Sericin promotes chondrogenic proliferation and differentiation via glycolysis and Smad2/3 TGF-ß signaling inductions and alleviates inflammation in three-dimensional models.

Knee osteoarthritis is a chronic joint disease mainly characterized by cartilage degeneration. The treatment is challenging due to the lack of blood vessels and nerve supplies in cartilaginous tissue, causing a prominent limitation of regenerative capacity. Hence, we investigated the cellular promotional and anti-inflammatory effects of sericin, Bombyx mori-derived protein, on three-dimensional chondrogenic ATDC5 cell models. The results revealed that a high concentration of sericin promoted chondrogenic proliferation and differentiation and enhanced matrix production through the increment of glycosaminoglycans, COL2A1, COL X, and ALP expressions. SOX-9 and COL2A1 gene expressions were notably elevated in sericin treatment. The proteomic analysis demonstrated the upregulation of phosphoglycerate mutase 1 and triosephosphate isomerase, a glycolytic enzyme member, reflecting the proliferative enhancement of sericin. The differentiation capacity of sericin was indicated by the increased expressions of procollagen12a1, collagen10a1, rab1A, periostin, galectin-1, and collagen6a3 proteins. Sericin influenced the differentiation capacity via the TGF-ß signaling pathway by upregulating Smad2 and Smad3 while downregulating Smad1, BMP2, and BMP4. Importantly, sericin exhibited an anti-inflammatory effect by reducing IL-1ß, TNF-α, and MMP-1 expressions and accelerating COL2A1 production in the early inflammatory stage. In conclusion, sericin demonstrates potential in promoting chondrogenic proliferation and differentiation, enhancing cartilaginous matrix synthesis through glycolysis and TGF-ß signaling pathways, and exhibiting anti-inflammatory properties.

Vinpocetine alleviated alveolar epithelial cells injury in experimental pulmonary fibrosis by targeting PPAR-γ/NLRP3/NF-κB and TGF-ß1/Smad2/3 pathways.

This study aimed to investigate the potential anti-fibrotic activity of vinpocetine in an experimental model of pulmonary fibrosis by bleomycin and in the MRC-5 cell line. Pulmonary fibrosis was induced in BALB/c mice by oropharyngeal aspiration of a single dose of bleomycin (5 mg/kg). The remaining induced animals received a daily dose of pirfenidone (as a standard anti-fibrotic drug) (300 mg/kg/PO) and vinpocetine (20 mg/kg/PO) on day 7 of the induction till the end of the experiment (day 21). The results of the experiment revealed that vinpocetine managed to alleviate the fibrotic endpoints by statistically improving (P ≤ 0.05) the weight index, histopathological score, reduced expression of fibrotic-related proteins in immune-stained lung sections, as well as fibrotic markers measured in serum samples. It also alleviated tissue levels of oxidative stress and inflammatory and pro-fibrotic mediators significantly elevated in bleomycin-only induced animals (P ≤ 0.05). Vinpocetine managed to express a remarkable attenuating effect in pulmonary fibrosis both in vivo and in vitro either directly by interfering with the classical TGF-ß1/Smad2/3 signaling pathway or indirectly by upregulating the expression of Nrf2 enhancing the antioxidant system, activating PPAR-γ and downregulating the NLRP3/NF-κB pathway making it a candidate for further clinical investigation in cases of pulmonary fibrosis.

Exosomal let-7a-5p derived from human umbilical cord mesenchymal stem cells alleviates coxsackievirus B3-induced cardiomyocyte ferroptosis via the SMAD2/ZFP36 signal axis. / 人脐带间å 质干细胞来源的外泌体let-7a-5p通过SMAD2/ZFP36ä¿¡å·è½´å‡è½»æŸ¯è¨å¥‡ç— æ¯’B3诱导的心肌细胞铁死亡.

Viral myocarditis (VMC) is one of the most common acquired heart diseases in children and teenagers. However, its pathogenesis is still unclear, and effective treatments are lacking. This study aimed to investigate the regulatory pathway by which exosomes alleviate ferroptosis in cardiomyocytes (CMCs) induced by coxsackievirus B3 (CVB3). CVB3 was utilized for inducing the VMC mouse model and cellular model. Cardiac echocardiography, left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were implemented to assess the cardiac function. In CVB3-induced VMC mice, cardiac insufficiency was observed, as well as the altered levels of ferroptosis-related indicators (glutathione peroxidase 4 (GPX4), glutathione (GSH), and malondialdehyde (MDA)). However, exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) could restore the changes caused by CVB3 stimulation. Let-7a-5p was enriched in hucMSCs-exo, and the inhibitory effect of hucMSCs-exolet-7a-5p mimic on CVB3-induced ferroptosis was higher than that of hucMSCs-exomimic NC (NC: negative control). Mothers against decapentaplegic homolog 2 (SMAD2) increased in the VMC group, while the expression of zinc-finger protein 36 (ZFP36) decreased. Let-7a-5p was confirmed to interact with SMAD2 messenger RNA (mRNA), and the SMAD2 protein interacted directly with the ZFP36 protein. Silencing SMAD2 and overexpressing ZFP36 inhibited the expression of ferroptosis-related indicators. Meanwhile, the levels of GPX4, solute carrier family 7, member 11 (SLC7A11), and GSH were lower in the SMAD2 overexpression plasmid (oe-SMAD2)+let-7a-5p mimic group than in the oe-NC+let-7a-5p mimic group, while those of MDA, reactive oxygen species (ROS), and Fe2+ increased. In conclusion, these data showed that ferroptosis could be regulated by mediating SMAD2 expression. Exo-let-7a-5p derived from hucMSCs could mediate SMAD2 to promote the expression of ZFP36, which further inhibited the ferroptosis of CMCs to alleviate CVB3-induced VMC.

Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

The Presence of TGFß3 in Human Ovarian Intrafollicular Fluid and Its Involvement in Thromboxane Generation in Follicular Granulosa Cells through a Canonical TGFßRI, Smad2/3 Signaling Pathway and COX-2 Induction.

Ovarian follicular fluid (FF) has a direct impact on oocyte quality, playing key roles in fertilization, implantation, and early embryo development. In our recent study, we found FF thromboxane (TX) to be a novel factor inversely correlated with oocyte maturation and identified thrombin, transforming growth factor ß (TGFß), TNF-α, and follicular granulosa cells (GCs) as possible contributors to FF TX production. Therefore, this study sought to investigate the role of TGFß3 in regulating TX generation in human ovarian follicular GCs. TGFß3 was differentially and significantly present in the FF of large and small follicles obtained from IVF patients with average concentrations of 68.58 ± 12.38 and 112.55 ± 14.82 pg/mL, respectively, and its levels were correlated with oocyte maturity. In an in vitro study, TGFß3 induced TX generation/secretion and the converting enzyme-COX-2 protein/mRNA expression both in human HO23 and primary cultured ovarian follicular GCs. While TGFßRI and Smad2/3 signaling was mainly required for COX-2 induction, ERK1/2 appeared to regulate TX secretion. The participation of Smad2/3 and COX-2 in TGFß3-induced TX generation/secretion could be further supported by the observations that Smad2/3 phosphorylation and nuclear translocation and siRNA knockdown of COX-2 expression compromised TX secretion in GCs challenged with TGFß3. Taken together, the results presented here first demonstrated that FF TGFß3 levels differ significantly in IVF patients' large preovulatory and small mid-antral follicles and are positively associated with oocyte maturation. TGFß3 can provoke TX generation by induction of COX-2 mRNA/protein via a TGFßR-related canonical Smad2/3 signaling pathway, and TX secretion possibly by ERK1/2. These imply that TGFß3 is one of the inducers for yielding FF TX in vivo, which may play a role in folliculogenesis and oocyte maturation.

Exosomes derived from adipose tissue-derived stem cells alleviated H2O2-induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway.

Hypertrophic scar (HS) is characterized by excessive collagen deposition and myofibroblasts activation. Endothelial-to-mesenchymal transition (EndoMT) and oxidative stress were pivotal in skin fibrosis process. Exosomes derived from adipose tissue-derived stem cells (ADSC-Exo) have the potential to attenuate EndoMT and inhibit fibrosis. The study revealed reactive oxygen species (ROS) levels were increased during EndoMT occurrence of dermal vasculature of HS. The morphology of endothelial cells exposure to H2O2, serving as an in vitro model of oxidative stress damage, transitioned from a cobblestone-like appearance to a spindle-like shape. Additionally, the levels of endothelial markers decreased in H2O2-treated endothelial cell, while the expression of fibrotic markers increased. Furthermore, H2O2 facilitated the accumulation of ROS, inhibited cell proliferation, retarded its migration and suppressed tube formation in endothelial cell. However, ADSC-Exo counteracted the biological effects induced by H2O2. Subsequently, miRNAs sequencing analysis revealed the significance of mir-486-3p in endothelial cell exposed to H2O2 and ADSC-Exo. Mir-486-3p overexpression enhanced the acceleration of EndoMT, its inhibitors represented the attenuation of EndoMT. Meanwhile, the target regulatory relationship was observed between mir-486-3p and Sirt6, whereby Sirt6 exerted its anti-EndoMT effect through Smad2/3 signaling pathway. Besides, our research had successfully demonstrated the impact of ADSC-Exo and mir-486-3p on animal models. These findings of our study collectively elucidated that ADSC-Exo effectively alleviated H2O2-induced ROS and EndoMT by inhibiting the mir-486-3p/Sirt6/Smad axis.

FOXF1 reverses lung fibroblasts transdifferentiation via inhibiting TGF-ß/SMAD2/3 pathway in silica-induced pulmonary fibrosis.

Silicosis is one of the most common and severe types of pneumoconiosis and is characterized by lung dysfunction, persistent lung inflammation, pulmonary nodule formation, and irreversible pulmonary fibrosis. The transdifferentiation of fibroblasts into myofibroblasts is one of the main reasons for the exacerbation of silicosis. However, the underlying mechanism of transcription factors regulating silicosis fibrosis has not been clarified. The aim of this study was to investigate the potential mechanism of transcription factor FOXF1 in fibroblast transdifferentiation in silica-induced pulmonary fibrosis. Therefore, a silicosis mouse model was established, and we found that FOXF1 expression level was significantly down-regulated in the silicosis group, and after overexpression of FOXF1 by adeno-associated virus (AAV), FOXF1 expression level was up-regulated, and silicosis fibrosis was alleviated. In order to further explore the specific regulatory mechanism of FOXF1 in silicosis, we established a fibroblasts transdifferentiation model induced by TGF-ß in vitro. In the model, the expression levels of SMAD2/3 and P-SMAD2/3 were up-regulated, but the expression levels of SMAD2/3 and P-SMAD2/3 were down-regulated, inhibiting transdifferentiation and accumulation of extracellular matrix after the overexpressed FOXF1 plasmid was constructed. However, after silencing FOXF1, the expression levels of SMAD2/3 and P-SMAD2/3 were further up-regulated, aggravating transdifferentiation and accumulation of extracellular matrix. These results indicate that the activation of FOXF1 in fibroblasts can slow down the progression of silicosis fibrosis by inhibiting TGF-ß/SMAD2/3 classical pathway, which provides a new idea for further exploration of silicosis treatment.

Recruitment or activation of mast cells in the liver aggravates the accumulation of fibrosis in carbon tetrachloride-induced liver injury.

Liver diseases caused by viral infections, alcoholism, drugs, or chemical poisons are a significant health problem: Liver diseases are a leading contributor to mortality, with approximately 2 million deaths per year worldwide. Liver fibrosis, as a common liver disease characterized by excessive collagen deposition, is associated with high morbidity and mortality, and there is no effective treatment. Numerous studies have shown that the accumulation of mast cells (MCs) in the liver is closely associated with liver injury caused by a variety of factors. This study investigated the relationship between MCs and carbon tetrachloride (CCl4)-induced liver fibrosis in rats and the effects of the MC stabilizers sodium cromoglycate (SGC) and ketotifen (KET) on CCl4-induced liver fibrosis. The results showed that MCs were recruited or activated during CCl4-induced liver fibrosis. Coadministration of SCG or KET alleviated the liver fibrosis by decreasing SCF/c-kit expression, inhibiting the TGF-ß1/Smad2/3 pathway, depressing the HIF-1a/VEGF pathway, activating Nrf2/HO-1 pathway, and increasing the hepatic levels of GSH, GSH-Px, and GR, thereby reducing hepatic oxidative stress. Collectively, recruitment or activation of MCs is linked to liver fibrosis and the stabilization of MCs may provide a new approach to the prevention of liver fibrosis.

Adipocyte-derived exosomes from obstructive sleep apnoea rats aggravate MASLD by TCONS_00039830/miR-455-3p/Smad2 axis.

A correlation exists between obstructive sleep apnoea (OSA) and the severity of metabolic dysfunction-associated steatotic liver disease (MASLD), OSA can induce more severe MASLD. However, the underlying regulatory mechanism between the two is unclear. To this end, this study explored the role and possible molecular mechanisms of adipocyte-derived exosomes under OSA in aggravating MASLD. Through sequencing technology, miR-455-3p was identified as a co-differentially expressed miRNA between the MASLD + OSA and Control groups and between the MASLD + OSA and MASLD groups. Upregulation of TCONS-00039830 and Smad2 and downregulation of miR-455-3p in the MASLD and MASLD + OSA groups were validated in vivo and in vitro. TCONS-00039830, as a differentially expressed LncRNA in exosomes found in the sequencing results, transfection notably downregulated miR-455-3p and upregulated Smad2 in hepatocytes. TCONS_00039830 overexpression increased fat, triglyceride and cholesterol levels, while miR-455-3p overexpression decreased these levels. Furthermore, exosome administration promoted the accumulation of fat, triglyceride and cholesterol, upregulated TCONS_00039830 and Smad2, and downregulated miR-455-3p. Overexpression of miR-455-3p reversed the increased fat accumulation and upregulated TCONS_00039830 and Smad2. In conclusion, OSA-derived exosomes promoted hepatocyte steatosis by regulating TCONS_00039830/miR-455-3p/Smad2 axis, thereby aggravating liver damage in MASLD.

The antiprotozoal drug nitazoxanide improves experimental liver fibrosis in mice.

Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous studies find that nitazoxanide and its metabolite tizoxanide affect AMPK, STAT3, and Smad2/3 signals which are involved in the pathogenesis of liver fibrosis, therefore, in the present study, we examined the effect of nitazoxanide on experimental liver fibrosis and elucidated the potential mechanisms. The in vivo experiment results showed that oral nitazoxanide (75, 100 mg·kg-1) significantly improved CCl4- and bile duct ligation-induced liver fibrosis in mice. Oral nitazoxanide activated the inhibited AMPK and inhibited the activated STAT3 in liver tissues from liver fibrosis mice. The in vitro experiment results showed that nitazoxanide and its metabolite tizoxanide activated AMPK and inhibited STAT3 signals in LX-2 cells (human hepatic stellate cells). Nitazoxanide and tizoxanide inhibited cell proliferation and collagen I expression and secretion of LX-2 cells. Nitazoxanide and tizoxanide inhibited transforming growth factor-ß1 (TGF-ß1)- and IL-6-induced increases of cell proliferation, collagen I expression and secretion, inhibited TGF-ß1- and IL-6-induced STAT3 and Smad2/3 activation in LX-2 cells. In mouse primary hepatic stellate cells, nitazoxanide and tizoxanide also activated AMPK, inhibited STAT3 and Smad2/3 activation, inhibited cell proliferation, collagen I expression and secretion. In conclusion, nitazoxanide inhibits liver fibrosis and the underlying mechanisms involve AMPK activation, and STAT3 and Smad2/3 inhibition.

Stigmasterol alleviates airway inflammation in OVA-induced asthmatic mice via inhibiting the TGF-ß1/Smad2 and IL-17A signaling pathways.

Stigmasterol is a common dietary phytosterol with high nutritional value and physiological activity. In this study, we evaluated the effects of stigmasterol on inflammatory cytokines and the TGF-ß1/Smad2 and IL-17A signaling pathway in an ovalbumin (OVA)-induced asthma mouse model. Stigmasterol treatment improved airway remodeling. In addition, it significantly attenuated the symptoms of asthma attacks, reduced the number of macrophages, lymphocytes, neutrophils, and eosinophils in BALF and inflammatory cytokines, including IL-1ß, IL-5, IL-6, and IL-13. It further decreased the level of IL-17A in BALF, serum and spleen. Spleen single-cell suspension analysis via flow cytometry showed that IL-17A level was consistent with the results obtained in BALF, serum and spleen. Stigmasterol decreased the protein expression levels of TGF-ß, p-Smad2 and IL-17A in the spleen, by increasing the protein expression level of IL-10. After 24 h of co-culture of TGF-ß, IL-6 and stigmasterol, the level of IL-17 in CD4+ T cell supernatant was lower relative to levels in the group without stigmasterol. Meanwhile, stigmasterol treatment attenuated the expression level of TGF- ß, p-Smad2 and IL-17A proteins in CD4+ T cells and enhanced the expression levels of IL-10 protein. These data suggested that stigmasterol inhibited the TGF-ß1/Smad2 and IL-17A signaling pathway to achieve anti-asthmatic effects in the OVA-induced asthma mouse model. Collectively, the results of this study are that stigmasterol has achieved preliminary efficacy in the non-clinical laboratory, further studies are needed to consider the clinical application of stigmasterol.

Neuroprotective effect of Withania somnifera leaves extract nanoemulsion against penconazole-induced neurotoxicity in albino rats via modulating TGF-ß1/Smad2 signaling pathway.

Penconazole (PEN) is a systemic triazole fungicide used to control various fungal diseases on grapes, stone fruits, cucurbits, and strawberries. Still, it leaves residues on treated crops after collection with many hazardous effects on population including neurotoxicity. Withania somnifera leaves extract (WSLE) is known for its memory and brain function enhancing ability. To evoke such action efficiently, WSLE bioactive metabolites are needed to cross the blood-brain barrier, that could limit the availability of such compounds to be localized within the brain. Therefore, in the present study, the association between PEN exposure and neurotoxicity was evaluated, and formulated WSLE nanoemulsion was investigated for improving the permeability of the plant extract across the blood-brain barrier. The rats were divided into five groups (n = 6). The control group was administered distilled water, group II was treated with W. somnifera leaves extract nanoemulsion (WSLE NE), group III received PEN, group IV received PEN and WSLE, and group V received PEN and WSLE NE. All rats were gavaged daily for 6 weeks. Characterization of compounds in WSLE using LC-MS/MS analysis was estimated. Neurobehavioral disorders were evaluated in all groups. Oxidative stress biomarkers, antioxidant enzyme activities, and inflammatory cytokines were measured in brain tissue. Furthermore, the gene expression patterns of GFAP, APP, vimentin, TGF-ß1, Smad2 and Bax were measured. Histopathological changes and immunohistochemical expression in the peripheral sciatic nerve and cerebral cortex were evaluated. A total of 91 compounds of different chemo-types were detected and identified in WSLE in both ionization modes. Our data showed behavioral impairment in the PEN-treated group, with significant elevation of oxidative stress biomarkers, proinflammatory cytokines, neuronal damage, and apoptosis. In contrast, the PEN-treated group with WSLE NE showed marked improvement in behavioral performance and histopathological alteration with a significant increase in antioxidant enzyme activity and anti-inflammatory cytokines compared to the group administered WSLE alone. The PEN-treated group with WSLE NE in turn significantly downregulated the expression levels of GFAP, APP, vimentin, TGF-ß1, Smad2 and Bax in brain tissue. In conclusion, WSLE NE markedly enhanced the permeability of plant extract constituents through the blood brain barrier to boost its neuroprotective effect against PEN-induced neurotoxicity.

Knockdown of the UL-16 binding protein 1 promotes osteoblast differentiation of human mesenchymal stem cells by activating the SMAD2/3 pathway.

Osteoporosis is caused by the imbalance of osteoblasts and osteoclasts. The regulatory mechanisms of differentially expressed genes (DEGs) in pathogenesis of osteoporosis are of significant and needed to be further investigated. GSE100609 dataset downloaded from Gene Expression Omnibus (GEO) database was used to identified DEGs in osteoporosis patients. KEGG analysis was conducted to demonstrate signaling pathways related to enriched genes. Osteoporosis patients and the human mesenchymal stem cells (hMSCs) were obtained for in vivo and in vitro resaerch. Lentivirus construction and viral infection was used to knockdown genes. mRNA expression and protein expression were detected via qRT-PCR and western blot assay separately. Alkaline phosphatase (ALP) activity detection, alizarin Red S (ARS) staining, and expression of bone morphogenetic protein 2 (BMP2), osteocalcin (OCN) and Osterix were evaluated to determine osteoblast differentiation capacity. UL-16 binding protein 1 (ULBP1) gene was upregulated in osteoporosis and downregulated in differentiated hMSCs. Knockdown of ULBP1 increased ALP activity, mineralization ability evaluated by ARS staining, expression of BMP2, OCN and Osterix in differentiated hMSCs. Furthermore, rescue experiment demonstrated that suppressed ULBP1 boosted osteoblast differentiation by activating TNF-ß signaling pathway. Knockdown of ULBP1 gene could promoted osteoblast differentiation by activating TNF-ß signaling pathway in differentiated hMSCs. ULBP1 may be a the Achilles' heel of osteoporosis, and suppression of ULBP1 could be a promising treatment for osteoporosis.

Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF-ß/SMAD2/3 pathway.

Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin-dependent kinase inhibitor p21. The activation of the TGF-ß/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF-ß/SMAD2/3 pathway.

Exosomes from adipose-derived mesenchymal stem cell improve diabetic wound healing and inhibit fibrosis via miR-128-1-5p/TGF-ß1/Smad axis.

OBJECTIVE: Difficult-to-heal wound is a prevalent and significant complication of diabetes, characterized by impaired functionality of epithelial cells such as fibroblasts. This study aims to investigate the potential mechanism of ADSC-Exos promoting diabetic wound healing by regulating fibroblast function. MATERIALS AND METHODS: ADSC-Exos were confirmed through TEM, NTA, and Western Blot techniques. The study conducted on rat skin fibroblasts (RSFs) exposed to 33 mmol/L glucose in vitro. We used cck-8, EDU, transwell, and scratch assays to verify the proliferation and migration of RSFs. Furthermore, levels of TGF-ß1 and α-SMA proteins were determined by immunofluorescence and Western Blot. RSFs were transfected with miR-128-1-5p mimics and inhibitors, followed by quantification of TGF-ß1, α-SMA, Col I and Smad2/3 protein levels using Western Blot. In vivo, the effects of ADSC-Exos on diabetic wounds were assessed using digital imaging, histological staining, as well as Western Blot analysis. RESULTS: In vitro, ADSC-Exos significantly enhanced proliferation and migration of RSFs while reducing the expression of TGF-ß1 and α-SMA. In vivo, ADSC-Exos effectively promoted diabetic wound healing and mitigated scar fibrosis. Additionally, ADSC-Exos exhibited elevated levels of miR-128-1-5p, which targets TGF-ß1, resulting in a notable reduction in TGF-ß1, α-SMA, Col I and smad2/3 phosphorylation in RSFs. CONCLUSION: In conclusion, our results demonstrated that ADSC-Exos promoted diabetic wound healing, and inhibited skin fibrosis by regulating miR-128-1-5p/TGF-ß1/Smad signaling pathway, which provides a promising innovative treatment for diabetic wound healing.

Maternal malnutrition associated with postnatal sugar consumption increases inflammatory response and prostate disorders in rat offspring.

Maternal malnutrition can alter developmental biology, programming health and disease in offspring. The increase in sugar consumption during the peripubertal period, a worldwide concern, also affects health through adulthood. Studies have shown that maternal exposure to a low protein diet (LPD) is associated with an increase in prostate disease with aging. However, the combined effects of maternal LPD and early postnatal sugar consumption on offspring prostate disorders were not investigated. The effects on aging were evaluated using a maternal gestational model with lactational LPD (6% protein) and sugar consumption (10%) from postnatal day (PND) 21-90, associating the consequences on ventral prostate (VP) rats morphophysiology on PND540. An increase was shown in mast cells and in the VP of the CTR + SUG and Gestational and Lactational Low Protein (GLLP) groups. In GLLP + SUG, a significant increase was shown in TGF-ß1 expression in both the systemic and intra-prostatic forms, and SMAD2/3p had increased. The study identified maternal LPD and sugar consumption as risk factors for prostatic homeostasis in senility, activating the TGFß1-SMAD2/3 pathway, a signaling pathway with potential markers for prostatic disorders.

Hypoxia-induced TREM1 promotes mesenchymal-like states of glioma stem cells via alternatively activating tumor-associated macrophages.

The mesenchymal subtype of glioblastoma (GBM) cells characterized by aggressive invasion and therapeutic resistance is thought to be dependent on cell-intrinsic alteration and extrinsic cellular crosstalk. Tumor-associated macrophages (TAMs) are pivotal in tumor progression, chemo-resistance, angiogenesis, and stemness maintenance. However, the impact of TAMs on the shifts in glioma stem cells (GSCs) states remains largely uncovered. Herein, we showed that the triggering receptor expressed on myeloid cells-1 (TREM1) preferentially expressed by M2-like TAMs and induced GSCs into mesenchymal-like states by modulating the secretion of TGFß2, which activated the TGFßR/SMAD2/3 signaling in GSCs. Furthermore, we demonstrated that TREM1 was transcriptionally regulated by HIF1a under the hypoxic environment and thus promoted an immunosuppressive type of TAMs via activating the TLR2/AKT/mTOR/c-MYC axis. Collectively, this study reveals that cellular communication between TAMs and GSCs through the TREM1-mediated TGFß2/TGFßR axis is involved in the mesenchymal-like transitions of GSCs. Our study provides valuable insights into the regulatory mechanisms between the tumor immune microenvironment and the malignant characteristics of GBM, which can lead to potential novel strategies targeting TAMs for tumor control.

Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

The PIEZO1/miR-155-5p/GDF6/SMAD2/3 signaling axis is involved in inducing the occurrence and progression of osteoarthritis under excessive mechanical stress.

OBJECTIVE: To elucidate the molecular mechanism of overloading-induced osteoarthritis (OA) and to find a novel therapeutic target. METHODS: We utilized human cartilage specimens, mouse chondrocytes, a destabilization of the medial meniscus (DMM) mouse model, and a mouse hindlimb weight-bearing model to validate the role of overloading on chondrocyte senescence and OA development. Then, we observed the effect of PIEZO1-miR-155-5p-GDF6-SMAD2/3 signaling axis on the preservation of joint metabolic homeostasis under overloading in vivo, in vitro and ex vivo by qPCR, Western blot, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence, SA-ß-gal staining, CCK8 assay, et al. Finally, we verified the therapeutic effects of intra-articular injection of miR-155-5p inhibitor or recombinant GDF6 on the murine overloading-induced OA models. RESULTS: Chondrocytes sensesed the mechanical overloading through PIEZO1 and up-regulated miR-155-5p expression. MiR-155-5p mimics could copy the effects of overloading-induced chondrocyte senescence and OA. Additionally, miR-155-5p could suppress the mRNA expression of Gdf6-Smad2/3 in various tissues within the joint. Overloading could disrupt joint metabolic homeostasis by downregulating the expression of anabolism indicators and upregulating the expression of catabolism indicators in the chondrocytes and synoviocytes, while miR-155-5p inhibition or GDF6 supplementation could exert an antagonistic effect by preserving the joint homeostasis. Finally, in the in vivo overloading models, intra-articular injection of miR-155-5p inhibitor or recombinant GDF6 could significantly mitigate the severity of impending OA and lessened the progression of existing OA. CONCLUSION: GDF6 overexpression or miR-155-5p inhibition could attenuate overloading-induced chondrocyte senescence and OA through the PIEZO1-miR-155-5p-GDF6-SMAD2/3 signaling pathway. Our study provides a new therapeutic target for the treatment of overloading-induced OA.

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.