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 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

Animals ; Epithelial-Mesenchymal Transition ; Lung/metabolism* ; Male ; Pulmonary Disease, Chronic Obstructive ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad2 Protein/metabolism* ; Syndecan-1 ; Transforming Growth Factor beta1/metabolism*

OBJECTIVES: Stably expressed transforming growth factor -beta 1(TGF-β1)MCs were obtained and the effects of centellaasiatica (CA) granule on the expressions of Smad 2/3, Smad 7 and collagen Ⅳ and the level of Smad 2/3 phosphorylation were observed. METHODS: Lipofectin method was used to transfect TGF-β1 vector into MC, and the stably expressed TGF-β1 cell lines were selected by G418. The cells were divided into three groups. Control group:normal MC + RPMI 1640 + 10% normal rat serum; TGF-β1 group:stably expressed TGF-β1 MC + RPMI 1640 + 10% normal rat serum; CA group:stably expressed TGF-β1 MC + RPMI 1640 + 10% rat serum containing high CA. The experiments were repeated for five times. The contents of TGF-β1 and collagen Ⅳ in the culture medium were detected with ELISA, the expressions of mRNA and protein of TGF-β1, Smad 2/3, Smad 7 and the level of Smad 2/3 phosphorylation were detected by using real time quantitative polymerase chain reaction and Western blot. RESULTS: The contents of TGF-β1 and collagen Ⅳ in the culture medium of stably-expressed TGF-β1 MC were increased significantly, and the CA could reverse the effects of TGF-β1. The expressions of mRNA and protein of TGF-β1, Smad 2/3 and the level of Smad 2/3 phosphorylation were increased significantly in TGF-β1 transfected MC, and CA could dramatically reduce the expressions of mRNA and protein of TGF-β1, Smad 2/3 and the level of Smad 2/3 phosphorylation. The high expression of TGF-β1 decreased the expression of Smad 7 mRNA and protein, and the CA could antagonize the effect of mRNA expression. CONCLUSIONS The MCs stably-expressed TGF-β1 can activate the TGF-β1/Smad signal pathway and increase the expression of collagen Ⅳ. CA can decrease the occurrence of diabetic nephropathy(DN) by reducing the production of collagen Ⅳ through inhibiting the TGF-β1/Smad signal pathway.
Animals ; Cells, Cultured ; Centella ; chemistry ; Collagen Type IV ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Mesangial Cells ; drug effects ; metabolism ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVE: To investigate the effects of centella asiatica (CA) granule on the expression of transform growth factor-β(TGF-β) and related down-stream signals in rats with early diabetic nephropathy(DN) and to clarify the molecular mechanisms of CA molecular mechanism of on preventing and curing early diabetic kidney disease DN by studying the effects of centella asiatica on TGF-β expression and related down-stream signals. METHODS: Sixty male SD rats were divided into control group(=10) and DN model group(=50). The model rats were made a right nephrectomy. One week later, diabetic nephropathy was induced by intraperitoneal injection of streptocozin(30 mg/kg) for three consecutive days. High blood glucose level of Tail vein (fasting glucose ≥ 16.7 mmol/L) and high urinary protein level(total protein level in DN group was more than twice higher than the control group) were measured to confirm early DN in rats. In the sham operation group, the right renal capsule was damaged and the corresponding amount of saline was injected. The model rats were administrated by the means of intragastric administration. The DN model group were divided into DN group, DN+fosinopril group(1.6 mg/kg·d), DN+high CA group(16.8 mg/kg·d), DN+medium CA group(11.2 mg/kg·d) and DN+low CA group(5.6 mg/kg·d), and each group was intragastric administration one time every morning last for 16 weeks. The expressions of mRNA and protein of TGF-β, TβR1, TβR2, Smad2/3, Smad7 and the level of Smad2/3 phosphorylation were detected by using real time quantitative polymerase chain reaction and Western blot. RESULTS: The expressions of mRNA and protein of TGF-β, TβR1, TβR2, Smad2/3 and the level of Smad2/3 phosphorylation were significantly increased, the expressions of mRNA and protein of Smad7 were dramatically decreased. The fosinopril and high dosage CA could reverse the effects of DN. CONCLUSIONS CA plays an important role in preventing and curing DN through regulating the TGF-β/Smad signaling pathways.
Animals ; Centella ; chemistry ; Diabetes Mellitus, Experimental ; Diabetic Nephropathies ; chemically induced ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; physiopathology ; Male ; Rats ; Rats, Sprague-Dawley ; Receptor, Transforming Growth Factor-beta Type I ; metabolism ; Receptor, Transforming Growth Factor-beta Type II ; metabolism ; Signal Transduction ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

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

OBJECTIVETo investigate the effect of tumor necrosis factor-α-induced protein 8 like-2 (TIPE2) on apoptosis of CD4T lymphocytes in a murine model of severe burn injury.

METHODSA total of 140 male mice were randomly allocated into 6 groups. Small RNA interference technique was used to construct a siTIPE2-overexpressing lentivirus, and severe burn injury models were established in the mice. CD4T cells were purified from spleen of the mice, and the expressions of TIPE2, Smad2/Smad3, P-Smad2/P-Smad3 and Bcl-2/Bimprotein in CD4Tregs were detected. The changes in mitochondrial membrane potential and cytochrome C in CD4T cells were detected, and the activities of caspase-3, caspase-8, and caspase-9 were analyzed.

RESULTSDown-regulation of TIPE2 promoted the apoptosis of CD4T lymphocytes in siTIPE2-burn group, in which the protein expressions of P-smad2/P-Smad3 decreased, Bcl-2 expression increased and Bim expression decreased significantly as compared with the other groups (P<0.01 or 0.05). The mitochondrial membrane potential and cytochrome C expression in CD4T cells were down-regulated in siTIPE2-burn group (P<0.05) with a lowered caspase-3 activity compared with TIPE2-burn group (P<0.01) and decreased caspase-8 and caspase-9 compared with the other groups (P<0.05). The apoptosis rate was the highest in TIPE2-burn group, whose Smad2/Smad3 was higher than that in the sham group (P<0.05) and the expression of P-smad2/P-Smad3 significantly increased compared with the other groups (P<0.05). In TIPE2-burn group, the mitochondrial membrane potential in CD4T cells was decreased (P<0.01), the expression of cytochrome C increased markedly (P<0.01), and the activities of caspase-3, caspase-8, and caspase-9 were all obviously higher than those in the other groups (P<0.05).

CONCLUSIONAs an important immunoregulatory molecule, TIPE2 can promote the apoptosis of CD4T lymphocyte in mice with sever burn injury.

Animals ; Apoptosis ; Burns ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Down-Regulation ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Mice ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Spleen

OBJECTIVETo explore the effects of Wenyang Huazhuo Tongluo Recipe (WYHZTLR) containing serum on transforming growth factor β1 (TGF-β1)/Smad signaling pathway of skin fibroblasts in systemic sclerosis (SSc).

METHODSTotally 36 SSc patients were randomly assigned to Chinese medicine (CM) group, Western medicine (WM) group, and integrative medicine (IM) group according to random digit table, 12 in each group. Patients in the CM group took WYHZTLR decoction (one dose per day). Patients in the WM group took penicillamine tablet (0. 125 g each time, bid) and Prednisone Acetate Tablet (PAT 20 mg, qd). Patients in the IM group took penicillamine, PAT, and WYHZTLR decoction (in the same dosage of corresponding drugs as aforesaid). All patients were treated for one month to get drug containing serum. Besides, 10 untreated SSc patients' serum was taken as the control group. Healthy subjects' skin fibroblasts were originated from healthy skin tissue of the upper arms of 2 female patients undergoing plastic surgery. Corresponding serum of each group was added in the culture system of SSc patients' and healthy subjects' dermal fibroblasts respectively. Expression levels of TGF-β1 receptor type I (TGF-β1 RI), TGF-β1 receptor II (TGF-β1 RII), p-Smad2/3 and Smad7 protein were examined by Western blot. Expression levels of collagen type I and collagen type III (Col-I, Col-III) mRNA were examined by reverse transcription PCR. Contents of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in the supernatant of SSc, skin fibroblasts were examined by ELISA.

RESULTSCompared with the control group, expression levels of TGF-β1 R I and p-Smad2/3 protein significantly decreased, but expression levels of Smad7 protein significantly increased in skin fibroblasts of SSc patients and healthy subjects of WM, CM, and IM groups (P <0.05, P <0. 01). Meanwhile, the expression level of TGF-β1 RII decreased in the IM group (P <0. 05, P <0. 01). Compared with the WM group, expression levels of TGF-β1 RI and p-Smad2/ 3 protein significantly decreased, but that of Srnad7 protein significantly increased in IM groups (P <0. 01). mRNA expression levels of Col-I and Col-II in SSc skin fibroblasts significantly decreased more in WM, CM, and IM groups than in the control group (P <0. 05, P <0. 01). Besides, the expression level of Col-III mRNA was significantly lower in the IM group than in the WM group (P <0.01). Compared with the control group, serum levels of MMP-9 and MMP-9/TIMP-1 ratios increased more obviously in WM, CM, and IM groups (P <0.05, P <0.01). But expression levels of TIMP-1 decreased significantly in CM and IM groups (P <0.01). Expression levels of MMP-9 and MMP-9/TIMP-1 ratios increased more in the IM group than in the WM group (P <0. 01). Expression levels of TIMP-1 decreased more in CM and IM groups than in the WM group (P <0.01).

CONCLUSIONWYHZTLR containing serum could reduce expression levels of Col-I and Col-III possibly through regulating key signal molecules, such as TGF-β1 RI, p-Smad2/3, and Smad7 in TGF-β1/Smad signaling pathway of SSc skin fibroblasts, and inhibiting transduction of TGF-β1/Smad signaling pathway.

Collagen Type I ; Collagen Type III ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Fibroblasts ; Humans ; Matrix Metalloproteinase 9 ; Scleroderma, Systemic ; drug therapy ; metabolism ; Signal Transduction ; drug effects ; Smad2 Protein ; Smad7 Protein ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo study the effect of Tangshenkang Granule (TG) containing serum on renal mesangial cells' (RMCs) proliferation and TGF-β1/Smad2/3 pathway in the high glucose condition.

METHODSTwelve SD rats were randomly divided into four groups, i.e., the low dose TG group, the middle dose TG group, the high dose TG group, and the blank control group, 3 in each group. After 7-day gastrogavage via portal vein blood, rats were sacrificed and their serum samples were collected. RMCs were cultured in common rat serum and TG containing serum respectively. The proliferation of mesangial cells was determined by methly thiazolyl tetrazolium (MTT) assay to determine the optimal TG containing serum concentration. Expression levels of TGF-β1 mRNA and protein were determined by real time quantitative PCR and ELISA. Smad2/3 protein expression and phosphorylation were determined by Western blot and immunofluorescence.

RESULTSTG containing serum at different doses could inhibit high glucose induced RMC cells' proliferation, TGF-β1 over-expression and Smad2/3 phosphorylation.

CONCLUSIONTG containing serum could inhibit high glucose induced RMC cells' proliferation, and its mechanism might be possibly associated with inhibiting TGF-β1/Smad2/3 signaling pathway.

Animals ; Cell Proliferation ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; Mesangial Cells ; Phosphorylation ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Serum ; Signal Transduction ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism