Marrow mesenchymal stem cell mediates diabetic nephropathy progression via modulation of Smad2/3/WTAP/m6A/ENO1 axis.

Diabetic nephropathy (DN) is one of the common microvascular complications in diabetic patients. Marrow mesenchymal stem cells (MSCs) have attracted attention in DN therapy but the underlying mechanism remains unclear. Here, we show that MSC administration alleviates high glucose (HG)-induced human kidney tubular epithelial cell (HK-2 cell) injury and ameliorates renal injury in DN mice. We identify that Smad2/3 is responsible for MSCs-regulated DN progression. The activity of Smad2/3 was predominantly upregulated in HG-induced HK-2 cell and DN mice and suppressed with MSC administration. Activation of Smad2/3 via transforming growth factor-ß1 (TGF-ß1) administration abrogates the protective effect of MSCs on HG-induced HK-2 cell injury and renal injury of DN mice. Smad2/3 has been reported to interact with methyltransferase of N6-methyladenosine (m6A) complex and we found a methyltransferase, Wilms' tumor 1-associating protein (WTAP), is involved in MSCs-Smad2/3-regulated DN development. Moreover, WTAP overexpression abrogates the improvement of MSCs on HG-induced HK-2 cell injury and renal injury of DN mice. Subsequently, α-enolase (ENO1) is the downstream target of WTAP-mediated m6A modification and contributes to the MSCs-mediated regulation. Collectively, these findings reveal a molecular mechanism in DN progression and indicate that Smad2/3/WTAP/ENO1 may present a target for MSCs-mediated DN therapy.

Dang-Gui-Si-Ni decoction facilitates wound healing in diabetic foot ulcers by regulating expression of AGEs/RAGE/TGF-ß/Smad2/3.

Diabetic foot ulcer (DFU) is a predominant complication of diabetes mellitus with poor prognosis accompanied by high amputation and mortality rates. Dang-Gui-Si-Ni decoction (DSD), as a classic formula with a long history in China, has been found to improve DFU symptoms. However, mechanism of DSD for DFU therapy remains unclear with no systematic elaboration. In vivo, following establishment of DFU rat model, DSD intervention with low, medium and high doses was done, with Metformin (DM) as a positive control group. With wound healing detection, pathological changes by HE staining, inflammatory factor expression by ELISA and qRT-PCR, oxidative stress levels by ELISA, and AGEs/RAGE/TGF-ß/Smad2/3 expression by Western blot were performed. In vitro, intervention with LY2109761 (TGF-ß pathway inhibitor) based on DSD treatment in human dermal fibroblast-adult (HDF-a) cells was made. Cell viability by CCK8, migration ability by cell scratch, apoptosis by flow cytometry, and AGEs/RAGE/TGF-ß/Smad2/3 expression by Western blot were measured. DFU rats exhibited elevated AGEs/RAGE expression, whereas decreased TGF-ß1 and p-Smad3/Smad3 protein expression, accompanied by higher IL-1ß, IL-6, TNF-α levels, and oxidative stress. DSD intervention reversed above effects. Glucose induction caused lower cell viability, migration, TGF-ß1 and p-Smad3/Smad3 protein expression, with increased apoptosis and AGEs/RAGE expression in HDF-a cells. These effects were reversed after DSD intervention, and further LY2109761 intervention inhibited DSD effects in cells. DSD intervention may facilitate wound healing in DFU by regulating expression of AGEs/RAGE/TGF-ß/Smad2/3, providing scientific experimental evidence for DSD clinical application for DFU therapy.

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.

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.

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.

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.

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.

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.

Renoprotective effect of rosmarinic acid by inhibition of indoxyl sulfate-induced renal interstitial fibrosis via the NLRP3 inflammasome signaling.

We previously reported that rosmarinic acid (RA) ameliorated renal fibrosis in a unilateral ureteral obstruction (UUO) murine model of chronic kidney disease. This study aimed to determine whether RA attenuates indoxyl sulfate (IS)-induced renal fibrosis by regulating the activation of the NLRP3 inflammasome/IL-1ß/Smad circuit. We discovered the NLRP3 inflammasome was activated in the IS treatment group and downregulated in the RA-treated group in a dose-dependent manner. Additionally, the downstream effectors of the NLRP3 inflammasome, cleaved-caspase-1 and cleaved-IL-1ß showed similar trends in different groups. Moreover, RA administration significantly decreased the ROS levels of reactive oxygen species in IS-treated cells. Our data showed that RA treatment significantly inhibited Smad-2/3 phosphorylation. Notably, the effects of RA on NLRP3 inflammasome/IL-1ß/Smad and fibrosis signaling were reversed by the siRNA-mediated knockdown of NLRP3 or caspase-1 in NRK-52E cells. In vivo, we demonstrated that expression levels of NLRP3, c-caspase-1, c-IL-1ß, collagen I, fibronectin and α-SMA, and TGF- ß 1 were downregulated after treatment of UUO mice with RA or RA + MCC950. Our findings suggested RA and MCC950 synergistically inhibited UUO-induced NLRP3 signaling activation, revealing their renoprotective properties and the potential for combinatory treatment of renal fibrosis and chronic kidney inflammation.

Neferine mitigates angiotensin II-induced atrial fibrillation and fibrosis via upregulation of Nrf2/HO-1 and inhibition of TGF-ß/p-Smad2/3 pathways.

BACKGROUND: Atrial fibrillation (AF) is often associated with atrial fibrosis and oxidative stress. Neferine, a bisbenzylisoquinoline alkaloid, has been reported to exert an antiarrhythmic effect. However, its impact on Angiotensin II (Ang II) infusion-induced AF and the underlying mechanism remains unclear. This study aimed to investigate whether neferine alleviates Ang II-induced AF and explore the underlying mechanisms. METHODS: Mice subjected to Ang II infusion to induce AF were concurrently treated with neferine or saline. AF incidence, myocardial cell size, fibrosis, and oxidative stress were then examined. RESULTS: Neferine treatment inhibited Ang II-induced AF, atrial size augmentation, and atrial fibrosis. Additionally, we observed that Ang II increased reactive oxygen species (ROS) generation, induced mitochondrial membrane potential depolarization, and reduced glutathione (GSH) and superoxide dismutase (SOD) levels, which were reversed to some extent by neferine. Mechanistically, neferine activated the Nrf2/HO-1 signaling pathway and inhibited TGF-ß/p-Smad2/3 in Ang II-infused atria. Zinc Protoporphyrin (ZnPP), an HO-1 inhibitor, reduced the anti-oxidative effect of neferine to some extent and subsequently abolished the beneficial effect of neferine on Ang II-induced AF. CONCLUSIONS: These findings provide hitherto undocumented evidence that the protective role of neferine in Ang II-induced AF is dependent on HO-1.

Liebig's law of the minimum in the TGF-ß/SMAD pathway.

Cells use signaling pathways to sense and respond to their environments. The transforming growth factor-ß (TGF-ß) pathway produces context-specific responses. Here, we combined modeling and experimental analysis to study the dependence of the output of the TGF-ß pathway on the abundance of signaling molecules in the pathway. We showed that the TGF-ß pathway processes the variation of TGF-ß receptor abundance using Liebig's law of the minimum, meaning that the output-modifying factor is the signaling protein that is most limited, to determine signaling responses across cell types and in single cells. We found that the abundance of either the type I (TGFBR1) or type II (TGFBR2) TGF-ß receptor determined the responses of cancer cell lines, such that the receptor with relatively low abundance dictates the response. Furthermore, nuclear SMAD2 signaling correlated with the abundance of TGF-ß receptor in single cells depending on the relative expression levels of TGFBR1 and TGFBR2. A similar control principle could govern the heterogeneity of signaling responses in other signaling pathways.

Verteporfin inhibits TGF-ß signaling by disrupting the Smad2/3-Smad4 interaction.

Transforming growth factor-ß (TGF-ß) signaling plays a crucial role in pathogenesis, such as accelerating tissue fibrosis and promoting tumor development at the later stages of tumorigenesis by promoting epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion. Targeting TGF-ß signaling is a promising therapeutic approach, but nonspecific inhibition may result in adverse effects. In this study, we focus on the Smad2/3-Smad4 complex, a key component in TGF-ß signaling transduction, as a potential target for cancer therapy. Through a phase-separated condensate-aided biomolecular interaction system, we identified verteporfin (VP) as a small-molecule inhibitor that specifically targets the Smad2/3-Smad4 interaction. VP effectively disrupted the interaction between Smad2/3 and Smad4 and thereby inhibited canonical TGF-ß signaling, but not the interaction between Smad1 and Smad4 in bone morphogenetic protein (BMP) signaling. Furthermore, VP exhibited inhibitory effects on TGF-ß-induced EMT and cell migration. Our findings indicate a novel approach to develop protein-protein interaction inhibitors of the canonical TGF-ß signaling pathway for treatments of related diseases.

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.

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.

Thbs1 regulates skeletal muscle mass in a TGFß-Smad2/3-ATF4-dependent manner.

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor ß (TGFß)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFß-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFß-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFß-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.

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.

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.

Intrauterine infusion of platelet-rich plasma improves fibrosis by transforming growth factor beta 1/Smad pathway in a rat intrauterine adhesion model.

This study aims to elucidate the effects of Platelet-rich plasma (PRP) in fibrosis development in intrauterine adhesion (IUA), and the associated underlying mechanisms are also explored, which are expected to be a potential therapeutic scheme for IUA. In this research, PRP was obtained and prepared from the peripheral venous blood of rats. A rat model was induced by mechanical injury. Further, PRP was directly injected into the uterus for treatment. The appearance and shape of the uterus were assessed based on the tissues harvested. The fibrosis biomarker levels were analyzed. The transforming growth factor beta 1 (TGF-ß1) and Mothers against decapentaplegic homolog 7 (Smad7) levels, the phosphorylation of Smad2 (p-Smad2), and the phosphorylation of Smad3 (p-Smad3) were analyzed, and the molecular mechanism was investigated by rescue experiments. It was found that PRP improved the appearance and shape of the uterus in IUA and increased endometrial thickness and gland numbers. The administration of PRP resulted in a decrease in the expressions of fibrosis markers including collagen I, α-SMA, and fibronectin. Furthermore, PRP increased Smad7 levels and decreased TGF-ß1 levels, p-Smad2, and p-Smad3. Meanwhile, administration of TGF-ß1 activator reversed the therapeutic effects of PRP in IUA. Collectively, the intrauterine infusion of PRP can promote endometrial damage recovery and improve endometrial fibrosis via the TGF-ß1/Smad pathway. Hence, PRP can be a potential therapeutic strategy for IUA.