Activation of SIRT1 by Resveratrol Alleviates Pressure Overload-Induced Cardiac Hypertrophy via Suppression of TGF-ß1 Signaling.

INTRODUCTION: Silent information regulator 1 (SIRT1) has been extensively investigated in the cardiovascular system and has been shown to play a pivotal role in mediating cell death/survival, energy production, and oxidative stress. However, the functional role of SIRT1 in pressure overload-induced cardiac hypertrophy and dysfunction remains unclear. Resveratrol (Rsv), a widely used activator of SIRT1, has been reported to protect against cardiovascular disease. We here examine whether activation of SIRT1 by Rsv attenuate pressure overload-induced cardiac hypertrophy and to identify the underlying molecular mechanisms. METHODS: In vivo, rat model of pressure overload-induced myocardial hypertrophy was established by abdominal aorta constriction (AAC) procedure. In vitro, Angiotensin II (Ang II) was applied to induce hypertrophy in cultured neonatal rat cardiomyocytes (NCMs). Hemodynamics and histological analyses of the heart were evaluated. The expression of SIRT1, transforming growth factor-ß1 (TGF-ß1)/phosphorylated (p)-small mother against decapentaplegic (Smad)3 and hypertrophic markers were determined by immunofluorescence, real-time PCR, and Western blotting techniques. RESULTS: In the current study, Rsv treatment improved left ventricular function and reduced left ventricular hypertrophy and cardiac fibrosis significantly in the pressure overload rats. The expression of SIRT1 was significantly reduced, while the expression of TGF-ß1/p-Smad3 was significantly enhanced in AAC afflicted rat heart. Strikingly, treatment with Rsv restored the expressions of SIRT1 and TGF-ß1/p-Smad3 under AAC influence. However, SIRT1 inhibitor Sirtinol (Snl) markedly prevented the effects of Rsv, which suggest that SIRT1 signaling pathway was involved in the cardiac protective effect of Rsv. In vitro studies performed in Ang II-induced hypertrophy in NCMs confirmed the cardiac protective effect of Rsv. Furthermore, the study presented that SIRT1 negatively correlated with the cardiac hypertrophy, cardiac fibrosis, and the TGF-ß1/p-Smad3 expression. CONCLUSIONS: Taken together, these results indicated that activation of SIRT1 by Rsv attenuates cardiac hypertrophy, cardiac fibrosis, and improves cardiac function possibly via regulation of the TGF-ß1/p-Smad3 signaling pathway. Our study may provide a potential therapeutic strategy for cardiac hypertrophy.

CSE/H2S system alleviates uremic accelerated atherosclerosis by regulating TGF-ß/Smad3 pathway in 5/6 nephrectomy ApoE-/- mice.

BACKGROUND: Hydrogen sulfide (H2S) has been shown to inhibit the atherosclerosis development and progression. It is produced by cystathionine γ-lyase (CSE) in the cardiovascular system. In our previous study, it has been shown that CSE/H2S system plays a significant role in the changes of uremic accelerated atherosclerosis (UAAS), but the mechanism is not known clearly. METHODS: In this study, we explored the antagonism of CSE/H2S system in UAAS and identified its possible signaling molecules in ApoE-/- mice with 5/6 nephrectomy and fed with atherogenic diet. Mice were divided into sham operation group (sham group), UAAS group, sodium hydrosulfide group (UAAS+NaHS group) and propargylglycine group (UAAS+PPG group). Serum creatinine, urea nitrogen, lipid levels and lesion size of atherosclerotic plaque in the aortic roots were analyzed. Meanwhile, the expression of CSE, TGF-ß and phosphorylation of Smad3 were detected. RESULTS: Compared with sham group, the aortic root of ApoE-/- mice in the UAAS group developed early atherosclerosis, the levels of total cholesterol, triglyceride, low-density lipoprotein-cholesterol, serum creatinine and urea nitrogen were also higher than that in the sham group. NaHS administration can inhibit the development of atherosclerosis, but PPG administration can accelerate the atherosclerosis development. Meanwhile, the protein expression levels of CSE and TGF-ß and phosphorylation of Smad3 significantly decreased in the UAAS mice. Treatment of UAAS mice with NaHS inhibited TGF-ß protein expression and Smad3 phosphorylation decrease, but PPG treatment had the opposite effect. CONCLUSIONS: The CSE/H2S system is of great importance for treating atherosclerosis in patients with chronic kidney disease, and it may protect the vascular from atherosclerosis through the TGF-ß/Smad pathway.

Arsenic trioxide inhibits the differentiation of fibroblasts to myofibroblasts through nuclear factor erythroid 2-like 2 (NFE2L2) protein and the Smad2/3 pathway.

BACKGROUND: Tissue contraction and the extracellular matrix deposition are part of the pathogenesis of hypertrophic scars. The transcriptional factor NFE2L2 inhibits fibroblast differentiation in idiopathic pulmonary fibrosis and promotes myofibroblast dedifferentiation. Our previous study showed that the transcription factor NFE2L2 was strongly induced on treatment with arsenic trioxide (ATO). OBJECTIVE: The present study sought to investigate the effect of ATO on myofibroblast formation to determine its potential role in hypertrophic scar treatment. METHODS: Small interfering RNA against NFE2L2 was used on treatment with ATO in human skin myofibroblasts. The expression levels of fibrosis markers were assessed by reverse transcription polymerase chain reaction, western blot, and immunofluorescence staining. The transforming growth factor-ß1 (TGF-ß1)/Smad2/3 signaling was detected by western blot. A rabbit ear model was used to evaluate the antifibrotic role of ATO. RESULTS: At the cellular level, ATO abolished fibroblast differentiation in response to TGF-ß1. ATO reduced TGF-ß1-induced reactive oxygen species accumulation through increased expression of the antioxidant gene HO-1 in fibroblasts. In addition, ATO promoted the nuclear translocation of NFE2L2 and inhibited the phosphorylation of Smad2/3. In the rabbit ear model, ATO prevented the progression of hypertrophic scar formation. CONCLUSIONS: This study provides the first evidence implying that ATO inhibits the formation of myofibroblasts in vivo and in vitro and provides a possible treatment for hypertrophic scars.

Lysyl Hydroxylase Inhibition by Minoxidil Blocks Collagen Deposition and Prevents Pulmonary Fibrosis via TGF-ß1/Smad3 Signaling Pathway.

BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a deadly disease characterized by excessive collagen in the extracellular matrix (ECM) of the lungs. Collagen is the primary protein component of the ECM. However, the exact mechanisms underlying the formation and deposition of collagen in the ECM under normal and pathological conditions remain unclear. Previous studies showed that lysyl hydroxylase (LH) plays a crucial role in the formation of collagen. Minoxidil is an FDA-approved anti-hypertensive agent that inhibits LH that reduces fibrosis. In this study, we investigated the functional roles of LHs (LH1, LH2, and LH3) in pulmonary fibrosis and the anti-fibrotic effects of minoxidil. MATERIAL AND METHODS Patient serum samples were examined for their expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLOD) 1-3, the genes encoding LH 1-3. Mice with bleomycin (BLM 2.5 mg/kg)-induced pulmonary fibrosis were administered a minoxidil solution (30 mg/kg) by oral gavage. RESULTS The PLOD mRNA levels were significantly higher in the IPF patients than in the healthy control subjects. Minoxidil suppressed the BLM-induced pulmonary fibrosis in vivo. These effects were associated with blocking TGF-ß1/Smad3 signal transduction and attenuating the expression and activity of LHs, resulting in decreased collagen formation, thus reducing the pulmonary fibrosis. The anti-fibrotic effects of minoxidil may be mediated through competitive inhibition of LHs activity, resulting in decreased pyridine cross-link formation and collagen production and deposition. CONCLUSIONS The results of this study suggest that LH represents a target to prevent or treat pulmonary fibrosis, and minoxidil may provide an effective agent to inhibit LHs.

Inhibitory effects of low decibel infrasound on the cardiac fibroblasts and the involved mechanism.

INTRODUCTION: Infrasound is a mechanical vibration wave with frequency between 0.0001 and 20 Hz. It has been established that infrasound of 120 dB or stronger is dangerous to humans. However, the biological effects of low decibel infrasound are largely unknown. The purpose of this study was to investigate the effects of low decibel infrasound on the cardiac fibroblasts. MATERIALS AND METHODS: The cardiac fibroblasts were isolated and cultured from Sprague-Dawley rats. The cultured cells were assigned into the following four groups: control group, angiotensin II (Ang II) group, infrasound group, and Ang II+infrasound group. The cell proliferation and collagen synthesis rates were evaluated by means of [3H]-thymidine and [3H]-proline incorporation, respectively. The levels of TGF-ß were determined by enzyme-linked immunosorbent assay. Moreover, RNAi approaches were used for the analysis of the biological functions of miR-29a, and the phosphorylation status of Smad3 was detected using western blotting analysis. RESULTS: The results showed that low decibel infrasound significantly alleviated Ang II-induced enhancement of cell proliferation and collagen synthesis. DISCUSSION: Compared with the control, Ang II markedly decreased the expression of miR-29a levels and increased the secretion of TGF-ß and phosphorylation of Smad3, which was partly reversed by the treatment with low decibel infrasound. Importantly, knockdown of miR-29a diminished the effects of infrasound on the cardiac fibroblasts. In conclusion, low decibel infrasound inhibits Ang II-stimulated cardiac fibroblasts via miR-29a targeting TGF-ß/Smad3 signaling.

TGF-ß is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5-Smad2/3 pathway. Potential role in OA related pain?

OBJECTIVE: Pain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-ß (TGF-ß), which in turn stimulates chondrocytes to produce NGF. DESIGN: Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-ß1 and/or Interleukin-1 (IL-1)ß. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-ß pathway inducing NGF. RESULTS: NGF expression was consistently induced in higher levels by TGF-ß than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-ß-induced NGF whereas it fully blocked IL-1ß-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-ß-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-ß exposure led to a consistent high level of NGF induction. CONCLUSION: We show for the first time that TGF-ß induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.

Xanthohumol attenuates collagen synthesis in scleroderma skin fibroblasts by ROS/Nrf2/TGFß1/Smad3 pathway.

Skin fibrosis, the most obvious clinical manifestation of systemic sclerosis (SSc), has a high unmet need for treatment. Xanthohumol (Xn) has been shown to have beneficial effects on fibrotic diseases, but its efficacy in SSc remains unreported. This study aims to elucidate the effects and mechanisms of Xn on collagen synthesis in SSc skin fibroblasts (SScF). We found increased collagen production in SScF cultured in vitro, accompanied by dysregulated levels of oxidative stress. Cell experiments showed that Xn inhibited cell proliferation and promoted apoptosis. In addition, Xn was shown for the first time to upregulate reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2)levels in SScF, and when combined with the ROS scavenger N-acetylcysteine (NAC), Nrf2 expression was decreased. Importantly, we demonstrated that Xn significantly attenuated collagen synthesis by blocking the fibrotic classical transforming growth factor beta 1 (TGFß1)/Smad3 pathway, which interestingly was upregulated when combined with the Nrf2 inhibitor 385. Taken together, Xn suppressed the TGFß1/Smad3 pathway to ameliorate collagen overproduction by promoting ROS-induced oxidative stress damage and activating Nrf2, suggesting that Xn administration may be an emerging therapeutic strategy for skin fibrosis in SSc.

Imperatorin ameliorates kidney injury in diabetic mice by regulating the TGF-ß/Smad2/3 signaling axis, epithelial-to-mesenchymal transition, and renal inflammation.

Diabetic nephropathy (DN) is a serious concern in patients with diabetes mellitus. Prolonged hyperglycemia induces oxidative damage, chronic inflammation, and build-up of extracellular matrix (ECM) components in the renal cells, leading to kidney structural and functional changes. Imperatorin (IMP) is a naturally occurring furanocoumarin derivative with proven antioxidative and anti-inflammatory properties. We investigated whether IMP could improve DN and employed high glucose (HG)-induced HK-2 cells and high-fat diet-fed streptozotocin (HFD/STZ)-generated DN experimental model in C57BL/6 mice. In vitro, IMP effectively reduced the HG-activated reactive oxygen species generation, disturbance in the mitochondrial membrane potential (MMP) and epithelial-to-mesenchymal transition (EMT)-related markers, and the transforming growth factor (TGF)-ß and collagen 1 expression in HK-2 cells. In vivo, we found an elevation of serum creatinine, kidney histology alterations, and collagen build-up in the kidneys of the DN control group. Also, we found an altered expression of EMT-related markers, upregulation of the TGF-ß/Smad2/3 axis, and elevated pro-inflammatory molecules, TNF-α, IL-1ß, IL-18 and phospho-NF-kB (p65) in the DN control group. IMP treatment did not significantly reduce the blood glucose level compared to the DN control group. However, IMP treatment effectively improved renal damage by ameliorating kidney histological changes and serum renal injury markers. IMP treatment restored renal antioxidants and exhibited anti-inflammatory effects in the kidneys. Moreover, the abnormal manifestation of EMT-related attributes and elevated levels of TGF-ß, phospho-Smad2/3, and collagen 1 were also normalized in the IMP treatment group. Our findings highlight that IMP may be a potential candidate for treating DN.

Inhibition of TGF-ß signaling by the fungal lactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione and galiellalactone.

TGF-ß is a multifunctional cytokine that regulates cell proliferation, differentiation, apoptosis and extracellular matrix production. Deregulation of TGF-ß production or signaling plays a pivotal role in a variety of pathological processes such as cancer, metastasis, angiogenesis and fibrosis. Therefore, TGF-ß inhibitors should be promising therapeutic agents for the suppression of cancer progression and metastasis as well as fibrotic disorders. In a screening program of natural compounds from fungi inhibiting the TGF-ß dependent expression of a reporter gene in HepG2 cells, we found that the fungal lactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione and galiellalactone inhibited the binding of the activated Smad2/3 transcription factors to the DNA and antagonized the cellular effects of TGF-ß including reporter gene activation and expression of TGF-ß induced genes in HepG2 and MDA-MB-231 cells. The most active compound oxacyclododecindione inhibited TGF-ß dependent reporter activity with IC50-values of 190-217 nM. In an in vitro angiogenesis assay, the fungal lactones strongly decreased the formation of capillary-like tubules of MDA-MB-231 cells on Matrigel.

Proteomics Analysis Revealed Smad3 as a Potential Target of the Synergistic Antitumor Activity of Disulfiram and Cisplatin in Ovarian Cancer.

INTRODUCTION: Among gynecological cancers, ovarian cancer has a high mortality rate. Cisplatin-based chemotherapy is commonly used for the treatment of ovarian cancer. However, the clinical efficacy of cisplatin in ovarian cancer is limited due to the development of chemo-resistance during treatment. OBJECTIVE: In the study, we aimed to investigate the synergistic anti-cancer activity and targets of the FDA-approved drug disulfiram combined with cisplatin in ovarian cancer. METHODS: The cell viability was determined by Celltier-Glo luminescent assay. The synergistic anti-cancer activity was assessed by combination index. Cell cycle and apoptosis were detected by flow cytometry. The in vivo anti-tumor activity and side effects were evaluated using a xenografted mice model. The synergistic anti-cancer targets were identified by a mass spectrometry-based proteomics analysis. RESULTS: In this study, we first found that disulfiram synergistically enhanced the anti-tumor activity of cisplatin in chemo-resistant ovarian cancer cells, which was accompanied by the enhanced induction of cellular apoptosis. Secondly, the in vivo study demonstrated that the combination treatment of disulfiram and cisplatin dramatically inhibited tumor growth and had no apparent side effects in ovarian cancer xenografted mice. Finally, proteomics analysis identified SMAD3 as a potential target of disulfiram-cisplatin combined treatment, and the down-regulation of SMAD3 could increase cisplatin-induced cell death in ovarian cancer. CONCLUSION: Combination treatment of disulfiram and cisplatin synergistically inhibited the growth of ovarian cancer through down-regulating SMAD3. As a repurposed drug, disulfiram could be quickly transformed into a clinic to overcome cisplatin resistance for the treatment of ovarian cancer.

TGF-ß and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells.

Transforming growth factor-ß (TGF-ß) is upregulated at the time of arterial injury; however, the mechanism through which TGF-ß enhances the development of intimal hyperplasia is not clear. Recent studies from our laboratory suggest that in the presence of elevated levels of Smad3, TGF-ß stimulates smooth muscle cell (SMC) proliferation. This is a novel phenomenon in that TGF-ß has traditionally been known as a potent inhibitor of cellular proliferation. In these studies we explore the signaling pathways through which TGF-ß mediates its proliferative effect in vascular SMCs. We found that TGF-ß phosphorylates and activates Akt in a time-dependent manner, and this effect is significantly enhanced by overexpression of Smad3. Furthermore, both chemical and molecular inhibition of Smad3 can reverse the effect of TGF-ß on Akt. Although we found numerous signaling pathways that might function as intermediates between Smad3 and Akt, p38 appeared the most promising. Overexpression of Smad3 enhanced p38 phosphorylation and inhibition of p38 with a chemical inhibitor or a small interfering RNA blocked TGF-ß-induced Akt phosphorylation. Moreover, TGF-ß/Smad3 enhancement of SMC proliferation was blocked by inhibition of p38. Phosphorylation of Akt by TGF-ß/Smad3 was not dependent on gene expression or protein synthesis, and immunoprecipitation studies revealed a physical association among p38, Akt, and Smad3 suggesting that activation requires a direct protein-protein interaction. Our findings were confirmed in vivo where overexpression of Smad3 in a rat carotid injury model led to enhancement of p-p38, p-Akt, as well as SMC proliferation. Furthermore, inhibition of p38 in vivo led to decreased Akt phosphorylation and SMC proliferation. In summary, our studies reveal a novel pathway whereby TGF-ß/Smad3 stimulates SMC proliferation through p38 and Akt. These findings provide a potential mechanism for the substantial effect of TGF-ß on intimal hyperplasia and suggest new targets for chemical or molecular prevention of vascular restenosis.

Qingda granule attenuates cardiac fibrosis via suppression of the TGF-ß1/Smad2/3 signaling pathway in vitro and in vivo.

Cardiac fibrosis plays an important role in hypertension-related contractile dysfunction and heart failure. Qingda granule (QDG), derived from the Qingxuan Jiangya decoction, has been used clinically for more than 60 years to treat hypertension. However, the effect of QDG on hypertensive cardiac fibrosis remains largely unknown. The objective of this study was to investigate the effect of QDG on cardiac fibrosis and explore the underlying mechanism in vivo and in vitro. For in vivo experiments, 30 male spontaneously hypertensive rats were randomly divided into groups that received no QDG or one of three doses (0.45, 0.9 or 1.8 g/kg/day). Positive-control animals received valsartan (VAL, 7.2 mg/kg/day). Treatments were administered by gavage for 10 weeks. All three doses of QDG and VAL led to significantly lower blood pressure than in SHR animals. Besides, all three doses of QDG and VAL attenuated pathological changes in SHR animals. However, only intermediate, high concentrations of QDG and VAL led to significantly lower left ventricle ejection fraction and left ventricle fractional shortening than in SHR animals. Therefore, the minimum and effective QDG dose (intermediate concentration of QDG) was selected for subsequent animal experiments in this study. Our results showed that intermediate concentration of QDG also significantly mitigated the increases in levels of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), collagen III, transforming growth factor-ß1 (TGF-ß1) and in the ratio of phospho-Smad2/3 to total Smad2/3 protein in cardiac tissue, based on immunohistochemistry, Western blotting, and Masson staining. For in vitro experiments, primary cardiac fibroblasts were stimulated with 100 nM angiotensin II in the presence or absence of QDG. And we tested different concentrations of QDG (3.125, 6.25, 12.5, 25, 50 µg/mL) in the cell viability experiment. Our results showed that 3.125, 6.25 and 12.5 µg/mL of QDG treatment for 24 h didn't affect the cell viability of cardiac fibroblasts. Consistently, QDG at 6.25 or 12.5 µg/mL significantly reduced cell viability and down-regulated α-SMA in primary cardiac fibroblasts were stimulated with 100 nM angiotensin II. Therefore, QDG at 12.5 µg/mL was chosen for the following cell experiment. Our results showed that QDG at 12.5 µg/mL alleviated the increase of PCNA, collagen Ⅲ, TGF-ß1 expression, and the ratio of phospho-Smad2/3 to total Smad2/3 protein. Our studies in vitro and in vivo suggest that QDG reduces blood pressure and cardiac fibrosis as well as protecting cardiac function, and that it exerts these effects in part by suppressing TGF-ß1/Smad2/3 signaling.

Inhibitory Effect of the LY2109761 on the Development of Human Keloid Fibroblasts.

Keloids are scars characterized by abnormal proliferation of fibroblasts and overproduction of extracellular matrix components including collagen. We previously showed that LY2109761, a transforming growth factor- (TGF-) ß receptor inhibitor, suppressed the secretion of matrix components and slowed the proliferation of fibroblasts derived from human hypertrophic scar tissue. However, the exact mechanism underlying this effect remains unclear. Here, we replicated the above results in keloid-derived fibroblasts and show that LY2109761 promoted apoptosis, decreased the phosphorylation of Smad2 and Smad3, and suppressed TGF-ß1. These results suggest that the development and pathogenesis of keloids are positively regulated by the Smad2/3 signaling pathway and the upregulation of TGF-ß1 receptors. LY2109761 and other inhibitors of these processes may therefore serve as therapeutic targets to limit excessive scarring after injury.

Network model-based screen for FDA-approved drugs affecting cardiac fibrosis.

Cardiac fibrosis is a significant component of pathological heart remodeling, yet it is not directly targeted by existing drugs. Systems pharmacology approaches have the potential to provide mechanistic frameworks with which to predict and understand how drugs modulate biological systems. Here, we combine network modeling of the fibroblast signaling network with 36 unique drug-target interactions from DrugBank to predict drugs that modulate fibroblast phenotype and fibrosis. Galunisertib was predicted to decrease collagen and α-SMA expression, which we validated in human cardiac fibroblasts. In vivo fibrosis data from the literature validated predictions for 10 drugs. Further, the model was used to identify network mechanisms by which these drugs work. Arsenic trioxide was predicted to induce fibrosis by AP1-driven TGFß expression and MMP2-driven TGFß activation. Entresto (valsartan/sacubitril) was predicted to suppress fibrosis by valsartan suppression of ERK signaling and sacubitril enhancement of PKG activity, both of which decreased Smad3 activity. Overall, this study provides a framework for integrating drug-target mechanisms with logic-based network models, which can drive further studies both in cardiac fibrosis and other conditions.

Single-cell transcriptome dissection of the toxic impact of Di (2-ethylhexyl) phthalate on primordial follicle assembly.

Rationale: Accumulated evidence indicates that environmental plasticizers are a threat to human and animal fertility. Di (2-ethylhexyl) phthalate (DEHP), a plasticizer to which humans are exposed daily, can trigger reproductive toxicity by acting as an endocrine-disrupting chemical. In mammals, the female primordial follicle pool forms the lifetime available ovarian reserve, which does not undergo regeneration once it is established during the fetal and neonatal period. It is therefore critical to examine the toxicity of DEHP regarding the establishment of the ovarian reserve as it has not been well investigated. Methods: The ovarian cells of postnatal pups, following maternal DEHP exposure, were prepared for single cell-RNA sequencing, and the effects of DEHP on primordial follicle formation were revealed using gene differential expression analysis and single-cell developmental trajectory. In addition, further biochemical experiments, including immunohistochemical staining, apoptosis detection, and Western blotting, were performed to verify the dataset results. Results: Using single-cell RNA sequencing, we revealed the gene expression dynamics of female germ cells and granulosa cells following exposure to DEHP in mice. Regarding germ cells: DEHP impeded the progression of follicle assembly and interfered with their developmental status, while key genes such as Lhx8, Figla, and others, strongly evidenced the reduction. As for granulosa cells: DEHP likely inhibited their proliferative activity, and activated the regulation of cell death. Furthermore, the interaction between ovarian cells mediated by transforming growth factor-beta signaling, was disrupted by DEHP exposure, since the expression of GDF9, BMPR1A, and SMAD3 was affected. In addition, DNA damage and apoptosis were elevated in germ cells and/or somatic cells. Conclusion: These findings offer substantial novel insights into the reproductive toxicity of DEHP exposure during murine germ cell cyst breakdown and primordial follicle formation. These results may enhance the understanding of DEHP exposure on reproductive health.

Inhibition of c-Jun NH2-terminal kinase switches Smad3 signaling from oncogenesis to tumor- suppression in rat hepatocellular carcinoma.

UNLABELLED: Transforming growth factor beta (TGF-beta) signaling involves both tumor-suppression and oncogenesis. TGF-beta activates the TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TbetaRI-dependent pSmad3C transmits a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promotes carcinogenesis in human chronic liver disorders. The aim of this study is to elucidate how SP600125, a JNK inhibitor, affected rat hepatocellular carcinoma (HCC) development, while focusing on the domain-specific phosphorylation of Smad3. The rats received subcutaneous injections of either SP600125 or vehicle 11 times weekly together with 100 ppm N-diethylnitrosamine (DEN) administration for 56 days and were sacrificed in order to evaluate HCC development 28 days after the last DEN administration. The number of tumor nodules greater than 3 mm in diameter and the liver weight/body weight ratio were significantly lower in the SP600125-treated rats than those in the vehicle-treated rats (7.9 +/- 0.8 versus 17.7 +/- 0.9: P < 0.001; 6.3 +/- 1.2 versus 7.1 +/- 0.2%: P < 0.05). SP600125 significantly prolonged the median survival time in rats with DEN-induced HCC (113 versus 97 days: log-rank P = 0.0018). JNK/pSmad3L/c-Myc was enhanced in the rat hepatocytes exposed to DEN. However, TbetaRI/pSmad3C/p21(WAF1) was impaired as DEN-induced HCC developed and progressed. The specific inhibition of JNK activity by SP600125 suppressed pSmad3L/c-Myc in the damaged hepatocytes and enhanced pSmad3C/p21(WAF1), acting as a tumor suppressor in normal hepatocytes. CONCLUSION: Administration of SP600125 to DEN-treated rats shifted hepatocytic Smad3-mediated signal from oncogenesis to tumor suppression, thus suggesting that JNK could be a therapeutic target of human HCC development and progression.

Levosimendan up-regulates transforming growth factor-beta and smad signaling in the aorta in the early stage of sepsis.

BACKGROUND: This prospective, controlled experimental study was planned to investigate the effects of levosimendan on transforming growth factor (TGF)-beta3 and Smad1, Smad2 and Smad3 expression in the early stages of sepsis. METHODS: Twenty-four rats were randomized into four groups: (1) sham-operated controls, (2) dobutamine group--subjected to abdominal hypertension and peritonitis-induced sepsis using cecal ligation and puncture (CLP), then treated with 10 microg x kg(-1) min(-1) intravenous (IV) dobutamine infusion, (3) levosimendan group--as in 2, then treated with levosimendan IV bolus 200 microg x kg(-1) followed by 200 microg x kq(-1) min(-1) IV infusion, and (4) a control group as in 2, with no treatment. All rats were killed 8 hours after CLP. Aorta tissue samples were analyzed by immunohistochemical staining. RESULTS: CLP caused mild interleukin (IL)-1 immunostaining in both control and dobutamine groups. Immunoreactivity of tumor necrosis factor (TNF)-alpha was mild in both sham and control groups. TGF-beta3 immunostaining was mildly increased in groups sham, control and dobutamine, whereas it was found moderate in group levosimendan. Smad1, Smad2 and Smad3 were found moderately increased only in group levosimendan. CONCLUSION: Beneficial effects of levosimendan on hemodynamics and global oxygen transport were reported in experimental and clinical trials. Besides its potency on C++ ion sensitivity, it should influence inflammatory cytokine production by diminishing TGF-beta3 and Smad1, Smad2 and Smad3 expression.

LncRNA-OBFC2A targeted to Smad3 regulated Cyclin D1 influences cell cycle arrest induced by 1,4-benzoquinone.

Long-term exposure to benzene is associated with adverse health effects such as leukemia. Abnormal cell cycle progression has been reported participating in tumorigenesis. Our previous study found that lncRNA-OBFC2A was involved in benzene toxicity through regulating cell proliferation. However, the function of lncRNA-OBFC2A in the regulation of cell cycle remains obscure and the precise mechanisms need to be explored. In vitro study, results showed that benzene metabolic, 1,4-Benzoquinone (1,4-BQ), induced cell cycle arrest at the G1 phase accompanied with decreased expression of Cyclin D1 in a dose-dependently manner. Interestingly, lncRNA-OBFC2A overexpression was found in AHH-1 cells treated with 1,4-BQ and while interference with lncRNA-OBFC2A, the expression of Cyclin D1 were reversed. Further, we found that lncRNA-OBFC2A can interact with Smad3 to control cell cycle via modulating Cyclin D1 expression. In benzene exposed workers, the expression of lncRNA-OBFC2A and Smad3 increased while cyclin D1 decreased which was consistent with the in vitro experiment, meanwhile, the significant associations among them were also found. Thus, these findings indicate that lncRNA-OBFC2A targeted to Smad3 regulated cyclin D1 influences cell cycle arrest induced by 1,4-BQ. LncRNA-OBFC2A, Smad3 and Cyclin D1 as a set of biomarkers play important roles in benzene haematotoxicity.

Quercetin regulates fibrogenic responses of endometrial stromal cell by upregulating miR-145 and inhibiting the TGF-ß1/Smad2/Smad3 pathway.

OBJECTIVES: Aim of this study is to explore whether quercetin can inhibit the enlarged fibrogenic responses of endometrial stromal cells by increasing the level of microRNA-145 (miR-145) and mediating the TGFß1/Smad2/Smad3 signaling pathway, and to discuss the mechanism of signal transduction, further to provide experimental basis for revealing the pathophysiological mechanism and seeking new strategies for effective prevention and treatment of endometrial fibrosis. METHODS: The expression levels of miR-145 and TGF-ß receptor 2 (TGFBR2) were detected by RT-qPCR analysis. Expressions of α-smooth muscle actin (α-SMA) and vimentin were examined by immunofluorescence staining. Cell viability was measured by MTT assay. The protein expression of collagen type 1 alpha 1 (Col1a1), α-SMA, fibronectin (FN), TGFBR2, transforming growth factor (TGF-ß1), Smad2/3, phospho-Smad2/3 (p-Smad2/3) were detected by western blot analysis. The interaction between miR-145 and TGFBR2 was confirmed by dual-luciferase reporter gene assay. RESULTS: The expression level of miR-145 was decreased, whereas TGFBR2 was increased in intrauterine adhesion tissue. The expression levels of COL1A1, α-SMA, FN, TGFBR2, and p-Smad2/3 were increased, whereas miR-145 and cell proliferation were decreased in human endometrial stromal cells (hESCs) in response to TGF-ß1 stimulation in a time and dose-dependent manner, which could be reversed by quercetin. Furthermore, quercetin regulates cell fibrogenic responses of endometrial stromal cells via miR-145/TGF-ß1/Smad2/Smad3 pathway. CONCLUSIONS: These findings indicated that quercetin have a significant anti-fibrotic effect and could upregulate miR-145 and inhibit activation of TGF-ß1/Smad2/Smad3 pathway to regulate TGF-ß1 induced fibrogenic responses of endometrial stromal cells, which may serve as a potential therapeutic agent for endometrial fibrosis.

Melatonin Ameliorates Renal Fibrosis Through the Inhibition of NF-κB and TGF-ß1/Smad3 Pathways in db/db Diabetic Mice.

OBJECTIVE: To investigate the effects and molecular mechanism of melatonin (MT) on NF-κB and TGF-ß/Smad3 signaling pathways in db/db diabetic mice. METHODS: db/db diabetic mice were divided into five groups treated with melatonin at doses of 50, 100, 200 µg/kg, the urinary concentration was detected by ELISA, renal histology was observed in PAS paining. Mouse mesangial cells were divided into mannitol control group, normal control group, normal control + MT group, high glucose group, high glucose + different concentrations (10, 100, 1000) µmol/L MT group. The proliferation of mesangial cells was detected by EdU kit; the expression of NF-κBp65, ColⅣ and Fn were detected by laser confocal system; the concentrations and mRNA levels of ColⅣ and Fn were detected by ELISA and qRT-PCR. the expressions of ColⅣ, Fn, IκB, p-IκB, TGF-ß1, Smad3 and p-Smad3 were detected by Western blot in renal tissues and mesangial cells. RESULTS: MT treatment could markedly improve the kidney histopathologic lesions. Compared with the db/m mice, 24 h urinary albumin excretion rate (UAER) and the expressions of ColIV, Fn, p-IκB/IκB, NF-κBp65, TGF-ß1 and p-Smad3/Smad3 were decreased after melatonin treatment (p <0.05). Compared with the control group, the proliferation function of mesangial cells in high glucose group was significantly enhanced, and the expressions of ColIV, Fn, p-IκB/IκB, NF-κBp65, TGF-ß1 and p-Smad3/Smad3 in mesangial cells were significantly up-regulated (p <0.05), and these changes were significantly lowered in MT treatment. CONCLUSION: Melatonin can inhibit renal inflammation and fibrosis by inhibiting the NF-κB and TGF-ß1/Smad3 signaling pathways, and melatonin may be a promising therapeutic target in diabetic nephropathy.