TGF-ß/Smad signaling pathway in fatty liver disease: a case-control study.

BACKGROUND: Fatty liver disease is a metabolic disorder that recently has been classified into two categories: metabolic dysfunction-associated fatty liver disease (MAFLD) and non-MAFLD. TGF-ß signaling pathway is likely a significant factor in the pathogenesis of this condition, exerting its effects through its downstream signaling proteins, Smad2/3. Accordingly, this study aimed to investigate the TGF-ß signaling pathway in the white blood cells (WBCs) of patients with MAFLD compared to those with non-MAFLD and control groups. METHODS AND RESULTS: In this study, 41 patients with fatty liver were evaluated, comprising 22 patients with MAFLD and 19 patients with non-MAFLD, and compared to 22 healthy controls. Gene expression of TGF-ß1, TGF-ß3, and CTGF were quantified using qRT-PCR, and the protein expressions of Smad2/3 and P-Smad2/3 were analyzed using western blotting. Gene expression analysis revealed a significant decrease in the gene expressions of the TGF-ß1 and TGF-ß3 and an increase in CTGF gene expression in patients with MAFLD and non-MAFLD compared to the control group. Notably, the Smad2/3 protein expression was significantly higher in the non-MAFLD group compared to the control group (P < 0.05). On the other hand, the P-smad2/3 protein expression was significantly elevated in the MAFLD group compared to the control group (P < 0.001). CONCLUSIONS: TGF-ß signaling pathway in WBCs of patients with fatty liver are affected by a complex signaling pathway. However, metabolic factors most probably affect TGF-ß1 gene expression and its downstream signaling proteins more than TGF-ß3.

The Myhre Syndrome Foundation as a global modern support group: The business of rare.

Advocacy support groups grow into national and international organizations, but they all begin with personal experiences. As the parents to a newly diagnosed two-year-old son with Myhre syndrome, my husband and I were overwhelmed with the journey ahead. Thanks to networking, primarily through social media, we located other families living with Myhre syndrome and were quickly immersed in the challenges and joy of this community. Myhre syndrome, caused by pathogenic missense variants in SMAD4, is a rare connective tissue disease characterized by short stature, hearing loss, neurodevelopmental challenges, and fibroproliferation. This personal essay, written with physician partners, describes the development of a global advocacy group for patients with Myhre syndrome. I have the honor of serving as the founding Executive Director and reflect proudly on the great strides that our marvelous support group has made. We empower the global community impacted by this rare condition by providing meaningful and accessible data, educational opportunities, and connections with others going through similar experiences. Utilizing the expertise of our Board of Directors and my corporate expertise, we discuss how we have been able to elevate our ultra-rare community into a broader, more comprehensive network.

TGF-ß and TNF-α interaction promotes the expression of MMP-9 through H3K36 dimethylation: implications in breast cancer metastasis.

Increased MMP-9 expression in the tumor microenvironment (TME) plays a crucial role in the extracellular matrix remodeling to facilitate cancer invasion and metastasis. However, the mechanism of MMP-9 upregulation in TME remains elusive. Since TGF-ß and TNF-α levels are elevated in TME, we asked whether these two agents interacted to induce/augment MMP-9 expression. Using a well-established MDA-MB-231 breast cancer model, we found that the synergy between TGF-ß and TNF-α led to MMP-9 upregulation at the transcriptional and translational levels, compared to treatments with each agent alone. Our in vitro findings are corroborated by co-expression of elevated MMP-9 with TGF-ß and TNF-α in human breast cancer tissues. Mechanistically, we found that the MMP-9 upregulation driven by TGF-ß/TNF-α cooperativity was attenuated by selective inhibition of the TGF-ßRI/Smad3 pathway. Comparable outcomes were observed upon inhibition of TGF-ß-induced phosphorylation of Smad2/3 and p38. As expected, the cells defective in Smad2/3 or p38-mediated signaling did not exhibit this synergistic induction of MMP-9. Importantly, the inhibition of histone methylation but not acetylation dampened the synergistic MMP-9 expression. Histone modification profiling further identified the H3K36me2 as an epigenetic regulatory mark of this synergy. Moreover, TGF-ß/TNF-α co-stimulation led to increased levels of the transcriptionally permissive dimethylation mark at H3K36 in the MMP-9 promoter. Comparable outcomes were noted in cells deficient in NSD2 histone methyltransferase. In conclusion, our findings support a cooperativity model in which TGF-ß could amplify the TNF-α-mediated MMP-9 production via chromatin remodeling and facilitate breast cancer invasion and metastasis.

PCSK9 inhibitor attenuates cardiac fibrosis in reperfusion injury rat by suppressing inflammatory response and TGF-ß1/Smad3 pathway.

Progressive cardiac fibrosis, a hallmark of heart failure, remains poorly understood regarding Proprotein convertase subtilisin/kexin type 9 (PCSK9) 's role. This study aims to elucidate PCSK9's involvement in cardiac fibrosis. After ischemia/reperfusion (I/R) injury surgery in rats, PCSK9 inhibitors were used to examine their effects on the transforming growth factor-ß1 (TGF-ß1)/small mother against decapentaplegic 3 (Smad3) pathway and inflammation. Elevated PCSK9, TGF-ß1, and Smad3 levels were observed in cardiac tissues post-I/R injury, indicating fibrosis. PCSK9 inhibition reduced pro-fibrotic protein expression, protecting the heart and mitigating I/R-induced damage and fibrosis. Additionally, it ameliorated cardiac inflammation and reduced post-myocardial infarction (MI) size, improving cardiac function and slowing heart failure progression. PCSK9 inhibitors significantly attenuate myocardial fibrosis induced by I/R via the TGF-ß1/Smad3 pathway.

Translation Initiation Factor-2S2 (eIF2S2) Contributes to Cervical Carcinogenesis by Inhibiting the TGF-ß/SMAD4 Signaling Pathway.

The deregulation of protein translational machinery and the oncogenic role of several translation initiation factors have been extensively investigated. This study aimed to investigate the role of eukaryotic translation initiation factor 2S2 (eIF2S2, also known as eIF2ß) in cervical carcinogenesis. Immunohistochemical analysis of human cervical carcinoma tissues revealed a stage-specific increase in eIF2S2 expression. The knockdown of eIF2S2 in human cervical cancer (SiHa) cells significantly reduced growth and migration properties, whereas its overexpression demonstrated the opposite effect. Immunoprecipitation and Bimolecular fluorescence complementation (BiFC) assay confirmed the previous photo array finding of the interaction between eIF2S2 and SMAD4 to understand the tumorigenic mechanism of eIF2S2. The results indicated that the N-terminus of eIF2S2 interacts with the MH-1 domain of SMAD4. The interaction effect between eIF2S2 and SMAD4 was further evaluated. The knockdown of eIF2S2 increased SMAD4 expression in cervical cancer cells without changing SMAD4 mRNA expression, whereas transient eIF2S2 overexpression reduced SMAD4 expression. This indicates the possibility of post-translational regulation of SMAD4 expression by eIF2S2. Additionally, eIF2S2 overexpression was confirmed to weaken the expression and/or promoter activity of p15 and p27, which are SMAD4-regulated antiproliferative proteins, by reducing SMAD4 levels. Therefore, our study indicated the pro-tumorigenic role of eIF2S2, which diminishes both SMAD4 expression and function as a transcriptional factor in cervical carcinogenesis.

LHPP deficiency aggravates liver fibrosis through TGF-ß/Smad3 signaling.

Liver fibrosis is characterized by a wound-healing response and may progress to liver cirrhosis and even hepatocellular carcinoma. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a tumor suppressor that participates in malignant diseases. However, the role of LHPP in liver fibrosis has not been determined. Herein, the function and regulatory network of LHPP were explored in liver fibrosis. The expression of LHPP in human and murine fibrotic liver tissues was assessed via immunohistochemistry and Western blot analysis. In addition, liver fibrosis was induced in wild-type (WT) and LHPP-/- (KO) mice after carbon tetrachloride (CCl4) or thioacetamide (TAA) treatment. The effect of LHPP was systematically assessed by using specimens acquired from the above murine models. The functional role of LHPP was further explored by detecting the pathway activity of TGF-ß/Smad3 and apoptosis after interfering with LHPP in vitro. To explore whether the function of LHPP depended on the TGF-ß/Smad3 pathway in vivo, an inhibitor of the TGF-ß/Smad3 pathway was used in CCl4-induced WT and KO mice. LHPP expression was downregulated in liver tissue samples from fibrosis patients and fibrotic mice. LHPP deficiency aggravated CCl4- and TAA-induced liver fibrosis. Moreover, through immunoblot analysis, we identified the TGF-ß/Smad3 pathway as a key downstream pathway of LHPP in vivo and in vitro. The effect of LHPP deficiency was reversed by inhibiting the TGF-ß/Smad3 pathway in liver fibrosis. These results revealed that LHPP deficiency exacerbates liver fibrosis through the TGF-ß/Smad3 pathway. LHPP may be a potential therapeutic target in hepatic fibrosis.

Qianggan Ruanjian Pill ameliorates liver fibrosis through regulation of the TGF-ß1/Smad and PI3K/AKT signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is a critical pathological process in the progression of chronic liver injury, ultimately resulting in cirrhosis, for which currently available therapeutic interventions remain inadequate. Among these, the Qianggan Ruanjian Pill (QGRJP) has emerged as a clinically experienced formula with notable therapeutic efficacy against liver fibrosis. However, the precise underlying mechanisms require further investigation. AIM OF THE STUDY: In this study, we investigated the key pathways and target genes of QGRJP that attenuate liver fibrosis and elucidated the underlying mechanisms. MATERIALS AND METHODS: High-performance liquid chromatography-mass spectrometry (HPLC-MS) was used to identify the major components of the QGRJP. Mouse models of liver fibrosis were established by injecting olive oil containing 25% carbon tetrachloride (CCl4), which was administered at different doses of QGRJP by gavage. Liver damage and function were assessed using serum biochemical detection, ultrasound imaging, and histopathological examination. The anti-fibrosis effect was assessed using immunohistochemistry, western blotting, and quantitative real-time PCR (qRT-PCR). The in vivo safety of the QGRJP was evaluated using weight monitoring and biopsy. Potential anti-liver fibrosis signalling pathways and key targets of QGRJP were identified using RNA-seq analysis and network pharmacology. The predicted targets and pathways were validated using in vitro and in vivo experiments. RESULTS: QGRJP significantly ameliorated CCl4-induced liver fibrosis, and its mechanism was correlated with the inhibition of hepatic stellate cell (HSC) activation and the inflammatory response via inhibition of the TGF-ß1/Smad and PI3K/AKT pathways, leading to a significant reduction in the expression of collagen and other fibrosis-related proteins. Additionally, no obvious toxic side effects were observed in the major organs of the mice or in activated HSCs (aHSCs). CONCLUSION: This study demonstrated that QGRJP mitigated liver injury, inflammation, and fibrosis by inhibiting the TGF-ß1/Smad and PI3K/AKT signalling pathways.

Schistosoma japonicum infection-mediated downregulation of lncRNA Malat1 contributes to schistosomiasis hepatic fibrosis by the Malat1/miR-96/Smad7 pathway.

BACKGROUND: Schistosoma japonicum infection causes hepatic fibrosis, a primary cause of morbidity and mortality associated with the disease, and effective treatments are still lacking. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenic process of various tissue fibroses. However, the role of lncRNAs in schistosomiasis hepatic fibrosis (HF) is poorly understood. Understanding the role of lncRNAs in schistosomiasis HF will enhance knowledge of disease processes and aid in the discovery of therapeutic targets and diagnostic biomarkers. METHODS: Differentially expressed lncRNA profiles in primary hepatic stellate cells (HSCs) of mice infected with S. japonicum were identified using high-throughput lncRNA sequencing. Primary HSCs were isolated from infected mice using collagenase digestion and density-gradient centrifugation, cultured in DMEM with 10% fetal bovine serum. Dual-luciferase reporter assays, nuclear cytoplasm fractionation and RIP assays were employed to assess the relationship between Malat1 and miRNA-96. Malat1 lentivirus and ASO-Malat1 were constructed for forced expression and downregulated expression of Malat1. The Malat1-KO mouse was constructed by CRISPR/Cas9 technology. Pathological features of the liver were evaluated by hematoxylin-eosin (HE), Masson's trichrome staining and immunohistochemistry (IHC). The expression levels of fibrosis-related genes were determined by quantitative real-time PCR (qRT-PCR) and Western blot. RESULTS: A total of 1561 differentially expressed lncRNAs were identified between infected and uninfected primary HSCs. Among the top altered lncRNAs, the downregulated Malat1 was observed in infected HSCs and verified by qPCR. Treatment of infected mice with praziquantel (PZQ) significantly increased the Malat1 expression. Elevated Malat1 expression in infected primary HSC reduced the expressions of profibrogenic genes, whereas Malat1 knockdown had the opposite effect. Moreover, Malat1 was found to interact with miR-96, a profibrotic miRNA, by targeting Smad7. Forced Malat1 expression reduced miR-96 levels in infected primary HSCs, attenuating fibrogenesis and showing negative correlation between Malat1 expression and the expression levels of miR-96 and profibrogenic genes α-SMA and Col1α1. Notably, in Malat1-KO mice, knockout of Malat1 aggravates schistosomiasis HF, while restored Malat1 expression in the infected HSCs reduced the expression of profibrogenic genes. CONCLUSIONS: We demonstrate that lncRNA is involved in regulation of schistosomiasis HF. Elevated lncRNA Malat1 expression in infected HSCs reduces fibrosis via the Malat1/miR-96/Smad7 pathway, thus providing a novel therapeutic target for schistosomiasis HF. Furthermore, Malat1 expression is sensitive to PZQ treatment, thus offering a potential biomarker for assessing the response to chemotherapy.

The fruit of Rosa odorata sweet var. gigantea (Coll. et Hemsl.) Rehd. et Wils attenuates chronic atrophic gastritis induced by MNNG and its potential mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosa odorata Sweet var. gigantea (Coll. et Hemsl.) Rehd. et Wils is a commonly utilized traditional medicine among the Yi nationality, also known as "Gugongguo", for the treatment of gastrointestinal disorders. Previous studies have indicated that the extract of Rosa odorata sweet var. gigantea (FOE) fruit has demonstrated a protective effect on the stomach; however, its impact on chronic atrophic gastritis (CAG) with severe disease remains unknown. AIM OF THE STUDY: This study aimed to investigate the impact of FOE on CAG and its underlying mechanisms both in vitro and in vivo. MATERIALS AND METHODS: By employing Ultra Performance Liquid Chromatography/Quadrupole-Time of Flight Mass Spectrometry (UPLC-QTOF-MS/MS) and network pharmacology, the primary active compounds and action targets of FOE were identified. In vitro, the impact of FOE on CAG was investigated through scratch, migration, and invasion assays. Subsequently, guided by network pharmacology, EMT and TGF-ß signaling pathway-related proteins were assessed using Western blot and immunofluorescence experiments. Additionally, an in vivo CAG rat model was established to validate the effects of FOE and confirm its mechanism of action through hematoxylin-eosin (H&E), immunohistochemistry, Western blot, as well as untargeted metabolomics analysis of rat serum. It was observed that FOE inhibited scratch healing abilities, migration, invasion capabilities, as well as the expression of EMT-related proteins (E-cadherin, N-cadherin, Snail, Vimentin) in CAG model cells (MC cells), providing initial evidence for its efficacy. RESULTS: Through the analysis of UPLC-QTOF-MS/MS, a total of 51 major compounds were identified in the FOE. Subsequent network pharmacological analysis suggested that FOE may regulate Epithelial mesenchymal transition (EMT) through the transforming growth factor ß (TGF-ß) pathway. Furthermore, experimental verification demonstrated that FOE inhibited the protein expression of TGF-ß1 and its downstream protein Smad2/3 in vitro. In vivo findings also indicated similar mechanisms in MC cells, suggesting a reversal of the CAG process and significant inhibition of EMT and TGF-ß signaling pathways. Additionally, untargeted metabolomics of rat serum confirmed the therapeutic effect of FOE on CAG and predicted its potential involvement in the arachidonic acid metabolic pathway. CONCLUSION: This study initially demonstrated that FOE effectively reverses the process of EMT through the TGF-ß1/Smad2/3 signaling pathway, thereby providing a therapeutic benefit for CAG.

6:2 Chlorinated polyfluoroalkyl ether sulfonate (F-53B) induced nephrotoxicity associated with oxidative stress, inflammation and fibrosis in mice.

6:2 Chlorinated polyfluoroalkyl ether sulfonate (trade name F-53B) is substitutes for perfluorooctane sulfonate (PFOS) used in the plating industry, and has been found in a range of environmental matrices and livings. There are numerous ways by which it is biotoxic to mammals. The kidneys are critical for maintaining homeostasis. However, little research has been conducted on how F-53B affects the kidneys. In this work, we investigated the renal toxicity of long-term oral F-53B treatment in C57BL/6J mice. Mice were allowed to drink F-53B freely at concentrations of 0, 0.057, 0.57, and 5.7 mg/L for 8 weeks. Renal oxidative stress, inflammation, and fibrosis were detected in mice exposed to F-53B, and the expression of related biochemical markers was significantly altered. Further investigations revealed that the TGF-ß1/Smad3 and NF-κB signaling pathways may be associated with F-53B-induced renal fibrotic damage and inflammation. Overall, this study suggested that F-53B causes renal injury possibly via oxidative stress, activating the TGF-ß1/Smad3 and NF-κB signaling pathways. This provides a foundation for further research into the harmful mechanism of F-53B in mammals.

Eucommiae cortex extract alleviates renal fibrosis in CKD mice induced by adenine through the TGF-ß1/Smad signaling pathway.

Research into the potential therapeutic benefits of herbal remedies for treating chronic kidney disease (CKD), a condition marked by renal fibrosis and persistent inflammation, has become popular. Eucommiae cortex (EC) is a vital herb for strengthening bones and muscles and tonifying the kidneys and liver. In the study, C57 BL/6 mice were given a diet containing 0.2% adenine to create a CKD model. The findings demonstrated that exogenous EC supplementation successfully decreased the levels of creatinine and urea nitrogen, down-regulated the TGF-ß1/Smad signaling pathway's expression levels of TGF-ß1, α-SMA, Smad3, and phospho-Smad3, and prevented renal fibrosis. Consequently, it was determined that EC might have a nephroprotective impact.

SMAD4 enhances the cytotoxic efficacy of human NK cells against colorectal cancer cells via the m6A reader YTHDF2.

Background: Colorectal cancer (CRC) ranks as the third most prevalent malignant neoplasm in terms of both morbidity and mortality. Within the tumor microenvironment (TME) of CRC, the diminished presence and diminished cytotoxic function of natural killer (NK) cells serve as important factors driving the advancement of CRC; however, the precise regulatory mechanisms governing this phenomenon remain incompletely understood. Consequently, the identification of novel, potential anti-CRC targets associated with NK cells emerges as a pressing and paramount concern warranting immediate attention. Methods: We examined the regulatory mechanism of SMAD4-mediated NK cell cytotoxicity on CRC by utilizing various experimental techniques, such as qRT-PCR, flow cytometry. Results: Our findings revealed that the expression of SMAD4 is decreased in NK cells within the TME of human CRC. Furthermore, we observed that enforced upregulation of SMAD4 resulted in enhanced cytotoxicity of NK cells towards CRC cells. Furthermore, our research has revealed that YTHDF2 functions as a downstream effector of SMAD4, playing a crucial role in the control of transcription and translation of m6A-modified RNA. Moreover, our investigation demonstrated that increased expression of SMAD4 promoted the activating receptor NKG2D by elevating levels of YTHDF2. Ultimately, the SMAD4-YTHDF2 regulatory axis significantly enhanced the cytotoxicity of NK cells against human CRC cells. Conclusion: Our study unveils a novel mechanism through which SMAD4 modulates the cytotoxicity of NK cells towards CRC cells, suggesting that SMAD4 may hold promise as a potential therapeutic target for NK cell therapy in CRC.

Impact of Suppressor of Mothers Against Decapentaplegic (SMAD) 7 Gene Single Nucleotide Polymorphisms on Colorectal Cancer Risk and Prognosis.

Colorectal cancer (CRC) is a prevalent diagnosis worldwide with significant associated mortality. Single nucleotide polymorphic (SNP) variants have been identified as being associated with CRC risk. Although SMAD7 SNPs have been associated with the risk of developing CRC, their prognostic effect is still unclear. We carried out a case-control study to establish an association between genotypes of the suppressor of mothers against decapentaplegic (SMAD) 7 SNP rs4464148, rs4939827, and rs12953717 and CRC risk. Furthermore, we retrospectively assessed whether these SNPs had prognostic implications in CRC patients by evaluating survival with Kaplan-Meier curves and Cox regression. Only the CT genotype of the rs4939827 variant showed an association with CRC risk, and no genotype (CC, CT, or TT) of any of the three SNPs was shown to have prognostic implications in overall survival. Our study failed to show an association between certain SNP genotypes and the risk of CRC, which has already been well documented in two meta-analyses. Furthermore, it showed no prognostic relevance for these SNPs. More studies are needed to understand whether there are population variations or haplotype effects that could disturb the evaluation of these results.

Downregulation of Krüppel-like factor 15 expression delays endochondral bone ossification during fracture healing.

OBJECTIVE: The role of Krüppel-like zinc finger transcription factor 15 (KLF15) in endochondral ossification during fracture healing remains unexplored. In this study, we aimed to elucidate the impact of KLF15 in a mouse model of tibial transverse fracture. METHODS: We created tamoxifen-inducible, cartilage-specific KLF15 knockout mice (KLF15 KO). KLF15 fl/fl Col2-CreERT mice from the same litters as the KLF15 KO mice, but not treated with 4-hydroxytamoxifen, were used as controls (CT). At 10 weeks, male KLF15 KO and CT mice underwent tibial fracture followed by intramedullary nailing. Both groups were administered tamoxifen at days 0, 3, and 7 after surgery. The tibiae were harvested on post-surgery days 7, 10, and 14 for radiological assessment using micro-computed tomography. Histological staining (Safranin-O) and immunohistochemistry for KLF15, SOX9, Indian hedgehog (IHH), RUNX2, and Osterix were performed. Additionally, cartilage from mouse fetus was cultured for qRT-PCR and western blot analyses of KLF15, SOX9, IHH, Col2, RUNX2, Osterix, TGF-ß, SMAD3, and phosphor-SMAD3. RESULTS: The radiological assessment revealed that immature callus formation was delayed in KLF15 KO, compared with that in CT, peaking on day 14 compared with that on day 10 in CT. KLF15 KO mice exhibited delayed fracture healing and reduced Safranin-O staining at days 7 and 10 post-surgery. The ratio of cells positive for KLF15 and SOX9 was significantly lower in KLF15 KO than in CT, whereas the ratios for IHH, RUNX2, and Osterix showed no significant difference. RT-PCR revealed reduced expression of KLF15, SOX9, and COL2, with no significant changes in IHH, Osterix, RUNX2, TGF-ß, and SMAD3. Western blot analysis indicated decreased SMAD3 phosphorylation in KLF15 KO mice. CONCLUSION: KLF15 regulates SOX9 via the TGF-ß-SMAD3-SOX9 pathway, independent of IHH, in endochondral ossification. The KLF15 deficiency decreases SOX9 expression through reduced SMAD3 phosphorylation, subsequently delaying fracture healing.

ß-Mangostin Alleviates Renal Tubulointerstitial Fibrosis via the TGF-ß1/JNK Signaling Pathway.

The epithelial-to-mesenchymal transition (EMT) plays a key role in the pathogenesis of kidney fibrosis, and kidney fibrosis is associated with an adverse renal prognosis. Beta-mangostin (ß-Mag) is a xanthone derivative obtained from mangosteens that is involved in the generation of antifibrotic and anti-oxidation effects. The purpose of this study was to examine the effects of ß-Mag on renal tubulointerstitial fibrosis both in vivo and in vitro and the corresponding mechanisms involved. As shown through an in vivo study conducted on a unilateral ureteral obstruction mouse model, oral ß-Mag administration, in a dose-dependent manner, caused a lesser degree of tubulointerstitial damage, diminished collagen I fiber deposition, and the depressed expression of fibrotic markers (collagen I, α-SMA) and EMT markers (N-cadherin, Vimentin, Snail, and Slug) in the UUO kidney tissues. The in vitro part of this research revealed that ß-Mag, when co-treated with transforming growth factor-ß1 (TGF-ß1), decreased cell motility and downregulated the EMT (in relation to Vimentin, Snail, and N-cadherin) and phosphoryl-JNK1/2/Smad2/Smad3 expression. Furthermore, ß-Mag co-treated with SB (Smad2/3 kinase inhibitor) or SP600125 (JNK kinase inhibitor) significantly inhibited the TGF-ß1-associated downstream phosphorylation and activation of JNK1/2-mediated Smad2 targeting the Snail/Vimentin axis. To conclude, ß-Mag protects against EMT and kidney fibrotic processes by mediating the TGF-ß1/JNK/Smad2 targeting Snail-mediated Vimentin expression and may have therapeutic implications for renal tubulointerstitial fibrosis.

Honokiol inhibits human osteosarcoma MG63 cell migration by upregulating FTO and Smad6 to promote autophagy.

BACKGROUND: Osteosarcoma (OS) is a common primary malignant tumor of bone, most commonly seen in children and adolescents, which has a low survival rate and is a serious threat to patients' lives. Honokiol (HKL) is the main active components of Magnolia officinalis, which have significant anti-tumor properties. The aim of this study was to observe the autophagic and migratory effects of HKL on MG63 cells and to investigate whether the mechanism of action was related to FTO and Smad6. METHODS: Firstly, we cultured MG63 cells in vitro and intervened with different concentrations of HKL to detect cell activity by CCK8, apoptosis by flow cytometry, cell migration ability by scratch assay, cell invasion ability by transwell assay and MMP2, P62, LC3 I/II, FTO and Smad6 protein expression by Western blot. RESULTS: HKL inhibited MG63 cells activity and that this effect was dose and time dependent. Although there was no significant effect on apoptosis and invasive ability, HKL could act through effects such as promoting cell autophagy and inhibiting migration. HKL increased the protein expression levels of FTO, Smad6, MMP2, LC3 I/II and P62, and this effect was reduced after silencing of Smad6. CONCLUSIONS: HKL induced autophagy and inhibited cell migration in MG63 cells by increasing the expression of FTP and Smad6. It can be seen that HKL may be a promising drug for the treatment of OS.

TGF-ß antagonism synergizes with PPARγ agonism to reduce fibrosis and enhance beige adipogenesis.

OBJECTIVE: Adipose tissue depots vary markedly in their ability to store and metabolize triglycerides, undergo beige adipogenesis and susceptibility to metabolic disease. The molecular mechanisms that underlie such heterogeneity are not entirely clear. Previously, we showed that TGF-ß signaling suppresses beige adipogenesis via repressing the recruitment of dedicated beige progenitors. Here, we find that TGF-ß signals dynamically regulate the balance between adipose tissue fibrosis and beige adipogenesis. METHODS: We investigated adipose tissue depot-specific differences in activation of TGF-ß signaling in response to dietary challenge. RNA-seq and fluorescence activated cell sorting was performed to identify and characterize cells responding to changes in TGF-ß signaling status. Mouse models, pharmacological strategies and human adipose tissue analyses were performed to further define the influence of TGF-ß signaling on fibrosis and functional beige adipogenesis. RESULTS: Elevated basal and high-fat diet inducible activation of TGF-ß/Smad3 signaling was observed in the visceral adipose tissue depot. Activation of TGF-ß/Smad3 signaling was associated with increased adipose tissue fibrosis. RNA-seq combined with fluorescence-activated cell sorting of stromal vascular fraction of epididymal white adipose tissue depot resulted in identification of TGF-ß/Smad3 regulated ITGA5+ fibrogenic progenitors. TGF-ß/Smad3 signal inhibition, genetically or pharmacologically, reduced fibrosis and increased functional beige adipogenesis. TGF-ß/Smad3 antagonized the beneficial effects of PPARγ whereas TGF-ß receptor 1 inhibition synergized with actions of rosiglitazone, a PPARγ agonist, to dampen fibrosis and promote beige adipogenesis. Positive correlation between TGF-ß activation and ITGA5 was observed in human adipose tissue, with visceral adipose tissue depots exhibiting higher fibrosis potential than subcutaneous or brown adipose tissue depots. CONCLUSIONS: Basal and high-fat diet inducible activation of TGF-ß underlies the heterogeneity of adipose tissue depots. TGF-ß/Smad3 activation promotes adipose tissue fibrosis and suppresses beige progenitors. Together, these dual mechanisms preclude functional beige adipogenesis. Controlled inhibition of TßR1 signaling and concomitant PPARγ stimulation can suppress adipose tissue fibrosis and promote beige adipogenesis to improve metabolism.

C23 ameliorates carbon tetrachloride-induced liver fibrosis in mice.

BACKGROUND: C23, an oligo-peptide derived from cold-inducible RNA-binding protein (CIRP), has been reported to inhibit tissue inflammation, apoptosis and fibrosis by binding to the CIRP receptor; however, there are few reports on its role in liver fibrosis and the underlying mechanism is unknown. AIM: To explore whether C23 plays a significant role in carbon tetrachloride (CCl4)-induced liver fibrosis. METHODS: CCl4 was injected for 6 weeks to induce liver fibrosis and C23 was used beginning in the second week. Masson and Sirius red staining were used to examine changes in fiber levels. Inflammatory factors in the liver were detected and changes in α-smooth muscle actin (α-SMA) and collagen I expression were detected via immunohistochemical staining to evaluate the activation of hematopoietic stellate cells (HSCs). Western blotting was used to detect the activation status of the transforming growth factor-beta (TGF-ß)/Smad3 axis after C23 treatment. RESULTS: CCl4 successfully induced liver fibrosis in mice, while tumor necrosis factor-alpha (TNF-α), IL (interleukin)-1ß, and IL-6 levels increased significantly and the IL-10 level decreased significantly. Interestingly, C23 inhibited this process. On the other hand, C23 significantly inhibited the activation of HSCs induced by CCl4, which inhibited the expression of α-SMA and the synthesis of collagen I. In terms of mechanism, C23 can block Smad3 phosphorylation significantly and inhibits TGF-ß/Smad3 pathway activation, thereby improving liver injury caused by CCl4. CONCLUSION: C23 may block TGF-ß/Smad3 axis activation, inhibit the expression of inflammatory factors, and inhibit the activation of HSCs induced by CCl4, alleviating liver fibrosis.

Niclosamide attenuates erectile dysfunction and corporal fibrosis via reversal of Smad signaling in diabetic rat model.

BACKGROUND: Diabetes mellitus-induced erectile dysfunction (DMED) is a common urological complication of diabetes, and current drugs often fail to provide an effective treatment. Smad2/3 signaling-mediated corporal fibrosis has a critical role in the molecular basis of DMED. AIM: We investigated the effect of Niclosamide (Nic), an antihelmintic drug with antifibrotic effects, on erectile function in a rat DMED model. METHODS: Male Sprague Dawley rats were injected intraperitoneally (i.p) with streptozotocin (75 mg/kg) to induce diabetes. At week 8, both diabetic and nondiabetic rats were treated with Nic (10 mg·kg-1/day; i.p) or vehicle for 4 weeks. At week 12, erectile function was evaluated as intracavernous pressure (ICP) response to the electrical stimulation of the cavernous nerve (CN). Penile tissues were harvested for Masson's trichrome staining or western blotting to determine corporal fibrosis and Smad2/3 pathway-related protein expression, respectively. OUTCOMES: At the end of the experimental protocol, in vivo erectile function was assessed by measuring the ratio of ICP/ mean arterial pressure (MAP) and total ICP following CN stimulation. Smooth muscle content and collagen fibers were evaluated by Masson's trichrome staining of the penile tissues. The expressions of fibrosis-related proteins (Smad2, Smad3, fibronectin) were determined using western blotting in the penile tissues. RESULTS: Erectile function, as determined by the maximum ICP/MAP and total ICP/MAP ratios, was drastically decreased in diabetic rats. Corporal tissues of diabetic rats were severely fibrotic with a significant increase in collagen fibers and a marked reduction in smooth muscle content. Also, the protein expressions of phosphorylated (p-)Smad2, p-Smad3 and fibronectin were significantly increased in the penis of diabetic rats. Both functional and molecular alterations in DMED were effectively reversed by Nic-treated diabetic rats without a glycemic alteration. CLINICAL IMPLICATIONS: Nic could be a promising candidate for the treatment of DMED due to its antifibrotic effects. STRENGTHS AND LIMITATIONS: The present study provides the first evidence that Nic has beneficial effect on erectile dysfunction by attenuating corporal fibrosis in a rat model of DMED. The effect of Nic on penile endothelial function and the other potential underlying mechanisms needs to be further elucidated. CONCLUSIONS: Nic improved erectile function in DMED rats possibly suppressing penile fibrosis by inhibiting Smad2/3 signaling. These results suggest a potential therapeutic repurposing of Nic as an adjuvant treatment in DMED.

NLRP3 deficiency improves bone healing of tooth extraction sockets through SMAD2/3-RUNX2 mediated osteoblast differentiation.

Impaired bone healing following tooth extraction poses a significant challenge for implantation. As a crucial component of the natural immune system, the NLRP3 inflammasome is one of the most extensively studied Pattern-Recognition Receptors (PRRs), and is involved in multiple diseases. Yet, the role of NLRP3 in bone healing remains to be clarified. Here, to investigate the effect of NLRP3 on bone healing, we established a maxillary first molar extraction model in wild-type (WT) and NLRP3KO mice using minimally invasive techniques. We observed that NLRP3 was activated during the bone repair phase, and its depletion enhanced socket bone formation and osteoblast differentiation. Moreover, NLRP3 inflammasome activation was found to inhibit osteogenic differentiation in alveolar bone-derived mesenchymal stem cells (aBMSCs), an effect mitigated by NLRP3 deficiency. Mechanistically, we established that SMAD2/3-RUNX2 signaling pathway is a downstream target of NLRP3 inflammasome activation, and SMAD2/3 knockdown partially reversed the significant decrease in expression of RUNX2, OSX, and ALP induced by NLRP3. Thus, our findings demonstrate that NLRP3 negatively modulates alveolar socket bone healing and contribute to the understanding of the NLRP3-induced signaling pathways involved in osteogenesis regulation.