Smad Pathway Research Articles

Estrogen receptor β attenuates renal fibrosis by suppressing the transcriptional activity of Smad3

Renal fibrosis (RF) is a common pathway leading to chronic kidney disease (CKD), which lacks effective treatment. While estrogen receptor beta (ERβ) is known to be present in the kidney, its role in RF remains unclear. The present study aimed to investigate the role and underlying mechanism of ERβ during RF progression in patients and animal models with CKD. We found that ERβ was highly expressed in the proximal tubular epithelial cells (PTECs) in healthy kidneys but its expression was largely lost in patients with immunoglobin A nephropathy (IgAN) and in mice with unilateral ureter obstruction (UUO) and subtotal nephrectomy (5/6Nx). ERβ deficiency markedly exacerbated, whereas ERβ activation by WAY200070 and DPN attenuated RF in both UUO and 5/6Nx mouse models, suggesting a protective role of ERβ in RF. In addition, ERβ activation inhibited TGF-β1/Smad3 signaling, while loss of renal ERβ was associated with overactivation of the TGF-β1/Smad3 pathway. Furthermore, deletion or pharmacological inhibition of Smad3 prevented the loss of ERβ and RF. Mechanistically, activation of ERβ competitively inhibited the association of Smad3 with the Smad-binding element, thereby downregulating the transcription of the fibrosis-related genes without altering Smad3 phosphorylation in vivo and in vitro. In conclusion, ERβ exerts a renoprotective role in CKD by blocking the Smad3 signaling pathway. Thus, ERβ may represent as a promising therapeutic agent for RF.

PDLIM2 can inactivate the TGF-β/Smad pathway to inhibit the malignant behavior of ovarian cancer cells.

PDZ-LIM domain-containing Protein 2 (PDLIM2) has been reported to be downregulated in ovarian cancer. However, its exact function and mechanism in regulating ovarian cancer progression have not been elucidated. This work researched the exert effect and mechanism of PDLIM2 on ovarian cancer progression. Briefly, PDLIM2 expression in clinical tissues of ovarian cancer patients and cells was investigated by qRT-PCR and Western blot. The function of PDLIM2 on the proliferation, colony formation, migration and invasion of ovarian cancer cells was explored via cell counting kit-8, colony formation and Transwell assays. To verify whether PDLIM2 regulates ovarian cancer progression via regulating the transforming growth factor-β (TGF-β)/Smad pathway, exogenous TGF-β (10 ng/mL) treatment was performed on the PDLIM2-overexpressed ovarian cancer cells. PDLIM2 effect on the in vivo growth of ovarian cancer cells was researched by establishing a xenograft tumor model. Immunohistochemistry and Western blot were performed to protein expression in cells and tissues. As a result, PDLIM2 was low-expressed in ovarian cancer tissues/cells. PDLIM2 upregulation attenuated the proliferation, colony formation, migration, invasion and epithelial-mesenchymal transition (EMT) of ovarian cancer cells, and inactivated the TGF-β/Smad pathway. The opposite results were found in the PDLIM2-silenced ovarian cancer cells. Exogenous TGF-β treatment abrogated the inhibition of PDLIM2 on the malignant behavior of ovarian cancer cells. PDLIM2 upregulation attenuated the in vivo growth and EMT of ovarian cancer cells. Thus, PDLIM2 attenuates the proliferation, migration, invasion and EMT of ovarian cancer cells via inactivating the TGF-β/Smad pathway. PDLIM2 may be a usefully target for ovarian cancer treatment.

Early growth response factor 1 upregulates pro-fibrotic genes through activation of TGF-β1/Smad pathway via transcriptional regulation of PAR1 in high-glucose treated HK-2 cells

Tubulointerstitial fibrosis (TIF) makes a key role in diabetic kidney disease (DKD). In this study, we revealed that the expressions of Egr1 and protease-activated receptor 1 (PAR1) were increased in renal tissues of DKD rats. In vitro experiments demonstrated that both Egr1 overexpression and high glucose (HG) condition could promote the expressions of PAR1, fibronectin (FN) and collagen I (COL I). Furthermore, HG stimulation enhanced the binding capacity of Egr1 to PAR1 promoter. Both HG condition and Egr1 upregulation could increase, and thrombin inhibitor did not affect activity of TGF-β1/Smad pathway via PAR1. Collectively, Egr1 is involved in TIF of DKD partly through activating TGF-β1/Smad pathway via transcriptional regulation of PAR1 in HG treated HK-2 cells.

MiR-135a Regulates Atrial Fibrillation by Targeting Smad3.

Atrial fibrillation (AF) is the most common arrhythmia in clinical. Atrial fibrosis is a hallmark feature of atrial structural remodeling in AF, which is regulated by the TGF-β1/Smad3 pathway. Recent studies have implicated that miRNAs are involved in the process of AF. However, the regulatory mechanisms of miRNAs remain largely unknown. This study is aimed at investigating the function and regulatory network of miR-135a in AF. In vivo, the plasma was collected from patients with AF and non-AF subjects. Adult SD rats were induced by acetylcholine (ACh) (66 μg/ml)-CaCl2 (10 mg/ml) to establish an AF rat model. In vitro, atrial fibroblasts (AFs), isolated from adult SD rats, were treated with high-frequency electrical stimulation (HES) (12 h) and hypoxia (24 h) to mimic the AF and atrial fibrosis, respectively. miR-135a expression was detected through quantitative real-time polymerase chain reaction (qRT-PCR). The association between miR-135a and Smad3 was speculated by the TargetScan database and confirmed by the luciferase reporter assay. Fibrosis-related genes, Smad3, and TRPM7 were all assessed. The expression of miR-135a was markedly decreased in the plasma of AF patients and AF rats, which was consistent with that in HES-treated and hypoxia-treated AFs. Smad3 was identified as a target of miR-135a. the downregulation of miR-135a was associated with the enhancement of Smad3/TRPM7 expressions in AFs. Additionally, the knockdown of Smad3 significantly reduced the expression of TRPM7 and further inhibited atrial fibrosis. Our study demonstrates that miR-135a regulates AF via Smad3/TRPM7, which is a potential therapeutic target for AF.

Dengzhan Shengmai capsule attenuates cardiac fibrosis in post-myocardial infarction rats by regulating LTBP2 and TGF-β1/Smad3 pathway

BackgroundCardiac fibrosis contributes to myocardial remodeling after myocardial infarction (MI), which may facilitate the progression to end-stage heart failure. Dengzhan Shengmai capsule (DZSMC), a traditional Chinese formula derived from Shen-mai powder, has shown remarkable therapeutic effects against cardiovascular diseases. However, the effect of DZSMC on cardiac fibrosis and its potential mechanism are ill-defined. PurposeTo evaluate the effects of DZSMC on cardiac fibrosis after myocardial infarction (MI) and investigate its underlying mechanism. MethodIn vivo, MI rat models were established by permanently ligation of left anterior descending coronary arteries (LAD) and then were intragastrically treated with DZSMC or captopril for 5 weeks. Ex vivo, an everted intestinal sac model was used to study the intestinal absorption components of DZSMC, which were further identified through an ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method. In vitro, a myocardium fibrotic model was constructed by stimulating primary cardiac fibroblasts (CFs) with 1 μM Ang II. Subsequently, the absorbent solution of DZSMC from the intestinal sac was performed on the cell models to further elucidate its anti-fibrotic effects and underling mechanism. ResultsIn vivo results showed that DZSMC significantly improved cardiac function and inhibited pathological myocardial fibrosis in post-MI rats in a dose dependent manner. Histological analysis and western blot results demonstrated that DZSMC treatment significantly reduced the expression of extracellular matrix (ECM)-related proteins, including LTBP2, TGF-βR1, Smad3 and pSmad3, in myocardial tissue of MI rats. Ex vivo results showed that 18 absorbed components were identified, mainly consisting of phenolic acids, flavonoids and lignans, which may be responsible for the anti-fibrotic effects. Further in vitro results validated that DZSMC attenuated myocardial fibrosis by suppressing the expression of LTBP2, TGF-β1 and pSmad3. ConclusionDZSMC ameliorates cardiac function and alleviates cardiac fibrosis, which may be mediated by inhibition of CFs activation and reduction of excessive ECM deposition via LTBP2 and TGF-β1/Smad3 pathways.

Geniposide alleviates pressure overload in cardiac fibrosis with suppressed TGF-β1 pathway

BackgroundCardiac fibrosis is one of the main contributors to the pathogenesis of heart failure. Geniposide (GE), a major iridoid in gardenia fruit extract, has recently been reported to improve skeletal muscle fibrosis through the modulation of inflammation response. This investigation aimed to illuminate the cardio-protective effect and the potential mechanism of GE in cardiac fibrosis. Material and methodsA transverse aortic contraction (TAC) induction mice model was established and GE (0 mg/kg; 10 mg/kg; 20 mg/kg; 40 mg/kg) was administered by oral gavage daily for 4 weeks. Hemodynamic parameters, Masson’s trichrome stain, and hematoxylin-eosin (HE) staining were estimated and cardiomyocyte fibrosis, interstitial collagen levels, and hypertrophic markers were analyzed using qPCR and western blot. In vitro, H9C2 cells were exposed to the Ang II (1 μM) pretreated with GE (0.1 μM, 1 μM, and 10 μM). Cardiomyocyte apoptosis was detected. Moreover, the transforming growth factor β1 (TGF-β1)/Smad2 pathway was assessed in vivo and in vitro. ResultsGE significantly ameliorated TAC-induced cardiac hypertrophy, ventricular remodeling, myocardial fibrosis, and improved cardiac function in vivo, and it inhibited Ang II-induced cardiomyocyte apoptosis in vitro. We further observed that the inflammatory channel TGF-β1/Smad2 pathway was suppressed by GE both in vivo and in vitro. ConclusionThese results indicate that GE inhibited myocardial fibrosis and improved hypertrophic cardiomyocytes with attenuated the TGF-β1/Smad2 pathway and proposed to be an important therapeutic of cardiac fibrosis reduced by TAC.

Activation of Kir2.1 improves myocardial fibrosis by inhibiting Ca 2+ overload and the TGF-β1/Smad signaling pathway.

The inwardly rectifying potassium channel Kir2.1 is closely associated with many cardiovascular diseases. However, the effect and mechanism of Kir2.1 in diabetic cardiomyopathy remain unclear. In vivo, we use STZ to establish the model, and ventricular structural changes, myocardial inflammatory infiltration, and myocardial fibrosis severity are detected by echocardiography, histological staining, immunohistochemistry, and western blot analysis, respectively. In vitro, a myocardial fibrosis model is established with high glucose. The Kir2.1 current amplitude, intracellular calcium concentration, fibrosis-related proteins, and TGF-β1/Smad pathway proteins are detected by whole-cell patch clamp, calcium probes, western blot analysis, and immunofluorescence, respectively. The in vivo results show that compared to diabetic cardiomyopathy, zacopride (a Kir2.1 selective agonist) significantly reduces the left ventricular systolic diameter and diastolic diameter, increases the left ventricular ejection fraction and left ventricular short-axis shortening, improves the degree of cell necrosis, and reduces the expression of myocardial interstitial fibrosis protein and collagen fibre deposition area. The in vitro results show that the current amplitude and protein expression of Kir2.1 are both decreased in the high glucose-induced myocardial fibrosis model. Additionally, zacopride significantly upregulates the expression of Kir2.1 and inhibits the expressions of the fibrosis-related proteins α-SMA, collagen I, and collagen III. Activation of Kir2.1 reduces the intracellular calcium concentration and inhibits the protein expressions of TGF-β1 and p-Smad 2/3. Activation of Kir2.1 can improve myocardial fibrosis induced by diabetic cardiomyopathy, and the possible mechanism may be related to inhibiting Ca 2+ overload and the TGF-β1/Smad signaling pathway.

Laminaria japonica polysaccharide attenuates podocyte epithelial-mesenchymal transformation via TGF-β1-mediated Smad3 and p38MAPK pathways

In the present work, we explored the interventional effect and potential mechanism of a purified Laminaria japonica polysaccharide (LJP61A) on podocyte epithelial-mesenchymal transition (EMT) in TGF-β1-induced podocytes and adriamycin-treated mice. Results showed that compared to the model groups, LJP61A significantly up-regulated the levels of epithelial markers (Nephrin, WT-1, podocin) and down-regulated the levels of mesenchymal markers (α-SMA, FN1) in vitro and in vivo, thus preventing EMT-like morphological changes of podocytes, proteinuria and kidney injury. Smad3 and p38MAPK are two central pathways mediating podocyte EMT activated by TGF-β1. We found that LJP61A suppressed TGF-β1-induced activation of Smad3, Smad4 and p38MAPK in vitro and in vivo. Moreover, the inhibitory actions of LJP61A on podocyte EMT were synergistically strengthened by Smad3 inhibitor SIS3 and p38MAPK inhibitor SB203580. Taken together, these findings revealed that LJP61A could prevent podocyte EMT, which might be related to the inhibition of TGF-β1-mediated Smad3 and p38MAPK pathways.

Retracted] TGF‑β1 induces peritoneal fibrosis by activating the Smad2 pathway in mesothelial cells and promotes peritoneal carcinomatosis.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the fluorescence microscopy data shown in Fig.6A and B were strikingly similar to data appearing in different form in Fig.7 in a previously published paper [LvZ‑D, NaD, LiuF‑N, DuZ‑M, SunZ, Li Z, MaX‑Y, WangZ‑N and XuH‑M: Induction of gastric cancer cell adhesion through transforming growth factor‑beta1‑mediated peritoneal fibrosis. J Exp Clin Cancer Res29: 139, 2010], which featured some of the same authors, although the data were shown to portray results obtained under different experimental conditions. Furthermore, the data in Fig.7A for the 'TGF‑β1' and the 'TGF‑β1+siRNAcon' experiments contained an overlapping section, such that these data appeared to have been derived from the same original source, even though they were intended to show the results from differently performed experiments. Owing to the fact that the contentious data in the above article had already been published prior to its submission to International Journal of Molecular Medicine, and due to a lack of overall confidence in the presented data, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 29: 373‑379, 2012; DOI: 10.3892/ijmm.2011.852].

MitoQ ameliorates PM2.5-induced pulmonary fibrosis through regulating the mitochondria DNA homeostasis

Pulmonary fibrosis is a severe pulmonary disease, and may related to PM2.5 exposure. Our study aims to explore the pathogenesis of PM2.5-induced pulmonary fibrosis, and MitoQ protective effect in this process. Our results find that inflammatory cells aggregation and pulmonary fibrosis in mice lung after PM2.5 exposure. Moreover, Collagen I/III overproduction, EMT and TGF-β1/Smad2 pathway activation in mice lung and BEAS-2B after PM2.5 exposure. Fortunately, these changes were partially ameliorated after MitoQ treatment. Meanwhile, severe oxidative stress, mitochondrial homeostasis imbalance, overproduction of 8-oxoG (7,8-dihydro-8-oxoguanine), as well as the inhibition of SIRT3/OGG1 pathway have founded in mice lung or BEAS-2B after PM2.5 exposure, which were alleviated by MitoQ treatment. Collectively, our study found that oxidative stress, especially mitochondrial oxidative stress participates in the PM2.5-induced pulmonary fibrosis, and MitoQ intervention had a protective effect on this progress. Moreover, mitochondrial DNA homeostasis might participate in the pulmonary fibrosis caused by PM2.5 exposure. Our study provides a novel pathogenesis of PM2.5-caused pulmonary fibrosis and a possible targeted therapy for the pulmonary diseases triggered by PM2.5.

Novel antiosteoporotic peptides purified from protein hydrolysates of taihe black-boned silky fowl: By larval zebrafish model and molecular docking

The black-boned silky fowl (BSF) muscle protein hydrolysate was gained by alcalase. The hydrolysate could stimulate MC3T3-E1 cell proliferation, as well as enhance alkaline phosphatas (ALP) activity and deposits of minerals. After isolation and purification, 55 peptide sequences with Mascot score over 40 were identified. Combined with molecular docking simulation and molecular dynamics analysis, two novel peptides (PASTGAAK and PGPPGTPF) were identified with the lowest binding energy of −4.99 kcal/mol and −3.07 kcal/mol with receptor BMPR1A of BMP-2/Smad pathway, showing the ability to increase BMPR1A stability. Moreover, both PASTGAAK and PGPPGTPF revealed strong anti-osteoporosis activities in the zebrafish model induced by dexamethasone. Additionally, the identified peptides could be beneficial for the differentiation of MC3T3-E1 cell for upregulating the expression of some osteoblast-related genes and proteins by stimulating BMP-2/Smad pathway. Overall, the two newly identified peptides could be the potential candidate to prevent osteoporosis.

SAA1 deficiency alleviates cardiac remodeling by inhibiting NF-κB/p38/JNK and TGFβ/Smad pathways.

Heart failure (HF) is the end stage of the progression of many cardiovascular diseases. Cardiac remodeling is the main pathophysiological process of cardiac function deterioration in HF patients. Inflammation is a key factor that stimulates cardiomyocyte hypertrophy, fibroblast proliferation, and transformation leading to myocardial remodeling, which severity is significantly related to the prognosis of patients. SAA1 (Serum amyloid A1) is a lipid-binding protein that was an important regulator involved in inflammation, whose biological functions in the heart remain rarely known. In this research, we intended to test the role of SAA1 in SAA1-deficient (SAA1-/- ), and wild-type mice were exposed to transverse aortic banding surgery to establish the model of cardiac remodeling. Besides, we assessed the functional effects of SAA1 on cardiac hypertrophy and fibrosis. The expression of SAA1 was increased in the mice transverse aortic banding model induced by pressure overload. After 8 weeks of transverse aortic banding, SAA1-/- mice displayed a lower level of cardiac fibrosis than wild-type mice, but did not significantly influence the cardiomyocyte hypertrophy. In addition, there was also no significant difference in cardiac fibrosis severity between wild-type-sham and knockout-sham mice. These findings are the first to reveal SAA1 absence hinders cardiac fibrosis after 8 weeks of transverse aortic banding. Furthermore, SAA1 deficiency had no significant effect on cardiac fibrosis and hypertrophy in the sham group in this study.

Abstract 3723: Identification and functional characterization of long noncoding RNAs that regulate TGF-<&946;>-Smad pathway as Smad co-factors

Abstract TGF-β-Smad pathway participates in various biological processes such as development, differentiation, growth regulation, and cancer progression including epithelial-mesenchymal transition (EMT). Long non-coding RNAs (lncRNAs) also involved in carcinogenesis and cancer malignancies. To identify lncRNAs regulating TGF-β-Smad pathway, we comprehensively identified lncRNAs induced by TGF-β and found and reported a lncRNA ELIT-1 (EMT-associated lncRNA induced by TGF-β-1) which was induced by TGF-β and promoted EMT by facilitating TGF-β-Smad signaling. ELIT-1 bound to Smad and participated in expression of TGF-β target genes including Snail. Prognosis of lung adenocarcinoma and gastric cancer patients with high expression of ELIT-1 was poor suggesting that ELIT-1 may be useful as a novel prognostic and therapeutic target. Recently, we found that lincNMR (long intergenic noncoding RNA-nucleotide metabolism regulation) was induced via TGF-β-Smad pathway in various cell lines. Moreover, RNA-seq analysis using lincNMRs-depleted cells indicated that lincNMRs was involved in expression of APOBEC3B, a cytidine deaminase, promoting C to U mutation and highly expressed in various human cancers. Although, regulatory mechanisms of APOBEC3B expression have not been fully elucidated, we proved that lincNMRs bound to Smad and promoted the activity of APOBEC3B promoter. These data suggest that lincNMRs participate in APOBEC3B expression by collaborating with TGF-β-Smad pathway. Additionally, high expression of lincNMRs was positively correlated with high expression of APOBEC3B in various cancer cell lines. The high expression of APOBEC3B as well as lincNMR was significantly associated with their poor prognosis in liver and lung cancer patients, suggesting that lincNMR may contribute to tumor malignancy by enhanced expression of APOBEC3B via TGF-β-Smad pathway. Citation Format: Masatoshi Kitagawa, Kosuke Ota, Tatsuya Ohhata, Satoshi Sakai. Identification and functional characterization of long noncoding RNAs that regulate TGF-<&946;>-Smad pathway as Smad co-factors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3723.

Abstract 3819: Naringin nanoparticles inhibit metastasis of lung cancer through TGF-β1-smad2/3 signaling pathway

Abstract Lung cancer causes high mortality due to its high metastasis. Naringin is a naturally occurring flavonoid that is widely found in the various citrus fruits. Naringin is known to have several therapeutic effects including anticancer, anti-pulmonary fibrosis, antimicrobial, anti-inflammatory. However, low bioavailability and solubility of naringin limit its application. This project designed a thermosensitive naringin nanoparticle to investigate whether it might inhibit metastasis of lung cancer by inhibiting TGF-β1-smad2/3 signaling pathway. Cells were treated with various concentrations of naringin nanoparticles and unwrapped naringin for 48 hours. Naringin nanoparticles of 100µmol/L showed obvious inhibitory effect on the larger wound surface of cultured human cancer cells. Westerm blot was used to detect the expression of P-Smad2, Smad2,and α-SMA in A549 cells. The results showed that naringin significantly reduced the expression of EMT-related protein α-SMA in A549 cells in a concentration-dependent manner. Naringin nanoparticles can reverse the EMT process. naringin nanoparticles can inhibit the expression of P-Smad2 protein in A549 cells, which may play a role in inhibiting lung cancer through TGF-β1/Smad signaling pathway.The animal experiment was performed. The mice were given various concentrations of naringin nanoparticles and unwrapped naringin by IV. the protein expression levels of α-SMA, TGF-β1 and P-Smad3 in mice lung tissue were detected by immunohistochemistry. The results showed that naringin nanoparticles 50um/ml,0.2mL/day/time had significant effect on the inhibition of lung cancer tissue migration, and the different doses of naringin nanoparticles showed a certain dose-dependent trend. The expression of TGF-β1 and P-Smad3 in mice treated with naringin nanoparticles was significantly decreased, suggesting that naringin nanoparticles could inhibit the migration of lung cancer cells by inhibiting TGF-β1/Smad pathway. Citation Format: Wen Chen. Naringin nanoparticles inhibit metastasis of lung cancer through TGF-β1-smad2/3 signaling pathway. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3819.

Bone mesenchymal stem cells promote gastric cancer progression through TGF-β1/Smad2 positive feedback loop

AimsBone marrow-derived mesenchymal stem cells (BMSCs) have been proven to be recruited into the tumor microenvironment and contribute to gastric cancer (GC) progression, but the underlying mechanism is still unclear. The purpose of this study is to explore the exact role and potential mechanism of BMSCs in the progression of GC. Materials and methodsBioinformatics analyzed were used to clarify the correlation between TGF-β1 and prognosis of gastric cancer. Cell co-culture were used to explore the interaction between gastric cancer cells (GCs) and BMSCs. Quantitative real time-PCR and Western blot assay were used to detect gene and protein expression, respectively. The biological characteristics of GCs and BMSCs were detected by immunofluorescence, Transwell migration, Elisa and invasion assay. Xenograft models in nude mice were constructed to evaluate GC development in vivo. Key findingsTGF-β1 was overexpressed in GC cells and tissues, and is positively related to the poor prognosis of patients. TGF-β1 from GCs activated the Smad2 pathway in BMSCs, promoting their differentiation into carcinoma-associated fibroblasts (CAFs) and TGF-β1 expression. Concomitantly, TGF-β1 secreted by CAFs activate Smad2 signaling in GC cells, thus inducing their epithelial-mesenchymal transition (EMT) and TGF-β1 secretion. BMSCs can dramatically promote the proliferation, migration, and invasion of GCs while blocking TGF-β1/Smad2 positive feedback loop can reverse these effects. SignificanceThe TGF-β1/Smad2 positive feedback loop between GCs and BMSCs, promotes the CAFs differentiation of BMSCs and the EMT of GCs, resulting in the progression of GC.

Inhibitory Effect of Verapamil on the Growth of Human Airway Granulation Fibroblasts.

To explore the inhibitory effect of verapamil, a calcium channel blocker, on the growth of human airway granulation fibroblasts to provide an experimental basis for the clinical use of calcium channel blockers in preventing and treating benign airway stenosis. Primary human airway normal fibroblasts and human airway granulation fibroblasts were cultured by tissue block attachment culture method, and the experimental studies were carried out using 3-8 generation cells. Cell Counting Kit-8 (CCK-8) was used to test the proliferation of human normal airway fibroblasts and human airway granulation fibroblasts and the semi-inhibitory concentration of verapamil on normal airway fibroblasts and airway granulation fibroblasts. A scratch test detected the migration effect of verapamil on human airway granulation fibroblasts. The mRNA relative expression levels of related factors were detected by PCR to compare the differences between normal airway fibroblasts and airway granulation fibroblasts. Western blot was used to detect the relative amount of related proteins and compare the differences between normal airway fibroblasts and granulation airway fibroblasts. After 48 hours of treatment with half of the inhibitory concentration of Vera Pammy for granulation airway fibroblasts, the relative expression levels of related factors on mRNA and protein were observed. Human normal airway fibroblasts and human airway granulation fibroblasts with a purity of more than 95% could be obtained from primary culture by tissue block adherence method. CCK8 results showed that the proliferation rate of human airway granulation fibroblasts was faster than that of the normal human airway fibroblasts. The semi-inhibitory concentration of verapamil on human normal airway fibroblasts was 92.81 ug/ml, while the semi-inhibitory concentration on human airway granulation fibroblasts was 69.57 ug/ml. The scratch test indicated that the cell migration rate of human airway granulation fibroblasts treated with verapamil decreased significantly (P < 0.05). PCR results showed that the mRNA relative expression levels of TGFβ1, COL1A1, Smad2/3, VEGFA, IL6, and IL8 in human airway granulation fibroblasts were significantly higher than those in normal human airway fibroblasts (P < 0.05). The mRNA relative expressions of TGFβ1, smad2/3, and COL1A1 in human airway granulation fibroblasts treated with semi-inhibited verapamil for 48h were down-regulated (P < 0.05), while the mRNA relative expressions of VEGFA, IL6 and IL8 had no significant changes (P > 0.05). WB test showed that the relative protein expressions of TGFβ1, Smad2, and VEGFC in human airway granulation fibroblasts were upregulated (P < 0.05) but downregulated after verapamil treatment compared with before treatment (P < 0.05). Calcium channel blockers can inhibit the proliferation of human airway granulation fibroblasts through TGFβ1/ Smad pathway, which may be a method to prevent and treat benign airway stenosis.

MiR-19a-3p affected ox-LDL-induced SDC-1/TGF-β1/Smad3 pathway in atherosclerosis.

Syndecan-1 (SDC-1) was a critical membrane proteoglycan and an important component of the glycocalyx in endothelial cells, but its role in atherosclerosis remains unknown. This study attempted to investigate the role of SDC-1 in atherosclerotic-related endothelial cell injury. Bioinformatics analyzed the differential miRNAs between atherosclerosis and healthy. Subjects with coronary atherosclerosis, which were diagnosed with intravascular atherosclerosis (IVUS), were enrolled as non-vulnerable plaque and vulnerable plaque in Changsha Central Hospital. Human aortic endothelial cells (HAECs) were induced by oxidized low-density lipoprotein (ox-LDL) to construct an in vitro model. A dual luciferase reporter assay was applied to analyze the target between miR-19a-3p and SDC-1. The cell proliferation and apoptosis were detected by CCK8 and flow cytometry, respectively. SDC-1 and cholesterol efflux was determined by ELISA. The expression of ATP-binding cassette (ABC) transports A1 (ABCA1), miR-19a-3p, ABCG1 and SDC-1 genes were detected by RT-qPCR. The expressions of SDC-1, ABCA1, ABCG1, TGF-β1, Smad3 and p-Smad3 proteins were detected by western blot. Our results found that miR-19a-3p was down-regulated in atherosclerosis. ox-LDL decreased miR-19a-3p expression, increased cholesterol efflux and the expression of ABCA1, ABCG1 and SDC-1 in HAECs. Vulnerable plaque tissues in patients with coronary atherosclerosis showed palpable fibrous necrosis and calcification with elevated blood SDC-1 levels. miR-19a-3p could bind to SDC-1. Overexpression of miR-19a-3p promoted cell proliferation, inhibited apoptosis and cholesterol efflux, down-regulated the expression of SDC-1, ABCA1, ABCG1, TGF-β1 and p-Smad3 proteins in ox-LDL-induced HAECs. In conclusion, miR-19a-3p targeting SDC-1 inhibited the ox-LDL-induced activation of the TGF-β1/Smad3 pathway in HAECs.

Black Tea Extract, via Modulation of TGF-β Pathway, Prevents Inorganic Arsenic-induced Development of Squamous Cell Carcinoma of the Skin in Swiss Albino Mice.

Chronic exposure to inorganic arsenic (iAs) elevates reactive oxygen species (ROS) generation and up-regulates TGF-β signalling. This promotes induction of epithelial to mesenchymal transition (EMT) and causes the development of squamous cell carcinoma (SCC) of skin. Black tea is a popular beverage worldwide and an effective antioxidant. Chemopreventive potential of black tea extract (BTE) against iAs induced carcinogenicity has been explored here. The study aims to investigate the role of BTE in prevention of iAs-induced SCC of skin in Swiss albino mice via the modulation of TGF-β signalling and EMT. Mice were divided into (1) control, (2) iAs, (3) iAs+BTE, and (4) BTE groups and were administered iAs and BTE alone, or in combination for 330 days. Histological studies were performed to assess development of SCC. ROS generation was estimated by flowcytometry. Expression of TGF-β and downstream proteins belonging to suppressor of mothers against decapentaplegic (Smad), phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) pathways was assessed by immunoblotting. Expression of EMT markers was evaluated by immunoblotting, immunohistochemistry and semi-quantitative reverse transcriptase-PCR. After 330 days of iAs treatment, development of invasive SCC of skin probably due to excess ROS generation, elevation of TGF-β, downregulation of the Smad pathway, upregulation of PI3K-AKT and MAPK signalling molecules and induction of EMT was observed. All these modulations were found to be reversed by BTE, which inhibits iAs induced SCC of skin by quenching excess ROS, promoting Smad mediated TGF-β signalling, downregulating signalling intermediates of PI3K-AKT and MAPK pathways and inhibiting EMT.

Roles of TGF-β1 in Viral Infection during Pregnancy: Research Update and Perspectives

Transforming growth factor-beta 1 (TGF-β1) is a pleiotropic growth factor playing various roles in the human body including cell growth and development. More functions of TGF-β1 have been discovered, especially its roles in viral infection. TGF-β1 is abundant at the maternal-fetal interface during pregnancy and plays an important function in immune tolerance, an essential key factor for pregnancy success. It plays some critical roles in viral infection in pregnancy, such as its effects on the infection and replication of human cytomegalovirus in syncytiotrophoblasts. Interestingly, its role in the enhancement of Zika virus (ZIKV) infection and replication in first-trimester trophoblasts has recently been reported. The above up-to-date findings have opened one of the promising approaches to studying the mechanisms of viral infection during pregnancy with links to corresponding congenital syndromes. In this article, we review our current and recent advances in understanding the roles of TGF-β1 in viral infection. Our discussion focuses on viral infection during pregnancy, especially in the first trimester. We highlight the mutual roles of viral infection and TGF-β1 in specific contexts and possible functions of the Smad pathway in viral infection, with a special note on ZIKV infection. In addition, we discuss promising approaches to performing further studies on this topic.

Sacha inchi albumin delays skin-aging by alleviating inflammation, oxidative stress and regulating gut microbiota in d-galactose induced-aging mice.

Sacha inchi albumin exhibits considerable functional activity with notable anti-inflammatory and antioxidation properties, which could delay skin aging. However, its underlying mechanisms for delaying skin aging have not been elucidated. The aim of the present study was to investigate the anti-skin-aging effect of sacha inchi albumin (SIA) in d-galactose induced-aging mice. Sacha inchi albumin improved moisture content, collagen level, and the state of aged skin in rats. Sacha inchi albumin intervention markedly increased the skin antioxidant enzymatic activities including those of glutathione peroxidase, and catalase, but decreased the malondialdehyde content. It also regulated inflammation by reducing the level of tumor necrosis factor-α (TNF-α) and increasing the level of interleukin-6 (IL-6). Administration of SIA also increased the expression level of collagen I and III, increased the expressionof tissue inhibitor of metalloprotease-1, and decreased the expression of metalloproteinases. Sacha inchi albumin can also activate the transforming growth factor-β (TGF-β)/Smad pathway. Meanwhile, 16S rRNA sequencing analysis revealed that SIA treatment altered the composition of microbiota, and increased the relative abundance of Lactobacillus, but decreased the relative abundance of Alloprevotella and Helicobacter, etc. Helicobacter was positively associated with malondialdehyde (MDA) content and was negatively related to IL-6. Sacha inchi albumin exhibits excellent anti-skin-aging effect, which provide a new insight for the development of functional sacha inchi albumin. © 2023 Society of Chemical Industry.