Silymarin and MSC-exosomes ameliorate thioacetamide-evoked renal fibrosis by inhibiting TGF-ß/SMAD pathway in rats.

BACKGROUND: TGF-ß1 and SMAD3 are particularly pathogenic in the progression of renal fibrosis. AIM: This study aimed to evaluate the kidney protective potentials of silymarin (SM) and exosomes of mesenchymal stem cells against the nephrotoxin thioacetamide (TAA) in rats. METHODS: 32 female rats were randomly assigned into four groups: the control group, the TAA group, the TAA + SM group, and the TAA + Exosomes group. The kidney homogenates from all groups were examined for expression levels of TGF-ß receptors I and II using real-time PCR, expression levels of collagen type I and CTGF proteins using ELISA, and the expression levels of nuclear SMAD2/3/4, cytoplasmic SMAD2/3, and cytoplasmic SMAD4 proteins using the western blot technique. RESULTS: Compared to the control group, the injection of TAA resulted in a significant increase in serum levels of urea and creatinine, gene expression levels of TßRI and TßRII, protein expression levels of both collagen I and CTGF proteins, cytoplasmic SMAD2/3 complex, and nuclear SMAD2/3/4 (p-value < 0.0001), with significantly decreased levels of the co-SMAD partner, SMAD4 (p-value < 0.0001). Those effects were reversed considerably in both treatment groups, with the superiority of the exosomal treatment regarding the SMAD proteins and the expression levels of the TßRI gene, collagen I, and CTGF proteins returning to near-control values (p-value > 0.05). CONCLUSION: Using in vitro and in vivo experimental approaches, the research discovered a reno-protective role of silymarin and exosomes of BM-MSCs after thioacetamide-induced renal fibrosis in rats, with the advantage of exosomes.

The Upregulation of Leucine-Rich Repeat Containing 1 Expression Activates Hepatic Stellate Cells and Promotes Liver Fibrosis by Stabilizing Phosphorylated Smad2/3.

Liver fibrosis poses a significant global health risk due to its association with hepatocellular carcinoma (HCC) and the lack of effective treatments. Thus, the need to discover additional novel therapeutic targets to attenuate liver diseases is urgent. Leucine-rich repeat containing 1 (LRRC1) reportedly promotes HCC development. Previously, we found that LRRC1 was significantly upregulated in rat fibrotic liver according to the transcriptome sequencing data. Herein, in the current work, we aimed to explore the role of LRRC1 in liver fibrosis and the underlying mechanisms involved. LRRC1 expression was positively correlated with liver fibrosis severity and significantly elevated in both human and murine fibrotic liver tissues. LRRC1 knockdown or overexpression inhibited or enhanced the proliferation, migration, and expression of fibrogenic genes in the human hepatic stellate cell line LX-2. More importantly, LRRC1 inhibition in vivo significantly alleviated CCl4-induced liver fibrosis by reducing collagen accumulation and hepatic stellate cells' (HSCs) activation in mice. Mechanistically, LRRC1 promoted HSC activation and liver fibrogenesis by preventing the ubiquitin-mediated degradation of phosphorylated mothers against decapentaplegic homolog (Smad) 2/3 (p-Smad2/3), thereby activating the TGF-ß1/Smad pathway. Collectively, these results clarify a novel role for LRRC1 as a regulator of liver fibrosis and indicate that LRRC1 is a promising target for antifibrotic therapies.

The immune regulation and therapeutic potential of the SMAD gene family in breast cancer.

Breast cancer is a serious threat to human health. The transforming growth factor-ß signaling pathway is an important pathway involved in the occurrence and development of cancer. The SMAD family genes are responsible for the TGF-ß signaling pathway. However, the mechanism by which genes of the SMAD family are involved in breast cancer is still unclear. Therefore, it is necessary to investigate the biological roles of the SMAD family genes in breast cancer. We downloaded the gene expression data, gene mutation data, and clinical pathological data of breast cancer patients from the UCSC Xena database. We used the Wilcox test to estimate the expression of genes of the SMAD family in cancers. And the biological functions of SMAD family genes using the DAVID website. The Pearson correlation method was used to explore the immune cell infiltration and drug response of SMAD family genes. We conducted in biological experiments vitro and vivo. In this study, we integrated the multi-omics data from TCGA breast cancer patients for analysis. The expression of genes of SMAD family was significantly dysregulated in patients with breast cancer. Except for SMAD6, the expression of other SMAD family genes was positively correlated. We also found that genes of the SMAD family were significantly enriched in the TGF-ß signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways. In addition, SMAD3, SMAD6, and SMAD7 were lowly expressed in stage II breast cancer, while SMAD4 and SMAD2 were lowly expressed in stage III cancer. Furthermore, the expression of genes of the SMAD family was significantly correlated with immune cell infiltration scores. Constructing a xenograft tumor mouse model, we found that SMAD3 knockdown significantly inhibited tumorigenesis. Finally, we analyzed the association between these genes and the IC50 value of drugs. Interestingly, patients with high expression of SMAD3 exhibited significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin. In addition, SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, and BAY-1161909 and XK-469 increased drug efficacy. In conclusion, genes of the SMAD family play a crucial role in the development of breast cancer.

Antler thymosin ß10 reduces liver fibrosis via inhibiting TGF-ß1/SMAD pathway.

Hepatic stellate cell (HSC) activation is a crucial step in the development of liver fibrosis. Previous studies have shown that antler stem cells (AnSCs) inhibited HSC activation, suggesting that this may be achieved through secreting or releasing peptides. This study aimed to investigate whether AnSC-derived peptides (AnSC-P) could reduce liver fibrosis. The results showed that AnSC-P effectively reduced liver fibrosis in rats. Furthermore, we found that thymosin ß10 (Tß-10) was rich in AnSC-P, which may be the main component of AnSC-P contributing to the reduction in liver fibrosis. A further study showed that Tß-10 reduced liver fibrosis in rats, with a reduction in HYP and MDA levels in the liver tissues, a decrease in the serum levels of ALP, ALT, AST, and TBIL and an increase in TP and ALB. Moreover, Tß-10 decreased the expression levels of the genes related to the TGF-ß/SMAD signaling pathway in vivo. In addition, Tß-10 also inhibited TGF-ß1-induced HSC activation and decreased the expression levels of the TGF-ß/SMAD signaling pathway-related genes in HSCs in vitro. In conclusion, antler Tß-10 is a potential drug candidate for the treatment of liver fibrosis, the effect of which may be achieved via inhibition of the TGFß/SMAD signaling pathway.

Rare flavanone-diarylheptanoid hybrids from Typha angustifolia shows anti breast cancer activity via activating TGF-ß1/Smad signaling pathway.

Four new flavanone-diarylheptanoid hetero dimers, typhatifolins A-D (1-4), were separated from the pollen of a widely distributed medicinal plant Typha angustifolia. Structures of these rare hybrids were elucidated by detailed interpretation of spectroscopic data, and their absolute configurations were determined on the basis of Mosher's method and ECD analyses. All the four compounds showed moderate to significant cytotoxicities against a panel of tumor cell lines with IC50 values ranging from 0.67 to 12.48 µM. Further in vitro antitumor evaluation for typhatifolin B (TTB, 2) on two breast cancer cells (4T1 and MDA-MB231) revealed that it could remarkably induce cell apoptosis and G0/G1 cycle arrest, as well as block cell migration and invasion. Mechanistically, TTB could exert its antitumor effect via activating the TGF-ß1 (transforming growth factor beta 1) signaling pathway as evidenced by RNA-seq analysis and immunoblotting experiments, which was further corroborated by treating cancer cells with a TGF-ß signaling inhibitor. Lastly, the in vivo anti breast cancer activity was demonstrated by applying the mixture of typhatifolins A-D to a preclinical animal model.

Microbubbles activated by low-frequency ultrasound enhance the anti-tumor effects of curcumin in glioma cells by suppressing the TGF-ß1/Smad/VEGF/NCAM signaling pathway.

This study investigated whether microbubbles activated by low-frequency ultrasound enhanced the anti-tumor effects of curcumin in glioma cells. CCK8 proliferation assay, scratch migration assay, and transwell invasion assay were performed to estimate the proliferation, migration, and invasion rates of the glioma cells in blank control and different treatment groups, respectively. Quantitative RT-PCR (qRT-PCR) analysis was performed to determine the relative expression levels of VEGF and NCAM mRNAs in the various experimental groups. Western blotting was performed to determine the activity status of the TGF-ß1/Smad signaling pathway in various groups of glioma cells by estimating the expression levels of p-SMAD2/3, VEGF, and NCAM proteins. Combined treatment (Cur-Us-MBs) with microbubbles activated by low-frequency ultrasound and curcumin significantly reduced the in vitro proliferation, migration, and invasiveness of glioma cells compared to the control and other treatment groups. Furthermore, Cur-Us-MBs significantly reduced the expression levels of VEGF and NCAM mRNAs and proteins and p-Smad2/3 proteins , including those cells stimulated with rhTGF-ß. These suggested that microbubbles activated by low-frequency ultrasound enhanced the inhibition of TGF-ß1/Smad/VEGF/NCAM signaling pathway by curcumin，and enhanced the antitumor effects of curcumin by significantly reducing in vitro proliferation, migration, and invasiveness of glioma cells through this pathway.

The Role of TGF-ß/SMAD Signaling in Hepatocellular Carcinoma: from Mechanism to Therapy and Prognosis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with high incidence and mortality, accounting for approximately 90% of liver cancer. The development of HCC is a complex process involving the abnormal activation or inactivation of multiple signaling pathways. Transforming growth factor-ß (TGF-ß)/Small mothers against decapentaplegic (SMAD) signaling pathway regulates the development of HCC. TGF-ß activates intracellular SMADs protein through membrane receptors, resulting in a series of biological cascades. Accumulating studies have demonstrated that TGF-ß/SMAD signaling plays multiple regulatory functions in HCC. However, there is still controversy about the role of TGF-ß/SMAD in HCC. Because it involves different pathogenic factors, disease stages, and cell microenvironment, as well as upstream and downstream relationships with other signaling pathways. This review will summary the regulatory mechanism of the TGF-ß/SMAD signaling pathway in HCC, involving the regulation of different pathogenic factors, different disease stages, different cell populations, microenvironments, and the interaction with microRNAs. In addition, we also introduced small molecule inhibitors, therapeutic vaccines, and traditional Chinese medicine extracts based on targeting the TGF-ß/SMAD signaling pathway, which will provide future research direction for HCC therapy targeting the TGF-ß/SMAD signaling pathway.

OLFM2 promotes epithelial-mesenchymal transition, migration, and invasion in colorectal cancer through the TGF-ß/Smad signaling pathway.

BACKGROUND: Colorectal cancer (CRC) is an aggressive tumor of the gastrointestinal tract, which is a major public health concern worldwide. Despite numerous studies, the precise mechanism of metastasis behind its progression remains elusive. As a member of the containing olfactomedin domains protein family, olfactomedin 2 (OLFM2) may play a role in tumor metastasis. It is highly expressed in colorectal cancer, and its role in the metastasis of CRC is still unclear. As such, this study seeks to explore the function of OLFM2 on CRC metastasis and its potential mechanisms. METHODS: Real-time fluorescence quantitative PCR and western blotting were used to study the expression of OLFM2 in human CRC and adjacent normal tissues. Knockdown and overexpression OLFM2 cell lines were constructed using siRNA and overexpression plasmids to explore the role of OLFM2 in the migration and invasion of CRC through transwell, and wound healing experiments. Finally, the expression of epithelial-mesenchymal transition (EMT) -related proteins and TGF-ß/Smad signaling pathway-related proteins was investigated using western blotting. RESULTS: In this study, we observed an elevation of OLFM2 expression levels in CRC tissues. To investigate the function of OLFM2, we overexpressed and knocked down OLFM2. We discovered that OLFM2 knockdown inhibited migration and invasion of colon cancer cells. Furthermore, E-cadherin expression increased while N-cadherin and Vimentin expression were opposite. It is no surprise that overexpressing OLFM2 had the opposite effects. We also identified that OLFM2 knockdown resulted in reduced TGF-ßR1 and downstream molecules p-Smad2 and p-Smad3, which are related to the TGF-ß / Smad pathway. In contrast, overexpressing OLFM2 significantly boosted their expression levels. CONCLUSION: The protein OLFM2 has been identified as a crucial determinant in the progression of CRC. Its mechanism of action involves the facilitation of EMT through the TGF-ß/Smad signaling pathway. Given its pivotal role in CRC, OLFM2 has emerged as a promising diagnostic and therapeutic target for the disease. These results indicate the potential of OLFM2 as a valuable biomarker for CRC diagnosis and treatment and highlight the need for further research exploring its clinical significance.

Echinacoside Exerts Antihepatic Fibrosis Effects in High-Fat Mice Model by Modulating the ACVR2A-Smad Pathway.

SCOPE: Nonalcoholic steatohepatitis (NASH) is an increasingly common chronic liver disease in which hepatic fibrosis is the major pathological change. The transforming growth factor ß (TGF-ß)/mall mothers against decapentaplegic (Smad) signaling is the main effector of fibrosis. Although the antifibrotic effect of echinacoside (Ech) on the liver has been indicated previously, the cellular and molecular mechanisms remain unclear. This study aims to investigate both in vivo and in vitro antifibrotic properties of Ech. METHODS AND RESULTS: Cell viability and scratch/wound assays show that Ech significantly inhibits the proliferation, migration, and activation of human hepatic stellate LX-2 cells. In mice with high-fat diet-induced hepatic fibrosis, Ech treatment attenuates the progression of liver injury, inflammation, and fibrosis. Furthermore, transcriptome analysis and subsequent functional validation demonstrate that Ech achieves antifibrotic effects by the activin receptor type-2A (ACVR2A)-mediated TGF-ß1/Smad signaling pathway; ultimately, ACVR2A is demonstrated to be an important target for hepatic fibrosis by inhibiting and inducing the expression of ACVR2A in LX-2 cells. CONCLUSION: Ech exerts potent antifibrotic effects by inhibiting the ACVR2A-mediated TGF-ß1/Smad signaling axis and may serve as an alternative treatment for hepatic fibrosis.

Multifaceted TGF-ß signaling, a master regulator: From bench-to-bedside, intricacies, and complexities.

Physiological embryogenesis and adult tissue homeostasis are regulated by transforming growth factor-ß (TGF-ß), an evolutionarily conserved family of secreted polypeptide factors, acting in an autocrine and paracrine manner. The role of TGF-ß in inflammation, fibrosis, and cancer is complex and sometimes even contradictory, exhibiting either inhibitory or promoting effects depending on the stage of the disease. Under pathological conditions, especially fibrosis and cancer, overexpressed TGF-ß causes extracellular matrix deposition, epithelial-mesenchymal transition, cancer-associated fibroblast formation, and/or angiogenesis. In this review article, we have tried to dive deep into the mechanism of action of TGF-ß in inflammation, fibrosis, and carcinogenesis. As TGF-ß and its downstream signaling mechanism are implicated in fibrosis and carcinogenesis blocking this signaling mechanism appears to be a promising avenue. However, targeting TGF-ß carries substantial risk as this pathway is implicated in multiple homeostatic processes and is also known to have tumor-suppressor functions. There is a need for careful dosing of TGF-ß drugs for therapeutic use and patient selection.

miR-92a-3p-inspired shRNA exhibits pro-chondrogenic and chondrocyte protective effects in osteoarthritis treatment through targeting SMAD6/7.

INTRODUCTION: Osteoarthritis (OA) compromises patients' quality of life and requires further study. Although miR-92a-3p was reported to possess chondroprotective effects, the underlying mechanism requires further clarification. The objectives of this study were to elucidate the mechanism by which miR-92a-3p alleviates OA and to examine the efficacy of shRNA-92a-3p, which was designed based on mature miR-92a-3p. MATERIALS AND METHODS: TargetScan and luciferase reporter assay were used to predict the target of miR-92a-3p. Adipose-derived stem cells (ADSCs) were transfected with miR-92a-3p/miR-NC mimic for the analysis of chondrogenic biomarkers and SMAD proteins. ADSCs and osteoarthritic chondrocytes were transduced with shRNA-92a-3p for the analysis of chondrogenic biomarkers and SMAD proteins. OA was surgically induced in C57BL/6JJcl mice, and ADSCs with/without shRNA-92a-3p transduction were intra-articularly injected for the assessment of cartilage damage. RESULTS: SMAD6 and SMAD7 were predicted as direct targets of miR-92a-3p by TargetScan and luciferase reporter assay. Transfection of the miR-92a-3p mimic resulted in a decrease in SMAD6 and SMAD7 levels and an increase in phospho-SMAD2/3, phospho-SMAD1/5/9, SOX9, collagen type II, and aggrecan levels in ADSCs. Furthermore, shRNA-92a-3p decreased SMAD6 and SMAD7 levels, and increased phospho-SMAD2/3, phospho-SMAD1/5/9, SOX9, collagen type II, and aggrecan levels in ADSCs and osteoarthritic chondrocytes. Additionally, ADSC-shRNA-92a-3p-EVs reduced the rate of decrease of SOX9, collagen type II, and aggrecan in osteoarthritic chondrocytes. In mice with surgically induced OA, shRNA-92a-3p-treated ADSCs alleviated cartilage damage more effectively than nontreated ADSCs. CONCLUSIONS: miR-92a-3p and shRNA-92a-3p exhibit therapeutic effects in treating OA by targeting SMAD6 and SMAD7, thereby enhancing TGF-ß signaling.

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.

17ß-Estradiol mediates TGFBR3/Smad2/3 signaling to attenuate the fibrosis of TGF-ß1-induced bovine endometrial epithelial cells via GPER.

Abnormal function and fibrosis of endometrium caused by cows' endometritis pose difficult implantation of embryos and uterine cavity adhesions. 17ß-Estradiol (E2) serves as the most effective aromatized estrogen, and its synthetase and receptors have been detected in the endometrium. Studies have demonstrated the positive role of estrogen in combating pathological fibrosis in diverse diseases. However, it is still unknown whether E2 regulates endometrium fibrosis in bovine endometritis. Herein, we evaluated the expression patterns of transforming growth factor-ß1 (TGF-ß1), epithelial-mesenchymal transformation (EMT)-related proteins (α-SMA, vimentin N-cadherin and E-cadherin), cytochrome P450 19A1 (CYP19A1), and G protein-coupled estrogen receptor (GPER) in bovine healthy endometrium and Inflammatory endometrium. Our data showed that the inflamed endometrium presented low CYP19A1 and GPER expression, and significantly higher EMT process versus the normal tissue. Moreover, we established a TGF-ß1-induced fibrosis model in BEND cells, and found that E2 inhibited the EMT process of BEND cells in a dose-dependent manner. The anti-fibrotic effect of E2 was blocked by the GPER inhibitor G15, but not the estrogen nuclear receptors (ERs) inhibitor ICI182780. Moreover, the GPER agonist G1 inhibited fibrosis and Smad2/3 phosphorylation but increased the expression of TGFBR3 in BEND cells. Transfection with TGFBR3 small interfering RNA blocked the effect of G1 on fibrosis of BEND cells and upregulated the expression of P-Smad2/3. Our in vivo data also showed that E2 and G1 affected uterus fibrosis in mice endometritis model caused by LPS, which was associated with the inhibition of TGFBR3/Smad2/3 signaling. In conclusion, our data implied that E2 alleviates the fibrosis of TGF-ß1-induced BEND cells, which is associated with the GPER mediation of TGFBR3/Smad2/3 signaling.

Aloin Attenuates Oxidative Stress, Inflammation, and CCl4-Induced Liver Fibrosis in Mice: Possible Role of TGF-ß/Smad Signaling.

Liver fibrosis refers to the excessive buildup of extracellular matrix (ECM) components in liver tissue. It is considered a pathological response to liver damage for which there is no effective treatment. Aloin, an anthraquinone compound isolated from the aloe plant, has shown good pharmacological effects in the treatment of gastric cancer, ulcerative colitis, myocardial hypertrophy, traumatic brain injury, and other diseases; however, its specific impact on liver fibrosis remains unclear. To address this gap, we conducted a study to explore the mechanisms underlying the potential antifibrotic effect of aloin. We constructed a mouse liver fibrosis model using carbon tetrachloride (CCl4) dissolved in olive oil as a modeling drug. Additionally, a cellular model was developed by using transforming growth factor ß1 (TGF-ß1) as a stimulus applied to hepatic stellate cells. After aloin intervention, serum alanine aminotransferase, hepatic hydroxyproline, and serum aspartate aminotransferase were reduced in mice after aloin intervention compared to CCl4-mediated liver injury without aloin intervention. Aloin relieved the oxidative stress caused by CCl4 via reducing hepatic malondialdehyde in liver tissue and increasing the level of superoxide dismutase. Aloin treatment decreased interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α and increased the expression of IL-10, which inhibited the inflammatory response in liver injury. In addition, aloin inhibited the activation of hepatic stellate cells and reduced the level of α-smooth muscle actin (α-SMA) and collagen type I. In cell and animal experiments, aloin attenuated liver fibrosis, acting through the TGF-ß/Smad2/3 signaling pathway, and mitigated CCl4- and TGF-ß1-induced inflammation. Thus, the findings of this study provided theoretical data support and a new possible treatment strategy for liver fibrosis.

Periplaneta americana extract attenuates hepatic fibrosis progression by inhibiting collagen synthesis and regulating the TGF-ß1/Smad signaling pathway.

INTRODUCTION: Liver fibrosis is the damage repair response following chronic liver diseases. Activated hepatic stellate cells (HSCs) are the main extracellular matrix (ECM)-producing cells and key regulators in liver fibrosis. Periplaneta americana shows prominent antifibrotic effects in liver fibrosis; however, the underlying mechanisms remain undetermined. This study aimed to elucidate the therapeutic effects of P. americana extract (PA-B) on liver fibrosis based on the regulation of the TGF-ß1/Smad signal pathway. MATERIAL AND METHODS: HSCs and Sprague Dawley rats were treated with TGF-ß1 and CCl4, respectively, to establish the hepatic fibrosis model in vitro and in vivo. The effect of PA-B on liver rat fibrosis was evaluated by biochemical (serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), hyaluronic acid (HA), laminin (LN), collagen type IV (Col-IV), pro-collagen type III (PC-III)) and histological examinations. Further, fibrogenic markers expression of alpha smooth muscle actin (α-SMA), collagen type I (Col-I), and collagen type III (Col-III), and the TGF-ß1/Smad pathway-related factors were assessed by immunofluorescence (IF), real time quantitative polymerase chain reaction (RT-qPCR), and western blotting (WB). RESULTS: Treatment of HSC-T6 cells with PA-B suppressed the expression of α-SMA, Col-I, and Col-III, downregulated the expression of TGF-ß1 receptors I and II (TßR I and TßR II, respectively), Smad2, and Smad3, and upregulated Smad7 expression. PA-B mitigates pathologic changes in the rat model of liver fibrosis, thus alleviating liver index, and improving liver function and fibrosis indices. The effects of PA-B on the expression of α-SMA, Col-I, Col-III, TßR I, TßR II, Smad2, Smad3, and Smad7 were consistent with the in vitro results, including reduced TGF-ß1 expression. CONCLUSIONS: The therapeutic effect of PA-B on liver fibrosis might involve suppression of the secretion and expression of TGF-ß1, regulation of the TGF-ß1/Smad signaling pathway, and inhibition of collagen production and secretion.

Inhibition of keloid by 32P isotope radiotherapy through suppressing TGF-ß/Smad signaling pathway.

BACKGROUND: Keloid seriously affects the appearance, and is accompanied by some symptoms including pain, burning, itching. Radioactive nuclides such as 32P have been proved to be effective in inhibiting the formation of keloid, but the mechanism remains unclear. METHODS: The keloid animal model was established through keloid tissues implantation. Hematoxylin-Eosin (HE) and Masson staining were performed to investigate histological changes and collagen deposition. The mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. Cell apoptosis and cycle were evaluated through flow cytometry. RESULTS: Both 32P isotope injection and skin path significantly reduced the size of keloid, and inhibited TGF-ß/Smad signaling pathway. SRI-011381, the agonist of TGF-ß/Smad signaling pathway, markedly reversed the influence of 32P isotope on cell proliferation, cell apoptosis, cell cycle of LNCaP cells and TGF-ß/Smad signaling pathway. CONCLUSIONS: 32P isotope injection and skin path greatly reduced the size of keloid, and the TGF-ß/Smad signaling pathway was remarkably inhibited by 32P isotope treatment. The regulation of dermal fibroblast by 32P isotope was reversed by SRI-011381. 32P isotope might inhibit keloid through suppressing TGF-ß/Smad signaling pathway. Our study provides a novel therapeutic strategy for the treatment of keloid.

Alpha-mangostin alleviate renal interstitial fibrosis via suppression of TGF-ß1/Smad/ERK signaling axis in vitro and in vivo.

α-mangostin (α-MG), a natural derivative of coumarin, exhibits anti-inflammatory, antioxidant and anti-fibrotic effects. This study aimed to determine the effect of α-MG treatment in mediating the process of renal interstitial fibrosis. We found that α-MG could alleviate tubule-interstitial damage and decrease fibrotic (α-smooth muscle actin [α-SMA], fibronectin, and collagen I), and epithelial-mesenchymal transition (EMT) protein (N-cadherin, Snail, Slug, TGF-ß1 and vimentin) expression in unilateral ureteral obstruction (UUO) mice with chronic kidney disease. α-MG significantly decreased motility as well as inhibited expression of fibrotic- and EMT-related proteins in TGF-ß1-induced HK2 cells. To clarify the molecular mechanisms of α-MG in reducing renal interstitial fibrosis, we used a MEK inhibitor (U0126) or Smad inhibitor (SB431542) cotreatment with α-MG. This is the first study is to demonstrate the antifibrotic effects of α-MG by targeting the TGF-ß1/ERK/Smad-mediated EMT signaling pathway, is even more effective against renal interstitial fibrosis.

SETD2 deficiency promotes renal fibrosis through the TGF-ß/Smad signalling pathway in the absence of VHL.

BACKGROUND: Renal fibrosis is the final development pathway and the most common pathological manifestation of chronic kidney disease. Epigenetic alteration is a significant intrinsic factor contributing to the development of renal fibrosis. SET domain-containing 2 (SETD2) is the sole histone H3K36 trimethyltransferase, catalysing H3K36 trimethylation. There is evidence that SETD2-mediated epigenetic alterations are implicated in many diseases. However, it is unclear what role SETD2 plays in the development of renal fibrosis. METHODS: Kidney tissues from mice as well as HK2 cells were used as research subjects. Clinical databases of patients with renal fibrosis were analysed to investigate whether SETD2 expression is reduced in the occurrence of renal fibrosis. SETD2 and Von Hippel-Lindau (VHL) double-knockout mice were used to further investigate the role of SETD2 in renal fibrosis. Renal tubular epithelial cells isolated from mice were used for RNA sequencing and chromatin immunoprecipitation sequencing to search for molecular signalling pathways and key molecules leading to renal fibrosis in mice. Molecular and cell biology experiments were conducted to analyse and validate the role of SETD2 in the development of renal fibrosis. Finally, rescue experiments were performed to determine the molecular mechanism of SETD2 deficiency in the development of renal fibrosis. RESULTS: SETD2 deficiency leads to severe renal fibrosis in VHL-deficient mice. Mechanically, SETD2 maintains the transcriptional level of Smad7, a negative feedback factor of the transforming growth factor-ß (TGF-ß)/Smad signalling pathway, thereby preventing the activation of the TGF-ß/Smad signalling pathway. Deletion of SETD2 leads to reduced Smad7 expression, which results in activation of the TGF-ß/Smad signalling pathway and ultimately renal fibrosis in the absence of VHL. CONCLUSIONS: Our findings reveal the role of SETD2-mediated H3K36me3 of Smad7 in regulating the TGF-ß/Smad signalling pathway in renal fibrogenesis and provide an innovative insight into SETD2 as a potential therapeutic target for the treatment of renal fibrosis.

The long non-coding RNA LINC00707 interacts with Smad proteins to regulate TGFß signaling and cancer cell invasion.

BACKGROUND: Long non-coding RNAs (lncRNAs) regulate cellular processes by interacting with RNAs or proteins. Transforming growth factor ß (TGFß) signaling via Smad proteins regulates gene networks that control diverse biological processes, including cancer cell migration. LncRNAs have emerged as TGFß targets, yet, their mechanism of action and biological role in cancer remain poorly understood. METHODS: Whole-genome transcriptomics identified lncRNA genes regulated by TGFß. Protein kinase inhibitors and RNA-silencing, in combination with cDNA cloning, provided loss- and gain-of-function analyses. Cancer cell-based assays coupled to RNA-immunoprecipitation, chromatin isolation by RNA purification and protein screening sought mechanistic evidence. Functional validation of TGFß-regulated lncRNAs was based on new transcriptomics and by combining RNAscope with immunohistochemical analysis in tumor tissue. RESULTS: Transcriptomics of TGFß signaling responses revealed down-regulation of the predominantly cytoplasmic long intergenic non-protein coding RNA 707 (LINC00707). Expression of LINC00707 required Smad and mitogen-activated protein kinase inputs. By limiting the binding of Krüppel-like factor 6 to the LINC00707 promoter, TGFß led to LINC00707 repression. Functionally, LINC00707 suppressed cancer cell invasion, as well as key fibrogenic and pro-mesenchymal responses to TGFß, as also attested by RNA-sequencing analysis. LINC00707 also suppressed Smad-dependent signaling. Mechanistically, LINC00707 interacted with and retained Smad proteins in the cytoplasm. Upon TGFß stimulation, LINC00707 dissociated from the Smad complex, which allowed Smad accumulation in the nucleus. In vivo, LINC00707 expression was negatively correlated with Smad2 activation in tumor tissues. CONCLUSIONS: LINC00707 interacts with Smad proteins and limits the output of TGFß signaling, which decreases LINC00707 expression, thus favoring cancer cell invasion. Video Abstract.

LIPUS Alleviates Knee Joint Capsule Fibrosis in Rabbits by Regulating SOD/ROS Dynamics and Inhibiting the TGF-ß1/Smad Signaling Pathway.

OBJECTIVE: The aim of the work described here was to investigate the efficacy and potential mechanisms of low-intensity pulsed ultrasound (LIPUS) for the treatment of arthrogenic contracture induced by immobilization in rabbits. METHODS: The left knee joint of rabbits was immobilized for 6 wk to establish the model of extending knee joint contracture. The rabbits were divided into a control group (C), a group immobilized for 6 wk (IM-6w), a group remobilized for 1 wk (RM-1w), a group subjected to LIPUS intervention for 1 wk (LIPUS-1w), a group remobilized for 2 wk (RM-2w) and a group subjected to LIPUS intervention for 2 wk (LIPUS-2w). The degrees of arthrogenic contracture and joint capsule fibrosis were assessed, as were the levels of reactive oxygen species (ROS) and the activation status of the TGF-ß1/Smad signaling pathway in the joint capsule. RESULTS: After immobilization for 6 wk, the degrees of arthrogenic contracture and joint capsule fibrosis increased. The ROS level increased, as evidenced by an increase in malondialdehyde content and a decrease in superoxide dismutase content. In addition, the TGF-ß1/Smad signaling pathway was significantly activated. The degrees of knee joint contracture increased in the first week after remobilization and decreased in the second week. Furthermore, joint capsule fibrosis continued to develop during the 2 wk of remobilization, and the ROS level increased, while the TGF-ß1/Smad signaling pathway was significantly activated. LIPUS effectively reduced the level of ROS in the joint capsule, which further inhibited activation of the TGF-ß1/Smad signaling pathway, thereby improving joint capsule fibrosis and reducing arthrogenic contracture. CONCLUSION: The high ROS levels and overactivation of the TGF-ß1/Smad signaling pathway may be reasons why immobilization induces knee joint capsule fibrosis. LIPUS can alleviate the degree of knee joint capsule fibrosis induced by immobilization by inhibiting the production of ROS and the activation of the TGF-ß1/Smad signaling pathway.