Deletion of GPR81 activates CREB/Smad7 pathway and alleviates liver fibrosis in mice.

BACKGROUND: Enhanced glycolysis is a crucial metabolic event that drives the development of liver fibrosis, but the molecular mechanisms have not been fully understood. Lactate is the endproduct of glycolysis, which has recently been identified as a bioactive metabolite binding to G-protein-coupled receptor 81 (GPR81). We then questioned whether GPR81 is implicated in the development of liver fibrosis. METHODS: The level of GPR81 was determined in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and in transforming growth factor beta 1 (TGF-ß1)-activated hepatic stellate cells (HSCs) LX-2. To investigate the significance of GPR81 in liver fibrosis, wild-type (WT) and GPR81 knockout (KO) mice were exposed to CCl4, and then the degree of liver fibrosis was determined. In addition, the GPR81 agonist 3,5-dihydroxybenzoic acid (DHBA) was supplemented in CCl4-challenged mice and TGF-ß1-activated LX-2 cells to further investigate the pathological roles of GPR81 on HSCs activation. RESULTS: CCl4 exposure or TGF-ß1 stimulation significantly upregulated the expression of GPR81, while deletion of GPR81 alleviated CCl4-induced elevation of aminotransferase, production of pro-inflammatory cytokines, and deposition of collagen. Consistently, the production of TGF-ß1, the expression of alpha-smooth muscle actin (α-SMA) and collagen I (COL1A1), as well as the elevation of hydroxyproline were suppressed in GPR81 deficient mice. Supplementation with DHBA enhanced CCl4-induced liver fibrogenesis in WT mice but not in GPR81 KO mice. DHBA also promoted TGF-ß1-induced LX-2 activation. Mechanistically, GPR81 suppressed cAMP/CREB and then inhibited the expression of Smad7, a negative regulator of Smad3, which resulted in increased phosphorylation of Smad3 and enhanced activation of HSCs. CONCLUSION: GPR81 might be a detrimental factor that promotes the development of liver fibrosis by regulating CREB/Smad7 pathway.

Smad7 palmitoylation by the S-acyltransferase zDHHC17 enhances its inhibitory effect on TGF-ß/Smad signaling.

Intracellular signaling by the pleiotropic cytokine transforming growth factor-ß (TGF-ß) is inhibited by Smad7 in a feedback control mechanism. The activity of Smad7 is tightly regulated by multiple post-translational modifications. Using resin-assisted capture and metabolic labeling methods, we show here that Smad7 is S-palmitoylated in mammary epithelial cell models that are widely studied because of their strong responses to TGF-ß and their biological relevance to mammary development and tumor progression. S-palmitoylation of Smad7 is mediated by zDHHC17, a member of a family of 23 S-acyltransferase enzymes. Moreover, we identified four cysteine residues (Cys202, Cys225, Cys415, and Cys417) in Smad7 as palmitoylation acceptor sites. S-palmitoylation of Smad7 on Cys415 and Cys417 promoted the translocation of Smad7 from the nucleus to the cytoplasm, enhanced the stability of the Smad7 protein, and enforced its inhibitory effect on TGF-ß-induced Smad transcriptional response. Thus, our findings reveal a new post-translational modification of Smad7, and highlight an important role of S-palmitoylation to enhance inhibition of TGF-ß/Smad signaling by Smad7.

Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/-) mice.

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.

Mir-195-5p targets Smad7 regulation of the Wnt/ß-catenin pathway to promote osteogenic differentiation of vascular smooth muscle cells.

In this study, we sought to investigate the mechanisms of action of miR-195-5p in the osteogenic differentiation of vascular smooth muscle cells (VSMCs), and thereby provide novel insights and a reference for the targeted therapy of arterial media calcification. VSMC differentiation was induced using sodium ß-glycerophosphate, and we investigated the effects of transfecting cells with miR-195-5p mimics, vectors overexpressing Smad7, and the Wnt/ß-catenin pathway inhibitor (KYA1797K) on VSMC differentiation by determining cell viability and apoptosis, and the mRNA and protein expression of factors associated with osteogenic differentiation and the Wnt/ß-catenin pathway. The results revealed that miR-195-5p mimics enhanced the osteogenic differentiation of VSMCs induced by ß-glycerophosphate, whereas the overexpression of Smad7 reversed this phenomenon. In addition, KYA1797K was found to promote the effects of Smad7 overexpression. In conclusion, by targeting, Smad7, miR-195-5p promotes the Wnt/ß-catenin pathway. and thus the osteogenic differentiation of VSMCs. These findings will provide a reference for elucidating the mechanisms whereby miR-195-5p regulates osteogenic differentiation.

UHRF1 inhibition mitigates vascular endothelial cell injury and ameliorates atherosclerosis in mice via regulating the SMAD7/YAP1 axis.

BACKGROUND: Endothelial cell injury and dysfunction lead to cholesterol and lipid accumulation and atherosclerotic plaque formation in the arterial wall during atherosclerosis (AS) progression, Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), a DNA methylation regulator, was strongly upregulated in atherosclerotic plaque lesions in mice. This study aimed to investigate the precise biological functions and regulatory mechanisms of UHRF1 on endothelial dysfunction during AS development. METHODS: UHRF1 levels in the atherosclerotic plaque tissues and normal arterial intima from AS patients were tested with Western blot analysis and immunohistochemistry assays. Human umbilical vein endothelial cells (HUVECs) were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce an injury model and then transfected with short hairpin RNA targeting UHRF1 (sh-UHRF1). Cell proliferation, migration, apoptosis, the levels of inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and the protein levels adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were measured. Moreover, co-immunoprecipitation assay was used to determine the interactions between UHRF1 and DNA methyltransferases 1 (DNMT1), As well as mothers against DPP homolog 7 (SMAD7) and yes-associated protein 1 (YAP1). SMAD7 promoter methylation was examined with methylation-specific PCR. In addition, we established an AS mouse model to determine the in vivo effects of UHRF1 on AS progression. RESULTS: UHRF1 was upregulated in atherosclerotic plaque tissues and ox-LDL-treated HUVECs. UHRF1 knockdown mitigated ox-LDL-induced proliferation and migration inhibition, apoptosis and the production of TNF-α, IL-6, VCAM-1, and ICAM-1 in HUVECs. Mechanistically, UHRF1 promoted DNMT1-mediated SMAD7 promoter methylation and inhibited its expression. SMAD7 knockdown abolished the protective effects of UHRF1 knockdown on ox-LDL-induced HUVEC injury. Moreover, SMAD7 interacted with YAP1 and inhibited YAP1 expression by promoting YAP1 protein ubiquitination-independent degradation in HUVECs. YAP1 overexpression abrogated SMAD7 overexpression-mediated protective effects on ox-LDL-induced HUVEC injury. Finally, UHRF1 knockdown alleviated atherosclerotic plaque deposition and arterial lesions in AS mice. CONCLUSION: UHRF1 inhibition mitigates vascular endothelial cell injury and ameliorates AS progression in mice by regulating the SMAD7/YAP1 axis.

Neutrophil-Based Bionic Delivery System Breaks Through the Capillary Barrier of Liver Sinusoidal Endothelial Cells and Inhibits the Activation of Hepatic Stellate Cells.

The capillarization of hepatic sinusoids resulting from the activation of hepatic stellate cells poses a significant challenge, impeding the effective delivery of therapeutic agents to the Disse space for liver fibrosis treatment. Therefore, overcoming these barriers and achieving efficient drug delivery to activated hepatic stellate cells (aHSCs) are pressing challenge. In this study, we developed a synergistic sequential drug delivery approach utilizing neutrophil membrane hybrid liposome@atorvastatin/amlisentan (NCM@AtAm) and vitamin A-neutrophil membrane hybrid liposome @albumin (VNCM@Bai) nanoparticles (NPs) to breach the capillary barrier for targeted HSC cell delivery. Initially, NCM@AtAm NPs were successfully directed to the site of hepatic fibrosis through neutrophil-mediated inflammatory targeting, resulting in the normalization of liver sinusoidal endothelial cells (LSECs) and restoration of fenestrations under the combined influence of At and Am. Elevated tissue levels of the p-Akt protein and endothelial nitric oxide synthase (eNOS) indicated the normalization of LSECs following treatment with At and Am. Subsequently, VNCM@Bai NPs traversed the restored LSEC fenestrations to access the Disse space, facilitating the delivery of Bai into aHSCs under vitamin A guidance. Lastly, both in vitro and in vivo results demonstrated the efficacy of Bai in inhibiting HSC cell activation by modulating the PPAR γ/TGF-ß1 and STAT1/Smad7 signaling pathways, thereby effectively treating liver fibrosis. Overall, our designed synergistic sequential delivery system effectively overcomes the barrier imposed by LSECs, offering a promising therapeutic strategy for liver fibrosis treatment in clinical settings.

Carbonic Anhydrase 3 is required for cardiac repair post myocardial infarction via Smad7-Smad2/3 signaling pathway.

Appropriate fibrosis is required to prevent subsequent adverse remodeling and heart failure post myocardial infarction (MI), and cardiac fibroblasts (CFs) play a critical role during the process. Carbonic anhydrase 3 (CAR3) is an important mediator in multiple biological processes besides its CO2 hydration activity; however, the role and underlying mechanism of CAR3 on cardiac repair post MI injury remains unknown. Here, we found that CAR3 expression was up-regulated in cardiac tissue in infarct area at the reparative phase of MI, with a peak at 7 days post MI. The upregulation was detected mainly on fibroblast instead of cardiomyocyte, and primary cardiac fibroblasts treated with TGF-ß1 recaptured our observation. While CAR3 deficiency leads to weakened collagen density, enlarged infarct size and aggravated cardiac dysfunction post-MI. In fibroblast, we observed that CAR3 deficiency restrains collagen synthesis, cell migration and gel contraction of cardiac fibroblasts, whereas overexpression of CAR3 in CFs improves wound healing and cardiac fibroblast activation. Mechanistically, CAR3 stabilizes Smad7 protein via modulating its acetylation, which dampens phosphorylation of Smad2 and Smad3, thus inhibiting fibroblast transformation. In contrast, inhibition of Smad7 acetylation with C646 blunts CAR3 deficiency induced suppression of fibroblast activation and impaired cardiac healing. Our data demonstrate a protective role of CAR3 in cardiac wound repair post MI via promoting fibroblasts activation through Smad7-TGF-ß/Smad2/3 signaling pathway.

LncRNA ZFAS1 Alleviated NLRP3 Inflammasome-Mediated Pyroptosis through Regulating miR-96-5p/Smad7 Signaling in Allergic Rhinitis.

INTRODUCTION: The NLR family pyrin domain containing 3 (NLRP3)-mediated pyroptosis was positively correlated with the allergic rhinitis progression and was reported to be regulated by SMAD family member 7 (Smad7). Bioinformatics analysis revealed that Smad7 might be targeted by miR-96-5p, and miR-96-5p might be targeted by long noncoding RNA zinc finger antisense 1 (ZFAS1). However, the effects and regulatory mechanisms of the ZFAS1/miR-96-5p/Smad7 functional axis in allergic rhinitis have not been investigated. METHODS: Human nasal mucosa epithelial cell line RPMI 2650 and C57BL/6 mice were obtained for in vitro and in vivo studies. Dual-luciferase reporter assay and RNA immunoprecipitation were implemented for detecting molecular interactions. Cell counting kit-8 and flow cytometry were used for measuring cell viability and pyroptosis. ELISA was obtained for monitoring cytokine secretion. RT-qPCR and Western blot were examined for determining RNA and protein expression. RESULTS: In vitro studies revealed that ZFAS1 was downregulated in interleukin (IL)-13-treated RPMI 2650 cells, while overexpression of ZFAS1 enhanced cell viability and inhibited NLRP3-mediated pyroptosis and inflammatory response. ZFAS1 directly inhibited miR-96-5p to suppress NLRP3-mediated pyroptosis in IL-13-treated RPMI 2650 cells. MiR-96-5p bound to the 3'-untranslated region of Smad7 and knockdown of Smad7 significantly reversed the effects of miR-96-5p depletion. Moreover, in vivo experiments further confirmed the findings of in vitro studies and showed ZFAS1 overexpression or miR-96-5p inhibition alleviated allergic rhinitis in vivo. CONCLUSION: ZFAS1 downregulated the expression of miR-96-5p to upregulate Smad7 level, which subsequently inhibited NLRP3-mediated pyroptosis and inflammatory response to ameliorate allergic rhinitis.

Linc-smad7 is involved in the regulation of lipid synthesis in mouse mammary epithelial cells.

Long intergenic non-coding RNA(lincRNA) is transcribed from the intermediate regions of coding genes and plays a pivotal role in the regulation of lipid synthesis. N6-methyladenosine (m6A) modification is widely prevalent in eukaryotic mRNAs and serves as a regulatory factor in diverse biological processes. This study aims to delineate the mechanism by which Linc-smad7 mediates m6A methylation to regulate milk fat synthesis. Tissue expression analysis in this study revealed a high expression of Linc-smad7 in breast tissue during pregnancy. Cell proliferation assays, including CCK8 and EdU assays, demonstrated that Linc-smad7 had no significant impact on the proliferation of mammary epithelial cells. However, during mammary epithelial cell differentiation, the overexpression of Linc-smad7 led to reduced lipid formation, whereas interference with Linc-smad7 promoted lipogenesis. Mechanistically, Linc-smad7 was found to modulate RNA m6A levels, as evidenced by dot blot assays and methylated RNA immunoprecipitation sequencing (MeRIP-Seq). Subsequent validation through RT-qPCR corroborated these findings, aligning with the m6A sequencing outcomes. Furthermore, co-transfection experiments elucidated that Linc-smad7 regulates lipid synthesis in mammary epithelial cells by influencing the expression of METTL14. In summary, these findings underscore the regulatory role of Linc-smad7 in controlling METTL14 gene expression, thereby mediating m6A modifications to regulate lipid synthesis in mammary epithelial cells.

2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione isolated from Averrhoa carambola L. root inhibits high glucose-induced EMT in HK-2 cells through targeting the regulation of miR-21-5p/Smad7 signaling pathway.

OBJECTIVE: 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) isolated from Averrhoa carambola L. root, has been proven therapeutic effects on diabetic kidney disease (DKD). This research aims to assess DMDD's effects on DKD and to investigate its underlying mechanisms, to establish DMDD as a novel pharmaceutical agent for DKD treatment. METHODS: The human renal tubular epithelial (HK-2) cells were induced by high glucose (HG) to mimic DKD and followed by DMDD treatment. The cytotoxicity of DMDD was assessed using the Cell Counting Kit-8 (CCK-8) assay. The migratory capacity of HK-2 cells was evaluated through transwell and scratch-wound assays. To investigate the effect of Smad7 and miR-21-5p, lentiviral transfection was employed in HK-2 cells. Additionally, the expression of proteins related to epithelial-mesenchymal transition (EMT) and TGFß1/Smad2/3 pathway was checked by QRT-PCR, Western blot, and immunofluorescence techniques. RESULTS: This study has shown that DMDD significantly suppresses cell migration and the expression of Vimentin, α-SMA, TGFß1, and p-Smad2/3 in HK-2 cells under HG conditions. Concurrently, DMDD enhances the protein expression of E-cadherin and Smad7. Intriguingly, the therapeutic effect of DMDD was abrogated upon Smad7 silencing. Further investigations revealed that DMDD effectively inhibits miR-21-5p expression, which is upregulated by HG. Downregulation of miR-21-5p inhibits the activation of the TGFß1/Smad2/3 pathway and EMT induced by HG. In contrast, overexpression of miR-21-5p negates DMDD's therapeutic benefits. CONCLUSION: DMDD mitigates EMT in HG-induced HK-2 cells by modulating the miR-21-5p/Smad7 pathway, thereby inhibiting renal fibrosis in DKD. These findings suggest that DMDD holds promise as a potential therapeutic agent for DKD.

Expression of SMADs in orthotopic human endometrium, ovarian endometriosis, and endometriotic lesions in a murine model.

Activin A promotes the development of endometriotic lesions in a murine model of endometriosis, and the immunohistochemical localization of phosphorylated suppressor of mothers against decapentaplegic homolog 2/3 (pSMAD2/3) complex in endometriotic lesions has been reported. Activin may therefore be involved in the development and proliferation of endometriotic cells via the SMAD signaling pathway. However, few detailed reports exist on SMAD7 expression in endometriosis. The purpose of this study was to investigate the expression of pSMAD2/3 or pSMAD3 and SMAD7 in the orthotopic human endometrium, ovarian endometriosis, and endometriotic lesions in a murine model and the effect of activin A on pSMAD2/3 and SMAD7 expression. We established an endometriosis murine model via the intraperitoneal administration of endometrial tissue and blood from donor mice. Activin A was intraperitoneally administered to the activin group. We immunohistochemically evaluated orthotopic endometria, ovarian endometriotic tissues, and endometriotic lesions in the murine model followed by western blotting. We found that pSMAD3 and SMAD7 were expressed in ovarian endometriosis and orthotopic endometria from patients with and without endometriosis. In the murine model, endometriotic lesions expressed pSMAD2/3 and SMAD7 in the activin and control groups, and higher SMAD7 expression was found in the activin group. To the best of our knowledge, this study is the first to show that SMAD7 expression is upregulated in endometriosis. In conclusion, these results suggest that activin A activates the SMAD signaling pathway and promotes the development of endometriotic lesions, thus identifying SMAD7 as a potential therapeutic target for endometriosis.

SMAD7 expression in CAR-T cells improves persistence and safety for solid tumors.

Despite the tremendous progress of chimeric antigen receptor T (CAR-T) cell therapy in hematological malignancies, their application in solid tumors has been limited largely due to T-cell exhaustion in the tumor microenvironment (TME) and systemic toxicity caused by excessive cytokine release. As a key regulator of the immunosuppressive TME, TGF-ß promotes cytokine synthesis via the NF-κB pathway. Here, we coexpressed SMAD7, a suppressor of TGF-ß signaling, with a HER2-targeted CAR in engineered T cells. These novel CAR-T cells displayed high cytolytic efficacy and were resistant to TGF-ß-triggered exhaustion, which enabled sustained tumoricidal capacity after continuous antigen exposure. Moreover, SMAD7 substantially reduced the production of inflammatory cytokines by antigen-primed CAR-T cells. Mechanistically, SMAD7 downregulated TGF-ß receptor I and abrogated the interplay between the TGF-ß and NF-κB pathways in CAR-T cells. As a result, these CAR-T cells persistently inhibited tumor growth and promoted the survival of tumor-challenged mice regardless of the hostile tumor microenvironment caused by a high concentration of TGF-ß. SMAD7 coexpression also enhanced CAR-T-cell infiltration and persistent activation in patient-derived tumor organoids. Therefore, our study demonstrated the feasibility of SMAD7 coexpression as a novel approach to improve the efficacy and safety of CAR-T-cell therapy for solid tumors.

The translational potential of miR-26 in atherosclerosis and development of agents for its target genes ACC1/2, COL1A1, CPT1A, FBP1, DGAT2, and SMAD7.

Atherosclerosis is one of the leading causes of death worldwide. miR-26 is a potential biomarker of atherosclerosis. Standardized diagnostic tests for miR-26 (MIR26-DX) have been developed, but the fastest progress has been in predicting the efficacy of IFN-α therapy for hepatocellular carcinoma (HCC, phase 3). MiR-26 slows atherosclerosis development by suppressing ACC1/2, ACLY, ACSL3/4, ALDH3A2, ALPL, BMP2, CD36, COL1A1, CPT1A, CTGF, DGAT2, EHHADH, FAS, FBP1, GATA4, GSK3ß, G6PC, Gys2, HMGA1, HMGB1, LDLR, LIPC, IL-1ß, IL-6, JAG2, KCNJ2, MALT1, ß-MHC, NF-κB, PCK1, PLCß1, PYGL, RUNX2, SCD1, SMAD1/4/5/7, SREBF1, TAB3, TAK1, TCF7L2, and TNF-α expression. Many agents targeting these genes, such as the ACC1/2 inhibitors GS-0976, PF-05221304, and MK-4074; the DGAT2 inhibitors IONIS-DGAT2Rx, PF-06427878, PF-0685571, and PF-07202954; the COL1A1 inhibitor HT-100; the stimulants 68Ga-CBP8 and RCT-01; the CPT1A inhibitors etomoxir, perhexiline, and teglicar; the FBP1 inhibitors CS-917 and MB07803; and the SMAD7 inhibitor mongersen, have been investigated in clinical trials. Interestingly, miR-26 better reduced intima-media thickness (IMT) than PCSK9 or CT-1 knockout. Many PCSK9 inhibitors, including alirocumab, evolocumab, inclisiran, AZD8233, Civi-007, MK-0616, and LIB003, have been investigated in clinical trials. Recombinant CT-1 was also investigated in clinical trials. Therefore, miR-26 is a promising target for agent development. miR-26 promotes foam cell formation by reducing ABCA1 and ARL4C expression. Multiple materials can be used to deliver miR-26, but it is unclear which material is most suitable for mass production and clinical applications. This review focuses on the potential use of miR-26 in treating atherosclerosis to support the development of agents targeting it.

Circular RNA circWNK1 inhibits the progression of gastric cancer via regulating the miR-21-3p/SMAD7 axis.

Gastric cancer (GC) is a highly aggressive malignancy with limited treatment options for advanced-stage patients. Recent studies have highlighted the role of circular RNA (circRNA) as a novel regulator of cancer progression in various malignancies. However, the underlying mechanisms by which circRNA contributes to the development and progression of GC remain poorly understood. In this study, we utilized microarrays and real-time quantitative polymerase chain reaction (qRT-PCR) to identify and validate a downregulated circRNA, hsa_circ_0003251 (referred to as circWNK1), in paired GC and normal tissues. Through a series of in vitro and in vivo gain-of-function and loss-of-function assays, we demonstrated that circWNK1 exerts inhibitory effects on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of GC cells. Additionally, we discovered that circWNK1 acts as a competitive endogenous RNA (ceRNA) for SMAD7 by sequestering miR-21-3p. Our findings were supported by comprehensive biological information analysis, as well as RNA pull-down, luciferase reporter gene, and western blot assays. Notably, the downregulation of circWNK1 in GC cells resulted in reduced SMAD7 expression, subsequently activating the TGF-ß signaling pathway. Collectively, our study reveals that circWNK1 functions as a tumor suppressor in GC by regulating the miR-21-3p/SMAD7-mediated TGF-ß signaling pathway. Furthermore, circWNK1 holds promise as a potential biomarker for the diagnosis and treatment of GC.

Nicotinamide phosphoribose transferase facilitates macrophage-mediated pulmonary fibrosis through the Sirt1-Smad7 pathway in mice.

Pulmonary fibrosis is a challenging lung disease characterized by a bleak prognosis. A pivotal element in the progression of this disease is the dysregulated recruitment of macrophages. Nicotinamide phosphoribose transferase (NAMPT), secreted by alveolar epithelial cells and inflammatory cells, has been previously identified to influence macrophage inflammation in acute lung injury through the nicotinamide adenine dinucleotide (NAD) rescue synthesis pathway. Nonetheless, the exact role of NAMPT in the regulation of lung fibrosis is yet to be elucidated. In our research, we employed bleomycin (BLM) to induce pulmonary fibrosis in Namptflox/flox;Cx3cr1CreER mice, using Namptflox/flox mice as controls. Our findings revealed an augmented expression of NAMPT concurrent with a marked increase in the secretion of NAD and inflammatory cytokines such as IL-6, TNF-α, and TGF-ß1 post-BLM treatment. Furthermore, an upsurge in NAMPT-positive macrophages was observed in the lungs of BLM-treated Namptflox/flox mice. Notably, a conditional knockout of NAMPT (NAMPT cKO) in lung macrophages curtailed the BLM-induced inflammatory responses and significantly mitigated pulmonary fibrosis. This was associated with diminished phospho-Sirt1 (p-Sirt1) expression levels and a concomitant rise in mothers against decapentaplegic homolog 7 (Smad7) expression in BLM-treated mouse lungs and murine RAW 264.7 macrophage cells. Collectively, our data suggests that NAMPT exacerbates macrophage-driven inflammation and pulmonary fibrosis via the Sirt1-Smad7 pathway, positioning NAMPT as a promising therapeutic target for pulmonary fibrosis intervention.

Colorectal Cancer Risk Prediction Using the rs4939827 Polymorphism of the SMAD7 Gene in the Romanian Population.

Colorectal cancer (CRC) is globally recognized as a prevalent malignancy known for its significant mortality rate. Recent years have witnessed a rising incidence trend in colorectal cancer, emphasizing the necessity for early diagnosis. Our study focused on examining the impact of the SMAD7 gene variant rs4939827 on the risk of colorectal cancer occurrence. The composition of our study group included 340 individuals, comprising 170 CRC diagnosed patients and 170 healthy controls. We performed genotyping of all biological samples using the TaqMan assay on the ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). This investigation focused on the rs4939827 gene variant, assessing its association with CRC risk and clinicopathological characteristics. Genotyping results for the SMAD7 gene variant rs4939827 revealed a 42.6% prevalence of the C allele in CRC patients (p = 0.245) and a 22.8% prevalence of the T allele in control subjects (p = 0.109). This study concluded that there was an elevated risk of CRC in the dominant model for CC/CT+TT, with a p-value of 0.113 and an odds ratio (OR) of 2.781, within a 95% confidence interval (CI) of 0.998 to 3.456. The findings of our research indicate a correlation between variants of the SMAD7 gene and the likelihood of developing colorectal cancer in our study population. Consequently, these results could be instrumental in facilitating early diagnosis of colorectal cancer utilizing information on single-nucleotide polymorphism (SNP) and genetic susceptibility to the disease.

Vascular smooth muscle cell-specific miRNA-214 deficiency alleviates simulated microgravity-induced vascular remodeling.

The human cardiovascular system has evolved to accommodate the gravity of Earth. Microgravity during spaceflight has been shown to induce vascular remodeling, leading to a decline in vascular function. The underlying mechanisms are not yet fully understood. Our previous study demonstrated that miR-214 plays a critical role in angiotensin II-induced vascular remodeling by reducing the levels of Smad7 and increasing the phosphorylation of Smad3. However, its role in vascular remodeling evoked by microgravity is not yet known. This study aimed to determine the contribution of miR-214 to the regulation of microgravity-induced vascular remodeling. The results of our study revealed that miR-214 expression was increased in the forebody arteries of both mice and monkeys after simulated microgravity treatment. In vitro, rotation-simulated microgravity-induced VSMC migration, hypertrophy, fibrosis, and inflammation were repressed by miR-214 knockout (KO) in VSMCs. Additionally, miR-214 KO increased the level of Smad7 and decreased the phosphorylation of Smad3, leading to a decrease in downstream gene expression. Furthermore, miR-214 cKO protected against simulated microgravity induced the decline in aorta function and the increase in stiffness. Histological analysis showed that miR-214 cKO inhibited the increases in vascular medial thickness that occurred after simulated microgravity treatment. Altogether, these results demonstrate that miR-214 has potential as a therapeutic target for the treatment of vascular remodeling caused by simulated microgravity.

Salvianolic acid B ameliorates myocardial fibrosis in diabetic cardiomyopathy by deubiquitinating Smad7.

BACKGROUND: Salvianolic acid B (Sal B), a water-soluble phenolic compound derived from Salvia miltiorrhiza Bunge, is commonly used in Traditional Chinese Medicine to treat cardiovascular disease. In our previous study, Sal B protected against myocardial fibrosis induced by diabetic cardiomyopathy (DCM). This study aimed to investigate the ameliorative effects and potential mechanisms of Sal B in mitigating myocardial fibrosis induced by DCM. METHODS: Various methods were used to investigate the effects of Sal B on myocardial fibrosis induced by DCM in vivo and in vitro. These methods included blood glucose measurement, echocardiography, HE staining, Masson's trichrome staining, Sirius red staining, cell proliferation assessment, determination of hydroxyproline levels, immunohistochemical staining, evaluation of fibrosis-related protein expression (Collagen-I, Collagen-III, TGF-ß1, p-Smad3, Smad3, Smad7, and α-smooth muscle actin), analysis of Smad7 gene expression, and analysis of Smad7 ubiquitin modification. RESULTS: The animal test results indicated that Sal B significantly improved cardiac function, inhibited collagen deposition and phenotypic transformation, and ameliorated myocardial fibrosis in DCM by upregulating Smad7, thereby inhibiting the TGF-ß1 signaling pathway. In addition, cell experiments demonstrated that Sal B significantly inhibited the proliferation, migration, phenotypic transformation, and collagen secretion of cardiac fibroblasts (CFs) induced by high glucose (HG). Sal B significantly decreased the ubiquitination of Smad7 and stabilized the protein expression of Smad7, thereby increasing the protein expression of Smad7 in CFs and inhibiting the TGF-ß1 signaling pathway, which may be the potential mechanism by which Sal B mitigates myocardial fibrosis induced by DCM. CONCLUSION: This study revealed that Sal B can improve myocardial fibrosis in DCM by deubiquitinating Smad7, stabilizing the protein expression of Smad7, and blocking the TGF-ß1 signaling pathway.

miR-423 sponged by lncRNA NORHA inhibits granulosa cell apoptosis.

BACKGROUND: Atresia and degeneration, a follicular developmental fate that reduces female fertility and is triggered by granulosa cell (GC) apoptosis, have been induced by dozens of miRNAs. Here, we report a miRNA, miR-423, that inhibits the initiation of follicular atresia (FA), and early apoptosis of GCs. RESULTS: We showed that miR-423 was down-regulated during sow FA, and its levels in follicles were negatively correlated with the GC density and the P4/E2 ratio in the follicular fluid in vivo. The in vitro gain-of-function experiments revealed that miR-423 suppresses cell apoptosis, especially early apoptosis in GCs. Mechanically speaking, the miR-423 targets and interacts with the 3'-UTR of the porcine SMAD7 gene, which encodes an apoptosis-inducing factor in GCs, and represses its expression and pro-apoptotic function. Interestingly, FA and the GC apoptosis-related lncRNA NORHA was demonstrated as a ceRNA of miR-423. Additionally, we showed that a single base deletion/insertion in the miR-423 promoter is significantly associated with the number of stillbirths (NSB) trait of sows. CONCLUSION: These results demonstrate that miR-423 is a small molecule for inhibiting FA initiation and GC early apoptosis, suggesting that treating with miR-423 may be a novel approach for inhibiting FA initiation and improving female fertility.

SMAD7 gene polymorphisms and their influence on patients with colorectal cancer.

Colorectal cancer (CRC) is a prevalent malignant tumor, and its pathogenesis is still not fully understood. Studies have shown that SMAD7 gene polymorphisms can affect CRC susceptibility, but the results have been inconsistent and require additional confirmation. Our study aimed to evaluate the effect of SMAD7 variants on the risk of CRC in the Chinese Han population. A total of five single nucleotide polymorphisms (SNPs) in SMAD7 were genotyped among 696 CRC patients and 696 healthy participants using the MassARRAY iPLEX platform. SNPs were evaluated for their associations with CRC using logistic regression analysis under multiple genetic models. The false-positive report probability (FPRP) analysis was used to validate the positive findings. Our study indicated that rs11874392 showed an increased association with CRC risk (odds ratio, 1.31; 95% confidence interval, 1.04-1.67; p = 0.024). Stratified analysis showed that rs11874392 might increase the risk of CRC in females (OR = 1.70, p = 0.028), individuals with smoking (OR = 1.87, p = 0.026), and drinking (OR = 1.38, p = 0.027). The rs11874392 was found to be related to an elevated risk of rectal cancer (OR = 1.73, p = 0.003), but not with colon cancer. FPRP analysis demonstrated that all of these associations were statistically significant (FPRP <0.2). Additionally, rs11874392 was the strongest predictive model for CRC. This study provides evidence that the SMAD7 rs11874392 is related to an increased susceptibility to CRC.