Downregulation Of TGF-β1 Research Articles

Meglumine Antimoniate and Miltefosine Combined With Allopurinol Sustain Pro-inflammatory Immune Environments During Canine Leishmaniosis Treatment.

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic disease of global concern. Antileishmanial drug therapies commonly used to treat sick dogs improve their clinical condition, although when discontinued relapses can occur. Thus, the current study aims to evaluate the effect of CanL treatments in peripheral blood, lymph node, and bone marrow cytokine profile associated with clinical recovery. Two groups of six dogs diagnosed with CanL were treated with miltefosine combined with allopurinol and meglumine antimoniate combined with allopurinol (MT+A and MG+A), respectively. At diagnosis and after treatment, during a 3-month follow-up, clinical signs, hematological and biochemical parameters, urinalysis results and antileishmanial antibody titers were registered. Furthermore, peripheral blood, popliteal lymph node, and bone marrow samples were collected to assess the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β, and IFN-γ by qPCR. In parallel, were also evaluated samples obtained from five healthy dogs. Both treatment protocols promoted the remission of clinical signs as well as normalization of hematological and biochemical parameters and urinalysis values. Antileishmanial antibodies returned to non-significant titers in all dogs. Sick dogs showed a generalized upregulation of IFN-γ and downregulation of IL-2, IL-4, and TGF-β, while gene expression of IL-12, TNF-α, IL-5, and IL-10 varied between groups and according to evaluated tissue. A trend to the normalization of cytokine gene expression was induced by both miltefosine and meglumine antimoniate combined therapies. However, IFN-γ gene expression was still up-regulated in the three evaluated tissues. Furthermore, the effect of treatment in the gene expression of cytokines that were not significantly changed by infection, indicates that miltefosine and meglumine antimoniate combined therapy directly affects cytokine generation. Both combined therapies are effective in CanL treatment, leading to sustained pro-inflammatory immune environments that can compromise parasite survival and favor dogs' clinical cure. In the current study, anti-inflammatory and regulatory cytokines do not seem to play a prominent role in CanL or during clinical recovery.

P1.01-29 MLPH Promotes Epithelial–Mesenchymal Transition and Brain Metastasis via Cdc42/PAK1 Signaling in Non-Small Cell Lung Cancer

Brain metastasis (BM) is associated with poor prognosis, recurrence, and death in patients with non-small cell lung cancer (NSCLC). Therefore, a better understanding of molecular mechanisms underlying NSCLC development and progression could provide helpful insights for NSCLC prevention and effective treatment. Melanophilin (MLPH), an actin-based transport binding partner, involves in cancer progression. However, the role of MLPH in NSCLC remains unclear. Here we elucidated the functional significance, molecular mechanisms and clinical impact of MLPH in NSCLC. RNA-Sequencing was performed to identify differentially expressed genes in lung tissues of NSCLC patients with (BM+) and without BM (BM-). The expression of MLPH was examined in the serum of BM+ and BM- patients by PCR. Integrative database analysis was used to examine MLPH levels in NSCLC tissues and analyze the relationship between MLPH levels and patient survival. Lentivirus containing small hairpin (sh) RNA targeting MLPH or empty vector was designed to explore its role in NSCLC. The cell counting kit-8 assay, wound healing assay, transwell assay, flow cytometry analysis, Phalloidin staining, xenografted tumor model and brain metastasis model were used to determine the effects of MLPH on the proliferation, migration, invasion, EMT, tumorigenesis and brain metastasis of NSCLC. Western blot analysis was used to explore the underlying mechanism. High-throughput sequencing showed that MLPH mRNA was significantly differentially expressed in lung tumors between BM+ and BM- NSCLC patients. MLPH was frequently overexpressed in NSCLC tissues and cells, and high levels of MLPH correlated with poor prognosis of NSCLC patients. Silencing MLPH by shRNA suppressed NSCLC cell proliferation, migration, invasion and TGF-β-induced EMT, and triggered cell cycle arrest and apoptosis. Being in consistent with the in vitro findings, the in vivo experiment exhibited that knockdown of MLPH inhibited xenograft tumorigenesis and brain metastasis in nude mice. Mechanically, we identified TGF-β as a key downstream effector of MLPH. MLPH silencing attenuated Cdc42/PAK1 signaling activation at least in part through the downregulation of TGF-β. Furthermore, EMT phenotypes changes caused by MLPH knockdown were partially dependent on TGF-β inhibition. Our findings uncovered the role of MLPH in NSCLC progression and provided evidence for MLPH positively modulating the Cdc42/PAK1 signaling pathway to promote EMT and metastasis via TGF-β in NSCLC cells. MLPH may have the potential as a therapeutic target against metastatic NSCLC.

All-Trans Retinoic Acid Attenuates Fibrotic Processes by Downregulating TGF-β1/Smad3 in Early Diabetic Nephropathy.

Diabetic nephropathy (DN) involves damage associated to hyperglycemia and oxidative stress. Renal fibrosis is a major pathologic feature of DN. The aim of this study was to evaluate anti-fibrogenic and renoprotective effects of all-trans retinoic acid (ATRA) in isolated glomeruli and proximal tubules of diabetic rats. Diabetes was induced by single injection of streptozotocin (STZ, 60 mg/Kg). ATRA (1 mg/Kg) was administered daily by gavage, from days 3–21 after STZ injection. ATRA attenuated kidney injury through the reduction of proteinuria, renal hypertrophy, increase in natriuresis, as well as early markers of damage such as β2-microglobulin, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL). The following parameters increased: macrophage infiltration, localization of alpha-smooth muscle actin (αSMA)-positive cells in renal tissue, and pro-fibrotic proteins such as transforming growth factor-β (TGF-β1), laminin beta 1 (LAM-β1), and collagens IV and I. Remarkably, ATRA treatment ameliorated these alterations and attenuated expression and nuclear translocation of Smad3, with increment of glomerular and tubular Smad7. The diabetic condition decreased expression of retinoic acid receptor alpha (RAR-α) through phosphorylation in serine residues mediated by the activation of c-Jun N-terminal kinase (JNK). ATRA administration restored the expression of RAR-α and inhibited direct interactions of JNK/RAR-α. ATRA prevented fibrogenesis through down-regulation of TGF-β1/Smad3 signaling.

Exogenous IL-19 mediates downregulation of TGF-β through Erk and p38 pathway to inhibit epidural fibrosis.

To evaluate the effect of interleukin-19 (IL-19) treatment on epidural fibrosis and its mechanism of action with transforming growth factor β (TGF-β). Initially, IL-19 (10, 20, 50 and 100 ng/L) was used to pretreat rat fibroblasts. TGF-β (10 μg/L) was then applied to activate fibroblasts. The protein expression levels of TGF-β receptor, extracellular-signal-regulated kinase (Erk) and p-38 were measured by Western blotting. In addition, we performed laminectomy at T10 vertebral plate in rats, followed by injection of IL-19 in caudal vein one week after injury. Furthermore, IL-19, TGF-β and fibrosis indexes were measured by quantitative Real-time polymerase chain reaction (qRT-PCR) and Western blotting at 7 and 28 days after injury, respectively. Concentration-dependent IL-19 significantly down-regulated TGF-β receptor expression and inhibited phosphorylated Erk (p-Erk) and phosphorylated p38 (p-p38). In vivo, IL-19 reduced the expressions of TGF-β and connective tissue growth factor (CTGF) at 7 days. Furthermore, IL-19 significantly suppressed extracellular matrix productions formation, including α smooth muscle actin (α-SMA) and collagen-1 (COL-1), and fibronectin at 28 days. IL-19 inhibited TGF-β expression via Erk and p38 pathway. Moreover, it decreased CTGF expression to suppress α-SMA, COL-1 and fibronectin in scar tissues, thereby preventing spinal cord from compression of scar tissues.

The impact of Lactobacillus acidophilus on hepatic and colonic fibrosis induced by ethephon in a rat model.

Objective(s):The study is aimed to elucidate the impact of antioxidant, anti-inflammatory and antifibrosis properties of Lactobacillus acidophilus (L. acidophilus) on liver and colon in ethephon treated rats through measuring Pro- inflammatory cytokines, oxidative stress index, lysosomal cathepsin-D enzyme activity and fibrosis markers.Materials and Methods:Rats divided into three groups; Group 1: distilled water control, Group 2: rats at day 16 from experiment beginning were orally received ethephon 50 mg/ kg BW in distilled water once daily for 60 days. Group 3: rats were orally received L. acidophilus enriched diet 1% (w/w) for 15 days as prophylactic, then received both L. acidophilus enriched diet 1% (w/w) and ethephon 50 mg/kg BW for 60 days. Results:Ethephon exerts hepatic and colonic oxidative stress, inflammatory response and fibrosis through NF-κB activation. On contrary, L. acidophilus supplementation evokes hepatoprotective properties as revealed by decreased serum AST, ALT, γ -GT and increased IGF-1. L. acidophilus exerts antioxidant and anti-inflammatory properties as indicated by decreased TOS, OSI, TNF-α, IL-1β, cathepsins D activity, NF-κB expression and increased TAC, lysosomal membrane stability. L. acidophilus shows antifibrotic activity as demonstrated by down-regulation of TGF-β1, α-SMA, collagen expression. Conclusion: L. acidophilus possess antioxidant, anti -inflammatory and antifibrotic activity through inhibition of NF-kB.

Pirfenidone suppresses the abnormal activation of human Müller cells after platelet-derived growth factor-BB stimulation.

To determine the effect of pirfenidone on the activated human Müller cells by platelet-derived growth factor-BB (PDGF-BB). The primary human Müller cells were separated from retinal tissues and established the pathogenic model by stimulated with PDGF-BB. The Müller cells behaviour of normal group and the model group was measured by MTT assay, Trypan blue assay, cell migration assay, and collagen contraction assay. The expression of transforming growth factor (TGF)-β1, -β2, and pigment epithelium-derived factor (PEDF) was estimated with real-time polymerase chain reaction (PCR), Western blot and immunofluorescence analyses. A pathogenic/proliferative model of Müller cells was established by stimulating normal cultured Müller cells with 10 ng/mL PDGF-BB for 48h. After treated with 0.2 and 0.3 mg/mL pirfenidone, the proliferation, migration and collagen contraction was statistically significantly depressed in the model group compared with the normal groups. The expression levels of TGF-β1 and TGF-β2 were significantly down-regulated, while the PEDF expression was significantly up-regulated after treated with 0.2 and 0.3 mg/mL pirfenidone in the model group. Pirfenidone effectively suppress the proliferation, migration and collagen contraction of the human Müller cells stimulated with PDGF-BB through down-regulation of TGF-β1/TGF-β2 and up-regulation of PEDF.

Transcriptome Analysis of Small Molecule-Mediated Astrocyte-to-Neuron Reprogramming.

Chemical reprogramming of astrocytes into neurons represents a promising approach to regenerate new neurons for brain repair, but the underlying mechanisms driving this trans-differentiation process are not well understood. We have recently identified four small molecules – CHIR99021, DAPT, LDN193189, and SB431542 – that can efficiently reprogram cultured human fetal astrocytes into functional neurons. Here we employ the next generation of RNA-sequencing technology to investigate the transcriptome changes during the astrocyte-to-neuron (AtN) conversion process. We found that the four small molecules can rapidly activate the hedgehog signaling pathway while downregulating many glial genes such as FN1 and MYL9 within 24 h of treatment. Chemical reprogramming is mediated by several waves of differential gene expression, including upregulation of hedgehog, Wnt/β-catenin, and Notch signaling pathways, together with downregulation of TGF-β and JAK/STAT signaling pathways. Our gene network analyses reveal many well-connected hub genes such as repulsive guidance molecule A (RGMA), neuronatin (NNAT), neurogenin 2 (NEUROG2), NPTX2, MOXD1, JAG1, and GAP43, which may coordinate the chemical reprogramming process. Together, these findings provide critical insights into the molecular cascades triggered by a combination of small molecules that eventually leads to chemical conversion of astrocytes into neurons.

Ferroptosis inhibitor alleviates Radiation-induced lung fibrosis (RILF) via down-regulation of TGF-\u03b21

BackgroundRadiation-induced lung fibrosis (RILF) is a severe and life-threatening complication of thoracic radiotherapy. Cell death is the key issue in RILF. Ferroptosis is a form programmed cell death implicated in the pathologies of inflammation. This study aimed to investigate the role of ferroptosis in RILF, and the effectiveness and the potential underlying mechanism of ferroptosis inhibitor on RILF.MethodsImmunofluorescence, western blot and RT-PCR assays were performed to examine the ferroptosis maker glutathione peroxidase 4 (GPX4) in a mice RILF model. The lung tissue sections were stained with hematoxylin and eosin (H&E), Masson trichrome staining and Sirius-Red staining to evaluate the histopathological changes in RILF mice. Reactive oxygen species (ROS) and hydroxyproline (HYP) in lungs were measured by the relevant kits. The serum levels of inflammatory cytokines (TNF-α, IL-6, IL-10, and TGF-β1) were measured with Elisa. The protein and mRNA levels of GPX4, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), hemeoxygenase-1 (HO1) and quinone oxidoreductase 1 (NQO1) in lungs were examined by western blot and RT-PCR.ResultsGPX4 levels of the irradiated lungs were significantly down-regulated than the groups with no irradiation, and the ferroptosis inhibitor, liproxstatin-1, increased GPX4 levels significantly in RILF mice. Treatment with liproxstatin-1 lowered the Szapiel and Ashcroft scores significantly, down-regulated the levels of ROS and HYP in lungs and reduced the serum inflammatory cytokines levels in RILF mice. The protein and the mRNA levels of Nrf2, HO1 and NQO1 were up-regulated by liproxsratin-1 in RILF.ConclusionsOur data suggested that ferroptosis played a critical role in RILF, ferroptosis inhibitor liproxstatin-1 alleviated RILF via down-regulation of TGF-β1 by the activation of Nrf2 pathway. The effectiveness of ferroptosis inhibition on RILF provides a novel therapeutic target for RILF.

The microRNA-141-3p/ CDK8 pathway regulates the chemosensitivity of breast cancer cells to trastuzumab.

This study was conducted to explore the function ofmicroRNA-141-3p/cyclin-dependent kinase 8 (miR-141-3p/CDK8) in regulating trastuzumab resistance of breast cancer cells. Microarray analysis was performed to screen microRNAs that are differentially expressed in wild type and trastuzumab-resistant (TR) breast cancer cell lines. TargetScan helped predict the target gene of miR-141-3p. The regulatory relationship was confirmed through a luciferase reporter assay, quantitative reverse transcriptase polymerase chain reaction, and Western blot analysis. The MTT assay, transwell invasion assay, and wound scratch assay were performed to measure the proliferative, invasive, and migratory ability of breast cancer cells, respectively. Tumor cell xenografts in nude mice were conducted to observe the effect of miR-141-3p on trastuzumab resistance in breast cancer cells in vivo. The enzyme-linked immunosorbent assay was used to detect protein secretion. miR-141-3p was downregulated in the drug-resistant cell lines. CDK8 was proved to be a target gene of miR-141-3p. Transfection of miR-141-3p or CDK8 small interfering RNA (siRNA) reversed the resistance to trastuzumab in TR cell lines and suppressed cell invasion and migration. Dysregulation of transforming growth factor beta (TGF-β) was detected when the expression of CDK8 was silenced by CDK8 siRNA, and downregulation of TGF-β had a notable effect on reducing the phosphorylation of SMAD2/SMAD3. miR-141-3p could restore the sensitivity to trastuzumab in breast cancer cells by repressing CDK8, which might regulate the phosphorylation levels of SMAD2/SMAD3 via TGF-β.

EB1-3 Chain of IL-35 Along With TGF-β Synergistically Regulate Anti-leishmanial Immunity.

Immunosuppression is a characteristic feature of chronic leishmaniasis. The dynamicity and the functional cross talks of host immune responses during Leishmania infection are still not clearly understood. Here we explored the functional aspects of accumulation of immune suppressive cellular and cytokine milieu during the progression of murine visceral leishmaniasis. In addition to IL-10 and TGF-β, investigation on the responses of different subunit chains of IL-12 family revealed a progressive elevation of EBI-3 and p35 chains of IL-35 with Leishmania donovani infection in BALB/c mice. The expansion of CD25 and FoxP3 positive T cells is associated with loss of IFN-γ and TNF-α response in advanced disease. Ex-vivo and in vivo neutralization of TGF-β and EBI-3 suggests a synergism in suppression of host anti-leishmanial immunity. The down-regulation of EBI-3 and TGF-β is crucial for re-activation of JAK-STAT pathway for induction as well as restoration of protective immunity against L. donovani infection.

The protective efficacy of soursop fruit extract against hepatic injury associated with acetaminophen exposure is mediated through antioxidant, anti-inflammatory, and anti-apoptotic activities.

In the current report, we examined the potential beneficial role of soursop fruit extract (SSFE) on liver injury induced by a single paracetamol (APAP) overdose (2000mg/kg). Thirty-five Wistar albino rats were randomly divided into five groups as follows: control, SSFE, APAP, SSFE+APAP, and silymarin (SIL)+APAP. APAP intoxication was found to elevate alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin levels. Moreover, it increased the levels of malondialdehyde, nitrites, and nitrates and depleted glutathione, superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase. APAP intoxication inactivated the nuclear factor erythroid 2-related factor 2 (Nrf2) defense pathway and upregulated the expression of heme oxygenase-1 (HO-1). APAP administration enhanced the activation of nuclear factor-kappa B (NF-κB), the elevation of tumor necrosis factor-alpha and interleukin 1-beta levels, and the upregulation of inducible nitric oxide synthase mRNA expression. In addition, APAP activated the overexpression of Bax protein, increased release of cytochrome c, and the downregulation of Bcl-2 protein. Finally, APAP-induced overexpression of transforming growth factor-beta (TGF-β) further suggested enhanced liver damage. On the other hand, SSFE pretreatment attenuated these biochemical, molecular, and histopathological alterations in the liver, which might be partially due to the regulation of hepatic Nrf2/HO-1 and downregulation of NF-κB and TGF-β.

MicroRNA-29a Disrupts DNMT3b to Ameliorate Diet-Induced Non-Alcoholic Steatohepatitis in Mice.

MicroRNA-29 (miR-29) has been found to reduce liver inflammation and fibrosis following a liver injury. Meanwhile, DNA methyltransferase has been reported to participate in the development of non-alcoholic steatohepatitis (NASH). The aim of this study is to investigate the miR-29a regulation of methyltransferase signaling and epigenetic program in NASH progression. Methods: miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates were subjected to the methionine-choline-deficient (MCD) diet-induced animal model of NASH. Primary hepatic stellate cells were transfected with a miR-29a mimic and antisense inhibitor. We then analyzed gene expressions with qRT-PCR, immunohistochemical stain, Western blot, and luciferase reporter assay. The results demonstrated that increased miR-29a alleviated the MCD diet-induced body weight loss and steatosis and decreased aspartate aminotransferase (AST) levels in mice. Furthermore, hepatic tissue in miR-29aTg mice displayed a weak fibrotic matrix, as shown with Sirius Red staining concomitant with low fibrotic α-SMA expression within affected tissues compared to the wild-type mice fed the MCD diet. Forced miR-29a expression reduced the MCD diet exaggeration of reactive oxygen species (ROS) production by immunohistochemically staining 8-OHdG. Increased miR-29a signaling also resulted in the downregulation of DNMT3b, TGF-β, IL-6, heme oxygenase-1 (HO-1), p-SMAD3, PI3K, and L3BII expression within the liver tissue. An in vitro luciferase reporter assay further confirmed that miR-29a mimic transfection reduced DNMT3b expression in primary HSCs. Our data provide new insights that miR-29a improves MCD diet-induced liver inflammation, steatosis and fibrosis, and highlight the potential of miR-29a targeted therapy for treating NASH.

Long non-coding RNA-neighboring enhancer of FOXA2 inhibits the migration and invasion of small cell lung carcinoma cells by downregulating transforming growth factor-β1.

Long non-coding RNA-neighboring enhancer of FOXA2 (lncRNA-NEF) is a recently identified tumor suppressor in hepatocellular carcinoma. The present study aimed to investigate the role of lncRNA-NEF in small cell lung carcinoma (SCLC). Expression levels of lncRNA-NEF in the lung biopsy tissues and plasma samples from patients with SCLC and from healthy controls were detected using reverse transcription-quantitative polymerase chain reaction. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic value of lncRNA-NEF as a marker of SCLC. The association between plasma levels of lncRNA-NEF and the clinical data of patients was analyzed using the χ2 test. An lncRNA-NEF expression vector was prepared and transfected into SCLC cells, and cellular migration and invasion were detected using Transwell migration and invasion assays, respectively. The expression of transforming growth factor β1 (TGF-β1) was detected using western blotting. The results demonstrated that the expression level of lncRNA-NEF was lower in patients with SCLC compared with that in healthy controls. The expression level of lncRNA-NEF in the plasma was associated with distant tumor metastasis. lncRNA-NEF overexpression inhibited SCLC cell migration and invasion, resulting in TGF-β1 downregulation, while treatment with exogenous TGF-β1 reduced the inhibitory effects of lncRNA-NEF overexpression on migration and invasion. Therefore, it was concluded that lncRNA-NEF inhibited the migration and invasion of SCLC cells, which was potentially associated with the downregulation of TGF-β1.

Kangxianling decoction prevents renal fibrosis in rats with 5/6 nephrectomy and inhibits Ang II-induced ECM production in glomerular mesangial cells

Renal fibrosis is a common pathological change in all stages of kidney disease. Kangxianling decoction was widely used in patients with chronic kidney disease, which could improve symptoms such as poor appetite, edema, and fatigue. However, its effect on renal fibrosis remains to be studied. In this study, we investigated its effects on renal fibrosis in a rat model of 5/6 Nephrectomy (5/6 N) in vivo and in angiotensin II (Ang II)-treated rat glomerular mesangial cells (HBZY-1) in vitro. Our data showed that 5/6 N induced renal fibrosis and combined with the activation of JNK signaling, the upregulation of transforming growth factor-β (TGF-β), collagen I (Col-I) and fibronectin (FN). The administration of kangxianling decoction inhibited the activation of JNK signaling and attenuated the deposition of extracellular matrix (ECM) proteins in damaged kidneys. In HBZY-1 cells, Ang II increased the protein expression of Col-I and FN. It also activates JNK signaling and TGF-β in a time-dependent manner. Treatment of the HBZY-1 cells with kangxianling decoction blocked Ang II-induced JNK activation and ECM overproduction. Our results indicated that Kangxianling Decoction could reduce renal fibrosis, accompanied by inhibiting the production of ECM proteins and JNK, along with downregulation of TGF-β, Ang II.

Mycobacterium smegmatis But Not Mycobacterium avium subsp. hominissuis Causes Increased Expression of the Long Non-Coding RNA MEG3 in THP-1-Derived Human Macrophages and Associated Decrease of TGF-β.

Pathogenic mycobacteria are able to persist intracellularly in macrophages, whereas non-pathogenic mycobacteria are effectively combated and eliminated after their phagocytosis. It is known that TGF-β plays an important role in this context. Infection with pathogenic mycobacteria such as Mycobacterium tuberculosis or M. avium leads to production of active TGF-β, which blocks the ability of IFN-γ and TNF-α to inhibit intracellular replication. On the other hand, it is known that the long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) is involved in the regulation of TGF-β. In this study, we show how the infection of THP-1-derived human macrophages with the saprophytic M. smegmatis but not with the facultatively pathogenic M. avium subsp. hominissuis leads to increased MEG3 expression. This is associated with the downregulation of DNA methyltransferases (DNMT) 1 and 3b, which are known to regulate MEG3 expression via promoter hypermethylation. Consequently, we observe a significant downregulation of TGF-β in M. smegmatis-infected macrophages but not in M. avium subsp. hominissuis pointing to lncRNAs as novel mediators of host cell response during mycobacterial infections.

KLF4 Regulates Corneal Epithelial Cell Cycle Progression by Suppressing Canonical TGF-β Signaling and Upregulating CDK Inhibitors P16 and P27.

PurposeKrüppel-like factor 4 (KLF4) promotes corneal epithelial (CE) cell fate while suppressing mesenchymal properties. TGF-β plays a crucial role in cell differentiation and development, and if dysregulated, it induces epithelial-mesenchymal transition (EMT). As KLF4 and TGF-β regulate each other in a context-dependent manner, we evaluated the role of the crosstalk between KLF4 and TGF-β-signaling in CE homeostasis.MethodsWe used spatiotemporally regulated ablation of Klf4 within the adult mouse CE in ternary transgenic Klf4Δ/ΔCE (Klf4LoxP/LoxP/ Krt12rtTA/rtTA/ Tet-O-Cre) mice and short hairpin RNA (shRNA)-mediated knockdown or lentiviral vector-mediated overexpression of KLF4 in human corneal limbal epithelial (HCLE) cells to evaluate the crosstalk between KLF4 and TGF-β-signaling components. Expression of TGF-β signaling components and cyclin-dependent kinase (CDK) inhibitors was quantified by quantitative PCR, immunoblots, and/or immunofluorescent staining.ResultsCE-specific ablation of Klf4 resulted in (1) upregulation of TGF-β1, -β2, -βR1, and -βR2; (2) downregulation of inhibitory Smad7; (3) hyperphosphorylation of Smad2/3; (4) elevated nuclear localization of phospho-Smad2/3 and Smad4; and (5) downregulation of CDK inhibitors p16 and p27. Consistently, shRNA-mediated knockdown of KLF4 in HCLE cells resulted in upregulation of TGF-β1 and -β2, hyperphosphorylation and nuclear localization of SMAD2/3, downregulation of SMAD7, and elevated SMAD4 nuclear localization. Furthermore, overexpression of KLF4 in HCLE cells resulted in downregulation of TGF-β1, -βR1, and -βR2 and upregulation of SMAD7, p16, and p27.ConclusionsCollectively, these results demonstrate that KLF4 regulates CE cell cycle progression by suppressing canonical TGF-β signaling and overcomes the undesirable concomitant decrease in TGF-β–dependent CDK inhibitors p16 and p27 expression by directly upregulating them.

Effect of Miao medicine, Jinwujiangu decoction, on IL- 17/IL-23 inflammatory axis of fibroblast-like synoviocytes in rheumatoid arthritis

Purpose: To explore the influence of the Miao medicine, Jinwujiangu decoction, on the interleukin (IL)- 17/IL-23 inflammatory axis of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA).Methods: Synovial tissue samples were randomly divided into a blank control group, high-dose (0.06mg/mL), medium-dose (0.6mg/mL), and low-dose (6.0mg/mL) groups of Jinwujiangu decoction, a leflunomide group, and a tripterygium glycosides group. Proliferation of RA synovial cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion of IL-6, transforming growth factor beta (TGF-β), and IL-17. Real-time polymerase chain reaction was used to evaluate the expression of IL-23R, IL-17R, RAR-related orphan receptor alpha (RORα), RORγt, and signal transducer and activator of transcription (STAT3) mRNA. The protein activities of IL-17R, STAT3 and pSTAT3 were assessed by Western blot assay.Results: Jinwujiangu decoction inhibited the proliferation of RA synovial cells. Treatment with different drug concentrations resulted in downregulation of IL-6, TGF-β, and IL-17 secretion. The expression levels of IL-23R, IL-17R, RORα, RORγt, and STAT3 mRNA in RA-FLS were significantly reduced after intervention with different drugs. Protein expression levels of STAT3, pSTAT3, and IL-17 in the different drug treatment groups were significantly decreased.Conclusion: Jinwujiangu decoction inhibits the secretion of IL-6 and TGF-β in RA-FLS, and intervenes to regulate gene expression of IL-23/IL-17 inflammation axis and suppress immune inflammation. The results of this study provide new evidence for the study of anti-inflammatory mechanism of TCM compound prescription.Keywords: Jinwujiangu decoction, IL-17/IL-23, Fibroblast-like synoviocytes, Rheumatoid arthritis, Ethnomedicine

Protein arginine methyltransferase 1 promotes epithelial-mesenchymal transition via TGF-β1/Smad pathway in hepatic carcinoma cells.

Protein arginine methyltransferase 1 (PRMT1) is dysregulated in a number of human cancers. Herein, we report that PRMT1 expression is directly associated with epithelial-mesenchymal transition (EMT) in hepatic carcinoma cells. Firstly, we find that PRMT1 expression is higher in hepatic carcinoma tissues than that in normal liver tissues at both mRNA and protein levels, and higher expression of PRMT1 correlates with poor survival in liver tumors. The data in vitro reveals that PRMT1 knockdown inhibits the abilities of proliferation, migration and invasion, while PRMT1 overexpression promotes the above behaviors in hepatic carcinoma cells. Further studies indicate that PRMT1 knockdown remarkably decreases the expression of mesenchymal markers including Vimentin, Snail and N-cadherin, and upregulates expression of epithelial markers E-cadherin. Conversely, PRMT1 overexpression results in the opposite effects. Additionally, we identified that PRMT1 knockdown resulted in downregulation of TGF-β1, p-Smad2 and p-Smad3, while PRMT1 overexpression activated TGF-β1, p-Smad2 and p-Smad3. These findings suggest that PRMT1 promotes EMT in hepatic carcinoma cells probably via TGF-β1/Smad pathway, and might represent a novel anti-liver cancer strategy.

Decreased expression of transforming growth factor-β1 and α-smooth muscle actin contributes to the protection of lotensin against chronic renal failure in rats

Background: Lotensin has been shown to have a protective function in the early stage of chronic renal failure. However, its role in the intermediate and late stages of chronic renal failure remains largely unknown. The present study aimed to investigate the role and underlying mechanism of lotensin in advanced chronic kidney disease.Methods: Female Wistar rats were randomly divided into three groups (n = 10): sham group, 5/6 nephrectomy (5/6 Nx) group, and lotensin group (oral administration of lotensin for 9 weeks following 5/6 Nx). Rats were sacrificed and pathological parameters were measured. Western blot assay and immunohistochemical staining were performed to detect the expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in kidney tissues.Results: Compared to the 5/6 Nx group, lotensin administration significantly decreased 5/6 Nx-induced elevation in blood urea nitrogen, serum creatinine and 24-h urinary protein excretion (UPE) rates, but markedly increased red blood cell count, plasma albumin and hemoglobin levels, along with improved renal morphology. Mechanistically, lotensin dramatically downregulated the renal expression of TGF-β1 and α-SMA induced by 5/6 Nx.Conclusions: Lotensin protects against advanced chronic kidney disease in rats with 5/6 Nx through the downregulation of TGF-β1 and α-SMA.

Suppressive effect of bacoside A on hypertrophic scar formation by downregulation of TGF-β1

Purpose: To determine the anti-scarring property of bacoside A (BA) in a rabbit model of post-burns hypertrophic scar (HS). Methods: A total of 16 healthy male rabbits were divided into four groups (4 rabbits/group). The rabbits were subjected to full-thickness burn wound by placing a 10-mm brass rod at 90 oC on their ears for 20s, and were untreated (HS group). The remaining rabbits also underwent HS process but were topically treated daily with 0.2, 0.4 or 0.6 % BA gel (HS + 0.2, 0.4, and 0.6 % BA groups) from day 8 to day 34. Results: The mean values of collagen (types I & III), hydroxyproline (precursor of collagen) and hexosamine, as well as scar index significantly (p < 0.001) decreased after 28 days of topical treatment with BA. Moreover, BA application resulted in improved histological changes with proper arrangement of collagen fibers. Significant (p < 0.001) upregulation or downregulation of transforming growth factor beta-1 (TGF-β1) and matrix metalloproteinase-1 (MMP-1) were observed in the BA-treated rabbits. Conclusion: Treatment with BA significantly lowers the scarring area of burn wounds by downregulation of TGF-β1. Thus, BA can potentially be developed into a drug for managing injury scars. Keywords: Hypertrophic scar, Bacoside A, Scar elevation index, Transforming growth factor beta-1, Collagen