This study aimed to explore the effects and mechanisms of total extracts from Anthriscus sylvestris on pulmonary hypertension in rats. Sixty male SD rats were divided into normal(NC) group, model(M) group, positive drug sildenafil(Y) group, low-dose A. sylvestris(ES-L) group, medium-dose A. sylvestris(ES-M) group, and high-dose A. sylvestris(ES-H) group. On day 1, rats were intraperitoneally injected with monocrotaline(60 mg·kg~(-1)) to induce pulmonary hypertension, and the rat model was established on day 28. From days 15 to 28, intragastric administration of the respective treatments was performed. After modeling and treatment, small animal echocardiography was used to detect the right heart function of the rats. Arterial blood gas was measured using a blood gas analyzer. Hematoxylin and eosin(HE) staining and Masson staining were performed to observe cardiopulmonary pathological damage. Flow cytometry was used to detect apoptosis in the lung and myocardial tissues and reactive oxygen species(ROS) levels. Western blot was applied to detect the expression levels of transforming growth factor-β1(TGF-β1), phosphorylated mothers against decapentaplegic homolog 3(p-Smad3), Smad3, tissue plasminogen activator(t-PA), and plasminogen activator inhibitor-1(PAI-1) in lung tissue. A blood routine analyzer was used to measure inflammatory immune cell levels in the blood. Enzyme-linked immunosorbent assay(ELISA) was used to detect the expression levels of P-selectin and thromboxane A2(TXA2) in plasma. The results showed that, compared with the NC group, right heart hypertrophy index, right ventricular free wall thickness, right heart internal diameter, partial carbon dioxide pressure(PaCO_2), apoptosis in cardiopulmonary tissue, and ROS levels were significantly increased in the M group. In contrast, the ratio of pulmonary blood flow acceleration time(PAT)/ejection time(PET), right cardiac output, change rate of right ventricular systolic area, systolic displacement of the tricuspid ring, oxygen partial pressure(PaO_2), and blood oxygen saturation(SaO_2) were significantly decreased in the M group. After administration of the total extract of A. sylvestris, right heart function and blood gas levels were significantly improved, while apoptosis in cardiopulmonary tissue and ROS levels significantly decreased. Further testing revealed that the total extract of A. sylvestris significantly decreased the levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and PAI-1 proteins in lung tissue, while increasing the expression of t-PA. Additionally, the extract reduced the levels of inflammatory cells such as leukocytes, lymphocytes, granulocytes, and monocytes in the blood, as well as the levels of P-selectin and TXA2 in plasma. Metabolomics results showed that the total extract of A. sylvestris significantly affected metabolic pathways, including arginine biosynthesis, tyrosine metabolism, and taurine and hypotaurine metabolism. In conclusion, the total extract of A. sylvestris may exert an anti-pulmonary hypertension effect by inhibiting the TGF-β1/Smad3 signaling pathway, thereby alleviating immune-inflammatory responses and thrombosis.
Animals ; Male ; Smad3 Protein/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Rats, Sprague-Dawley ; Rats ; Signal Transduction/drug effects* ; Hypertension, Pulmonary/genetics* ; Thrombosis/immunology* ; Drugs, Chinese Herbal/administration & dosage* ; Humans ; Apoptosis/drug effects*

Tissue formation and organ homeostasis are achieved by precise coordination of proliferation and differentiation of stem cells and progenitors. While deregulation of these processes can result in degenerative disease or cancer, their molecular interplays remain unclear. Here we show that the switch of human pluripotent stem cell (hPSC) self-renewal to differentiation is associated with the induction of distinct cyclin-dependent kinase inhibitors (CDKIs). In hPSCs, Activin/Nodal/TGFβ signaling maintains CDKIs in a poised state via SMAD2/3-NANOG-OCT4-EZH2-SNON transcriptional complex. Upon gradual differentiation, CDKIs are induced by successive transcriptional complexes between SMAD2/3-SMYD2 and developmental regulators such as EOMES, thereby lengthening the G1 phase. This, in turn, induces SMAD2/3 transcriptional activity by blocking its linker phosphorylation. Such SMAD2/3-CDKI positive feedback loops drive the exit from pluripotency and stepwise cell-fate specification that could be harnessed for producing cells for therapeutic applications. Our study uncovers fundamental mechanisms of how cell-fate specification is interconnected to cell-cycle dynamics and provides insight into autonomous circuitries governing tissue self-formation.
Humans ; Smad2 Protein/genetics* ; Smad3 Protein/genetics* ; Cell Differentiation ; Pluripotent Stem Cells/metabolism* ; Signal Transduction ; Octamer Transcription Factor-3/genetics* ; Enhancer of Zeste Homolog 2 Protein/genetics* ; Nanog Homeobox Protein/genetics* ; Phosphorylation

This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.
Animals ; Smad3 Protein/genetics* ; CRISPR-Cas Systems/genetics* ; Mice ; Podocytes/metabolism* ; Transforming Growth Factor beta1/pharmacology* ; Cell Line ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics*

This study aimed to investigate the potential mechanism and the compatibility significance of Tanyu Tongzhi Formula in treating atherosclerosis(AS) in mice based on the transforming growth factor-β(TGF-β)/Smad2/3 signaling pathway. Eight C57BL/6J mice were as assigned to a normal control group and fed a regular diet, while 35 ApoE~(-/-) mice of the same strain were fed a high-fat diet for 8 weeks to establish an AS model. The model mice were randomly divided into a model group, a Tanyu Tongzhi group(18.2 mg·kg~(-1)), a Huatan(phlegm-resolving) group(10.4 mg·kg~(-1)), and a Quyu(blood stasis-resolving) group(7.8 mg·kg~(-1)), with 8 mice in each group. Except for the normal group, all other groups continued to be fed a high-fat diet for 8 weeks to maintain the AS model, and then the mice were treated by gavage for 8 weeks. Plasma levels of total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), interleukin-1β(IL-1β), and interleukin-18(IL-18) were measured using enzyme-linked immunosorbent assay(ELISA). Hematoxylin and eosin(HE) staining, oil red O staining, and Russell-Movat pentachrome staining were performed to observe the pathological changes in the aortic tissue. The proportions of aortic plaque area, lipid-stained area, collagen fibers, and elastic fibers were calculated. Immunofluorescence was used to detect the protein expression levels of matrix metalloproteinase 2(MMP2) and tissue inhibitor of metalloproteinases 2(TIMP2). Western blot was used to detect the protein expression levels of TGF-β1, TGF-β2, Smad2/3, and Smad7 in aortic tissue. Real-time fluorescence quantitative PCR(RT-qPCR) was used to measure the mRNA expression levels of TGF-β receptor(TGF-βR), TGF-β1, Smad2/3, Smad7, intercellular adhesion molecule-1(ICAM-1), and vascular cell adhesion molecule-1(VCAM-1) in aortic tissue. The results showed that compared with the normal control group, the model group had increased plasma TC and LDL-C, significantly decreased HDL-C, and significantly elevated plasma IL-1β and IL-18 levels. The model group also exhibited an increased proportion of aortic plaque area, lipid-stained area, and collagen fiber area, along with significantly upregulated MMP2 and downregulated TIMP2 expression in the aortic arch. Additionally, the expression levels of TGF-βR, TGF-β1, and p-Smad2/3 proteins and mRNA in the aortic tissue were significantly elevated, while Smad7 expression was decreased. Compared with the model group, the Tanyu Tongzhi group showed significantly reduced plasma TC and LDL-C levels, significantly increased HDL-C levels, and significantly decreased plasma IL-1β and IL-18 levels. The Tanyu Tongzhi group also exhibited a significant reduction in aortic plaque size and severity, a significant downregulation of MMP2 expression in the aortic arch, and significantly decreased ICAM-1 and VCAM-1 mRNA expression levels. Moreover, the Tanyu Tongzhi group demonstrated significantly reduced expression levels of TGF-β1 and p-Smad2/3 proteins and mRNA in the aortic tissue, and an increased expression level of Smad7 protein to varying degrees. Compared with the Tanyu Tongzhi group, the Quyu group had significantly higher LDL-C levels and elevated plasma IL-1β and IL-18 levels. The Huatan group showed upregulated MMP2 expression and downregulated TIMP2 expression in the aortic arch. In conclusion, Tanyu Tongzhi Formula, which is composed based on the pathogenesis of phlegm and blood stasis, maintains vascular homeostasis by primarily regulating lipid metabolism and controlling inflammatory factors through the Huatan group, and maintaining vascular wall permeability, inhibiting plaque development, and stabilizing plaques through the Quyu group. The mechanism of action may involve inhibiting TGF-β1 expression in the aorta, reducing Smad2/3 phosphorylation, and simultaneously increasing Smad7 expression.
Animals ; Atherosclerosis/metabolism* ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Mice, Inbred C57BL ; Male ; Transforming Growth Factor beta/genetics* ; Humans ; Homeostasis/drug effects* ; Aorta/metabolism* ; Smad2 Protein/genetics* ; Smad3 Protein/genetics*

OBJECTIVE: To investigate the intervention of melatonin (MT) in the expression of circadian genes in patients with pulmonary fibrosis and to analyze the mechanism by which it alleviates the progression of pulmonary fibrosis. METHODS: By utilizing the Gene Expression Omnibus (GEO) database, we identified differentially expressed circadian genes between patients with pulmonary fibrosis and controls. We analyzed the correlation between circadian genes and pulmonary function as well as genes related to pulmonary fibrosis. A bleomycin-induced mouse model of pulmonary fibrosis (BLM group) was constructed to observe the expression differences of PER2 and CRY2 by sequencing and immunohistochemical staining in the BLM group and after MT intervention (BLM+MT group). Hematoxylin and eosin (HE) staining and Masson staining were used to observe the effects of MT on fibrosis. We used Western blot to detect the expression of P-smad2/3 in lung epithelial cells induced by transforming growth factor β (TGF-β). Reverse transcription quantitative real-time PCR technology was employed to investigate the rhythmic expression changes of circadian genes in the control group, TGF-β group, and TGF-β+MT group. Finally, luzindole, a MT receptor antagonist, was used to intervene in TGF-β+MT group, and Western blot was used to explore the receptor dependence of MT in alleviating TGF-β-induced epithelial-mesenchymal transition. RESULTS: (1) Analysis of the GEO dataset (GSE) revealed a negative correlation between circadian genes PER2 and CRY2 and the expression of TGF-β, and a positive correlation with pulmonary function indicators in patients. (2) Transcriptome sequencing analysis of lung tissue in BLM group found that the expression of PER2 and CRY2 was significantly reduced compared with the normal group. Histopathological staining results showed that the lung tissue structure of the normal group was intact and clear, with thin alveolar septa; in the BLM group, there was a large increase in collagen fibers and disordered alveolar structure; compared with the BLM group, the BLM+MT group had reduced collagen fiber proliferation and inflammatory cell infiltration; the expression of PER2 and CRY2 in the BLM group was lower than in the normal group, and the expression in the BLM+MT group was increased compared with the BLM group. (3) In vitro lung epithelial cell experiments with TGF-β intervention showed that compared with the control group, the expression of P-smad2/3 increased in the TGF-β group, and MT intervention inhibited the inducing effect of TGF-β on P-smad2/3, while intervention with the MT receptor antagonist reversed this phenomenon. The results indicated that MT could inhibit the activation of the TGF-β pathway, and this process was dependent on MT receptors. (4) The 48-hour rhythm experiment in lung epithelial cells showed that the mRNA rhythm of PER2 and CRY2 in the TGF-β+MT group was close to 24 hours and showed a trend towards restoring the rhythm of the control group, while the addition of the MT receptor blocker tended to make the rhythm duration and amplitude of both groups approach that of the TGF-β group. CONCLUSION MT, by binding to its receptors, can restore the periodic expression of the circadian genes PER2 and CRY2, thereby inhibiting the activation of the TGF-β classical pathway and suppressing the pathological process of epithelial-mesenchymal transition in pulmonary fibrosis. This finding provides new molecular targets and potential therapeutic strategies for the treatment of pulmonary fibrosis.
Melatonin/pharmacology* ; Animals ; Mice ; Pulmonary Fibrosis/chemically induced* ; Bleomycin ; Humans ; Transforming Growth Factor beta/metabolism* ; Period Circadian Proteins/metabolism* ; Smad3 Protein/genetics* ; Disease Models, Animal ; Lung/pathology* ; Cryptochromes/metabolism* ; Smad2 Protein/genetics* ; Epithelial Cells/metabolism* ; Mice, Inbred C57BL

Objective: To investigate the clinicopathological features, immunophenotypes and molecular genetics of EWSR1-SMAD3 positive fibroblastic tumor (ESFT) with an emphasis on differential diagnosis. Methods: The clinicopathological data, immunohistochemical profiles and molecular profiles of 3 ESFT cases diagnosed at the Department of Pathology, Fudan University Shanghai Cancer Center from 2018 to 2021were analyzed. The related literature was also reviewed. Results: There were two males and one female. The patients were 24, 12 and 36 years old, respectively. All three tumors occurred in the subcutis of the foot with the disease duration of 6 months to 2 years. The tumors were presented with a slowly growing mass or nodule, accompanied with pain in 1 patient. The tumors ranged in size from 0.1 to 1.6 cm (mean, 1.0 cm). Microscopically, the tumors were located in the subcutaneous tissue with a nodular or plexiform growth pattern. They were composed of cellular fascicles of bland spindle cells with elongated nuclei and fine chromatin. One of the tumors infiltrated into adjacent adipose tissue. There was no nuclear atypia or mitotic activities. All three tumors showed prominent stromal hyalinization with zonal pattern present in one case. Focal punctate calcification was noted in two cases. The immunohistochemical studies showed that tumor cells were diffusely positive for ERG and negative for CD31 and CD34, with Ki-67 index less than 2%. Fluorescence in situ hybridization on the two tested cases identified EWSR1 gene rearrangement. The next generation sequencing analysis demonstrated EWSR1-SMAD3 fusion in all three cases. During the follow up, one patient developed local recurrence 24 months after the surgery. Conclusions: ESFT is a benign fibroblastic neoplasm and has a predilection for the foot, characterized by ERG immunoreactivity and EWSR1-SMAD3 fusion. Local recurrence might occur when incompletely excised. Familiarity with its clinicopathological features is helpful in distinguishing it from other spindle cell neoplasms that tend to occur at acral sites.
Adult ; Child ; Female ; Humans ; Male ; Biomarkers, Tumor/analysis* ; China ; In Situ Hybridization, Fluorescence ; Neoplasms, Fibrous Tissue/pathology* ; RNA-Binding Protein EWS/genetics* ; Smad3 Protein/genetics* ; Soft Tissue Neoplasms/surgery*

Objective To investigate the effect of SMAD family member 3(SMAD3) silenced by small interfering RNA (siRNA) on macrophage polarization and transforming growth factor β1 (TGF-β1)/ SMAD family signaling pathway in rheumatoid arthritis (RA). Methods RA macrophages co-cultured with rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were used as a cell model. TGF-β1 was used to stimulate macrophages, and SMAD3-specific siRNA (si-SMAD3) and negative control siRNA (si-NC) were transfected into human RA macrophages co-cultured in Transwell^TM chamber. The expression of SMAD3 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of TGF-β1, SMAD3 and SMAD7 protein was detected by Western blot analysis. The contents of TGF-β1 and IL-23 in cell culture supernatant were determined by ELISA. Cell proliferation was detected by CCK-8 assay. Transwell^TM chamber was used to measure cell migration. Results Compared with the model group and the si-NC group, the expression of TGF-β1, SMAD3 mRNA and protein in RA macrophages decreased significantly after silencing SMAD3. In addition, the secretion of IL-23 decreased significantly, and the cell proliferation activity and cell migration were inhibited, with high expression of SMAD7. Conclusion Knockdown of SMAD3 can promote M2 polarization and SMAD7 expression in RA macrophages.
Humans ; Arthritis, Rheumatoid/genetics* ; Interleukin-23 ; Macrophages ; RNA, Messenger ; RNA, Small Interfering/genetics* ; Smad7 Protein/genetics* ; Transforming Growth Factor beta1/genetics* ; Smad3 Protein/genetics* ; Gene Silencing

Humans ; Nerve Tissue Proteins ; Epithelial-Mesenchymal Transition/genetics* ; Constriction, Pathologic ; Receptors, Immunologic ; Epithelial Cells/metabolism* ; Smad3 Protein/metabolism* ; Transforming Growth Factor beta1/metabolism*

Animals ; Carbon Tetrachloride/toxicity* ; Drugs, Chinese Herbal/pharmacology* ; Liver/pathology* ; Liver Cirrhosis/pathology* ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Signal Transduction ; Smad3 Protein ; Smad7 Protein ; Transforming Growth Factor beta1/genetics*

To explore the influence for combination of nourishing yin and tonifying yang sequential therapy (NYTYST) with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve (DOR) based on TGF-β1/Smads signaling pathway. 
 Methods: A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group (n=8 in each group). The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy (HRT) and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor β1 receptor (TGF-β1R) in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot.
 Results: Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group (P<0.01); the levels of TGF-β1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly (P<0.01). Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group (P<0.05 or P<0.01). The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group (P<0.05). Compared with the Western medicine group, the levels of TGF-β1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group (P<0.05 or P<0.01). 
 Conclusion: NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-β1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.
Animals ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Gene Expression Regulation ; drug effects ; Infertility ; therapy ; Medicine, Chinese Traditional ; Ovarian Reserve ; drug effects ; Rats ; Signal Transduction ; drug effects ; Smad2 Protein ; genetics ; metabolism ; Smad3 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism