OBJECTIVE: To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway. METHODS: Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR. RESULTS: Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist. CONCLUSION Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.
Animals ; Cell Differentiation/drug effects* ; Endoribonucleases/metabolism* ; Estradiol/pharmacology* ; Estrogens/metabolism* ; Interleukin-10 ; Interleukin-6/metabolism* ; Macrophages, Peritoneal/metabolism* ; Mice ; Phenotype ; Protein Serine-Threonine Kinases/metabolism* ; RNA, Messenger/metabolism* ; Signal Transduction/drug effects* ; Transforming Growth Factor beta/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Up-Regulation/drug effects* ; X-Box Binding Protein 1/metabolism*

This study aims to investigate the efficacy of forsythiaside A(FTA) against CCl_4-induced liver fibrosis and the mechanism. Specifically, activities of serum alanine/aspartate aminotransferase(ALT/AST) and hydroxyproline(HYP) level in liver were detected, and pathological morphology of liver was observed based on hematoxylin-eosin(HE) staining, Masson's trichrome staining, and Sirius red staining of liver. On this basis, the effect of FTA on liver fibrosis was evaluated. The mRNA expression of actin alpha 2/α-smooth muscle actin(Acta2/α-SMA), transforming growth factor β(Tgfβ), collagen Ⅰ alpha 1(Col1 a1), and collagen Ⅲ alpha 1(Col3 a1) in liver tissue and hepatic stellate cells(HSC) was determined by qPCR, and the protein expression of α-SMA in liver tissue and HSC was measured by Western blot to assess the inhibition of FTA on HSC activation. The protein expression of α-SMA, vi-mentin(Vim), vascular endothelial cadherin(Ve-cadherin), and platelet endothelial cell adhesion molecule-1(PECAM-1/CD31) was measured by Western blot to evaluate the reverse of endothelial-mesenchymal transition(EMT) by FTA. The efficacy of FTA in relieving CCl_4-induced liver fibrosis was evidenced by the alleviation of hepatocyte necrosis, liver inflammation, and hepatic collagen deposition. FTA decreased the mRNA expression of Acta2, Tgfβ, Col1 a1, and Col3 a1 and protein expression of α-SMA both in vivo and in vitro. FTA reversed the increase of α-SMA and Vim and the decrease of CD31 and Ve-cadherin in livers from mice treated with CCl_4. Therefore, FTA alleviated CCl_4-induced liver fibrosis in mice via suppressing HSC activation and reversing EMT.
Animals ; Mice ; Actins/metabolism* ; Alanine Transaminase/blood* ; Carbon Tetrachloride/metabolism* ; Collagen/metabolism* ; Hepatic Stellate Cells ; Liver/drug effects* ; Liver Cirrhosis/genetics* ; RNA, Messenger/metabolism* ; Transforming Growth Factor beta/metabolism* ; Glycosides/therapeutic use*

OBJECTIVE: Behcet's disease (BD) is an autoimmune disorder that causes most commonly mouth and genital ulcerations and erythema nodules of the skin and currently has limited options of therapeutic medicines. Metformin is recently reported to suppress immune reaction, and we hypothesized that metformin could be an option for treatment of BD. METHODS: Thirty patients with BD were enrolled in this perspective single-blinded, before-after study. We recorded the changes in the mucocutaneous activity index for BD (MAIBD), relapse frequency, C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR) after metformin treatment to assess the changes in the disease activity. We also analyzed the changes in the protein and mRNA expression levels of Foxp3, interleukin-35 (IL-35), transforming growth factor-β (TGF-β), Ror-γt, IL-17, and tumor necrosis factor- (TNF-) in these patients using ELISA and qRT-PCR. RESULTS: Of the 30 patients enrolled, 26 completed the trial. After the treatment, favorable responses were achieved in 88.46% (23/26) of the patients, and partial remission was obtained in 11.54% (4/26) of them. During the treatment, 8 patients complained of gastrointestinal side effects, for which 4 chose to withdraw from the study in the first week. Our results showed that metformin treatment decreased MAIBD and relapse frequency in the patients, and significantly lowered the clinical inflammatory indexes including CRP and ESR. The results of ELISA and qRT-PCR revealed that metformin treatment obviously increased Foxp3 and TGF-β expressions at both the protein and mRNA levels and significantly decreased the levels of ROR-γt, IL-17 and TNF- as well as IL-35 level in these patients. CONCLUSIONS Metformin treatment relieves the clinical symptoms, reduces the inflammatory reaction indexes and regulates the Treg/Th17 axis in patients with BD, suggesting the potential of metformin as a candidate medicine for treatment of BD.
Behcet Syndrome ; drug therapy ; metabolism ; Controlled Before-After Studies ; Forkhead Transcription Factors ; metabolism ; Humans ; Immunosuppressive Agents ; adverse effects ; therapeutic use ; Interleukin-17 ; metabolism ; Interleukins ; metabolism ; Metformin ; adverse effects ; therapeutic use ; Neoplasm Recurrence, Local ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; RNA, Messenger ; metabolism ; Recurrence ; Single-Blind Method ; T-Lymphocytes, Regulatory ; cytology ; Th17 Cells ; cytology ; Transforming Growth Factor beta ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE: To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis (EAE) in mice and explore the underlying mechanism. METHODS: An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low-dose (0.3 mg/kg) or high-dose (1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system (CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p- smad3 were investigated using Western blotting. RESULTS: High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group (43±1.83 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high- dose rapamycin group than in the model group (10.17 ± 0.68 3.52 ± 0.32). In the experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells (13.66±1.89) compared with the treatment with rapamycin (6.23±0.80) or TGF-β (4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes. CONCLUSIONS Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.
Animals ; Anti-Inflammatory Agents ; administration & dosage ; therapeutic use ; Cell Differentiation ; drug effects ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; metabolism ; Interferon-gamma ; metabolism ; Interleukins ; metabolism ; Lymphocytes ; cytology ; Mice ; Mice, Inbred C57BL ; Sirolimus ; administration & dosage ; therapeutic use ; Smad Proteins ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Transforming Growth Factor beta ; metabolism ; Up-Regulation

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor &beta;1 (TGF-&beta;1), hepatocyte growth factor (HGF), &beta;-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-&beta;1 and up-regulated HGF and &beta;-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-&beta;1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

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

Transforming growth factor (TGF)-β regulates a wide variety of cellular responses, including cell growth arrest, apoptosis, cell differentiation, motility, invasion, extracellular matrix production, tissue fibrosis, angiogenesis, and immune function. Although tumor-suppressive roles of TGF-β have been extensively studied and well-characterized in many cancers, especially at early stages, accumulating evidence has revealed the critical roles of TGF-β as a pro-tumorigenic factor in various types of cancer. This review will focus on recent findings regarding epithelial-mesenchymal transition (EMT) induced by TGF-β, in relation to crosstalk with some other signaling pathways, and the roles of TGF-β in lung and pancreatic cancers, in which TGF-β has been shown to be involved in cancer progression. Recent findings also strongly suggested that targeting TGF-β signaling using specific inhibitors may be useful for the treatment of some cancers. TGF-β plays a pivotal role in the differentiation and function of regulatory T cells (Tregs). TGF-β is produced as latent high molecular weight complexes, and the latent TGF-β complex expressed on the surface of Tregs contains glycoprotein A repetitions predominant (GARP, also known as leucine-rich repeat containing 32 or LRRC32). Inhibition of the TGF-β activities through regulation of the latent TGF-β complex activation will be discussed.
Drug Discovery ; Humans ; Lung Neoplasms ; drug therapy ; immunology ; metabolism ; Membrane Proteins ; metabolism ; Pancreatic Neoplasms ; drug therapy ; immunology ; metabolism ; Signal Transduction ; drug effects ; physiology ; T-Lymphocytes, Regulatory ; metabolism ; Transforming Growth Factor beta ; antagonists & inhibitors ; immunology ; metabolism

The traditional Chinese medicine Shensong Yangxin (SSYX) can improve the clinical symptoms of arrhythmia in an integrated manner. This study aimed to investigate the electrophysiological effect of SSYX on the hearts of myocardial-infarcted rabbits and further explore the mechanism by which SSYX alleviates myocardial fibrosis. Myocardial infarction (MI) was established in rabbits by ligation of the left circumflex coronary. The rabbits were treated with SSYX (0.5 g/kg/d) or saline for 8 weeks by oral administration. Microelectrode array (MEA) technology was used in vivo for extracellular electrophysiological recordings of the infarct border zone. Masson's trichrome staining was used to observe myocardial fibrosis. Western blotting was performed to evaluate the protein expression levels of collagen I (COL I) and collagen III (COL III). Quantitative real-time polymerase chain reaction (real-time PCR) was performed to evaluate the TGF-&beta;1 and MMP-2 mRNA expression levels. The results showed that the total activation time (TAT) and the dispersion of TAT were significantly increased and the excitation propagation markedly disordered after MI. SSYX could significantly decrease TAT and the dispersion of TAT, and significantly ameliorate the chaotic spread pattern of excitation. Furthermore, SSYX treatment could significantly decrease COL I and COL III protein levels and down-regulate TGF-&beta;1 and MMP-2 mRNA expression levels in MI rabbits. It was concluded that SSYX may ameliorate cardiac electrophysiological abnormalities in infarcted hearts by decreasing the protein levels of COL I and COL III, down-regulating the mRNA expression levels of TGF-&beta;1 and MMP2, and thereby reducing adverse cardiac remodeling.
Animals ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Female ; Heart Rate ; drug effects ; Hyperplasia ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Myocardial Infarction ; drug therapy ; pathology ; Myocardium ; metabolism ; pathology ; Rabbits ; Transforming Growth Factor beta ; genetics ; metabolism

Renal tubulointerstitial fibrosis is the common ending of progressive renal disease. It is worth developing new ways to stop the progress of renal fibrosis. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have been studied to treat diabetic nephropathy, cisplatin-induced acute renal injury, ischemia reperfusion injury and adriamycin nephropathy. In this study, unilateral ureteral obstruction (UUO) was used to establish a different renal fibrosis model. PPAR? agonist pioglitazone was administrated by oral gavage and saline was used as control. At 7th and 14th day after the operation, mice were sacrificed for fibrosis test and T lymphocytes subsets test. Unexpectedly, through MASSON staining, immunohistochemistry for α-SMA, and Western blotting for a-SMA and PDGFR-&beta;, we found that pioglitazone failed to attenuate renal fibrosis in UUO mice. However, flow cytometry showed that pioglitazone down-regulated Th1 cells, and up-regulated Th2 cells, Th17 cells and Treg cells. But the Th17/Treg ratio had no significant change by pioglitazone. Real-time PCR results showed that TGF-&beta; and MCP-1 had no significant changes, at the same time, CD4(+) T cells associated cytokines were partially regulated by pioglitazone pretreatment. Taken together, pioglitazone failed to suppress renal fibrosis progression caused by UUO.
Animals ; Chemokine CCL2 ; metabolism ; Fibrosis ; Kidney ; pathology ; Kidney Diseases ; drug therapy ; etiology ; Male ; Mice ; Mice, Inbred C57BL ; PPAR gamma ; agonists ; T-Lymphocyte Subsets ; drug effects ; Thiazolidinediones ; administration & dosage ; pharmacology ; therapeutic use ; Transforming Growth Factor beta ; metabolism ; Urethral Obstruction ; complications

OBJECTIVETo study the effect of ligustrazine nanoparticles nano spray (LNNS) on transforming growth factor &beta; (TGF-&beta;)/Smad signal protein of rat peritoneal mesothelial cells (RPMC) induced by tumor necrosis factor α (TNF-α), and the anti-adhesion mechanism of LNNS in the abdominal cavity.

METHODSThe primary culture and subculture of rat peritoneal mesothelial cells (RPMC) was processed by trypsin digestion method in vitro. The third generation was identifified for experiment and divided into 5 groups: a blank group: RPMC without treatment; a control group: RPMC stimulated with TNF-α; RPMC treated by a low-dosage LNNS group (2.5 mg/L); RPMC treated by a medium-dosage LNNS group (5 mg/L); and RPMC treated by a high-dosage LNNS group (10 mg/L). Reverse transcription-polymerase chain reaction was applied to test the expression of fifibronectin, collagen I (COL-I), TGF-&beta; mRNA, and Western blot method to test the Smad protein 7 expression of RPMC.

RESULTSCompared with the blank group, a signifificant elevation in fifibronectin (FN), COL-I and TGF-&beta; mRNA expression of RPMC were observed in the control group (P<0.05). Compared with the control group, LNNS suppressed the expressions of FN, COL-I and TGF-&beta; mRNA in a concentrationdependent manner (P<0.05). The expression of Smad7 protein of RPMC was down-regulated by TNF-α stimulation, and up-regulated with the increase of LNNS dose (P<0.05).

CONCLUSIONSTNF-α may induce changes in RPMC's viability, leading to peritoneal injury. LNNS could reverse the induction of fifibrosis related cytokine FN, COL-I and TGF-&beta;, up-regulating the expression of Smad7 by TNF-α in RPMC, thus attenuate peritoneal injury by repairing mesothelial cells.

Animals ; Collagen Type I ; genetics ; metabolism ; Epithelium ; drug effects ; metabolism ; Fibronectins ; metabolism ; Male ; Nanoparticles ; chemistry ; ultrastructure ; Particle Size ; Peritoneal Cavity ; cytology ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology