Recombinant Slit2 attenuates tracheal fibroblast activation in benign central airway obstruction by inhibiting the TGF-ß1/Smad3 signaling pathway.

Airway fibrosis is among the pathological manifestations of benign central airway obstruction noted in the absence of effective treatments and requires new drug targets to be developed. Slit guidance ligand 2-roundabout guidance receptor 1 (Slit2-Robo1) is involved in fibrosis and organ development. However, its significance in airway fibrosis has not yet been reported. The study explored how the recombinant protein Slit2 functions in transforming growth factor-ß1 (TGF-ß1)-mediated airway fibrosis in vivo and in vitro. In this study, Slit2 expression initially increased in the tracheal granulation tissues of patients with tracheobronchial stenosis but decreased in the fibrotic tissue. In primary rat tracheal fibroblasts (RTFs), recombinant Slit2 inhibited the expression of extracellular matrices such as Timp1, α-SMA, and COL1A2, whereas recombinant TGF-ß1 promoted the expression of Robo1, α-SMA, and COL1A2. Slit2 and TGF-ß1 played a mutual inhibitory role in RTFs. Slit2 supplementation and Robo1 downregulation inhibited excessive extracellular matrix (ECM) deposition induced by TGF-ß1 in RTFs via the TGF-ß1/Smad3 pathway. Ultimately, exogenous Slit2 and Robo1 knockdown-mediated attenuation of airway fibrosis were validated in a trauma-induced rat airway obstruction model. These findings demonstrate that recombinant Slit2 alleviated pathologic tracheobronchial healing by attenuating excessive ECM deposition. Slit2-Robo1 is an attractive target for further exploring the mechanisms and treatment of benign central airway obstruction.

Sleeve gastrectomy links the attenuation of diabetic kidney disease to the inhibition of renal tubular ferroptosis through down-regulating TGF-ß1/Smad3 signaling pathway.

PURPOSE: To investigate how sleeve gastrectomy (SG), a typical operation of bariatric surgery, attenuated symptom, and progression of diabetic kidney disease (DKD). METHODS: DKD model was induced by high-fat diet (HFD) combined with streptozocin in Wistar rats. SG was performed, and the group subjected to sham surgery served as control. The animals were euthanized 12 weeks after surgery, followed by sample collection for the subsequent experiment. The HK-2, a renal proximal tubular epithelial cell line derived from human, was utilized to investigate the potential mechanisms. RESULTS: SG improved metabolic parameters and glucose homeostasis, and could alleviate DKD in terms of renal function indices as well as histological and morphological structures in DM rats, accompanied with a significant reduction in renal tubular injury. Compared with sham group, SG reduced the renal tubular ferroptosis. To further clarify the mechanism involved, in vitro experiments were performed. In the presence of high glucose, renal tubular TGF-ß1 secretion was significantly increased in HK-2 cell line, which led to activation of ferroptosis through TGF-ß1/Smad3 signaling pathway. Inhibition of TGF-ß1 receptor and phosphorylation of Smad3 significantly ameliorated TGF-ß1-mediated ferroptosis. In vivo experiments also found that SG improved the hyperglycemic environment, reduced renal TGF-ß1 concentrations, and down-regulated the TGF-ß1/Smad3 signaling pathway. CONCLUSIONS: With the capacity to lower the glucose, SG could attenuate the ferroptosis by inhibiting TGF-ß1/Smad3 signaling pathway in DKD rats, and eventually attenuated DKD.

SIRT2-dependent DKK1 deacetylation aggravates polycystic ovary syndrome by targeting the TGF-ß1/Smad3 signaling pathway.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a prevalent metabolic and endocrine condition in females of reproductive age. This work was to discover the underlying role of Dickkopf 1 (DKK1) and its putative regulating mechanism in P COS. METHODS: Mice recieved dehydroepiandrosterone (DHEA) injection to establish the in vivo P COS model.Hematoxylin and eosin (H&E) staining was performed for histological analysis. RT-qP CR and Western blotting were used to detect gene and protein expression. CCK-8 and flow cytometry assays were applied to detect cell viability and apoptosis. Co-immunoprecipitation (Co-IP) and immunoprecipitation (IP) were applied to assess association between DKK1 and SIRT2. RESULTS: In this work, DKK1 is downregulated in P COS rats. It was revealed that DKK1 knockdown induced apoptosis and suppressed proliferation in KGN cells, whereas DKK1 overexpression had exactly the opposite effects. In addition, DKK1 deactivates the T GF-ß1/SMad3 signaling pathway, thereby controlling KGN cell proliferation and apoptosis. Besides, SIRT2 inhibition reversed the impact of DKK1 overexpression on KGN cell proliferation and apoptosis. Furthermore, SIRT2 downregulated DKK1 expression by deacetylating DKK1 in KGN cells. DISCUSSION: Altogether, we concluded that SIRT2-induced deacetylation of DKK1 triggers T GF-ß1/Smad3 hyperactivation, thereby inhibiting proliferation and promoting apoptosis of KGN cells. The above results indicated that DKK1 might function as a latent target for P COS treatment.

Sinomenine attenuates pulmonary fibrosis by downregulating TGF-ß1/Smad3, PI3K/Akt and NF-κB signaling pathways.

BACKGROUND: Since COVID-19 became a global epidemic disease in 2019, pulmonary fibrosis (PF) has become more prevalent among persons with severe infections, with IPF being the most prevalent form. In traditional Chinese medicine, various disorders are treated using Sinomenine (SIN). The SIN's strategy for PF defense is unclear. METHODS: Bleomycin (BLM) was used to induce PF, after which inflammatory factors, lung histological alterations, and the TGF-/Smad signaling pathway were assessed. By administering various dosages of SIN and the TGF- receptor inhibitor SB-431,542 to human embryonic lung fibroblasts (HFL-1) and A549 cells, we were able to examine proliferation and migration as well as the signaling molecules implicated in Epithelial-Mesenchymal Transition (EMT) and Extra-Cellular Matrix (ECM). RESULTS: In vivo, SIN reduced the pathological changes in the lung tissue induced by BLM, reduced the abnormal expression of inflammatory cytokines, and improved the weight and survival rate of mice. In vitro, SIN inhibited the migration and proliferation by inhibiting TGF-ß1/Smad3, PI3K/Akt, and NF-κB pathways, prevented the myofibroblasts (FMT) of HFL-1, reversed the EMT of A549 cells, restored the balance of matrix metalloenzymes, and reduced the expression of ECM proteins. CONCLUSION: SIN attenuated PF by down-regulating TGF-ß/Smad3, PI3K/Akt, and NF-κB signaling pathways, being a potential effective drug in the treatment of PF.

Integrating network pharmacology and experimental verification to explore the mechanisms of salidroside against myocardial fibrosis.

Myocardial fibrosis (MF) is the manifestation of a variety of cardiovascular diseases. Salidroside (SAL) has been proved to have a certain effect on anti-fibrosis in various organs. However, the mechanism of SAL in the treatment of MF remains unclear. Network pharmacology showed that there were 1228 SAL-related target genes and 2793 MF-related target genes. The intersection of these genes resulted in 271 drug-disease interactions, and 15 core active targets were filtered from protein-protein interaction mapping. The top 20 Gene ontology biological processes analysis showed that the involved processes were close to the pathogenesis of MF. Among the top 20 enriched KEGG pathways, Wnt/ß-catenin and TGF-ß1/Smad3 signaling pathways were identified. In vivo, MI rats exhibited thinning of the myocardial region and the formation of fibrous scars, the expression of smad3 and ß-catenin were increased. After SAL treatment, there was a significant reduction in collagen area and a decrease in the ratio of collagen type I to type III. The expression of smad3 and ß-catenin was suppressed and positively correlated with the dosage of SAL. SAL may contribute to the progression of MF through the TGF-ß1/Smad3 and Wnt/ß-catenin signaling pathways.

Nicotinamide N-methyltransferase ameliorates renal fibrosis by its metabolite 1-methylnicotinamide inhibiting the TGF-ß1/Smad3 pathway.

Chronic kidney disease (CKD), a disease involving damage to the kidney structure and function, is a global public health problem. Tubulointerstitial fibrosis (TIF) is both an inevitable pathological change in individuals with CKD and a driving force in the progression of renal fibrosis. Nicotinamide N-methyltransferase (NNMT) and its metabolite 1-methylnicotinamide (MNAM) have been shown to protect against lipotoxicity-induced kidney tubular injury. However, the biological roles of NNMT and MNAM in regulating TIF remain elusive. This study aimed to investigate the protective effect of NNMT and MNAM on TIF and the mechanisms involved. We explored the functions and mechanisms of NNMT and MNAM in TIF, as well as the interaction between NNMT and MNAM, using unilateral ureteral obstruction (UUO) mice and cultured mouse tubular epithelial cells (mTECs) stimulated with transforming growth factor-ß1 (TGF-ß1). Several important findings were obtained as follows: (1) NNMT expression was upregulated in the kidneys of UUO mice and TGF-ß1-induced mTECs, and this upregulation was proposed to be a protective compensatory response to TIF. (2) MNAM was a potentially effective antifibrotic and anti-inflammatory medication in UUO mice. (3) The antifibrotic effect of NNMT overexpression was exerted by increasing the concentration of MNAM. (4) The renoprotective role of MNAM depended on the selective blockade of the interaction of Smad3 with TGFß receptor I. Overall, our study shows that NNMT is involved in the development and progression of CKD and that its metabolite MNAM may be a novel inhibitor of the TGF-ß1/Smad3 pathway with great therapeutic potential for CKD.

Anlotinib Attenuates Liver Fibrosis by Regulating the Transforming Growth Factor ß1/Smad3 Signaling Pathway.

BACKGROUND: Hepatic stellate cell hyperactivation is a central link in liver fibrosis development, transforming growth factor ß1 (TGF-ß1) is a key activator of HSCs. AIMS: This study investigated whether anlotinib attenuates CCl4 induced liver fibrosis in mice and explored its antifibrotic mechanism. METHODS: We used the human hepatic stellate cell line LX-2 for in vitro assays and used TGF-ß1 to induce hepatic fibrosis in LX-2 cells. We analyzed cytotoxicity using a cell-counting kit-8 and transwell chambers to detect the migratory ability of LX-2 cells. Western blotting was used to detect the protein levels of collagen type I, α-smooth muscle actin, and p-Smad3. In addition, mice with CCl4-induced hepatic fibrosis were used as in vivo models. Histopathological examination was performed using H&E staining, Masson's trichrome staining, and immunohistochemistry. RESULTS: Anlotinib significantly reversed TGF-ß1-induced protein levels of Col I, α-SMA and p-Smad3 and inhibits migratory and proliferative abilities in vitro using LX-2 cells. CCl4 cause F4 grade (Ishak) hepatic fibrosis, liver inflammatory scores ranged from 12 to 14 (Ishak), a mean ALT measurement of 130 U/L and a mean measurement AST value of 119 U/L in mice. However, the CCl4-induced changes were markedly attenuated by anlotinib treatment, which returned to F2 grade (Ishak) hepatic fibrosis, liver inflammatory scores ranged from 4 to 6 (Ishak), a mean ALT measurement of 40 U/L and a mean measurement AST value of 56 U/L in mice. CONCLUSIONS: Our results suggest that anlotinib-mediated suppression of liver fibrosis is related to the inhibition of TGF-ß1 signaling pathway. Hepatic stellate cell hyper activation is a central link in liver fibrosis development, transforming growth factor ß1 is a key activator of HSCs. Anlotinib is a multi-targeted tyrosine kinase inhibitor that has similar targets to nintedanib, a clinically used anti-pulmonary fibrosis drug. Our study demonstrates an FDA-approved drug-anlotinib-that could prevent liver fibrosis and inflammation. Experiments in cell cultures and mice show that anlotinib can inhibit the activation of hepatic stellate cells by down-regulating the TGFß1/smad3 pathway, thereby reversing liver fibrosis. In animal experiments, anlotinib showed protective effects on the CCl4-induced liver damage, including ameliorating liver inflammation, reversing liver fibrosis and reducing liver enzymes. This is a very good signal, anlotinib may be useful for halting or reversing the progression of liver fibrosis and could be employed in the development of novel therapeutic drugs for the management of chronic liver diseases.

Simulated microgravity altered the proliferation, apoptosis, and extracellular matrix formation of L929 fibroblasts and the transforming growth factor-ß1/Smad3 signaling pathway.

Exposure to microgravity can adversely affect the fitness of astronauts. The integrity of the skin plays a crucial role in protecting against mechanical forces and infections, fluid imbalance, and thermal dysregulation. In brief, the skin wound may cause unknown challenges to the implementation of space missions. Wound healing is a physiological process that relies on the synergistic action of inflammatory cells, extracellular matrix (ECM), and various growth factors to maintain the integrity of skin after trauma. Fibroblasts are present almost throughout the entire process of wound repair, especially in the scar formation at the endpoint of wound healing. However, there is limited knowledge about the extent to which fibroblasts are affected by the lack of gravity during wound healing. In this study, we utilized the rotary cell culture system, a ground-based facility that mimics the weightless condition, to study the alterations of L929 fibroblast cells under simulated microgravity (SMG). Our results demonstrated that the SM condition exerted negative influences on the proliferation and ECM formation of the L929 fibroblast. Whereas, the apoptosis of fibroblast was significantly upregulated upon exposure to SMG conditions. Moreover, the transforming growth factor-ß1/Smad3 (TGF-ß1/smad3) signaling pathway of L929 fibroblast related to wound repair was also altered significantly under a weightless environment. Overall, our study provided evidence that fibroblasts are strongly sensitive to SMG and elucidated the potential value of the TGF-ß1/Smad3 signaling pathway modulating wound healing in the future practice of space medicine.

Antifibrotic mechanism of avitinib in bleomycin-induced pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by alveolar epithelial cell injury and lung fibroblast overactivation. At present, only two drugs are approved by the FDA for the treatment of IPF, including the synthetic pyridinone drug, pirfenidone, and the tyrosine kinase inhibitor, nintedanib. Avitinib (AVB) is a novel oral and potent third-generation tyrosine kinase inhibitor for treating non-small cell lung cancer (NSCLC). However, the role of avitinib in pulmonary fibrosis has not yet been established. In the present study, we used in vivo and in vitro models to evaluate the role of avitinib in pulmonary fibrosis. In vivo experiments first verified that avitinib significantly alleviated bleomycin-induced pulmonary fibrosis in mice. Further in vitro molecular studies indicated that avitinib inhibited myofibroblast activation, migration and extracellular matrix (ECM) production in NIH-3T3 cells, mainly by inhibiting the TGF-ß1/Smad3 signalling pathways. The cellular experiments also indicated that avitinib improved alveolar epithelial cell injury in A549 cells. In conclusion, the present findings demonstrated that avitinib attenuates bleomycin-induced pulmonary fibrosis in mice by inhibiting alveolar epithelial cell injury and myofibroblast activation.

Exploration of the mechanism of NORAD activation of TGF-ß1/Smad3 through miR-136-5p and promotion of tacrolimus-induced renal fibrosis.

BACKGROUND: Tacrolimus is a potent immunosuppressant, but has various side effects, with nephrotoxicity being the most common. Renal fibrosis is an important process of tacrolimus nephrotoxicity. Therefore, it is important to identify the factors that contribute to renal fibrosis after tacrolimus nephrotoxicity, and control its development. METHODS: The present study aims to determine whether tacrolimus may speed up the course of renal fibrosis by upregulating noncoding RNA activated by DNA damage (NORAD) to compete with miR-136-5p, and activating the TGF-ß1/Smad3 pathway. Furthermore, in vivo rat models and in vitro cell models were established. Then, the expression levels of NORAD and miR-136-5p were determined by RT-qPCR, while the expression of the TGF-ß1/Smad3 pathway was determined by western blot and RT-qPCR. In order to investigate the interaction between NORAD and miR-136-5p, as well as miR-136-5p and SYK, two luciferase reporters were employed. The renal fibrosis of mice was observed using Masson and PAS staining. The expression of inflammatory factors IL-1, IL-6, MCP-1 and TNF-α was detected by ELISA. RESULTS: In the in vitro experiments, NORAD was upregulated, while miR-136-5p was downregulated after tacrolimus induction. The expression of the TGF-ß1/Smad3 pathway correspondingly changed after the induction by tacrolimus. In the in vivo experiments, the expression of NORAD and miR-136-5p, and the trend for renal fibrosis were consistent with the results in the in vitro experiments. Furthermore, the inflammatory factors correspondingly changed with the severity of renal fibrosis. Moreover, the expression trend of the TGF-ß1/Smad3 pathway in tacrolimus-induced rats was consistent with that in the in vitro experiments. CONCLUSION: Through in vitro and in vivo experiments, the present study was able to successfully prove that tacrolimus upregulates NORAD to compete with miR-136-5p, resulting in a decrease in miR-136-5p expression, which in turn activates the TGF-ß1/smad3 pathway, and finally induces the aggravation of renal fibrosis.

Trichinella spiralis-Secreted Products Promote Collagen Capsule Formation through TGF-ß1/Smad3 Pathway.

Trichinella spiralis (T. spiralis) muscle larvae colonize in the host's skeletal muscle cells, which are surrounded by collagen capsules. The mechanism underlying muscle stage larva-induced collagen capsule formation remains unknown. To clarify the mechanism, a T. spiralis muscular-infected mouse model was established by a single lateral tail vein injection with 20,000 T. spiralis newborn larvae (NBL). The infected mice were treated with or without SB525334 (TGF-ß1 receptor type I inhibitor). Diaphragms were obtained post-infection, and the expression levels of the TGF-ß1/Smad3 pathway-related genes and collagen genes (type IV and VI) were observed during the process of collagen capsule formation. The changes in myoblasts under stimulation of the excretory-secretory (ES) products of NBL with or without SB525334 were further investigated. Results showed that the expression levels of type IV collagen gene, type VI collagen gene, Tgfb1, and Smad3 were significantly increased in infected mice muscle cells. The expression levels of all the above genes were enhanced by the products of NBL in myoblast cells. These changes were reversed by co-treatment with SB525334 in vivo and in vitro. In conclusion, the TGF-ß1/Smad3 pathway can be activated by T. spiralis infection in muscle cells. The activated TGF-ß1/Smad3 pathway can stimulate the secretion of collagens by myocytes and plays a promoting role in the process of collagen capsule formation. The research has the limitation that the protein identification of the products of NBL has yet to be performed. Therefore, the specific components in the T. spiralis ES products that induce collagen synthesis should be further investigated.

Pterostilbene, a Resveratrol Derivative, Improves Ectopic Lipid Deposition in the Kidneys of Mice Induced by a High-Fat Diet.

BACKGROUND: Diabetic kidney disease is a major cause of global end-stage renal diseases. Ectopic lipid deposition in the renal tissues of diabetic kidney disease is one major factor leading to renal fibrosis and chronic kidney disease. Pterostilbene has been reported to display lipid-lowing activity and participate in many kidney diseases. However, the influence of pterostilbene on the ectopic lipid deposition is unclear. We intend to explore the influence of pterostilbene on the ectopic lipid deposition in the kidneys of mice induced by high fat. METHODS: A high-fat diet-induced diabetic mouse model was established to detect the alleviative effect of pterostilbene on the ectopic lipid deposition in the kidneys of diabetic mice. A biochemical analysis was performed to examine the levels of urine albumin, urine creatinine, serum creatinine, and blood urea nitrogen in mice after pterostilbene treatment. Histological analysis was conducted to detect the degree of renal injury and fibrosis. Oil red O staining and immunohistochemical staining were carried out to evaluate lipid droplets and the expression of adipose differentiation-related protein in renal tissues of the mice treated by pterostilbene. The protein levels were assessed by Western blotting. RESULTS: Pterostilbene inhibits the expression of the TGF-ß1 and p-smad3 and suppresses the protein levels of SREBP-1 and FAS, and it ultimately reduces the ectopic lipid deposition, alleviates the renal tubular damage and renal fibrosis in the kidneys of diabetic mice induced by high fat, and improves kidney function. CONCLUSION: Pterostilbene alleviates renal fibrosis and ectopic lipid deposition in the kidneys of diabetic mice induced by high-fat diet by inhibiting the TGF-ß1/smad3 signaling.

Dimethyl fumarate ameliorates stress urinary incontinence by reversing ECM remodeling via the Nrf2-TGF-ß1/Smad3 pathway in mice.

INTRODUCTION AND HYPOTHESIS: Mechanical trauma and oxidative injury are involved in the pathogenesis of stress urinary incontinence (SUI), and oxidative stress (OS) is considered a potential therapeutic target. The antioxidant properties of dimethyl fumarate (DMF), a potent activator of Nrf2, have been highlighted recently. We therefore predicted that DMF might have therapeutic effects on mechanical trauma-induced SUI. METHODS: The SUI mice model was established by vaginal distension (VD). Leak point pressure (LPP), serum OS biomarkers, cell proliferation and apoptosis, collagen, elastin, matrix metalloproteinases (MMP), Nrf2, the TGF-ß1/Smad3 signaling pathway, and the associated tissue growth factors in the anterior vaginal wall were measured in either wild-type or Nrf2-knockout (Nrf2-/-) female C57BL/6 mice. RESULTS: The results showed that DMF improved the VD-induced LPP reduction, alleviated oxidative injury, stimulated cell proliferation and inhibited apoptosis in the anterior vaginal wall tissue of mice. Moreover, DMF treatment reduced the hydrolysis of ECM proteins by MMP2 and MMP9. The above effects may be mediated by a series of tissue growth factors, including α-SMA, PAI-1, and TIMP-2, with the TGF-ß1/Smad3 signaling pathway as the core regulatory mechanism. In further study, Nrf2-/- mice were used to replicate the SUI model. And the difference is that DMF failed to reactivate the TGF-ß1/Smad3 pathway, nor did it improve LPP. CONCLUSIONS: Dimethyl fumarate can ameliorate urethra closure dysfunction in the VD-induced SUI mice model, and the therapeutic effect of DMF is mediated by the Nrf2-dominated antioxidant system and its downstream TGF-ß1/Smad3 signaling pathway.

The impact of empagliflozin on cardiac physiology and fibrosis early after myocardial infarction in non-diabetic rats.

BACKGROUND: Myocardial fibrosis is a multistep process, which results in collagen deposition in the injured muscle. Empagliflozin, a sodium-glucose cotransporter 2 inhibitor (SGLT2i), decreases cardiovascular events risk. Little is known on the effects of empagliflozin in non-diabetic patients early post myocardial infarction. METHODS: Fourteen non-diabetic rats underwent myocardial infarction induction, and treated or not (control)immediately after myocardial infarction by daily empagliflozin (30 mg/kg/day). We evaluated cardiac function at baseline, 2 and 4 weeks after myocardial infarction by echocardiography, and prior to sacrifice by Millar pressure-volume system. We performed histological and biochemical evaluation of fibrosis and humoral factors promoting fibrosis. RESULTS: Baseline ejection fractions were 69.9 ± 5.3% and 76.4 ± 5.4%, and dropped to final values of 40.1 ± 5.8% and 39.4 ± 5.4% in the control and empagliflozin groups, respectively (P < 0.001 vs. baseline, P > 0.05 between groups). Collagen deposition, measured as collagen volume fraction, was higher in both the scar and the remote cardiac areas of the control group 79.1 ± 6.2% and 4.6 ± 2.5% for control, and 53.8 ± 5.4% and 2.5 ± 1.3% for empagliflozin group, respectively (P < 0.05 for each). Remote cardiac muscle collagen, measured by hydroxyproline, was 4.1 ± 0.4 µg/µl and 3.6 ± 0.2 µg/µl (P = 0.07). TGF-ß1 and Smad3 expression decreased by empagliflozin-18.73 ± 16.32%, 9.16 ± 5.69% and 16.32 ± 5.4%, 7.00 ± 5.28% in the control and empagliflozin groups, respectively (P < 0.05). CONCLUSION/INTERPRETATION: Empagliflozin administered early after myocardial infarction reduce myocardial fibrosis and inhibit the TGF-ß1/Smad3 fibrotic pathway, probably prior to exerting any hemodynamic or physiological effect.

LINC01116 regulates proliferation, migration, and apoptosis of keloid fibroblasts by the TGF-ß1/SMAD3 signaling via targeting miR-3141.

BACKGROUND: Keloids are benign fibroproliferative skin tumors. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of keloid formation. In this paper, we explored the precise actions of LINC01116 in keloid formation. METHODS: The targeted relationship between microRNA (miR)-3141 and LINC01116 or transforming growth factor ß1 (TGF-ß1) was verified by dual-luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays. The expression levels of LINC01116, miR-3141, TGF-ß1, and SMAD family member 3 (SMAD3) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Cell proliferation, migration, and apoptosis were assessed by the Cell Counting Kit-8 (CCK-8) assay, wound-healing assay, and flow cytometry, respectively. Animal studies were used to assess the role of LINC01116 in the subcutaneous keloid growth in vivo. RESULTS: Our data showed that LINC01116 targeted miR-3141 by directly binding to miR-3141. LINC01116 was up-regulated and miR-3141 was down-regulated in human keloid tissues and fibroblasts. LINC01116 knockdown or miR-3141 overexpression suppressed keloid fibroblast proliferation, migration, and promoted cell apoptosis. Moreover, miR-3141 was a downstream mediator of LINC01116 function. MiR-3141 regulated the TGF-ß1/SMAD3 signaling by directly targeting TGF-ß1. Furthermore, TGF-ß1 was identified as a direct and functional target of miR-3141. LINC01116 regulated the TGF-ß1/SMAD3 signaling through miR-3141. Additionally, LINC01116 knockdown diminished the subcutaneous keloid growth in vivo. CONCLUSION: Our findings demonstrated a novel mechanism, the miR-3141/TGF-ß1/SMAD3 regulatory pathway, at least partially for the oncogenic role of LINC01116 in keloid formation.

ß-acetoxyisovaleryl alkannin (AAN-II) from Alkanna tinctoria promotes the healing of pressure-induced venous ulcers in a rabbit model through the activation of TGF-ß/Smad3 signaling.

Alkannin-based pharmaceutical formulations for improving wound healing have been on the market for several years. However, detailed molecular mechanisms of their action have yet to be elucidated. Here, we investigated the potential roles of AAN-II in improving the healing of pressure-induced venous ulcers using a rabbit model generated by combining deep vein thrombosis with a local skin defect/local skin defect. The extent of healing was evaluated using hematoxylin and eosin (HE) or vimentin staining. Rabbit skin fibroblasts were cultured for AAN-II treatment or TGFB1-sgRNA lentivirus transfection. ELISA was used to evaluate the levels of various cytokines, including IL-1ß, IL-4, IL-6, TNF-α, VEGF, bFGF, TGF-ß and PDGF. The protein levels of TGF-ß sensors, including TGF-ß, Smad7 and phosphor-Smad3, and total Smad3, were assayed via western blotting after TGF-ß knockout or AAN-II treatment. The results show that, for this model, AAN-II facilitates ulcer healing by suppressing the development of inflammation and promoting fibroblast proliferation and secretion of proangiogenic factors. AAN-II enhances the activation of the TGF-ß1-Smad3 signaling pathway during skin ulcer healing. In addition, the results demonstrate that AAN-II and TGF-ß have synergistic effects on ulcer healing. Our findings indicate that AAN-II can promote healing of pressure-induced venous skin ulcers via activation of TGF-ß-Smad3 signaling in fibroblast cells and provide evidence that could be used in the development of more effective treatments.

A novel role of VEPH1 in regulating AoSMC phenotypic switching.

Abdominal aortic aneurysm (AAA) is a potentially lethal disease featured by focal dilatation in the aorta. The transition of vascular smooth muscle cells (SMCs) from a contractile/differentiated to a synthetic/dedifferentiated phenotype is considered to contribute to AAA formation and expansion. Our previous gene microarray data showed that Ventricular Zone Expressed PH Domain Containing 1 (VEPH1) expression increased in angiotensin II (Ang II)-infused aortic tissues. This study was thus performed to further explore the role of VEPH1. Herein, we first demonstrate that VEPH1 increases in the SMCs of Ang II-treated abdominal aortas. As in vivo, Ang II also upregulated VEPH1 expression in cultured hAoSMCs. The dedifferentiation of human aortic SMCs (hAoSMCs) was induced by a 24-hr stimulation of Ang II (1 µM)-the expression of contractile SMC markers, MYH11 and α-smooth muscle actin (α-SMA) decreased and that of synthetic markers, proliferating cell nuclear antigen and Vimentin increased. Inhibition of VEPH1 prevented Ang II-induced pathological dedifferentiation of hAoSMCs as indicated by the restored expression of MYH11 and α-SMA. In contrast, the forced overexpression of VEPH1 aggravated Ang II's effects. Furthermore, we demonstrated that VEPH1 and transforming growth factor-ß1 (TGF-ß1), a key regulator responsible for vascular SMC differentiation, negatively regulated each other's transcription. In contrast to VEPH1 silencing, its overexpression inhibited recombinant TGF-ß1-induced increases in MYH11 and α-SMA and suppressed Smad3 phosphorylation and nuclear accumulation. Collectively, our study demonstrates that VEPH1 elevation promotes the synthetic phenotype switching of AoSMCs and suppressed the TGF-ß1/Smad3 signaling pathway. Identification of VEPH1 as a pathogenic molecule for AAA formation provides novel insights into this disease.

LncRNA SOX2OT/Smad3 feedback loop promotes myocardial fibrosis in heart failure.

Long noncoding RNA SOX2OT is associated with myocardial fibrosis (MF) in heart failure (HF). This article aims to investigate the role of SOX2OT in MF. We constructed HF mouse models by subcutaneous injection of isoprenaline (ISO). Cardiac fibroblasts (CFs) were treated with ISO to induce MF.Hematoxylin-eosin, Masson, and Sirius-red staining were used to identify myocardial injury and collagen deposition in heart tissues. The relationship among SOX2OT, miR-138-5p, TGF-ß1, and Smad3 were evaluated by chromatin immunoprecipitation and luciferase reporter assay. The gene and protein expression were verified by quantitative real-time PCR and western blot. We found that SOX2OT was up-regulated in HF mice and ISO-induced CFs. SOX2OT knockdown reduced myocardial injury and collagen deposition in HF mice. The expression of collagen I, α-SMA, TGF-ß1, and p-Smad3 were inhibited by SOX2OT down-regulation in HF mice and ISO-induced CFs. Furthermore, TGF-ß1 was a target gene of miR-138-5p and indirectly regulated by SOX2OT. SOX2OT promoted MF in HF by activating TGF-ß1/Smad3, and then Smad3 interacted with the SOX2OT promoter and formed a positive feedback loop. In conclusion, our work verifies that SOX2OT/Smad3 feedback loop promotes MF in HF. Thus, SOX2OT is potentially a novel therapeutic target for MF in HF.

FOXF1 ameliorates angiotensin II-induced cardiac fibrosis in cardiac fibroblasts through inhibiting the TGF-ß1/Smad3 signaling pathway.

Cardiac fibrosis is a pathological feature common to a variety of heart diseases such as myocardial infarction, arrhythmias, cardiomyopathies and heart failure. The molecular mechanism underlying the cardiac fibrosis is still unclear. Forkhead box F1 (FOXF1), a member of the forkhead transcription factor superfamily, plays critical roles in the development of hepatic fibrosis. However, whether FOXF1 is involved in the pathogenesis of cardiac fibrosis remains to be elucidated. The present study aimed to investigate the role of FOXF1 and its mechanisms in regulating cardiac fibrosis. The results demonstrated that FOXF1 was downregulated in Ang II-induced CFs. Overexpression of FOXF1 inhibited angiotensin II (Ang II)-induced proliferation, migration and oxidative stress in cardiac fibroblasts (CFs). Overexpression of FOXF1 also reduced the expression of alpha-smooth muscle actin (a-SMA) in Ang II-induced CFs, suggesting that overexpression of FOXF1 prevented the differentiation of CFs to myofibroblasts. Furthermore, the production of extracellular matrix (ECM) components including type I collagen and fibronectin were reduced by overexpression of FOXF1 in Ang II-induced CFs. Furthermore, overexpression of FOXF1 prevented Ang II-induced activation of transforming growth factor beta 1 (TGF-ß1)/Smad3 pathway in CFs. In conclusion, the results of the present study indicated that FOXF1 acted as a key regulator of pathological cardiac fibrosis, and overexpression of FOXF1 ameliorated cardiac fibrosis by inhabiting the TGF-ß1/Smad3 signaling pathway. These results indicated that FOXF1 may be a novel target for attenuating cardiac fibrosis.

MiR-424 Inhibits neuronal apoptosis in rats with cerebral infarction through regulating TGF-ß1/Smad3 signaling pathway.

It has been reported that micro ribonucleic acid (miR)-424 is an important molecule in cerebral ischemia. However, the precise mechanism of action and biological effects of miR-424 remain to be further explored. miR-424 mimic and miR-424 inhibitor were injected via the caudal vein in rats, and the effect of miR-424 expression on brain tissue damage induced by middle cerebral artery occlusion (MCAO) was detected. The miR-424 mimic-induced changes in genomic levels were detected via the gene chip assay, and the signaling pathways regulated by miR-424 and its potential targets were explored combined with target prediction. Then the effect of miR-424 mimic on apoptosis of PC12 cells induced by oxygen-glucose deprivation (OGD) was determined using Annexin V/PI assay. Finally, drosophila mothers against decapentaplegic protein 7 (Smad7) was overexpressed to further verify the mechanism of action of miR-424 mimic. Compared with that in the sham group, the expression of miR-424 in brain tissues significantly declined in the model group. The results of 2,3,5-triphenyltetrazolium chloride (TTC) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed that the miR-424 mimic obviously reduced the cerebral infarction area and apoptosis level of brain tissues, while the miR-424 inhibitor obviously increased the cerebral infarction area and apoptosis level of brain tissues. It was found, using bioinformatics and KEGG enrichment analysis, that differentially expressed genes induced by miR-424 were significantly enriched in the transforming growth factor-ß (TGF-ß) signaling pathway. According to the results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting, the miR-424 mimic could evidently lower the expression of Smad7, thus activating the TGF-ß1/Smad3 signaling pathway. Overexpression of Smad7 could weaken the protective effect of miR-424 mimic on ischemic-hypoxic cells. Increasing the expression of miR-424 can inhibit Smad7 to activate the TGF-ß1/Smad3 signaling pathway, thereby exerting a protective effect against the brain tissue damage induced by MCAO.