Antioxidant therapy against TGF-ß/SMAD pathway involved in organ fibrosis.

Fibrosis refers to excessive build-up of scar tissue and extracellular matrix components in different organs. In recent years, it has been revealed that different cytokines and chemokines, especially Transforming growth factor beta (TGF-ß) is involved in the pathogenesis of fibrosis. It has been shown that TGF-ß is upregulated in fibrotic tissues, and contributes to fibrosis by mediating pathways that are related to matrix preservation and fibroblasts differentiation. There is no doubt that antioxidants protect against different inflammatory conditions by reversing the effects of nitrogen, oxygen and sulfur-based reactive elements. Oxidative stress has a direct impact on chronic inflammation, and as results, prolonged inflammation ultimately results in fibrosis. Different types of antioxidants, in the forms of vitamins, natural compounds or synthetic ones, have been proven to be beneficial in the protection against fibrotic conditions both in vitro and in vivo. In this study, we reviewed the role of different compounds with antioxidant activity in induction or inhibition of TGF-ß/SMAD signalling pathway, with regard to different fibrotic conditions such as gastro-intestinal fibrosis, cardiac fibrosis, pulmonary fibrosis, skin fibrosis, renal fibrosis and also some rare cases of fibrosis, both in animal models and cell lines.

Investigating the therapeutic effects and mechanisms of Roxadustat on peritoneal fibrosis Based on the TGF-ß/Smad pathway.

Peritoneal fibrosis (PF) is particularly common in individuals undergoing peritoneal dialysis (PD). Fibrosis of the parenchymal tissue typically progresses slowly. Therefore, preventing and reducing the advancement of fibrosis is crucial for effective patient treatment. Roxadustat is a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), primarily used to treat and improve renal anemia. Recent studies have found that HIF-1α possesses antioxidant activity and exerts a certain protective effect in ischemic heart disease and spinal cord injury, while it can also delay the progression of pulmonary and renal fibrosis. This study establishes the mice model through intraperitoneal injection of 4.25 % peritoneal dialysate fluid (PDF) and explores the therapeutic effects of Roxadustat by inducing TGF-ß1-mediated epithelial-mesenchymal transition (EMT) in Met-5A cells. The aim is to investigate the protective role and mechanisms of Roxadustat against PD-related PF. We observed thicker peritoneal tissue and reduced permeability in animals with PD-related PF samples. This was accompanied by heightened inflammation, which Roxadustat alleviated by lowering the levels of inflammatory cytokines (IL-6, TNF-α). Furthermore, Roxadustat inhibited EMT in PF mice and TGF-ß1-induced Met-5A cells, as evidenced by decreased expression of fibrotic markers, such as fibronectin, collagen I, and α-SMA, alongside an elevation in the expression of the epithelial marker, E-cadherin. Roxadustat also significantly decreased the expression of TGF-ß1 and the phosphorylation of p-Smad2 and p-Smad3. In conclusion, Roxadustat ameliorates peritoneal fibrosis through the TGF-ß/Smad pathway.

The novel peptide DR4penA attenuates the bleomycin- and paraquat-induced pulmonary fibrosis by suppressing the TGF-ß/Smad signaling pathway.

Pulmonary fibrosis (PF), which is caused by continuous alveolar epithelial cell injury and abnormal repair, is referred to as a difficult disease of the lung system by the World Health Organization due to its rapid progression, poor prognosis, and high mortality rate. However, there is still a lack of ideal therapeutic strategies. The peptide DR8 (DHNNPQIR-NH2 ), which is derived from rapeseed, exerted antifibrotic activity in the lung, liver, and kidney in our previous studies. By studying the structure-activity relationship and rational design, we introduced an unnatural hydrophobic amino acid (α-(4-pentenyl)-Ala) into DR8 and screened the novel peptide DR4penA (DHNα-(4-pentenyl)-APQIR-NH2 ), which had higher anti-PF activity, higher antioxidant activity and a longer half-life than DR8. Notably, DR4penA attenuated bleomycin- and paraquat-induced PF, and the anti-PF activity of DR4penA was equivalent to that of pirfenidone. Additionally, DR4penA suppressed the TGF-ß/Smad pathway in TGF-ß1-induced A549 cells and paraquat-induced rats. This study demonstrates that the novel peptide DR4penA is a potential candidate compound for PF therapy, and its antifibrotic activity in different preclinical models of PF provides a theoretical basis for further study.

Tanshinone IIA alleviates bleomycin-induced pulmonary fibrosis by inhibiting Zbtb16.

Pulmonary fibrosis is a complex disease that can occur in a variety of clinical settings. The Zinc Finger and BTB Domain Containing 16 (Zbtb16) is a transcription factor and has not been studied in pulmonary fibrosis. Lung tissues from rats which were treated with bleomycin and Tanshinone IIA (Tan IIA) were collected for mRNA sequencing. Zbtb16, a differentially expressed gene, was screened. Using adeno-associated virus to knock down Zbtb16 in rats, it was found that the lung index and the content of hydroxyproline in lung tissue were decreased. HE and Masson staining revealed that pathological symptoms of lung histopathology were relieved after Zbtb16 knockdown. Protein expressions of α-SMA, Collagen I and Fibronectin were significantly decreased after Zbtb16 knockdown in vivo and in vitro. Meanwhile, the protein content of TGF-ß1 and the phosphorylation of Smad2/3 were inhibited by Zbtb16 knockdown. Conversely, under the treatment of Tan IIA and TGF-ß1, overexpression of Zbtb16 improved cell viability, increased the expression of fibrosis-related proteins, and promoted the phosphorylation of Smad 2/3. All above demonstrates that Zbtb16 inhibition ameliorates pulmonary fibrosis and suppresses the TGF-ß/Smad pathway. Furthermore, Zbtb16 mediates the inhibitory process of Tan IIA on pulmonary fibrosis. This study provides a novel candidate therapeutic target for pulmonary fibrosis.

GL-V9 ameliorates liver fibrosis by inhibiting TGF-ß/smad pathway.

Liver fibrosis is a wound-healing response that arises from various aetiologies. Flavonoid compounds have been proved of their anti-liver fibrosis effects. This study aimed to elucidate the protective effect and mechanism of flavonoid compound GL-V9 on CCl4-induced and DDC-induced liver fibrosis. Treatment with GL-V9 alleviated hepatic injury and exhibited a dramatic protection effect of liver fibrosis. Further experiments found that GL-V9 treatment inhibited extracellular matrix (ECM) expression. Activation of hepatic stellate cells (HSCs) is a central driver of fibrosis. GL-V9 could inhibit the activation of HSCs through directly binding to TGFßRI, subsequently inhibit TGF-ß/Smad pathway. In conclusion, this study proved that GL-V9 executed a protective effect on liver fibrosis by inhibiting TGF-ß/Smad pathway.

Diterpenoids with Schistosomula-Killing and Anti-Fibrosis Activities In Vitro from the Leaves of Croton tiglium.

The leaves of C. tiglium have been comprehensively researched for their structurally novel bioactive natural compounds, especially those with anti-schistosomiasis liver fibrosis activity, because ethyl acetate extract, which can be extracted from the leaves of C. tiglium, has good anti-schistosomiasis liver fibrosis effects. One new tigliane-type diterpene, 20-acetyl-13-O-(2-metyl)butyryl-phorbol (1), and nine known (2-10) analogues were isolated from the leaves of C. tiglium. Their structures were elucidated on the basis of spectroscopic analysis and ECD analysis. All diterpenoids had a stronger insecticidal effect on schistosomula, and compounds 2, 4, and 10 had good anti-liver-fibrosis effects. Furthermore, compared with the model group, compound 2 significantly downregulated the protein and mRNA expression of COL-I, COL-III, α-SMA, and TGF-ß1 on TGF-ß1-induced liver fibrosis in LX-2 cells. Meanwhile, compound 2 also regulated the expression of TGF-ß/Smad-pathway-related proteins. The results suggest that diterpenoids from C. tiglium may serve as potential schistosomula-killing and anti-liver-fibrosis agents in the future.

Miglustat, a glucosylceramide synthase inhibitor, mitigates liver fibrosis through TGF-ß/Smad pathway suppression in hepatic stellate cells.

Transforming growth factor (TGF)-ß/Smad pathway is implicated in the pathogenesis of liver fibrosis, a condition characterized by excessive deposition of extracellular matrix (ECM) proteins such as collagen in response to chronic inflammation. It has been reported that ceramide regulates collagen production through TGF-ß/Smad pathway activation. In this study, we examined whether miglustat, an inhibitor of glucosylceramide synthase, can suppress liver fibrosis by reducing TGF-ß/Smad pathway activity. Human hepatic stellate cells (HHSteCs) were cultured with TGF-ß and multiple miglustat concentrations to examine dose-dependent effects on the expression levels of ECM-related genes and Smad proteins. To evaluate the efficacy of miglustat for fibrosis mitigation, C57BL/6 mice were treated with carbon tetrachloride (CCl4) for 4 weeks to induce liver fibrosis, followed by combined CCl4 plus miglustat for a further 2 weeks. To examine if miglustat can also prevent fibrosis, mice were treated with CCl4 for 2 weeks, followed by CCl4 plus miglustat for 2 weeks. Miglustat dose-dependently downregulated expression of α-smooth muscle actin and ECM components in TGF-ß-treated HHSteCs. Both phosphorylation and nuclear translocation of Smad2 and Smad3 were also suppressed by miglustat treatment. Sirius-Red staining and hydroxyproline assays of model mouse liver samples revealed that miglustat reduced fibrosis, an effect accompanied by decreased expression of ECM. Our findings suggest that miglustat can both prevent and reverse liver fibrosis by inhibiting TGF-ß/Smad pathway.

Application of recombinant TGF-ß1 inhibitory peptide to alleviate isoproterenol-induced cardiac fibrosis.

Cardiac fibrosis is a remodeling process of the cardiac interstitium, characterized by abnormal metabolism of the extracellular matrix, excessive accumulation of collagen fibers, and scar tissue hyperplasia. Persistent activation and transdifferentiation into myofibroblasts of cardiac fibroblasts promote the progression of fibrosis. Transforming growth factor-ß1 (TGF-ß1) is a pivotal factor in cardiac fibrosis. Latency-associated peptide (LAP) is essential for activating TGF-ß1 and its binding to the receptor. Thus, interference with TGF-ß1 and the signaling pathways using LAP may attenuate cardiac fibrosis. Recombinant full-length and truncated LAP were previously constructed, expressed, and purified. Their effects on cardiac fibrosis were investigated in isoproterenol (ISO)-induced cardiac fibroblasts (CFs) and C57BL/6 mice. The study showed that LAP and tLAP inhibited ISO-induced CF activation, inflammation, and fibrosis, improved cardiac function, and alleviated myocardial injury in ISO-induced mice. LAP and tLAP alleviated the histopathological alterations and inhibited the elevated expression of inflammatory and fibrosis-related markers in cardiac tissue. In addition, LAP and tLAP decreased the ISO-induced elevated expression of TGF-ß, αvß3, αvß5, p-Smad2, and p-Smad3. The study indicated that LAP and tLAP attenuated ISO-induced cardiac fibrosis via suppressing TGF-ß/Smad pathway. This study may provide a potential approach to alleviate cardiac fibrosis. KEY POINTS: • LAP and tLAP inhibited ISO-induced CF activation, inflammation, and fibrosis. • LAP and tLAP improved cardiac function and alleviated myocardial injury, inflammation, and fibrosis in ISO-induced mice. • LAP and tLAP attenuated cardiac fibrosis via suppressing TGF-ß/Smad pathway.

Follistatin (FST) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.

Follistatin (FST), a member of the transforming growth factor-ß (TGF-ß) superfamily, has been identified as an inhibitor of follicle-stimulating hormone. Previous studies showed that it plays an important role in animal reproduction. Therefore, this study aims to investigate its effect on the maturation of buffalo oocytes in vitro, and the underlying mechanism of FST affecting oocyte maturation was also explored in buffalo cumulus cells. Results showed that FST was enriched in the ovary and expressed at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. The FST expression level was up-regulated in MII buffalo oocytes compared with the GV stage (p < .05). To study the effects of FST on buffalo oocytes' maturation and early embryonic development, we added the pcD3.1 skeleton vector and PCD3.1-EGFP-FST vector into the maturation fluid of buffalo oocytes, respectively. It was demonstrated that FST promoted the in vitro maturation rate of buffalo oocytes and the blastocyst rate of embryos cultured in vitro (p < .05). By interfering with FST expression, we discovered that FST in cumulus cells plays a crucial role in oocyte maturation. Interference with the FST expression during the buffalo oocyte maturation did not affect the first polar body rate of buffalo oocyte (p > .05). In contrast, the location of mitochondria in oocytes was abnormal, and the cumulus expansion area was reduced (p < .05). After parthenogenetic activation, the cleavage and blastocyst rates of the FST-interfered group were reduced (p < .05). Furthermore, RT-qPCR was performed to investigate further the underlying mechanism by which FST enhances oocyte maturation. We found that overexpression of FST could up-regulate the expression level of apoptosis suppressor gene Bcl-2 and TGF-ß/SMAD pathway-related genes TGF-ß, SMAD2, and SMAD3 (p < .05). In contrast, the expression levels of SMAD4 and pro-apoptotic gene BAX were significantly decreased (p < .05). The FST gene could affect buffalo oocyte maturation by regulating the oocyte mitochondria integrity, the cumulus expansion, cumulus cell apoptosis, and the expression levels of TGF-ß/SMAD pathway-related genes.

Recombinant truncated latency-associated peptide alleviates liver fibrosis in vitro and in vivo via inhibition of TGF-ß/Smad pathway.

BACKGROUND: Liver fibrosis is a progressive liver injury response. Transforming growth factor ß1 (TGF-ß1) is oversecreted during liver fibrosis and promotes the development of liver fibrosis. Therapeutic approaches targeting TGF-ß1 and its downstream pathways are essential to inhibit liver fibrosis. The N-terminal latency-associated peptide (LAP) blocks the binding of TGF-ß1 to its receptor. Removal of LAP is critical for the activation of TGF-ß1. Therefore, inhibition of TGF-ß1 and its downstream pathways by LAP may be a potential approach to affect liver fibrosis. METHODS: Truncated LAP (tLAP) plasmids were constructed. Recombinant proteins were purified by Ni affinity chromatography. The effects of LAP and tLAP on liver fibrosis were investigated in TGF-ß1-induced HSC-T6 cells, AML12 cells and CCl4-induced liver fibrosis mice by real time cellular analysis (RTCA), western blot, real-time quantitative PCR (RT-qPCR), immunofluorescence and pathological staining. RESULTS: LAP and tLAP could inhibit TGF-ß1-induced AML12 cells inflammation, apoptosis and EMT, and could inhibit TGF-ß1-induced HSC-T6 cells proliferation and fibrosis. LAP and tLAP could attenuate the pathological changes of liver fibrosis and inhibit the expression of fibrosis-related proteins and mRNAs in CCl4-induced liver fibrosis mice. CONCLUSION: LAP and tLAP could alleviate liver fibrosis in vitro and in vivo via inhibition of TGF-ß/Smad pathway. TLAP has higher expression level and more effective anti-fibrosis activity compared to LAP. This study may provide new ideas for the treatment of liver fibrosis.

TRIM66 hastens the malignant progression of non-small cell lung cancer via modulating MMP9-mediated TGF-ß/SMAD pathway.

OBJECTIVE: To investigate regulatory function and underlying mechanism of TRIM66 in non-small cell lung cancer (NSCLC). METHODS: TRIM66 and MMP9 expression in NSCLC cells and tissues was assayed via qRT-PCR and western blot. CCK-8, colony formation, Transwell and flow cytometry assays were conducted to measure cell functional alternations in NSCLC. Western blot was employed to measure expression as well as phosphorylation levels of epithelial-mesenchymal transition-(EMT) and TGF-ß/SMAD pathways-related proteins. Co-immunoprecipitation (Co-IP) assay was done to probe interaction between TRIM66 and MMP9. Xenograft in vivo experiment and tumor metastasis model in nude mice were utilized to investigate effects of TRIM66 on tumor growth of NSCLC. RESULTS: TRIM66 and MMP9 were conspicuously highly expressed in NSCLC cells and tissues. High TRIM66 level was markedly correlated with metastasis. Silencing TRIM66 prominently repressed the proliferation, migration and invasion of transfected cells, while inducing cell apoptosis. Whereas forced expression of TRIM66 exerted the opposite effect. The aberrant expression of TRIM66 modulated EMT pathway. TRIM66 also regulated MMP9 expression, and the interaction between them was validated by Co-IP assay. Overexpression of MMP9 could activate TGF-ß/SMAD pathway. Rescue experiments manifested that si-MMP9 or SB431542 could partially reverse phenotypes induced by TRIM66. In vivo experiments revealed that silencing TRIM66 could hamper NSCLC tumor growth and metastasis. CONCLUSION: TRIM66 and MMP9 were up-regulated in NSCLC. TRIM66 facilitated the malignant progression of NSCLC through modulating MMP9-mediated TGF-ß/SMAD pathway.

Mesenchymal stem cells ameliorate silica-induced pulmonary fibrosis by inhibition of inflammation and epithelial-mesenchymal transition.

Silicosis is a devastating occupational disease caused by long-term inhalation of silica particles, inducing irreversible lung damage and affecting lung function, without effective treatment. Mesenchymal stem cells (MSCs) are a heterogeneous subset of adult stem cells that exhibit excellent self-renewal capacity, multi-lineage differentiation potential and immunomodulatory properties. The aim of this study was to explore the effect of bone marrow-derived mesenchymal stem cells (BMSCs) in a silica-induced rat model of pulmonary fibrosis. The rats were treated with BMSCs on days 14, 28 and 42 after perfusion with silica. Histological examination and hydroxyproline assays showed that BMSCs alleviated silica-induced pulmonary fibrosis in rats. Results from ELISA and qRT-PCR indicated that BMSCs inhibited the expression of inflammatory cytokines TNF-α, IL-1ß and IL-6 in lung tissues and bronchoalveolar lavage fluid of rats exposed to silica particles. We also performed qRT-PCR, Western blot and immunohistochemistry to examine epithelial-mesenchymal transition (EMT)-related indicators and demonstrated that BMSCs up-regulate E-cadherin and down-regulate vimentin and extracellular matrix (ECM) components such as fibronectin and collagen Ⅰ. Additionally, BMSCs inhibited the silica-induced increase in TGF-ß1, p-Smad2 and p-Smad3 and decrease in Smad7. These results suggested that BMSCs can inhibit inflammation and reverse EMT through the inhibition of the TGF-ß/Smad signalling pathway to exhibit an anti-fibrotic effect in the rat silicosis model. Our study provides a new and meaningful perspective for silicosis treatment strategies.

Moderate oxidative stress promotes epithelial-mesenchymal transition in the lens epithelial cells via the TGF-ß/Smad and Wnt/ß-catenin pathways.

The epithelial-mesenchymal transition (EMT) plays a significant role in fibrosis and migration of lens epithelial cells (LECs), and eventually induces posterior capsule opacification (PCO). In the past, it was generally believed that the TGF-ß/Smad pathway regulates lens EMT. A recent study found that attenuated glutathione level promotes LECs EMT via the Wnt/ß-catenin pathway, which suggests a more complex pathogenesis of PCO. To test the hypothesis, we used the mouse cataract surgery PCO model and tested both canonical Wnt/ß-catenin and TGF-ß/Smad signaling pathways. The results showed that both TGF-ß/Smad and Wnt/ß-catenin pathways were activated during the lens capsule fibrosis. Compared with the freshly isolated posterior capsule, the expression level of phosphorylated Smad2 was highest at day3 and then slightly decreased, but the expression level of Wnt10a gradually increased from day0 to day7. It shows that these two pathways are involved in the lens epithelium's fibrotic process and may play different roles in different periods. Subsequently, we established oxidative stress-induced EMT model in primary porcine lens epithelial cells and found that both the TGF-ß/Smad and Wnt/ß-catenin pathways were activated. Further study suggests that block Wnt/ß-catenin pathway using XAV939 alone or block TGF-ß/Smad pathway using LY2109761 could partially block pLECs fibrosis, but blocking Wnt/ß-catenin and TGF-ß/Smad pathway using combined XAV939 and LY2109761 could completely block pLECs fibrosis. In conclusion, this study demonstrates that both TGF-ß/Smad and canonical Wnt/ß-catenin pathways play a significant role in regulating epithelial-mesenchymal transformation of lens epithelial cells but might be in a different stage.

Endothelial to mesenchymal transition during neonatal hyperoxia-induced pulmonary hypertension.

Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, results from mechanical ventilation and hyperoxia, amongst other factors. Although most BPD survivors can be weaned from supplemental oxygen, many show evidence of cardiovascular sequelae in adulthood, including pulmonary hypertension and pulmonary vascular remodeling. Endothelial-mesenchymal transition (EndoMT) plays an important role in mediating vascular remodeling in idiopathic pulmonary arterial hypertension. Whether hyperoxic exposure, a known mediator of BPD in rodent models, causes EndoMT resulting in vascular remodeling and pulmonary hypertension remains unclear. We hypothesized that neonatal hyperoxic exposure causes EndoMT, leading to the development of pulmonary hypertension in adulthood. To test this hypothesis, newborn mice were exposed to hyperoxia and then allowed to recover in room air until adulthood. Neonatal hyperoxic exposure gradually caused pulmonary vascular and right ventricle remodeling as well as pulmonary hypertension. Male mice were more susceptible to developing pulmonary hypertension compared to female mice, when exposed to hyperoxia as newborns. Hyperoxic exposure induced EndoMT in mouse lungs as well as in cultured lung microvascular endothelial cells (LMVECs) isolated from neonatal mice and human fetal donors. This was augmented in cultured LMVECs from male donors compared to those from female donors. Using primary mouse LMVECs, hyperoxic exposure increased phosphorylation of both Smad2 and Smad3, but reduced Smad7 protein levels. Treatment with a selective TGF-ß inhibitor SB431542 blocked hyperoxia-induced EndoMT in vitro. Altogether, we show that neonatal hyperoxic exposure caused vascular remodeling and pulmonary hypertension in adulthood. This was associated with increased EndoMT. These novel observations provide mechanisms underlying hyperoxia-induced vascular remodeling and potential approaches to prevent BPD-associated pulmonary hypertension by targeting EndoMT. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PCBP2 promotes the development of glioma by regulating FHL3/TGF-ß/Smad signaling pathway.

The purpose of this study was to investigate the role of Poly (C)-binding protein 2 (PCBP2) and the related signaling pathway in glioma progression. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were performed to measure PCBP2 messenger RNA and protein expression in glioma tissues or cells. Cell transfection was completed using Lipofectamine 2000. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Transwell assay and flow cytometry assay were used to explore the effects of PCBP2 expression on biological behaviors of glioma cells. Western blot assay was used for the detection of pathway related proteins. Expression of PCBP2 in glioma tissues and cells were higher than that in paracancerous tissues and normal cells (both p < .01). Moreover, the elevated expression of PCBP2 was significantly correlated with tumor size (p = .001) and WHO stage (p = .010). Knockdown of PCBP2 could suppress proliferation, migration and invasion of glioma cells and promote apoptosis. Besides, the expression of transforming growth factor-ß (TGF-ß) pathway related proteins TGF-ß1, p-Smad2 and p-Smad7 were decreased following the downregulation of PCBP2. PCBP2 also inhibited FHL3 expression by binding to FHL3-3'UTR. The inhibition of FHL3 could reverse the antitumor action caused by PCBP2 silencing. In vivo assay, PCBP2 was also found to inhibit the tumor growth of glioma. PCBP2 activates TGF-ß/Smad signaling pathway by inhibiting FHL3 expression, thus promoting the development and progression of glioma.

miR-448-3p alleviates diabetic vascular dysfunction by inhibiting endothelial-mesenchymal transition through DPP-4 dysregulation.

Diabetes mellitus (DM) often causes vascular endothelial damage and alters vascular microRNA (miR) expression. miR-448-3p has been reported to be involved in the development of DM, but whether miR-448-3p regulates diabetic vascular endothelial dysfunction remains unclear. To investigate the molecular mechanism of diabetic vascular endothelial dysfunction and the role of miR-448-3p therein, Sprague-Dawley rats were injected with streptozotocin (STZ) to establish diabetic animal model and the rat aortic endothelial cells were treated with high glucose to establish diabetic cell model. For the treatment group, after the induction of diabetes, the miR-448-3p levels in vivo and in vitro were upregulated by adeno-associated virus serotype 2 (AAV2)-miR-448-3p injection and miR-448-3p mimic transfection, respectively. Our results showed that AAV2-miR-448-3p injection alleviated the body weight loss and blood glucose level elevation induced by STZ injection. The miR-448-3p level was significantly decreased and the dipeptidyl peptidase-4 (DPP-4) messenger RNA level was increased in diabetic animal and cell models, which was reversed by miR-448-3p treatment. Moreover, the diabetic rats exhibited endothelial damage and endothelial-mesenchymal transition (EndMT), while AAV2-miR-448-3p injection relieved those situations. In vitro experiments demonstrated that miR-448-3p overexpression in endothelial cells alleviated endothelial damage by inhibiting EndMT through blocking the transforming growth factor-ß/Smad pathway. We further proved that miR-448-3p negatively regulated DPP-4 by binding to its 3'-untranslated region, and DPP-4 overexpression reversed the effect of miR-448-3p overexpression on EndMT. Overall, we conclude that miR-448-3p overexpression inhibits EndMT via targeting DPP-4 and further ameliorates diabetic vascular endothelial dysfunction, indicating that miR-448-3p may serve as a promising therapeutic target for diabetic endothelial dysfunction.

MicroRNA-194 reduces inflammatory response and human dermal microvascular endothelial cells permeability through suppression of TGF-ß/SMAD pathway by inhibiting THBS1 in chronic idiopathic urticaria.

Chronic idiopathic urticaria (CIU) is a polyetiological dermatologic disease. Reports have stated that some microRNAs (miRNAs) have their roles to play in inflammatory response. In this present study, we aim to investigate whether miR-194 has an effect on attenuating inflammatory response and human dermal microvascular endothelial cells (HDMECs) permeability of CIU mast cells through TGF-ß/SMAD pathway by binding to thrombospondin 1 (THBS1). The Gene Expression Omnibus database was used to obtain the CIU-related microarray data, and then the analysis of differentially expressed genes was conducted and the miRNA regulated by THBS1 was predicted. After transfection of different mimic, inhibitor, or small interfering RNA, the effect of miR-194 on inflammatory reaction, mast cell degranulation, histamine release rate, HDMECs permeability, and the expression of THBS1, interferon γ (IFN-γ), TGF-ß, Smad3, and interleukin 4 (IL-4) were detected. THBS1 was verified to be the miR-194 target. After transfected with overexpressed miR-194 and si-THBS1, the degranulation rate, histamine release rate, and HDMECs permeability were significantly reduced, while the expression of IFN-γ was higher, and the expression of THBS1, TGF-ß, Smad3, IL-4 was significantly lower, accompanied with alleviated inflammatory reaction. Our study provides evidence that miR-194 negatively modulates THBS1 and inhibits the activation of TGF-ß/SMAD pathway, thereby alleviating the inflammatory response and HDMECs permeability of mast cells in CIU.

Theacrine alleviates chronic inflammation by enhancing TGF-ß-mediated shifts via TGF-ß/SMAD pathway in Freund's incomplete adjuvant-induced rats.

Rheumatoid arthritis is a chronic and systemic autoimmune disease, which affects approximately 1% of the adult population worldwide. The present study investigated the therapeutic effect of theacrine (TC) on arthritis and its mechanisms in Freund's incomplete adjuvant (FIA)-induced SD rats. Rats were randomly divided into 5 groups: i) healthy control; ii) model; iii) positive control with methotrexate (MTX); iv) treatment with 12.5 mg/kg TC; and v) treatment with 25.0 mg/kg TC. The apparent scores, including changes in body weights, degree of paw swelling and arthritis indicators, were analyzed to evaluate the anti-chronic inflammatory effect of TC. The levels of interleukin (IL)-6 and transforming growth factor-ß (TGF-ß) in serum were measured by enzyme-linked immunosorbent assay. The protein and RNA expression levels of the critical factors in rats were measured to elucidate the mechanisms responsible for chronic inflammation and to verify molecular indexes of chronic inflammatory conditions. TC notably suppressed the severity of FIA-induced rat by attenuating the apparent scores, animal weight and inflammatory indexes in the 25 mg/kg TC group compared with the FIA rat model. Furthermore, TC significantly decreased the levels of IL-6 and increased the levels of TGF-ß. Histopathological examinations indicated that TC rescued the synovial hyperplasia and inflammatory cell infiltration in joint tissues. In addition, TC enhanced TGF-ß-mediated shifts in inflammatory marker expression in joint tissue. Overall, the present study demonstrated that TC exerted a superior anti-arthritic effect via the suppression of IL-6 and the activation of TGF-ß by the TGF-ß/SMAD pathway.

Nimbolide abrogates cerulein-induced chronic pancreatitis by modulating ß-catenin/Smad in a sirtuin-dependent way.

Chronic pancreatitis (CP) is one of the leading causes of mortality worldwide with no clinically approved therapeutic interventions. The present study was designed to investigate the protective effect of nimbolide (NB), an active constituent of neem tree (Azadirachta indica), by targeting ß-catenin/Smad/SIRT1 in cerulein-induced CP model. The effects of NB was investigated on cerulein (50 µg/kg/hr*6 exposures /day, 3 days a week for 3 weeks) induced CP in mice. Amylase and lipase activity were measured and histopathological evaluation was performed. Collagen deposition in the pancreatic tissue was estimated by hydroxyproline assay, and collagen specific staining picrosirius red and Masson's trichrome. Cerulein-induced CP was significantly controlled by NB treatment, as shown by the downregulation of ß-catenin/Smad signaling in a SIRT1 dependent manner. NB treatment significantly decreased α-SMA, MMP-2, collagen1a, fibronectin, TGF-ß1, p-Smad-2/3 expression and extracellular matrix (ECM) deposition in pancreatic tissue. However, the protective effects of NB on cerulein-induced CP were undermined by nicotinamide (NMD) or splitomicin, sirtuin 1 (SIRT1) inhibitors treatment. NB treatment modulated protein expression by activating SIRT1 and decreasing the expression of ß-catenin/Smad proteins in CP mice. However, the expression of SIRT1 in pancreatic tissue was elevated by NB treatment and it was decreased by NMD or splitomicin treatment. In summary, our results strongly suggest that NB exerted promising protective effects in cerulein-induced CP model by inhibiting ß-catenin/Smad in a sirtuin-dependent manner, which could be attributed to its anti-inflammatory and antifibrotic effects. Our study suggests that NB could be an effective therapeutic intervention for the treatment of CP.

Placenta specific 8 gene induces epithelial-mesenchymal transition of nasopharyngeal carcinoma cells via the TGF-ß/Smad pathway.

The present study aimed to investigate the effects and mechanisms of PLAC8 on the epithelial-mesenchymal transition (EMT) of Nasopharyngeal carcinoma (NPC). The expression of PLAC8 in NPC and nasopharyngitis (NPG) tissues from 150 patients was determined using immunohistochemistry. The levels of PLAC8 in five NPC cell lines and nasopharyngeal permanent epithelial cell line were measured using western blotting. We then knocked out or overexpressed PLAC8 in CNE2 cells. Cell proliferation, wound healing, migration, and invasion assays were used to analyze the effects of PLAC8 on the proliferation, migration, and invasion in vivo and vitro. The results showed that the expression of PLAC8 was much higher in NPC tissues than in NPG tissues. The expression of PLAC8 was higher in all the cell lines than in the nasopharyngeal permanent epithelial cells. PLAC8 knockout resulted in significant decreases in cell proliferation, migration, and invasion; associated with lower protein levels of N-cadherin; and increased levels of E-cadherin. Overexpression of PLAC8 had the opposite effect. Furthermore, knockout of PLAC8 inactivated TGF-ß/SMAD signaling pathway and suppressed the growth of NPC xenografts. PLAC8 may promote the carcinogenesis and EMT of NPC via the TGF-ß/Smad pathway, which suggests that PLAC8 may be a potential biomarker for NPC.