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.

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.

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.

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.

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.

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.

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.

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.

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.

Wogonin Ameliorates Renal Inflammation and Fibrosis by Inhibiting NF-κB and TGF-ß1/Smad3 Signaling Pathways in Diabetic Nephropathy.

INTRODUCTION: Diabetic nephropathy (DN) has become an increasing threat to health, and inflammation and fibrosis play important roles in its progression. Wogonin, a flavonoid, has been proven to suppress inflammation and fibrosis in various diseases, including acute kidney injury. This study aimed at investigating the effect of wogonin on diabetes-induced renal inflammation and fibrosis. MATERIALS AND METHODS: Streptozotocin (STZ)-induced diabetic mouse models received gavage doses of wogonin (10, 20, and 40 mg/kg) for 12 weeks. Metabolic indices from blood and urine and pathological damage of glomerulus in the diabetic model were assessed. Glomerular mesangial cells SV40 were cultured in high glucose (HG) medium containing wogonin at concentrations of 1.5825, 3.125, and 6.25 µg/mL for 24 h. Inflammation and fibrosis indices were evaluated by histopathological, Western blotting, and PCR analyses. RESULTS: Wogonin treatment ameliorated albuminuria and histopathological lesions in diabetic mice. Inflammatory cytokines, such as monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and related signaling pathway NF-κB were downregulated after the administration of wogonin in vivo and in vitro. Furthermore, wogonin reduced the expression of extracellular matrix (ECM), including fibronectin (FN), collagen IV (Col-IV), α-smooth muscle actin (α-SMA), and transforming growth factor-ß1 (TGF-ß1) in the kidneys of diabetic mice and HG-induced mesangial cells. Moreover, the inhibition of TGF-ß1/Smad3 pathway might be responsible for these changes. CONCLUSION: Wogonin may ameliorate renal inflammation and fibrosis in diabetic nephropathy by inhibiting the NF-κB and TGF-ß1/Smad3 signaling pathways.

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.

Si-Miao-Yong-An Decoction attenuates isoprenaline-induced myocardial fibrosis in AMPK-driven Akt/mTOR and TGF-ß/SMAD3 pathways.

Myocardial fibrosis is well-known to be the aberrant deposition of extracellular matrix (ECM), which may cause cardiac dysfunction, morbidity, and death. Traditional Chinese medicine formula Si-Miao-Yong-An Decoction (SMYAD), which is used clinically in cardiovascular diseases has been recently reported to able to resist myocardial fibrosis. The anti-fibrosis effects of SMYAD have been evaluated; however, its intricate mechanisms remain to be clarified. Here, we found that SMYAD treatment reduced the fibrosis injury and collagen fiber deposition that could improve cardiac function in isoprenaline (ISO)-induced fibrosis rat models. Combined with our systematic RNA-seq data of SMYAD treatment, we demonstrated that the remarkable up-regulation or down-regulation of several genes were closely related to the functional enrichment of TGF-ß and AMPK pathways that were involved in myocardial fibrosis. Accordingly, we further explored the molecular mechanisms of SMYAD were mainly caused by AMPK activation and thereby suppressing its downstream Akt/mTOR and TGF-ß/SMAD3 pathways. Moreover, we showed that the ECM deposition and secretion process were attenuated, suggesting that the fibrosis pathological features are changed. Interestingly, we found the similar AMPK-driven pathways in NIH-3T3 mouse fibroblasts treated with ISO. Taken together, these results demonstrate that SMYAD may be a new candidate agent by regulating AMPK-driven Akt/mTOR and TGF-ß/SMAD3 pathways for potential therapeutic implications of myocardial fibrosis.

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.

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.

Positive Effects of a Young Systemic Environment and High Growth Differentiation Factor 11 Levels on Chondrocyte Proliferation and Cartilage Matrix Synthesis in Old Mice.

OBJECTIVE: To investigate the effects of a young systemic environment and growth differentiation factor 11 (GDF-11) on aging cartilage. METHODS: A heterochronic parabiosis model (2-month-old mouse and 12-month-old mouse [Y/O]), an isochronic parabiosis model (12-month-old mouse and 12-month-old mouse [O/O]), and 12-month-old mice alone (O) were evaluated. Knee joints and chondrocytes from old mice were examined by radiography, histology, cell proliferation assays, immunohistochemistry, Western blotting, and quantitative reverse transcriptase-polymerase chain reaction 16 weeks after parabiosis surgery. GDF-11 was injected into 12-month-old mouse joints daily for 16 weeks. Cartilage degeneration, cell proliferation, and osteoarthritis-related gene expression were evaluated. RESULTS: Osteoarthritis Research Society International scores in old mice were significantly lower in the Y/O group than in the O/O and O groups (both P < 0.05). The percentage of 5-ethynyl-2'-deoxyuridine-positive chondrocytes in old mice was significantly higher in the Y/O group than in the other groups (P < 0.05). Type II collagen (CII) and SOX9 messenger RNA levels differed in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). RUNX-2, CX, and matrix metalloproteinase 13 levels were significantly lower in cartilage from old mice in the Y/O group compared to the O/O and O groups (both P < 0.05). Similar results were obtained for protein expression levels and after GDF-11 treatment in vitro and in vivo. Phosphorylated Smad2/3 (pSmad2/3) levels were higher in the recombinant GDF-11-treated group than in the control group. CONCLUSION: A young systemic environment promotes chondrocyte proliferation and cartilage matrix synthesis in old mice. GDF-11, a "young factor," contributes to these effects through the up-regulation of pSmad2/3.

Cordyceps polysaccharide ameliorates airway inflammation in an ovalbumin-induced mouse model of asthma via TGF-ß1/Smad signaling pathway.

Allergic asthma is a chronic inflammatory disease characterized by airflow obstruction, airway hyperresponsiveness (AHR), airway inflammation, and mucus overproduction. Cordyceps polysaccharide (CPS) is one of the main bioactive compounds of Cordyceps militarisis, a traditional Chinese medicine. In this study, we established a mouse model of asthma using ovalbumin (OVA) challenge and evaluated the potential regulatory effect of CPS (25, 50, and 100 mg/kg) on asthmatic mice. These results showed that the asthmatic mice treated with CPS suppressed the secretion of eotaxin, IL-4, IL-5, IL-13, and IFN-γ in the blood and bronchoalveolar lavage fluid (BALF), and decreased serum IgE levels compared to the vehicle-treated mice. CPS also alleviated inflammatory cell infiltration, goblet cell hyperplasia, and the increases of inflammatory cells in the mouse model of asthma. In addition, OVA-induced AHR was inhibited by CPS treatment. Further analyses of protein expression revealed that CPS inhibited the activation of transforming growth factor ß1 (TGF-ß1)/Smad pathway in mice with asthma. These findings indicated that CPS might serve as a potential therapeutic agent for the management of allergic asthma.

Hypoxia and transforming growth factor ß1 regulation of long non-coding RNA transcriptomes in human pulmonary fibroblasts.

One of the key characteristics of idiopathic pulmonary fibrosis (IPF) is accumulation of excess fibrous tissue in the lung, which leads to hypoxic conditions. Transforming growth factor (TGF) ß is a major mediator that promotes the differentiation of fibroblasts to myofibroblasts. However, how hypoxia and TGFß together contribute the pathogenesis of IPF is poorly understood. Long non-coding RNAs (lncRNAs) have regulatory effects on certain genes and are involved in many diseases. In this study, we determined the effects of hypoxia and/or TGFß on mRNA and lncRNA transcriptomes in pulmonary fibroblasts. Hypoxia and TGFß1 synergistically increased myofibroblast marker expression. RNA sequencing revealed that hypoxia and TGFß1 treatment resulted in significant changes in 669 lncRNAs and 2,676 mRNAs compared to 150 lncRNAs and 858 mRNAs in TGFß1 alone group and 222 lncRNAs and 785 mRNAs in hypoxia alone group. TGFß1 induced the protein expression of HIF-1α, but not HIF-2α. On the other hand, hypoxia enhanced the TGFß1-induced phosphorylation of Smad3, suggesting a cross-talk between these two signaling pathways. In all, 10 selected lncRNAs (five-up and five-down) in RNA sequencing data were validated using real-time PCR. Two lncRNAs were primarily located in cytoplasm, three in nuclei and five in both nuclei and cytoplasm. The silencing of HIF-1α and Smad3, but not Smad2 and HIF-2α rescued the downregulation of FENDRR by hypoxia and TGFß1. In conclusion, hypoxia and TGFß1 synergistically regulate mRNAs and lncRNAs involved in several cellular processes, which may contribute to the pathogenesis of IPF.

Inhibition of the Progression of Skin Inflammation, Fibrosis, and Vascular Injury by Blockade of the CX3 CL1/CX3 CR1 Pathway in Experimental Mouse Models of Systemic Sclerosis.

OBJECTIVE: To assess the preclinical efficacy and mechanism of action of an anti-CX3 CL1 monoclonal antibody (mAb) in systemic sclerosis (SSc). METHODS: Cultured human dermal fibroblasts were used to evaluate the direct effect of anti-CX3 CL1 mAb on fibroblasts. In addition, bleomycin-induced and growth factor-induced models of SSc were used to investigate the effect of anti-CX3 CL1 mAb on leukocyte infiltration, collagen deposition, and vascular damage in the skin. RESULTS: Anti-CX3 CL1 mAb treatment significantly inhibited Smad3 phosphorylation (P < 0.05) and expression of type I collagen and fibronectin 1 (P < 0.01) in dermal fibroblasts stimulated with transforming growth factor ß1 (TGFß1). In the bleomycin model, daily subcutaneous bleomycin injection increased serum CX3 CL1 levels (P < 0.05) and augmented lesional CX3 CL1 expression. Simultaneous administration of anti-CX3 CL1 mAb or CX3 CR1 deficiency significantly suppressed the dermal thickness, collagen content, and capillary loss caused by bleomycin (P < 0.05). Injection of bleomycin induced expression of pSmad3 and TGFß1 in the skin, which was inhibited by anti-CX3 CL1 mAb. Further, the dermal infiltration of CX3 CR1+ cells, macrophages (inflammatory and alternatively activated [M2-like] subsets), and CD3+ cells significantly decreased following anti-CX3 CL1 mAb therapy (P < 0.05), as did the enhanced skin expression of fibrogenic molecules, such as thymic stromal lymphopoietin and secreted phosphoprotein 1 (P < 0.05). However, the treatment did not significantly reduce established skin fibrosis. In the second model, simultaneous anti-mCX3 CL1 mAb therapy significantly diminished the skin fibrosis induced by serial subcutaneous injection of TGFß and connective tissue growth factor (P < 0.01). CONCLUSION: Anti-CX3 CL1 mAb therapy may be a novel approach for treating early skin fibrosis in inflammation-driven fibrotic skin disorders such as SSc.

Effect of Dexamethasone on TGF-ß1/Smad3 Signalling Pathway in Airway Remodelling Model of Asthmatic Rats.

OBJECTIVE: To determine the effect of dexamethasone on regulating the TGF-ß1/Smad3 signalling pathway in airway remodelling model of asthmatic rats. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of Pathology, The First Hospital of Wuhan, Hubei Province, China, from February 2017 to April 2018. METHODOLOGY: Thirty male SPF Sprague-Dawley rats were randomly divided into the control group, the model group and the dexamethasone group, with 10 rats in each group. Rats were sensitised and excited by ovalbumin (OVA) to establish the model of bronchial asthma. The bronchial basement membrane perimeter (Pbm), the bronchial wall thickness (Wat), the smooth muscle thickness (Wam), collagenous fiber thickness (Wac) and other airway remodelling indices were measured and calculated by image analysis system. The expressions of TGF-ß1 and Smad3 mRNA and protein in lung tissue of each group of rats were detected by RT-PCR and Western blot. RESULTS: Wat/Pbm, Wai/Pbm and Wam/Pbm in the model group were higher compared with those in the control group (all p<0.001); Wat/Pbm, Wai/Pbm, and Wam/Pbm in the dexamethasone group were significantly lower than those in the model group (all p<0.001). Relative expression levels of TGF-ß1, Smad3 mRNA and protein in the model group were higher than those in the control group (all p<0.001); the relative expression levels of TGF-ß1, Smad3 mRNA and protein in the dexamethasone group were lower than those in the model group (all p<0.001). CONCLUSION: Dexamethasone may antagonize airway remodeling by regulating TGF-ß1/Smad3 signaling pathway, which likely to play a role in the treatment of bronchial asthma.

Dihydromyricetin attenuates hypertrophic scar formation by targeting activin receptor-like kinase 5.

Hypertrophic scar (HPS) is a manifestation of abnormal tissue repair, representing excessive extracellular matrix production and abnormal function of fibroblasts, for which no satisfactory treatment is available at present. Here we identified a natural product of flavonoid, dihydromyricetin, could effectively attenuate HPS formation. We showed that local intradermal injection of dihydromyricetin (50 µM) reduced the gross scar area, cross-sectional size of the scar and the scar elevation index in a mechanical load-induced mouse model. In addition, dihydromyricetin treatment also markedly decreased collagen density of the scar tissue. Furthermore, both in vitro and in vivo study both demonstrated that dihydromyricetin inhibited the proliferation, activation, contractile and migration abilities of hypertrophic scar-derived fibroblasts (HSFs) but did not affect HSFs apoptosis. Western blot analysis revealed that dihydromyricetin could down-regulate the phosphorylation of Smad2 and Smad3 of TGF-ß signaling. Such bioactivity of dihydromyricetin may result from its selective binding to the catalytic region of activin receptor-like kinase 5 (ALK5), as suggested by the molecular docking study and kinase binding assay (12.26 µM). Above all, dihydromyricetin may prove to be a promising agent for the treatment of HPS and other fibroproliferative disorders.