collagen I and FN expressions in kidney tissues. The PNS significantly inhibited changes of renal interstitial fibrosis in rats with renal failure, and this process was related to decreased expressions of TLR4 and NF-κB. The PNS also inhibited the expression of TLR4, and expressions of TGF-β1, α-SMA, Collagen I and FN were down-regulated in kidney cells after using TAK-242, especially in the PNS+TAK-242 group. The PNS can thus significantly inhibit the process of renal fibrosis, and this process is due to the fact that, PNS inhibits the activity of TLR4/NF-κB signaling pathway to down-regulate the expressions of fibrosis-related factors TGF-β1, α-SMA, etc., in exerting its effects.

Effect and mechanism of Jiming Powder on myocardial fibrosis in mice with myocardial infarction

Jiming Powder is a traditional ancient prescription with good therapeutic effect in the treatment of heart failure, but its mechanism lacks further exploration. In this study, a mouse model of coronary artery ligation was used to evaluate the effect and mechanism of Jiming Powder on myocardial fibrosis in mice with myocardial infarction. The study constructed a mouse model of heart failure after myocardial infarction using the method of left anterior descending coronary artery ligation. The efficacy of Jiming Powder was evaluated from multiple angles, including ultrasound imaging, hematoxylin-eosin(HE) staining, Masson staining, Sirius Red staining, and serum myocardial enzyme spectrum detection. Western blot analysis was performed to detect key proteins involved in ventricular remodeling, including transforming growth factor-β1(TGF-β1), α-smooth muscle actin(α-SMA), wingless-type MMTV integration site family member 3a(Wnt3a), β-catenin, matrix metallopeptidase 2(MMP2), matrix metallopeptidase 3(MMP3), TIMP metallopeptidase inhibitor 1(TIMP1), and TIMP metallopeptidase inhibitor 2(TIMP2). The results showed that compared with the model group, the high and low-dose Jiming Powder significantly reduced the left ventricular internal diameter in systole(LVID;s) and diastole(LVID;d), increased the left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), effectively improved cardiac function in mice after myocardial infarction, and effectively reduced the levels of myocardial injury markers such as creatine kinase(CK), creatine kinase isoenzyme(CK-MB), and lactic dehydrogenase(LDH), thus protecting ischemic myocardium. HE staining showed that Jiming Powder could attenuate myocardial inflammatory cell infiltration after myocardial infarction. Masson and Sirius Red staining demonstrated that Jiming Powder effectively inhibited myocardial fibrosis, reduced the collagen Ⅰ/Ⅲ ratio in myocardial tissues, and improved collagen remodeling after myocardial infarction. Western blot results showed that Jiming Powder reduced the expression of TGF-β1, α-SMA, Wnt3a, and β-catenin, decreased the levels of MMP2, MMP3, and TIMP2, and increased the level of TIMP1, suggesting its role in inhibiting cardiac fibroblast transformation, reducing extracellular matrix metabolism in myocardial cells, and lowering collagen Ⅰ and α-SMA content, thus exerting an anti-myocardial fibrosis effect after myocardial infarction. This study revealed the role of Jiming Powder in improving ventricular remodeling and treating myocardial infarction, laying the foundation for further research on the pharmacological effect of Jiming Powder.

Long Non-Coding RNA Dancr Affects Myocardial Fibrosis in Atrial Fibrillation Mice via the MicroRNA-146b-5p/Smad5 Axis.

Atrial fibrillation (AF) is the most frequent arrhythmia, and myocardial fibrosis (MF) has a close association with atrial remodeling and leads to AF. This study aimed to explore the function of the long non-coding RNA (lncRNA) differentiation antagonizing non-protein coding RNA (Dancr)/microRNA (miR)-146b-5p/Smad5 axis on MF in AF mice. AF mouse models were established. Overexpression Dancr lentivirus was injected into AF mice to increase Dancr expression in myocardial tissues. LncRNA Dancr, miR-146b-5p, and Smad5 expression levels and inflammatory factors (IL-18 and TNF-α) in the myocardial tissues were measured. MF was measured and the expression levels of MF-related genes (COL1A1, α-SMA, and FN1) were detected. In addition, in vitro HL-1 cell rapid pacing models were constructed, and after lncRNA Dancr and miR-146b-5p-related construct transfection, cell viability and cell apoptosis were determined. LncRNA Dancr up-regulation ameliorated MF in the AF mice, reduced IL-18 and TNF-α expression levels in myocardial tissues, and decreased COL1A1, α-SMA, and FN1 expression levels. The in vitro HL-1 cell rapid pacing models suggested that miR-146b-5p overexpression reversed the inhibitory effects of lncRNA Dancr overexpression on MF in HL-1 cells, and Smad5 interference reversed the ameliorative effects of miR-146b-5p interference on MF in HL-1 cells. LncRNA Dancr can sponge miR-146b-5p to promote Smad5 expression, thereby delaying MF in AF mice.

Pterostilbene exerts cytotoxicity on activated hepatic stellate cells by inhibiting excessive proliferation through the crosstalk of Sirt1 and STAT3 pathways.

Pterostilbene (PTE), a natural analogue of resveratrol, abundantly exists in blueberries and grapes and has several beneficial potentials against oxidative stress, inflammation, and cancer. In current study, we investigated the effects of PTE on hepatic fibrosis in vitro and in vivo. Activation of hepatic stellate cells (HSCs) is an initiating event in the initiation of hepatic fibrosis. MTT assay revealed that PTE (3.125-12.5 μM) displayed cytotoxicity on activated HSCs, no cytotoxicity on AML-12 and quiescent HSCs. PTE significantly inhibited the expressions of α-SMA, collagen Ⅰ and TIMP-1/MMP13 ratio; suppressed inflammatory cascade activation to reduce inflammatory cytokines release, such as Caspase-1, IL-1β and IL-6. PTE activated Sirt1 and decreased STAT3 phosphorylation, functioning as SRT1720 and Niclosamide. Sirt1 deficiency significantly elevated p-STAT3 expression, while STAT3 deficiency resulted in Sirt1 increasing and inhibited fibrosis and inflammatory cytokines expressions. In mice with hepatic fibrosis induced by thioacetamide (TAA), PTE significantly decreased ALT and AST activities, reduced fibrosis markers, STAT3 phosphorylation and activated Sirt1 expression. PTE showed cytotoxicity on activated HSCs to ameliorate hepatic fibrosis via regulating fibrogenesis, energy metabolism and inflammation and targeting the crosstalk of Sirt1 and STAT3. In conclusion, PTE could be potentially beneficial as a natural plant metabolite in preventing and treating hepatic fibrosis.