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ObjectiveTo observe the repair effect of Dahuanglingxian prescription （DHLX） on bile duct epithelial cells of rats. To explore whether its mechanism of action is to adjust the mutual binding of transforming growth factor -β （TGF-β） activated kinase 1（TAK1） and tumor necrosis factor receptor-associated factor 6 （TRAF6）， and regulate the activation of the nuclear transcription factor -κB （NF-κB）/mitogen-activated protein kinase （MAPK） signaling pathway. MethodThe 20 SD rats were randomly divided into normal group and DHLX group， 10 rats in each group， were given saline and DHLX （320 mg·kg-1·d-1） for 8 days， to prepare normal serum and DHLX serum. Biliary epithelial cells were extracted from normal SD rats and divided into 9 groups： Normal group， model group （20 mg·L-1）， LPS+DHLX group （20 mg·L-1+10% DHLX）， LPS+PDTC group （20 mg·L-1+200 μmol·L-1）， LPS+SB203580 group （20 mg·L-1+0.5 μmol·L-1）， LPS+PDTC+SB203580 group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1）， LPS+PDTC+DHLX group （20 mg·L-1+200 μmol·L-1+10% DHLX serum）， LPS+SB203580+DHLX group （20 mg·L-1+0.5 μmol·L-1+10% DHLX serum）， LPS+PDTC+SB203580 +DHLX group （20 mg·L-1+200 μmol·L-1+0.5 μmol·L-1+10% DHLX serum）. The microscopic observation of morphological changes in each group of cells after drug intervention. Enzyme-linked immunosorbent assay（ELISA） was used to detect the expression of （IL）-1β and IL-6 in each group of cells. Western blot detected the expression levels of TAK1 and TRAF6 proteins in each group of cells， Co-IP detected the interaction between TAK1 and TRAF6， and further observed the distribution and co-localization of TAK1 and TRAF6 using Laser confocal microscope. ResultAfter the action of LPS， the cell synapses are reduced， the cell body becomes significantly rounded and smaller， but the cell morphology of each group tends to be normal after medication. Compared with normal group， the expression levels of IL-1β and IL-6 in model group were significantly increased （P<0.05）， while the expression level of TAK1 was decreased while the expression level of TRAF6 was increased （P<0.05）. The content of TAK1-TRAF6 protein complex showed a decreasing trend， and the two proteins co-located in the cytoplasm. Compared with model group， the expression levels of IL-1β and IL-6 in LPS+DHLX group were significantly decreased （P<0.05）， the expression level of TAK1 was increased and the expression level of TRAF6 was decreased （P<0.05）， the content of TAK1-TRAF6 protein complex was significantly increased （P<0.01）， and the two proteins were significantly co-located in cytoplasm. Compared with LPS+DHLX group， the expression levels of IL-1β and IL-6 in other groups were significantly decreased （P<0.05，P<0.01）. TAK1-TRAF6 protein complex content in each group was significantly decreased after pathway blocker intervention （P<0.05）， while TAK1-TRAF6 protein complex content in each group was significantly increased after pathway blocker combined with DHLX intervention （P<0.05）. Co-localization of the TAK1-TRAF6 in cytoplasm was not obvious. ConclusionIn the LPS-induced inflammatory response of bile duct cells， the binding of TAK1 and TRAF6 showed a weakening trend， but DHLX could reverse the phenomenon， we think the mechanism of action may be related to promoting the mutual binding of TAK1 and TARF6 to inhibit the activation of the NF-κB/MAPK signaling pathway.
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Epiretinal membrane(ERM)is a retinal disease characterized by a fibrocell membranes that can develop on the inner surface of the retina. The existing clinical guidelines and literature have reached a consensus on the diagnosis and treatment of ERM, but the explanation of their mechanism is still controversial. Transforming growth factor-β(TGF-β)is a highly pleiotropic cytokine that plays an important role in wound healing, angiogenesis, immune regulation, cancer, inflammation and fibrosis diseases. Studies have increasingly shown that ERM is a kind of pathological changes in fibrosis that caused by the posterior vitreous detachment(PVD)and lead to the retinal inflammatory damage and epithelial to mesenchymal transition(EMT)of retinal pigment epithelial cells. A variety of cytokines regulate TGF-β-mediated EMT process by participating in the non-classical TGF-β-Snail pathway and the classical TGF-β-Smad pathway. At present, some drugs targeting cytokines related to the above pathway have entered the development stage, which is of great significance to provide new ideas for clinical treatment and prevention of ERM. This review reviews the progress of TGF-β related cytokines in ERM formation.
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Objective:To study the effect of Biejia Jianwan on expressions of signal molecules and target genes of transforming growth factor-β (TGF-β)/Smad pathway in diethylnitrosamine (DEN)-induced rat hepatocellular carcinoma, and explore the mechanisms of Biejia Jianwan suppressing the invasion of hepatocellular carcinoma. Method:The rats were divided into three group, namely normal group, model group and Biejia Jianwan group (2.2 g·kg-1·d-1). Rats in Biejia Jianwan group and model group received intraperitoneal injections of DEN to induce sequential chronic inflammation, cirrhosis and hepatocellular carcinoma. At the sign of cirrhosis, rats in Biejia Jianwan group began taking Biejia Jianwan by gavage for 6 weeks. Rat blood was collected to measure serum levels of biochemical markers of liver function tests, including alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bilirubin(TBIL), albumin(Alb), γ-glutamyl transpeptadase(GGT), alkaline phosphatase(ALP). Rat livers were fixed in formalin and stained with hematoxylin-eosin (HE)staining， quantitative real-time PCR was used to test the mRNA expressions of TGF-β1， and Western blot was used to test protein expressions of TGF-β1, Smad2/3, p-Smad2/3, N-cadherin, E-cadherin and Vimentin. Result:All of the levels of biochemical markers showed no difference in Biejia Jianwan group and model group. Biejia Jianwan could improve the pathological changes of balloon-like degeneration, edema, and necrosis in liver cancer tissues. Importantly, the treatment dramatically decreased the mRNA expression of TGF-β1(P<0.01), and the protein expressions of TGF-β1, p-Smad2(P<0.01). Besides, the protein expression of N-cadherin and Vimentin were decreased significantly (P<0.01). Conclusion:Biejia Jianwan can inhibit epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells activated via TGF-β/Smad pathway by reducing TGF-β1 expression, so as to suppress the metastasis and invasion of hepatocellular carcinoma.
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Objective • To explore the effect of inhibiting transforming growth factor β (TGF-β) signaling pathway on the stem cell-mediated liver regeneration in mice after partial hepatectomy (PH). Methods • Eighteen C57BL/6 male mice were selected to establish the model of hepatectomy. mRNA and protein levels of the signal molecules in TGF-β pathway, as well as stem cell markers α-fetoprotein (AFP) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) in the liver tissues were detected before (PH0) and on the 1st (PH1), 3rd (PH3) and 7th (PH7) day after operation. Then, additional 32 mice were assigned into inhibitor group [PH+SB-431542, 10 mg/(kg•d)] and control group (PH+normal saline). The mice were sacrificed on the 1st (PH1) and 3rd (PH3) day after operation and liver remnants were obtained. Realtime-PCR, Western blotting, and immunofluorescence staining were used to detect the expressing variation of TGF-β signaling pathway, AFP and LGR5. Results • The mRNA and protein expression of TGF-β1, phosphorylated SMAD2 (p-SMAD2) protein, Afp mRNA, the number of AFP immune-positive cells and Lgr5 mRNA were upregulated significantly after PH, peaked at PH3 (P<0.05) and recovered to pre-operative level at PH7. Compared with control group, the above indices were all obviously inhibited in inhibitor group at PH3 (P<0.05). Conclusion • TGF-β signaling pathway may regulate the stem cell-mediated liver regeneration in mice after PH.
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Objective: To explore the therapeutic effect of licochalcone A (Lico A) on pulmonary fibrosis (PF). Method: Thirty mice were divided into five group, namely sham, model, Lico A (15, 30 mg·kg-1) and pirfenidone (300 mg·kg-1) groups. All of the groups except for sham group were intratracheally given bleomycin (BLM, 5 mg·kg-1). The sham group was given normal saline. On day 2, the mice were treated with Lico A and pirfenidone, respectively. On day 28, all of the mice were put to death. Then, lung tissues were collected and weighted. Pathological changes in lung tissue were measured by htoxylin eosin(HE) and Masson staining. The α-smooth muscle actin(α-SMA), Collagen I, fibronectin p-Smad2/3 and Smad2/3 were analyzed by Western blot. Then, transforming growth factor-β1 (TGF-β1)-induced MRC-5 cells were employed for evaluating the inhibitory activity of Lico A in vitro. Result: Compared with normal group, several pathological changes, including alveolar space collapse, emphysema, infiltration of inflammatory cells, and collagen deposition were observed in the BLM-treated mice, and these pathological changes were markedly attenuated by subsequent treatment with Lico A. Lico A could significantly inhibit BLM-induced up-regulation of α-SMA and Collagen I and phosphorylation of Smad2/3 in lung tissues of mice(PPβ-induced α-SMA and fibronectin expression in MRC-5 cells(PPConclusion: The preliminary mechanisms of the anti-fibrosis effect of Lico A may inhibit TGF-β/Smad pathway.
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Asthma is a chronic disease of airway inflammation due to excessive T helper cell type 2 (Th2) response. Present treatment based on inhalation of synthetic glucocorticoids can only control Th2-driven chronic eosinophilic inflammation, but cannot change the immune tolerance of the body to external allergens. Regulatory T cells (Tregs) are the main negative regulatory cells of the immune response. Tregs play a great role in regulating allergic, autoimmune, graft-versus-host responses, and other immune responses. In this review, we will discuss the classification and biological characteristics, the established immunomodulatory mechanisms, and the characteristics of induced differentiation of Tregs. We will also discuss the progress of Tregs in the field of asthma. We believe that further studies on the regulatory mechanisms of Tregs will provide better treatments and control strategies for asthma.
Subject(s)
Humans , Antigens, CD/analysis , Apyrase/analysis , Asthma/immunology , Cell Differentiation , Cytokines/metabolism , Lymphocyte Transfusion , T-Lymphocytes, Regulatory/immunologyABSTRACT
Smad3 is a major transporter in the transforming growth factor β (TGF-β) signaling pathway.It is in charge of the transfer of TGF-β signal from the surface of the cell membrane into the nucleus.The TGF-β signal can be bound to the target gene in the nucleus and regulate its expression.Abnormalities in Smad3 expression level and functional status will lead to abnormal signal transduction,involving cell growth,proliferation,development,differentiation,migration,apoptosis and other basic life activities.This review focused on the differential expression of Smad3 in hepatocellular carcinoma (HCC)and the adjacent tissue.The character of Smad3 in HCC is outlined in three parts:Smad3 upstream signaling source,Smad3 self-assembly maturation and Smad3 downstream effects,which may provide a summary and reference for the follow-up study on Smad3.
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Objective To investigate the effect of sorafenib in ameliorating renal fibrosis and its possible mechanisms.Methods Rats were subjected to unilateral ureteral obstruction (UUO ) and intragastrically administered sorafenib.NRK-52E cells were treated with transforming growth factor-β1 (TGF-β1)and sorafenib. HE staining was used to visualize renal fibrosis.α-SMA and E-cadherin expressions in kidney tissue and NRK-52E cells were performed using immunofluorescence.The cell cycle of NRK-52E cells was determined by flow cytometry analysis.Smad3 and p-Smad3 protein expressions in NRK-52E cells were detected by Western blot analysis. Results HE staining showed that kidney interstitial fibrosis,tubular atrophy,and inflammatory cell infiltration in the sorafenib-treated UUO groups were significantly decreased compared with the vehicle-treated UUO group (P<0.05).Compared with those in UUO and TGF-β-stimulated NRK-52E groups,the expression of a-SMA decreased but E-cadherin expression increased in the UUO kidneys and NRK-52E cells of the sorafenib-treated groups (P<0.05).After 24 h stimulation with TGF-β1 5 ng/mL,the number of cell cycles arrested in G0/G1 phase was significantly increased and the number of cells that entered G2 ,S phase decreased (P<0 .0 5 ).Compared with that in TGF-β-stimulated NRK-52E groups, p-Smad3 decreased in the sorafenib-treated groups (P<0.05). Conclusion Our results suggest that sorafenib may be useful for the treatment of renal fibrosis through suppressing TGF-β/Smad3 signaling.
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The formation, maintenance, and regeneration of bone is a complex precess involving the interactions of many cellular elements with systemic and local regulators. TGF-β is one of growth factors that play an important role in the formation and remodeling of bone. In vitro studies have suggested that TGF-β regulates chondrogenesis and possibly osteogenesis by affecting replication, gene expression, and structural protein synthesis in bone formation. We investigated the effect of TGF-β1 upon fracture callus formation and maturation in mature rate. Closed femoral shaft fracture was made consistently by three point stress technique after percutaneous intramedullary nailing. TGF-β1 was injected subperiosteally at the fracture site daily for 2 weeks. We examined the effect of TGF-β1 on the fracture healing process with the radiographic, densitometric, histologic, and immunohistochemical methods. The following results were obtained. 1. Radiographic examination demonstrated that TGF-β1 injection group appeared to have more abundant callus formation and earlier callus maturation as compared to the control group. 2. Bone densitometric examination revealed that TGF-β1 injection group had higher bone mineral density and content that the control group. 3. Thermographic examination revealed that TGF-β1 injection group had higher local temperature at the injection area than the control group. 4. Histologic examination suggested that TGF-β1 stimulates and accelerates fracture callus formation and endochondral bone formation. 5. Immunohistochemical examination revealed that chondrocytes at the fracture site in the TGF-β1 injection group seemed to produce type I collagen.