Actin Levels Research Articles

Effect of exosome derived from ovarian cancer cell on the differentiation of fibroblast

Objective: To study the effects of exosome secreted by ovarian cancer (OC) cell on the differentiation and metastasis of normal fibroblasts (NFs). Methods: NFs were collected from patients who underwent hysteromyoma resection in the Affiliated Hospital of Qingdao University from May to December 2019. Exosome was extracted from the culture supernatant of SKOV3 cells by using ultra-high-speed centrifugation. The NFs were co-cultured with condition medium (CM), exosome of SKOV3 (SKOV3-exo) and control medium. The expression levels of fibroblast activation protein (FAP) and α-smooth muscle actin (α-SMA) were detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. The metastatic ability of NFs was detected by Transwell array. Results: Under the transmission electron microscope, the extracellular vesicles extracted from the culture supernatant of SKOV3 were 30-100 nm in diameter with cup holder-like bilayer membrane structure, and the protein expression levels of TSG101 and HSP27 in exosomes (1.00±0.05 and 1.12±0.13) were higher than those of ovarian cancer SKOV3 cells (0.22±0.21 and 0.36±0.14, respectively, P<0.05). PKH67 fluorescently labeled exosomes could be taken up by NFs. The expression levels of α-SMA and FAP mRNA in CM group(2.91±0.15 and 3.21±0.33)and SKOV3-exo group (3.50±0.21 and 4.63±0.24, respectively) were higher than that in blank group (1.00±0.06 and 1.00±0.13, P<0.05). The protein expression levels of α-SMA and FAP in CM group and SKOV3-exo group (0.89±0.11 and 1.25±0.09, 0.81±0.09 and 1.20±0.12) were higher than those in the blank group (0.12±0.31 and 0.11±0.19, respectively, P<0.05). The migrated numbers of cells in the CM group and SKOV3-exo group [(215.01±14.80) and (389.72±19.43), respectively] were higher than that in the blank group [(113.73±4.70), P<0.05]. Conclusion: The exosome secreted by SKOV3 cells can be taken up by NFs, which makes it to differentiate into cancer associated fibroblasts (CAFs) and significantly enhances its metastatic ability, indicating that OC cells may promote the transformation of normal ovarian mesenchymal fibroblasts to CAFs through exosome pathways, and then promote the development of ovarian cancer.

High Actin Expression in Thrombus of Acute Ischemic Stroke Can Be a Biomarker of Atherothrombotic Origin Stroke.

BackgroundAs the treatment target, the imaging information and histologic characteristics of the thrombus may differ according to the stroke subtype. This study aimed to provide the correlative study of stroke etiology with the non-contrast CT, and histological composition of retrieved clots in acute ischemic stroke (AIS).Materials and MethodsA total of 94 patients with AIS who underwent the endovascular treatment with successfully retrieved clots from January 2017 to October 2020 were enrolled in the present study. Histological analysis was performed using hematoxylin and eosin (H&E) staining and immunostaining with CD3, CD20, CD105, and actin antibodies. CT obtained at the patients' admission was to measure the attenuation and volume of all thrombus.ResultsA total of 94 subjects were included in this study. Fifty-six patients were classified as cardioembolic (CE), and 38 were classified with large-artery atherosclerosis (LAA). The subjects with LAA tend to exhibit higher actin and CD105 levels, and lower Hounsfield Unit (HU) values than subjects with CE. After adjusting for confounders, the actin was positively correlated with CD105 but not with HU values. Logistics regression shows actin was valuable for the prediction of LAA (OR, 1.148; 95% CI, 1.075–1.227; p < 0.001), even adjusted for age, sex, and intervention type (OR, 1.129; 95% CI, 1.048–1.216; p = 0.001), CT density and CD105 (OR, 1.161; 95% CI, 1.056–1.277; p = 0.002). Actin levels have a strong accuracy in differentiating LAA from CE, especially combined with CT density and CD105, which yielded a sensitivity of 63.2%, a specificity of 89.3%, with the area under the curve (AUC) at 0.821 (95% CI, 0.731–0.912).ConclusionOur findings suggest that actin's level was a major factor differentiating atherothrombotic origin strokes from the cardioembolic stroke.Clinical Trial RegistrationChiCTR2100051173.

Intrauterine neuromuscular and stromal dysplasia of the bladder in retinoic acid-induced myelomeningocele fetal rats

ObjectivesTo observe the changes in the bladder of fetal rats with myelomeningocele (MMC) induced by all-trans retinoic acid (atRA) during the embryonic development stages. MethodsThe fetal rat model of MMC was induced by intragastric administration of atRA to pregnant rats on embryonic day 10 (E10). Fetal rats were harvested at E16, E18, E20, and E21 for observation and further testing. Those with MMC were classified as the MMC group, while those without MMC as the RA group. The areas of MMC skin defect, the crown-rump length (CRL), and body weight in different groups were compared. The histopathological changes in the bladder were compared. The expression levels of alpha-smooth muscle actin (αSMA), smooth muscle myosin heavy chain (SMMHC), connexin 43 (Cx43), desmin, β3 tubulin, and vesicular acetylcholine transporter (VAChT) in the bladder were investigated by immunohistochemical staining and Western blotting. Pregnant rats given intragastric administration with olive oil (OIL group) at E10 were set as the blank control group. ResultsA total of 415 fetal rats of different gestational ages were harvested with an MMC incidence of 56.05 % (139/248). The incidence rate increased with embryonic days (p < 0.001). Compared with the other two control groups, the CRL and bodyweight of MMC fetal rats were significantly delayed at E21 (p < 0.001). The expression levels of αSMA, SMMHC, Cx43, desmin, β3 tubulin and VAChT in the bladder of MMC fetal rats were significantly decreased at E21 (p < 0.05). ConclusionsIn atRA-induced MMC fetal rats, there is neural, muscular, and stromal dysplasia in the bladder at an early gestational age. Further investigations on neurogenic bladder secondary to MMC are applicable using this animal model.

High abundance of Lachnospiraceae in the human gut microbiome is related to high immunoscores in advanced colorectal cancer.

The tumor microenvironment (TME) in colorectal cancer (CRC) includes the gut microbiome, immune cells, angiogenic factors, and fibroblasts and plays a major role in cancer progression. The Immunoscore (IS) is based on tumor infiltration by immune cells that are known prognostic biomarkers for CRC. However, the interrelation between the IS, microbiome, and other TME factors in human CRC remains unclear. A cohort of 94 patients with CRC was examined at the Shiga University of Medical Science Hospital in Japan. The expression levels of CD3, CD8, CD31, and alpha-smooth muscle actin (α-SMA) in the primary tumor were evaluated by immunohistochemistry. The IS was calculated based on the results of the CD3 and CD8 staining assays. Microbiomes in patients with CRC were examined by amplicon sequencing. The expression levels of α-SMA and tumor-infiltrating lymphocytes in patients with CRC were negatively correlated (P = 0.006). A high IS was associated with high abundance of Lachnospiraceae in the microbiomes of patients with CRC. Lymphocyte infiltration into the primary tumor was marked by reduced density of cancer-associated fibroblasts and enrichment of the Lachnospiraceae family in the gut microbiome, which may influence CRC progression.

Study of the negative regulation of transforming growth factor beta type II receptor to inhibit the occurrence and development of liver fibrosis with miR-217

Objective: To observe the effect of miR-217 on angiotensin II (AngII)-induced hepatic stellate cells (HSCs) activation, and carbon tetrachloride (CCl4)-induced overexpression in mice, so as to clarify miR-217 role in liver fibrosis. Methods: HSCs were stimulated with AngⅡ and the changes condition in the expression level of miR-217 were detected. HSCs were divided into control group, AngII-treated group and AngⅡ+miR-217-treated group. The expression levels of alpha-smooth muscle actin, fibroblast-specific protein 1 and collagen Ⅰ (Collagen Ⅰ) in each group were detected. The target gene of mir-217 was screened and verified by Targetscan and Dual luciferase gene reporter assay. Real-time quantitative PCR and Western blot were used to detect the effect of miR-217 on the expression level of transforming growth factor beta type Ⅱ receptor (TGFBR2). A CCl4-induced mouse liver fibrosis model was constructed. Masson staining and Sirius red staining were used to detect the effect of miR-217 overexpression on the progression of liver fibrosis in CCl4 mice. Data of two groups were compared using t-test. Data of multiple groups were statistically analyzed with one-way ANOVA. Results: The expression level of miR-217 was downregulated by AngⅡ-stimulated HSC cells. The expression levels of α-smooth muscle actin, fibroblast-specific protein 1 and Collagen Ⅰ induced by AngⅡ was inhibited by miR-217 mimics transfection. The 3'-UTR of TGFBR2 had specifically bind miR-217. The mRNA and protein expression levels of TGFBR2 was inhibited with miR-217 mimics transfection in HSCs. The overexpression of miR-217 had inhibited the expression levels of Collagen Ⅰ and Ⅲ in CCl4 mice and alleviated the progression of liver fibrosis . Conclusion: miR-217 regulates liver fibrosis by targeting TGFBR2, inhibits AngII-induced HSC activation, and slows down the process of liver fibrosis in CCl4 mice, suggesting that miR-217 may have an inhibitory effect on liver fibrosis.

Pharmacological Inhibition of STING/TBK1 Signaling Attenuates Myeloid Fibroblast Activation and Macrophage to Myofibroblast Transition in Renal Fibrosis.

Renal fibrosis is an important pathological biomarker of chronic kidney disease (CKD). Stimulator of interferon genes/TANK binding kinase 1 (STING/TBK1) axis has been identified as the main regulator of innate immune response and closely related to fibrotic disorder. However, the role of STING/TBK1 signaling pathway in kidney fibrosis is still unknown. In this study, we investigated the effect of pharmacological inhibition of STING/TBK1 signaling on renal fibrosis induced by folic acid (FA). In mice, TBK1 was significantly activated in interstitial cells of FA-injured kidneys, which was markedly inhibited by H-151 (a STING inhibitor) treatment. Specifically, pharmacological inhibition of STING impaired bone marrow-derived fibroblasts activation and macrophage to myofibroblast transition in folic acid nephropathy, leading to reduction of extracellular matrix proteins expression, myofibroblasts formation and development of renal fibrosis. Furthermore, pharmacological inhibition of TBK1 by GSK8612 reduced myeloid myofibroblasts accumulation and impeded macrophage to myofibroblast differentiation, resulting in less deposition of extracellular matrix protein and less severe fibrotic lesion in FA-injured kidneys. In cultured mouse bone marrow-derived monocytes, TGF-β1 activated STING/TBK1 signaling. This was abolished by STING or TBK1 inhibitor administration. In addition, GSK8612 treatment decreased levels of α-smooth muscle actin and extracellular matrix proteins and prevents bone marrow-derived macrophages to myofibroblasts transition in vitro. Collectively, our results revealed that STING/TBK1 signaling has a critical role in bone marrow-derived fibroblast activation, macrophages to myofibroblasts transition, and kidney fibrosis progression.

Wogonoside attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis through SOCS1/P53/SLC7A11 pathway.

Wogonoside (WG) is a flavonoid chemical component extracted from Scutellaria baicalensis, which exerts therapeutic effects on liver diseases. Ferroptosis, a novel form of programmed cell death, regulates diverse physiological/pathological processes. In this study, we attempted to investigate a novel mechanism by which WG mitigates liver fibrosis by inducing ferroptosis in hepatic stellate cells (HSCs). A CCl4 -induced mouse liver fibrosis model and a rat HSC line were employed for in vivo and in vitro experiments, both treated with WG. Firstly, the levels of the fibrotic markers α-smooth muscle actin (α-SMA) and α1(I)collagen (COL1α1) were effectively decreased by WG in CCl4 -induced mice and HSC-T6 cells. Additionally, mitochondrial condensation and mitochondrial ridge breakage were observed in WG-treated HSC-T6 cells. Furthermore, ferroptotic events including depletion of SLC7A11, GPX4 and GSH, and accumulation of iron, ROS and MDA were discovered in WG-treated HSC-T6 cells. Intriguingly, these ferroptotic events did not appear in hepatocytes or macrophages. WG-elicited HSC ferroptosis and ECM reduction were dramatically abrogated by ferrostatin-1 (Fer-1), a ferroptosis inhibitor. Importantly, our results confirm that SOCS1/P53/SLC7A11 is a signaling pathway which promotes WG attenuation of liver fibrosis. On the contrary, WG mitigated liver fibrosis and inducted HSC-T6 cell ferroptosis were hindered by SOCS1 siRNA and pifithrin-α (PFT-α). These findings demonstrate that SOCS1/P53/SLC7A11-mediated HSC ferroptosis is associated with WG alleviating liver fibrosis, which provides a new clue for the treatment of liver fibrosis.

4-Aminopyridine Induces Nerve Growth Factor to Improve Skin Wound Healing and Tissue Regeneration.

The discovery of ways to enhance skin wound healing is of great importance due to the frequency of skin lesions. We discovered that 4-aminopyridine (4-AP), a potassium channel blocker approved by the FDA for improving walking ability in multiple sclerosis, greatly enhances skin wound healing. Benefits included faster wound closure, restoration of normal-appearing skin architecture, and reinnervation. Hair follicle neogenesis within the healed wounds was increased, both histologically and by analysis of K15 and K17 expression. 4-AP increased levels of vimentin (fibroblasts) and alpha-smooth muscle actin (α-SMA, collagen-producing myofibroblasts) in the healed dermis. 4-AP also increased neuronal regeneration with increased numbers of axons and S100+ Schwann cells (SCs), and increased expression of SRY-Box Transcription Factor 10 (SOX10). Treatment also increased levels of transforming growth factor-β (TGF-β), substance P, and nerve growth factor (NGF), important promoters of wound healing. In vitro studies demonstrated that 4-AP induced nerve growth factor and enhanced proliferation and migration of human keratinocytes. Thus, 4-AP enhanced many of the key attributes of successful wound healing and offers a promising new approach to enhance skin wound healing and tissue regeneration.

Effect of Massage Therapy in Regulating Wnt/β-Catenin Pathway on Retarding Denervated Muscle Atrophy in Rabbits

ObjectiveThe purpose of this study was to investigate the effect of massage therapy on retarding denervated muscle atrophy in rabbits. MethodsThe denervated skeletal muscle atrophy rabbit model was established with the clamping rabbit tibial nerve method. On the third and first day after clamping the rabbit tibial nerve, the model rabbits were treated with massage or nerve growth factor (15 μg/mL) once a day for 8 weeks, respectively. Subsequently, gastrocnemius tissues were collected from rabbits and detected by electromyography. The gastrocnemius tissue sections were stained with hematoxylin and eosin and Sirius red staining to evaluate the histopathologic damage of denervated muscle atrophy in a rabbit model. Furthermore, the proliferation and differentiation-related targets of satellite cells in gastrocnemius tissues were detected by immunohistochemical, immunofluorescence, real-time quantitative reverse transcription polymerase chain reaction, and Western blot assays, respectively. Also, the effects of massage on the Wnt/β-catenin pathway were detected. Finally, gastrocnemius myocytes were isolated from rabbits to detect the expression levels of α-smooth muscle actin (α-SMA). ResultsThe nerve conduction deteriorated continuously with time in model rabbits. Massage significantly ameliorated the pathologic damages and motor endplate microstructure of gastrocnemius muscle, effectively prevented fibrosis, and regulated the proliferation and differentiation-related messenger RNA and protein expression of satellite cells with the time increased after denervation. Additionally, the number of differentiating satellite cells was increased after being treated with massage, and massage further kept denervated muscles from atrophy. Importantly, the effect of massage to attenuate muscle atrophy was associated with the activation of the Wnt/β-catenin pathway. Meanwhile, massage reduced the expression of α-SMA in gastrocnemius myocytes. ConclusionThis model demonstrated that massage delayed the atrophy of skeletal muscle. This was probably accomplished by regulating the proliferation and differentiation of the satellite cells via the Wnt/β-catenin signaling pathway.

To explore mechanism of Scabiosa comosa against liver fibrosis based on pharmacodynamics and network pharmacology

This study aims to systematically elucidate the pharmacodynamics and network pharmacological mechanism of Mongolian medicinal plants Scabiosa comosa, explore their key targets and related pathways, and further clarify the mechanism of the plants in treating liver fibrosis. Wistar rats were assigned into the blank group, carbon tetrachloride-induced liver fibrosis model group, and low-, medium-, and high-dose S. comosa groups. HE staining and Masson staining were performed for the observation of liver tissue under a microscope. Further, Wistar rats were assigned into a control group and a S. comosa group for administration. Seven days later, blood was collected from the abdominal aorta, and different doses of drug-containing serum samples were used to treat hepatic stellate cell-T6(HSC-T6). Flow cytometry was adopted to detect the apoptosis of HSC-T6 cells. Ultra-high performance liquid chromatography-time of flight-mass spectrometry(UHPLC-TOF-MS) was employed to determine the components in Scabiosa comosa. The target of S. comosa and liver fibrosis were obtained from SwissTargetPrediction and GeneCards, respectively, and the common targets were selected as the anti-liver fibrosis targets. Protein-protein interaction was analyzed via STRING. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment were carried out via Metascape. Phosphatidylinosital 3-kinase(PI3 K), protein kinase B(AKT), p-AKT, p38, and p-p38 targets which are involved in the top-ranked PI3 K/AKT and mitogen activated kinase-like protein(MAPK) signaling pathways were selected for validation via Western blot. The HE and Masson staining results showed that Scabiosa alleviated the hyperplasia of connective tissue and the fibrosis. The serum containing Scabiosa significantly promoted the apoptosis of HSC-T6 in a concentration-dependent manner. A total of 76 chemical components were identified by UHPLC-TOF-MS, among which flavonoids, alkaloids, terpenoids, phenols, and fatty acids were the main components. According to the prediction, there were 63 anti-liver fibrosis targets in Scabiosa comosa, the annotated GO terms of which involved biological processes, cell components, and molecular functions. The KEGG pathway enrichment showed that the targets were mainly involved in PI3 K/AKT, epidermal growth factor receptor(EGFR), RAS-associated protein 1(Rap1), hypoxia-inducible factor 1(HIF-1), resistance to audiogenic seizures(Ras), and MAPK signaling pathways. Western blot results showed that compared with the model group, S. comosa down-regulated the protein levels of α-smooth muscle actin(α-SMA), collagen Ⅰ, PI3 K, AKT, p-AKT, p38, and p-p38 in liver tissue. Compared with the control group, the low-, medium-, and high-dose S. comosa significantly down-regulated the protein levels of α-SMA, collagen Ⅰ, PI3 K, AKT, p-AKT, p38, and p-p38 in HSC-T6. The evidence of pharmacodynamics, network pharmacology, and molecular biology indicated that the plants of S. comosa had significant activity against liver fibrosis, the mechanism of which may involve the regulation of the key targets PI3 K, AKT, and MAPK14 p38 in the PI3 K/AKT and MAPK signaling pathways.

LPS-induced macrophage exosomes promote the activation of hepatic stellate cells and the intervention study of total astragalus saponins combined with glycyrrhizic acid.

Huangqi decoction, also known as Huangqi Liuyi decoction, was first recorded in the prescriptions of the Bureau of Taiping People's Welfare Pharmacy. It comprises astragalus and licorice, which is a commonly used prescription in traditional Chinese medicine for the clinical treatment of chronic liver disease, especially liver cirrhosis. Total astragalus saponins (AST) is the main component of astragalus, and glycyrrhizic acid (GA) is the main component of licorice. In this study, normal macrophage exosomes were extracted, and the exosomes incubated with lipopolysaccharides (LPS) and those incubated with LPS + AST + GA were co-cultured with JS1 cells (hepatic stellate cell line). The survival rate and the activation of key signaling pathways of JS1 cells in each group were detected and compared. We found that the co-culture of LPS-induced macrophage exosomes with JS1 cells could significantly increase the expression levels of Collagen-1 (Col-1) and Alpha smooth muscle actin (α-SMA)in JS1 cells. However, a significant reversal effect was observed after pretreatment with AST combined with GA. Further evaluation found that the expression levels of phospho (p)-Smad2 and p-Smad3 in the JS1 cells were significantly increased after macrophages were induced with LPS, whereas pretreatment with AST + GA could significantly decrease the expression levels of p-Smad2 and p-Smad3. Preliminary results of this study indicated that LPS-induced macrophage exosomes can promote the activation of hepatic stellate cells, and the pretreatment of AST combined with GA can exert a significant intervention effect. In this study, the new mechanism of anti-hepatic fibrosis effect of traditional Chinese medicine components of Huangqi Decoction was analyzed from the perspective of exosomes.

YAP activates pancreatic stellate cells and enhances pancreatic fibrosis

BackgroundPancreatic stellate cells (PSCs) foster the progression of pancreatic adenocarcinoma and chronic pancreatitis (CP) by producing a dense fibrotic stroma. However, the incomplete knowledge of PSCs biology hampers the exploration of antifibrotic therapies. Here, we explored the role of the Hippo pathway in the context of PSCs activation and experimental CP. MethodsCP model was created in rats with the tail vein injection of dibutyltin dichloride (DBTC). The expression of Yes-associated protein (YAP) in CP tissue was assessed. Primary and immortalized rats PSCs were treated with the YAP-inhibitor verteporfin. Furthermore, YAP siRNA was employed. Subsequently, DNA synthesis, cell survival, levels of α-smooth muscle actin (α-SMA) protein, presence of lipid droplets and PSCs gene expression were evaluated. Upstream regulators of YAP signaling were studied by reporter gene assays. ResultsIn DBTC-induced CP, pronounced expression of YAP in areas of tubular structures and periductal fibrosis was observed. Verteporfin diminished DNA replication in PSCs in a dose-dependent fashion. Knockdown of YAP reduced cell proliferation. Primary cultures of PSCs were characterized by a decrease of lipid droplets and increased synthesis of α-SMA protein. Both processes were not affected by verteporfin. At the non-cytotoxic concentration of 100 nmol/L, verteporfin significantly reduced mRNA levels of transforming growth factor-β1 (Tgf-β1) and Ccn family member 1 (Ccn1). YAP signaling was activated by TGF-β1, but repressed by interferon-γ. ConclusionsActivated YAP enhanced PSCs proliferation. The antifibrotic potential of Hippo pathway inhibitors warrants further investigation.

Melatonin modulates metabolic adaptation of pancreatic stellate cells subjected to hypoxia

Pancreatic stellate cells (PSCs), the main cell type responsible for the development of fibrosis in pancreatic cancer, proliferate actively under hypoxia. Melatonin has received attention as a potential antifibrotic agent due to its anti-proliferative actions on PSCs. In this work, we investigated the activation of the PI3K/Akt/mTOR pathway and the metabolic adaptations that PSCs undergo under hypoxic conditions, as well as the probable modulation by melatonin. Incubation of cells under hypoxia induced an increase in cell proliferation, and in the expression of alpha-smooth muscle actin and of collagen type 1. In addition, an increase in the phosphorylation of Akt was observed, whereas a decrease in the phosphorylation of PTEN and GSK-3b was noted. The phosphorylation of mTOR and its substrate p70 S6K was decreased under hypoxia. Treatment of PSCs with melatonin under hypoxia diminished cell proliferation, the levels of alpha-smooth muscle actin and of collagen type 1, the phosphorylation of Akt and increased phosphorylation of mTOR. Mitochondrial activity decreased in PSCs under hypoxia. A glycolytic shift was observed. Melatonin further decreased mitochondrial activity. Under hypoxia, no increase in autophagic flux was noted. However, melatonin treatment induced autophagy activation. Nevertheless, inhibition of this process did not induce detectable changes in the viability of cells treated with melatonin. We conclude that PSCs undergo metabolic adaptation under hypoxia that might help them survive and that pharmacological concentrations of melatonin modulate cell responses to hypoxia. Our results contribute to the knowledge of the mechanisms by which melatonin could modulate fibrosis within the pancreas.

Rapamycin-filgrastim combination therapy ameliorates portal hypertension-induced splenomegaly: Role of β actin and S100A9 proteins modulation.

Objective(s):Thioacetamide (TAA) was administered to induce an animal model of liver disease with secondary splenomegaly to assess the mechanisms underlying the effects of rapamycin and filgrastim when taken separately or in combination on the biochemical and histopathological aspects of the liver and spleen. Materials and Methods:Thirty adult male albino rats were divided into five groups (control, TAA-treated group, TAA+rapamycin, TAA+filgrastim, and TAA+rapamycin+filgrastim group). We measured relative liver and spleen weights, serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and albumin. Molecular docking modeling and histopathological examination of liver and spleen sections with hematoxylin and eosin and Masson trichrome staining with immunohistochemical detection of splenic CD3 and CD20 lymphocytes, S100A9 and β actin antibodies were detected. Morphometric and statistical analyses of the results were performed. Results:TAA administration altered the histological structure of the liver and spleen and impaired liver function. It increased the expression of splenic CD3, CD20 lymphocytes, and S100A9 while diminishing the expression of β actin. Each of rapamycin and filgrastim, when administered separately, improved liver and spleen indices and liver function, but rapamycin did not affect the albumin level. They lowered splenic B and T lymphocyte levels. Expression levels of S100A9 showed down-regulation while β actin levels were up-regulated when compared with TAA. Combination therapy improved liver and spleen tissue pathology and significantly ameliorated the expression of splenic lymphocytes through regulation of S100A9 and β actin expression. Conclusion:The synergistic effect of combination therapy was dependent on the regulation of splenic S100A9 and β actin levels.

Impact of SGLT2 inhibitors on cardiac fibroblasts properties

Clinical trials conducted over the past five years have reported significant cardioprotection conferred by antidiabetic SGLT2 inhibitors, including Cana-, Empa- and Dapagliflozin. These drugs showed significant reduction in hospitalizations and death for heart failure with reduced and preserved ejection fraction, in both diabetic and non-diabetic patients. The mechanisms underlying these protective effects cannot be fully explained by glucose lowering properties and remain poorly understood. We investigated the impact of SGLT2 inhibitors on cardiac fibroblasts properties, namely proliferation and myodifferentiation. For proliferation assessment, cultured human cardiac fibroblasts (HCFs) were serum starved for 24 h prior to stimulation with 1% fetal bovine serum and 1, 3 or 10 μM of Cana-, Empa- or Dapagliflozin. Proliferation was measured by flow cytometry, using 5-ethynyl-2′-deoxyuridine. To assess myodifferentiation, HCFs were serum starved for 2 h before stimulation with 1, 3 or 10 μM of Cana-, Empa- or Dapagliflozin for 72 hours. Transforming growth factor-β1 (TGFβ1) was added at the time of stimulation, to induce HCFs myodifferentiation. Then, we measured expression levels of the myodifferentiation marker α-smooth muscle actin (αSMA) and collagen type I by RT-qPCR. All SGLT2 inhibitors did not affect HCFs proliferation. Empa- and Dapagliflozin led to a significant decrease of 50% in the expression of αSMA ( P < 0.05) and collagen type I ( P < 0.01), compared to control. However, Canagliflozin barely affected myodifferentiation. In contrast to Canagliflozin, Empa- and Dapagliflozin significantly reduced myodifferentiation of HCFs. Our results suggest that all SGLT2 inhibitors will not have the same impact on extracellular matrix remodeling. Accordingly, Empa- and Dapagliflozin could be more potent in preventing extracellular matrix remodeling and interstitial fibrosis in the context of heart failure.

Dihydro-artemisinin inhibits angiotensin II-induced cardiac fibroblasts proliferation and induces apoptosis through phosphoinositide 3-kinase/akt pathway

Cardiac fibroblasts (CFs) abnormal proliferation can trigger myocardial fibrosis, which is closely related to a variety of cardiovascular diseases. This study investigated the effect of dihydroartemisinin (DHA) on CF growth through PI3K/AKT with angiotensin II (AngII). CCK-8 method and EdU were used detect cell proliferation, and flow cytometry assessed apoptosis of CFs. The PI3K and AKT expressions were determined, whilst immunofluorescence staining and RT-qPCR measured α-smooth muscle actin (α-SMA) level. With rat CFs exhibiting typical fibroblast morphology and nuclei in spindle, oval or polygonal shape, these cells were positive for the fibroblast marker vimentin. Treatment with AngII resulted in increased proliferation of CFs, but the proliferation declined in the presence of DHA (p &lt; 0.05). Meanwhile, the AngII group had lower apoptosis of CFs than blank and DHA groups with highest apoptosis observed in the DHA group. α-SMA increased at the mRNA and protein levels after Ang II treatment and decreased after DHA treatment (p &lt; 0.05). Importantly, the Ang II upregulated PI3K and AKT in CFs (p &lt; 0.05) and the increase was abrogated by DHA administration with lowest levels (p &lt; 0.05). Ang II activates PI3K/Akt in normal CFs, as administration of DHA can inhibit the effect of Ang II, thereby inhibiting proliferation of CFs induced by AngII and inducing apoptosis.

Ginsenoside Rb1 Alleviates Bleomycin-Induced Pulmonary Inflammation and Fibrosis by Suppressing Central Nucleotide-Binding Oligomerization-, Leucine-Rich Repeat-, and Pyrin Domains-Containing Protein Three Inflammasome Activation and the NF-κB Pathway.

PurposeIdiopathic pulmonary fibrosis is a chronic and irreversible fibrotic interstitial pneumonia of unknown etiology and therapeutic strategies are limited. Emerging evidence suggests that the continuous activation of the central nucleotide-binding oligomerization-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is involved in the pathogenesis of pulmonary fibrosis. Ginsenoside Rb1 (G-Rb1) is the most abundant component in the traditional Chinese herb ginseng and has anti-inflammatory and anti-fibrotic activities. The purpose of this study was to explore whether G-Rb1 exerts anti-inflammatory and anti-fibrotic activities in vivo and in vitro by suppressing the activation of the NLRP3 inflammasome and NF-κB pathway.MethodsForty-eight male C57BL/6 mice were randomly divided into four groups (n=12/group) as follows: control, bleomycin (BLM), BLM/G-Rb1, and G-Rb1. A pulmonary fibrosis model was developed via an intratracheal injection of BLM. Six mice from each group were euthanized on days 3 and 21. The degree of pulmonary fibrosis was examined by histological evaluation and assessing α-smooth muscle actin levels. THP-1 cells were differentiated into macrophages, and stimulated by lipopolysaccharide and adenosine triphosphate. Activation of the NLRP3 inflammasome and NF-κB pathway was determined by Western blotting. Interleukin-1 beta and interleukin-18 levels were measured by ELISA. MRC-5 cells were cultured in the conditioned medium of the treated macrophages, after which markers of myofibroblasts were determined by Western blotting.ResultsG-Rb1 ameliorated BLM-induced pulmonary inflammation and fibrosis in mice, and suppressed NLRP3 inflammasome activation and the NF-κB pathway in lung tissues. Moreover, interleukin-1 beta secreted after NLRP3 inflammasome activation in macrophages promoted fibroblast differentiation. G-Rb1 inhibited lipopolysaccharide- and adenosine triphosphate-induced NLRP3 inflammasome activation in macrophages and disturbed the crosstalk between macrophages and fibroblasts.ConclusionG-Rb1 ameliorates BLM-induced pulmonary inflammation and fibrosis by suppressing NLRP3 inflammasome activation and the NF-κB pathway. Hence, G-Rb1 is a potential novel therapeutic drug for idiopathic pulmonary fibrosis.

Regulation of endothelial nitric oxide synthase in cardiac remodeling

ObjectivesOur previous study demonstrated that endothelial nitric oxide synthase (eNOS) gene serves as a candidate for modifiers of hypertrophic cardiomyopathy (HCM), which alters severity of HCM phenotypes. Herein, we sought to further elucidate the role of eNOS on cardiac myocyte hypertrophy and fibrosis, the major phenotypes of HCM. MethodsMale eNOS-deficient mice (eNOS−/−) and wild type control mice (eNOS+/+, C57B1/6 J) were used in this study. Myocyte size was analyzed in hematoxylin/eosin stained sections using an image analyzing system. Cardiac β-myosin heavy chain (β-MHC) and α-skeletal actin (α-SKA) levels, markers of myocyte hypertrophy were evaluated by Western blot. Cardiac collagen volume fraction (CVF) was examined in picrosirius red stained section using an image analyzing system. Cardiac expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (TGF-β1), markers of fibrosis, were determined by Western blot. ResultsCompared to eNOS+/+ mice, we found that; 1) myocyte size was significantly increased in eNOS−/− mice; 2) cardiac expression of β-MHC was markedly elevated, while α-SKA levels remained unchanged in eNOS−/− mice; 3) cardiac total and interstitial CVF levels were significantly higher in eNOS−/− mice; and 4) cardiac TIMP-1 levels were significantly greater in eNOS−/− mice, however, cardiac TGF-β1 was not differently expressed between the two groups. ConclusionThe current study revealed that eNOS plays a beneficial role in cardiac remodeling, preventing the heart from development of myocyte hypertrophy and cardiac fibrosis. These findings support our previous report that eNOS may modify the severity of HCM phenotypes.

Morroniside, a novel GATA3 binding molecule, inhibits hepatic stellate cells activation by enhancing lysosomal acid lipase expression

BackgroundLiver fibrosis can be easily developed into irreversible liver cirrhosis or even liver cancer. Lysosomal acid lipase (LAL), encoded by the lipase A (Lipa) gene, is a critical enzyme involved in liver fibrosis development. Morroniside, an iridoid glycoside isolated from Cornus officinalis Sieb. et Zucc., exerts hepatic protective effects. However, the mechanism of action underling the anti-liver fibrosis effects of morroniside have not been fully elucidated. PurposeTo explore whether Lipa served as a biomarker for liver fibrosis and investigate the anti-liver fibrosis effects of morroniside and the underlying action mechanism in liver fibrosis cell models. MethodsLAL expression was examined in the liver tissues of CCl4 and high-fat diet (HFD)-induced liver fibrosis animal models. α-smooth muscle actin (α-SMA) level, collagen and GATA family expressions were analyzed by Real-time PCR and Western blot. Putative transcription factor binding sites in the DNA sequences of Lipa was identified by PROMO-ALGGEN v8.3 online software and ENCODE ChIP-Seq Significance Tool. MD simulation was performed to explore the protein-ligand interaction. ResultsWe found that the expression of hepatic LAL is lower in the liver fibrosis animal models than the control models. The reduced LAL expression is associated with HSCs activation, suggesting LAL is novel liver fibrosis biomarker. More importantly, our data showed that morroniside exerts anti-liver fibrosis effects in vitro. Mechanistic studies reveal that it binds to the hydrophobic sites of GATA3 and also reduces GATA3 expression, which increases LAL expression. ConclusionsThis study, for the first time, suggests LAL is a novel biomarker for liver fibrosis. Besides, morroniside exerts its anti-liver fibrosis effects by targeting GATA3 and LAL and hence inhibits HSC activation. These findings provide strong scientific evidence to support the development of morroniside as novel alternative or complementary therapeutics for liver injury prevention and treatment.

Extracellular Vesicular Transmission of miR-423-5p from HepG2 Cells Inhibits the Differentiation of Hepatic Stellate Cells.

Liver fibrosis (LF) is a major cause of morbidity and mortality worldwide. Hepatic stellate cells (HSCs) are the primary source of extracellular matrix in the liver and their activation is a central event in LF development. Extracellular vesicles (EVs) are intercellular communication agents, which play important roles in physiological processes in chronic liver diseases. The aim of this study was to examine the crosstalk between hepatocytes and HSCs mediated by hepatocyte-secreted EVs. EVs were purified from primary mouse hepatocytes, HepG2 cell lines, under normal or stressed conditions. The effect of EVs on primary HSCs (pHSCs) differentiation was evaluated by measuring of differentiation markers. In addition, their impact on the carbon tetrachloride (CCl4)-induced fibrosis mouse model was evaluated. The results demonstrated that HepG2-EVs regulate HSC differentiation and that under stress conditions, promoted pHSCs differentiation into the myofibroblast phenotype. The evaluation of miRNA sequences in the HepG2 secreted EVs demonstrated high levels of miR-423-5p. The examination of EV cargo following stress conditions identified a significant reduction of miR-423-5p in HepG2-EVs relative to HepG2-EVs under normal conditions. In addition, pHSCs transfected with miR-423-5p mimic and exhibit lower mRNA levels of alpha smooth muscle actin and Collagen type 1 alpha, and the mRNA expression level of genes targeted the family with sequence-similarity-3 (FAM3) and Monoacylglycerol lipase (Mgll). This study strengthened the hypothesis that EVs are involved in LF and that their cargo changes in stress conditions. In addition, miR-423-5p was shown to be involved in HSCs differentiation and hence, fibrosis development.