In order to investigate whether the effect of Yuxuebi Tablets on the peripheral and central inflammation resolution of mice with inflammatory pain is related to their regulation of G protein-coupled receptor 37(GPR37), an inflammatory pain model was established by injecting complete Freund's adjuvant(CFA) into the paws of mice, with a sham-operated group receiving a similar volume of normal saline. The mice were assigned randomly to the sham-operated group, model group, ibuprofen group(91 mg·kg~(-1)), and low-, medium-, and high-dose groups of Yuxuebi Tablets(60, 120, and 240 mg·kg~(-1)). The drug was administered orally from days 1 to 19 after modeling. Von Frey method and the hot plate test were used to detect mechanical pain thresholds and heat hyperalgesia. The levels of interleukin-10(IL-10) and transforming growth factor-beta(TGF-β) in the spinal cord were quantified using enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein expression of GPR37 in the spinal cord was measured by real-time quantitative reverse transcription PCR(qRT-PCR) and Western blot. Additionally, immunofluorescence was used to detect the expression of macrosialin antigen(CD68), mannose receptor(MRC1 or CD206), and GPR37 in dorsal root ganglia, as well as the expression of calcium-binding adapter molecule 1(IBA1), CD206, and GPR37 in the dorsal horn of the spinal cord. The results showed that compared with those of the sham-operated group, the mechanical pain thresholds and hot withdrawal latency of the model group significantly declined, and the expression of CD68 in the dorsal root ganglia and the expression of IBA1 in the dorsal horn of the spinal cord significantly increased. The expression of CD206 and GPR37 significantly decreased in the dorsal root ganglion and dorsal horn of the spinal cord, and IL-10 and TGF-β levels in the spinal cord were significantly decreased. Compared with those of the model group, the mechanical pain thresholds and hot withdrawal latency of the high-dose group of Yuxuebi Tablets significantly increased, and the expression of CD68 in the dorsal root ganglion and IBA1 in the dorsal horn of the spinal cord significantly decreased. The expression of CD206 and GPR37 in the dorsal root ganglion and dorsal horn of the spinal cord significantly increased, as well as IL-10 and TGF-β levels in the spinal cord. These findings indicated that Yuxuebi Tablets may reduce macrophage(microglial) infiltration and foster M2 macrophage polarization by enhancing GPR37 expression in the dorsal root ganglia and dorsal horn of the spinal cord of CFA-induced mice, so as to improve IL-10 and TGF-β levels, promote resolution of both peripheral and central inflammation, and play analgesic effects.
Inflammation/genetics* ; Pain/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Mice ; Freund's Adjuvant/pharmacology* ; Ibuprofen ; Pain Threshold/drug effects* ; Hyperalgesia/genetics* ; Ganglia, Spinal ; Interleukin-10/genetics* ; Transforming Growth Factor beta/genetics* ; Reverse Transcriptase Polymerase Chain Reaction ; Tablets ; Receptors, G-Protein-Coupled

The present study was designed to elucidate whether the mechanism by which osthole decreases collagenI/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL of osthole for 24 h, the mRNA and protein expression levels of collagensIand III were reduced. The collagen I/III ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRI, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen I and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.
Actins ; genetics ; Animals ; Cells, Cultured ; Collagen ; biosynthesis ; genetics ; Coumarins ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Mice ; Myocardium ; cytology ; Protein-Serine-Threonine Kinases ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Receptor, Transforming Growth Factor-beta Type I ; Receptors, Transforming Growth Factor beta ; genetics ; Signal Transduction ; drug effects ; Smad Proteins ; genetics ; Transforming Growth Factor beta1 ; genetics

Immunosuppressive regulatory T lymphocytes (Treg) expressing the transcription factor Foxp3 play a vital role in the maintenance of tolerance of the immune-system to self and innocuous non-self. Most Treg that are critical for the maintenance of tolerance to self, develop as an independent T-cell lineage from common T cell precursors in the thymus. In this organ, their differentiation requires signals from the T cell receptor for antigen, from co-stimulatory molecules, as well as from cytokine-receptors. Here we focus on the cytokines implicated in thymic development of Treg, with a particular emphasis on the roles of interleukin-2 (IL-2) and IL-15. The more recently appreciated involvement of TGF-β in thymic Treg development is also briefly discussed. Finally, we discuss how cytokine-dependence of Treg development allows for temporal, quantitative, and potentially qualitative modulation of this process.
Animals ; Cell Differentiation ; genetics ; Cytokines ; immunology ; Forkhead Transcription Factors ; genetics ; immunology ; Gene Expression Regulation ; Immune Tolerance ; genetics ; Interleukin-15 ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Mice ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta ; genetics ; immunology

Human telomerase reverse transcriptase (hTERT) plays a central role in telomere lengthening for continuous cell proliferation, but it remains unclear how extracellular cues regulate telomerase lengthening of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces the hTERT gene repression in a BMPRII receptor- and Smad3-dependent manner in human breast cancer cells. Chonic exposure of human breast cancer cells to BMP7 results in short telomeres, cell senescence and apoptosis. Mutation of the BMPRII receptor, but not TGFbRII, ACTRIIA or ACTRIIB receptor, inhibits BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres and continued cell proliferation. Expression of hTERT prevents BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging by a mechanism involving BMPRII receptor- and Smad3-mediated repression of the hTERT gene.
Actin-Related Protein 2 ; genetics ; metabolism ; Activin Receptors, Type II ; genetics ; metabolism ; Bone Morphogenetic Protein 7 ; genetics ; metabolism ; Bone Morphogenetic Protein Receptors, Type II ; genetics ; metabolism ; Breast Neoplasms ; genetics ; metabolism ; Cellular Senescence ; Female ; HeLa Cells ; Humans ; MCF-7 Cells ; Neoplasm Proteins ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Receptor, Transforming Growth Factor-beta Type II ; Receptors, Transforming Growth Factor beta ; genetics ; metabolism ; Smad3 Protein ; genetics ; metabolism ; Telomerase ; genetics ; metabolism ; Telomere Homeostasis

PURPOSE: This study aimed to investigate whether Mullerian inhibiting substance (MIS) in combination with calcitriol modulates proliferation and apoptosis of human ovarian cancer (OCa) cell lines (SKOV3, OVCAR3, and OVCA433) and identify the signaling pathway by which MIS mediates apoptosis. MATERIALS AND METHODS: OCa cell lines were treated with MIS in the absence or presence of calcitriol. Cell viability and proliferation were evaluated using the Cell Counting Kit-8 assay and apoptosis was evaluated by DNA fragmentation assay. Western blot and enzyme-linked immunosorbent assay were used to determine the signaling pathway. RESULTS: The cells showed specific staining for the MIS type II receptor. Treatment of OCa cells with MIS and calcitriol led to dose- and time-dependent inhibition of cell growth and survival. The combination treatment significantly suppressed cell growth, down-regulated the expression of B-cell lymphoma 2 (Bcl-2), and up-regulated the expressions of Bcl-2 associated X protein, caspase-3, and caspase-9 through the extracellular signal-regulated kinase signaling pathway. CONCLUSION: These results, coupled with a much-needed decrease in the toxic side effects of currently employed therapeutic agents, provide a strong rationale for testing the therapeutic potential of MIS, alone or in combination with calcitriol, in the treatment of OCa.
Anti-Mullerian Hormone/*pharmacology ; Apoptosis/*drug effects ; Calcitriol/*pharmacology ; Caspase 3/metabolism ; Caspase 9/metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Cell Survival/drug effects ; DNA Fragmentation/*drug effects ; Enzyme-Linked Immunosorbent Assay ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Female ; Growth Inhibitors/metabolism/pharmacology ; Humans ; Ovarian Neoplasms/*drug therapy/metabolism/*pathology ; Receptors, Peptide ; Receptors, Transforming Growth Factor beta ; Signal Transduction/*drug effects

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine involved in immune disorders, cancer, asthma, lung fibrosis, and chronic kidney disease, and its signal pathways are considered crucial mediators of a variety of cellular processes. In addition, several recent studies have reported that TGF-beta receptor (TGF-betaR) gene polymorphism is associated with chronic kidney disease. However, the association between end-stage renal disease (ESRD) and the TGF-beta gene polymorphism has not been sufficiently investigated. In this study, we hypothesized that polymorphisms of the TGF-beta ligands or their receptors may be related to ESRD. METHODS: We assessed the relationship between four single-nucleotide polymorphisms (SNPs) in the TGF-betaR2 and TGF-beta2 genes and ESRD, in 312 patients with ESRD and 258 controls. RESULTS: Compared with the control participants, the frequencies of the TGF-betaR2 (rs764522*C) and TGF-betaR2 (rs3087465*G) alleles were significantly higher in the patients with ESRD. Genotyping analysis demonstrated that two SNPs in TGF-betaR2 of the four SNPs included in the study were significantly associated with ESRD in the codominant 1 [rs764522, odds ratio (OR)=1.65; rs3087465, OR=1.63], dominant (rs764522, OR=1.63; rs3087465, OR=1.57), and log-additive (rs764522, OR=1.54; rs3087465, OR=1.39) models after adjusting for age and sex. CONCLUSION: We suggest that TGF-betaR2 polymorphisms (rs764522 and rs3087465) increase the risk of development of ESRD.
Alleles ; Asthma ; Fibrosis ; Humans ; Immune System Diseases ; Kidney Failure, Chronic* ; Ligands ; Lung ; Odds Ratio ; Polymorphism, Single Nucleotide ; Receptors, Transforming Growth Factor beta ; Renal Insufficiency, Chronic ; Signal Transduction ; Transforming Growth Factor beta ; Transforming Growth Factor beta2

OBJECTIVETo investigate the effect of nuclear factor I-C (NFI-C) on platelet-derived growth factor (PDGF)-induced up-regulation of TGF-β receptor II (TβRII) in dermal fibroblasts.

METHODSA lentiviral vector containing NFI-C sequence (Lenti-GFP-NFI-C) was transfected into a human foreskin fibroblast cell line (HFF-1). Cultured HFF-1 cells, cells transfected with Lenti-GFP-NFI-C, and cells transfected with a negative virus were stimulated with PDGF-BB, and Western blotting and RT-qPCR were used to detect the expression levels of TβRII in the treated cells.

RESULTSPDGF treatment significantly increased the expression level of T&beta;RII in HFF-1 cells (P<0.05). The cells transfected with Lenti-GFP-NFI-C expressed a significantly lower level of T&beta;RII than non-transfected cells in response to PDGF stimulation (P<0.05), but the negative virus showed no such inhibitory effect (P>0.05). No significant difference was found in the expression level of T&beta;RII protein between cells transfected with Lenti-GFP-NFI-C-transfection before PDGF stimulation and the blank control cells.

CONCLUSIONNFI-C can inhibit PDGF-induced up-regulation of T&beta;RII and thus reduce the sensitivity of the dermal fibroblasts to TGF-&beta;.

Cell Line ; Fibroblasts ; drug effects ; Genetic Vectors ; Humans ; Lentivirus ; NFI Transcription Factors ; genetics ; Platelet-Derived Growth Factor ; pharmacology ; Protein-Serine-Threonine Kinases ; metabolism ; Proto-Oncogene Proteins c-sis ; Receptors, Transforming Growth Factor beta ; metabolism ; Transfection ; Transforming Growth Factor beta ; pharmacology ; Up-Regulation

OBJECTIVETo explore the effects of blocking two sites of TGF-&beta;/Smads signaling on the formation of scar-related proteins in human skin fibroblasts.

METHODSTwo lentivirus vectors encoding soluble TGF-&beta; receptor II (sT&beta;RII) and mutant Smad 4-Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sT&beta;RII and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×10(4) cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-&beta;1 for 72 h; sT&beta;RII group, transfected with lenti-sT&beta;RII with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-&beta;1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-(q test).

RESULTS(1) HFF-1 cells transfected with lenti-sT&beta;RII and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sT&beta;RII protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sT&beta;RII group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4ΔM4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sT&beta;RII group and Smad 4ΔM4 group (with q values from 3.424 to 7.143, P values below 0.05).

CONCLUSIONSIn human skin fibroblasts, blockage of two sites of TGF-&beta;/Smad signaling can reduce the expression of scar related proteins which are up-regulated by TGF-&beta;1 to a greater extent than that of blocking one single site.

Cicatrix ; Connective Tissue Growth Factor ; Fibroblasts ; metabolism ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Protein-Serine-Threonine Kinases ; RNA, Messenger ; genetics ; Receptors, Transforming Growth Factor beta ; Signal Transduction ; drug effects ; Smad Proteins ; genetics ; metabolism ; Smad Proteins, Inhibitory ; genetics ; Transfection ; Transforming Growth Factor beta ; pharmacology ; Transforming Growth Factors

Cell Hypoxia ; Epithelial-Mesenchymal Transition ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Neoplasms ; metabolism ; pathology ; Receptors, Notch ; metabolism ; Signal Transduction ; Snail Family Transcription Factors ; Transcription Factors ; metabolism ; Transforming Growth Factor beta ; metabolism ; Twist-Related Protein 1 ; metabolism ; Wnt Proteins ; metabolism

OBJECTIVETo investigate the distribution and expression of transforming growth factor beta (TGF-&beta;) receptors I and II, p38 mitogen-activated protein kinase (p38 MAPK), and type I and type III collagen in the lungs of rats with silicosis and cultured pulmonary fibroblasts, and to investigate the relationship of the anti-fibrosis effect of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) with its inhibition of TGF-&beta; receptor-mediated p38 MAPK pathway activity.

METHODSRats were randomly divided into control group, silicosis model group, and AcSDKP treatment group (n = 10 for each group). For the model group and AcSDKP treatment group, rats were intratracheally instilled with silica to establish a silicosis model. Cultured pulmonary fibroblasts from neonatal rats were divided into control group, TGF-&beta;1 stimulation group, TGF-&beta; receptor inhibition group, p38 MAPK pathway inhibition group, and AcSDKP treatment group. The protein expression of TGF-&beta; receptors I and II, p38 MAPK, and type I and type III collagen were determined by immunohistochemistry and Western blot. The mRNA expression of TGF-&beta; receptors I and II were determined by real-time PCR. The distribution and nuclear translocation of phospho-p38 MAPK in cultured fibroblasts were determined by laser scanner confocal microscopy.

RESULTSIn the AcSDKP treatment group, AcSDKP reduced the expression of TGF-&beta; receptors I and II, phospho-p38 MAPK, and type I and type III collagen to 86.12%, 41.01%, 42.63%, 89.05%, and 52.71%, respectively, of those of the silicosis model group (P < 0.05). In cultured fibroblasts, AcSDKP reduced the mRNA expression of TGF-&beta; receptors I and II to 42.26% and 54.33%, respectively, of those of the TGF-&beta;1 stimulation group; the protein expression of TGF-&beta; receptors I and II, phospho-p38 MAPK, and type 1 and type III collagen was reduced to 58.14%, 51.40%, 45.6%, 58.04%, and 44.74%, respectively, of those of the TGF-&beta;1 stimulation group. The phospho-p38 MAPK translocation from plasma to the nucleus was also inhibited; the nucleus/plasma ratio of p38 MAPK and the protein expression of type I and type III collagen were reduced to 68.60%, 58.04%, and 44.74%, respectively, of those of the TGF-&beta; stimulation group (P < 0.05).

CONCLUSIONAcSDKP can inhibit the expression of collagen through inhibition of TGF-&beta; receptor-mediated p38 MAPK pathway activity, and is thus able to exert anti-fibrosis effect in rats with silicosis.

Animals ; Cells, Cultured ; Collagen ; metabolism ; Disease Models, Animal ; Fibroblasts ; drug effects ; metabolism ; MAP Kinase Signaling System ; drug effects ; Male ; Oligopeptides ; pharmacology ; Protein-Serine-Threonine Kinases ; metabolism ; Rats ; Rats, Wistar ; Receptors, Transforming Growth Factor beta ; metabolism ; Silicosis ; metabolism ; Transforming Growth Factor beta ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism